of mangroves - VLIZ · Mangroves are adapted to this direct salt impact by salt ﬁltration at the...

Growing on the edge

Hydraulic architecture of mangroves

Ecological plasticity and functional signifi cance of water conducting tissue

in Rhizophora mucronata and Avicennia marina

Nele Schmitz

Growing on the edge

Print: DCL Print & Sign, Zelzate

© 2008 Nele Schmitz

© 2008 Uitgeverij VUBPRESS Brussels University PressVUBPRESS is an imprint of ASP nv (Academic and Scientific Publishers nv)Ravensteingalerij 28B-1000 BrusselsTel. ++32 (0)2 289 26 50Fax ++32 (0)2 289 26 59E-mail: [email protected]

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Growing on the edgeHydraulic architecture of mangroves:

ecological plasticity and functional significance of water conducting tissue in Rhizophora mucronata

and Avicennia marina

Thesis

Submitted in fulfillment of the requirements for the degree of Doctor of Philosophy in Sciences

of the Vrije Universiteit Brussel

by

Nele Schmitz

May 2008

Promotors

Nico KoedamLaboratory of Plant Biology and Nature ManagementVrije Universiteit Brussel

Hans BeeckmanLaboratory of Wood Biology and XylariumRoyal Museum for Central Africa

This research project was a collaboration between the Laboratory of Plant Biology and Nature Management of the Vrije Universiteit Brussel and the Laboratory of Wood Biology and Xylarium of the Royal Museum for Central Africa, Tervuren and was funded by the Institute for the Promotion of Innovation through Science and Technology in Flanders. Research stays abroad were sponsored by the European Commission‘s Research Infrastructure Action via the Synthesys Project and by the European Cooperation in the field of Scientific and Technical Research programme (COST) on Cell wall macromolecules and reaction wood (CEMARE) via a Short Term Scientific Mission. Fieldwork expeditions were financially supported by the National Fund for Scientific Research (FWO, Belgium), the Schure-Beijerinck-Popping Fonds (Koninklijke Nederlandse Akademie van Wetenschappen, The Netherlands) and the Flemish Interuniversity Council (VLIR).

Photos: Nele Schmitz, Elisabeth Robert.

All men by nature desire to know.ARISTOTELES

A man should live if only to satisfy his curiosity.A YIDDISH PROVERB.

Contents

Outline of the thesis 9

General introduction 11

General objective & hypotheses 22

Part I Hydraulic architecture of mangroves: ecological plasticity & 24functional significance

Summarizing Abstract 25

Chapter 1 Influence of a salinity gradient on the vessel characters of the 27mangrove species Rhizophora mucronata Lam.

Chapter 2 Comparative anatomy of intervessel pits in two mangrove species 39growing along a natural salinity gradient in Gazi Bay, Kenya.

Chapter 3 Intervessel pit structure and histochemistry of two mangrove 53species as revealed by cellular UV microspectrophotometry and electron microscopy: intraspecific variation and functional significance

Part II Implications of successive cambia for the hydraulic architecture 64of Avicennia marina

Summarizing Abstract 65

Chapter 4 Successive cambia development in Avicennia marina (Forssk.) Vierh. 67 is not climatically driven in the seasonal climate at Gazi Bay, Kenya.

Chapter 5 A patchy growth via successive and simultaneous cambia: key to 79success of the most widespread mangrove species Avicennia marina?

General conclusion & perspectives 93

References 101

Samenvatting 123

Acknowledgements 127

Curriculum vitae 129

10

Outline of the thesis

Wonder is the beginning of wisdom.

11

Outline of the thesis

Outline of the thesisEcological wood anatomy is a challenging field of study. It is a quest for environmental information, which is a complex mix of manifold environmental variables intertwined with the trees genetic blueprint, to unravel how trees adapt their cellular make-up for survival under ambient and site-specific conditions, whether predictable or not. Sprouting ideas from the numerous, patiently and carefully observed and described wood samples give rise to carefully stated anatomy-environment relationships. Always alert for new findings in tree physiology fitting these observations, the researcher may be lead to new insights in the trees’ structure and functioning. Therefore, in a second stage, these hypotheses should be tested experimentally for physiological validation. This awareness of the not yet complete fundaments of ecological wood anatomical hypotheses is essential but certainly not undermining the value of ecological wood anatomy. In contrast to physiological studies, there is neither a restriction on the choice of the studied species nor on the study site, the number of samples analysed, the age or the size of the study objects, allowing profound knowledge on formation characteristics of the trees’ wood via comparison of environmentally different locations, species as well as individual trees.

Wood anatomy can be studied in view of the technical applications of wood, tree pathology, ecology, dendrochronology but also hydraulics. In this work, the introduction addresses the CHALLENGE for mangrove trees to transport water in their hydraulically stressful environment and the wood anatomical APPROACH to understand the way mangroves successfully respond to these high environmental demands. The current knowledge about the water transport of trees at risk of bubble formation is presented together with the possible solutions to minimize or to prevent these negative impacts. However, despite their ultimate position to study the regulation of the water transport under stress conditions, insight in the hydraulic structure of mangroves, its ecological plasticity and functional significance remains extremely scanty. The HYPOTHESES tested in this study are presented. The first part of the thesis highlights the hydraulic architecture of mangroves in relation to the specific requirements of the regularly flooded and saline environment. The study focuses on two species, Rhizophora mucronata and Avicennia marina, growing under contrasting conditions but with a certain overlap. The FIRST CHAPTER starts with the influence of soil water salinity on vessel characteristics and ends with the question why vessel diameters are nearly constant. The SECOND CHAPTER aims to answer this question by searching for a trend in intervessel pit anatomy. The remarkable variation in intervessel pit membrane thickness and electron density is the subject of the THIRD CHAPTER, presenting a study of the topochemistry of the intervessel pits of both mangrove species. The second part of the thesis focuses on the formation of wood by successive cambia and its implications for the hydraulic architecture. While the anatomical development of growth layers via successive cambia has been described for some species, the periodicity of the growth layer formation as a whole has never been addressed before. In the FOURTH CHAPTER the annual character of the growth layers of Avicennia marina is investigated while CHAPTER FIVE proposes a growth mechanism via successive cambia and the potential ecological and functional advantage of this peculiar anatomical structure for life in the mangrove environment. The thesis is completed with a general conclusion on the plasticity of the water transport system of mangrove trees, the functional advantage of having successive cambia and this all in view of their survival in an environment characterized by exceptional growth conditions, which are also to some extent unpredictable.

12

General Introduction

All things are difficult before they are easy.

13



Structure

The problem: water transport in mangroves, a risky business 12

▪ The mangrove environment ▪ Water transport and its limitations

Approach: the added value of ecological wood anatomy in clarifying tree functioning 16

▪ The tissue level▪ The cellular level▪ The sub-cellular level

General objective & hypotheses 22

▪ Hypothesis I: ecological plasticity within species▪ Hypothesis II: ecological significance of successive cambia

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From the origin of life on land, water uptake and transport against gravitational forces and under the restrictions imposed by the environment are one of the major challenges for plants (Kramer and Boyer, 1995; Kozlowski and Pallardy, 1997; Maseda and Fernández, 2006). Both for growth and species distribution water availability clearly is one of the most determining factors for land plants (e.g. Ball, 1998; Brodribb and Hill, 1999; Pockman and Sperry, 2000; Maherali et al., 2004). This is especially true for tall growing trees since tree height is constrained by water-controlled physiological factors (Woodward, 2004; Zaehle, 2005; Holbrook and Zwieniecki, 2008). Nevertheless, a successful forest ecosystem with trees reaching heights of several tens of meters developed along the coasts of the tropical and subtropical climate zone: mangroves (Tomlinson, 1994; Spalding et al., 1997; Kathiresan and Bingham, 2001). Spontaneously the question arises how mangrove trees manage to survive under these sometimes extreme but almost permanently limiting conditions for water transport. More specifically, what is the effect of the salty water and the alternation between periods of drought and periods of flooding on mangrove functioning, which is to a large extent the transport of water? To understand tree hydraulics the primary need is to understand the structure of the hydraulic network, which will at once clarify species distributions and limits on tree height (e.g. Tyree and Ewers, 1991; Hacke et al., 2001a; Anfodillo et al., 2006; Loepfe et al., 2007; Petit et al., 2007; Sperry et al., 2008).

This study wishes to contribute to unravel the hydraulic structure of mangrove trees with the aim to gain insight in its ecological plasticity and functionality. First, clarification for two basic questions must be sought: (1) “What makes the mangrove environment so stressful for transporting water?” and (2) “How can ecological wood anatomy help in understanding mangrove functioning under the stressful environmental conditions of the mangrove habitat?”.

The problem: water transport in mangroves, a risky business

The mangrove environment

What distinguishes mangrove trees from other plants prone to water stress? Halophytes (Munns, 2002), desert plants, trees of inundation forests (Kozlowski, 1984); they all are subjected to some kind of water stress. The difference with mangroves is that they have to endure a combination of salt, heat, periodic flooding and, depending on the specific location and tidal regime, periods of water shortage. And still, mangrove trees do not generally adopt a typical drought-stressed physiognomy with characters such as small, curling or deciduous leaves and trees of low stature. The negative impact of gravity and frictional resistance on water flow is of a higher order in trees compared to relatively short stemmed halophytic herbs (McDowell et al., 2002; Phillips et al., 2003; Woodruff et al., 2004; Meinzer et al., 2005). The unfavourable effect of tree height on water flow is illustrated by the dwarfed stature of mangroves under limiting growing conditions (Feller, 1995; Lovelock et al., 2004; Lovelock et al., 2006; Naidoo, 2006). Salt has a double negative impact on plant growth. In first instance, salt increases the osmotic potential of the soil water affecting plant growth by exerting a water stress (Naidoo, 1985, 1986; Lin and Sternberg, 1992). Only with time, sodium and chloride ions can accumulate to toxic levels leading to premature leaf senescence (Munns, 2002; Tester and Davenport, 2003). Mangroves are adapted to this direct salt impact by salt filtration at the roots or salt secretion via leaf glands (Scholander et al., 1962; Drennan and Pammenter, 1982; Sobrado, 2004). The stress on the water column, however, can be intensified by soil drought, which increases soil water salinity depending on tidal regime, temperature and relative humidity. A second amplifier of the salt stress mangroves have to cope with is the periodic inundation with sea water. First, flooding results in temporary or permanently waterlogged soils, affecting oxygen and nutrient availability and hence plant growth (Naidoo, 1985; Middelburg et al., 1996; Barrett-Lennard, 2003; Krauss et al., 2006a; Krauss et al., 2006b). Second, the tidal regime makes the mangrove habitat extraordinarily

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dynamic because of the daily but also the monthly rhythm (e.g. Hoguane et al., 1999; Bala Krishna Prasad and Ramanathan, 2005). A fluctuating salinity brings more suffering to plants than a constant salinity of the same mean (Lin and Sternberg, 1993). The degree of fluctuation in pore water salinity of the substrate depends on tidal amplitude, the position along the coastal slope, as well as on local hydrological characteristics (Kitheka, 1996, 1997, 1998) that can be affected by human action (Moritz-Zimmermann et al., 2002; Gopal and Chauhan, 2005; Liu, 2005).

The mangrove forest of Gazi Bay in Kenya, the study area, has a tidal amplitude of about 3.8 m with a maximum of 4.1 m (Kenya Ports Authority tide tables for Kilindini, Mombasa) and is characterized by a sloping topography (Matthijs et al., 1999). The resulting gradient in tidal elevation parallels the zoned species distribution. Two major species of the forest in Gazi are Rhizophora mucronata Lam., restricted to the seaward side of the forest with the exception of the upstream area of the river Kidogoweni where salinity is influenced by fresh river water, and Avicennia marina (Forssk.) Vierh. with a disjunct distribution in the forest, occurring both at the seaside and the landward side where trees are stunted (Beeckman et al., 1990; Dahdouh-Guebas et al., 2004). For most other mangrove species, the highly dynamic conditions of the landward zone are unbearable (Clough, 1984; Naidoo, 1985). Due to its upper tidal location and Kenya’s seasonal climate (McClanahan, 1988; Lieth et al., 1999), the trees that are growing here experience drought periods up to several weeks a month, associated with rising salinity levels from 34 ‰ (sea water salinity) to 100 ‰ and even higher during the dry season. In the rainy season, salinity drops to levels approaching fresh water during the weeks without inundation. At the seaward side of the forest, trees are flooded daily by seawater of almost constant salinity over the seasons. Trees growing at a different distance from the sea are thus characterized by an equally different water status, related with different risk levels for the water transport. A. marina, occurring at both extremes of the coastal slope, reflects the contrasting growth conditions in its morphology with dwarf trees towards the land and trees of impressive stature towards the sea. The meaning of water stress for trees is the chance of failure in keeping a continuous water flow from soil to canopy.

Water transport and its limitations

According to the cohesion-tension theory water is transported up the tree driven by a tension gradient created by transpirational water loss from the leaf surface (Kramer and Boyer, 1995; Milburn, 1996; Kozlowski and Pallardy, 1997; Tyree, 1997; Meinzer et al., 2001; Steudle, 2001). Although widely accepted, the theory has since long been challenged by alternative explanations questioning the existence of negative pressures or tensions in the xylem conduits (Steudle, 1995; Wei et al., 1999; Sperry et al., 2003; Tyree, 2003). The debate has been fostered by the technical difficulty in measuring xylem tensions without serious chance of artefacts. Nevertheless, rather than claiming the cohesion theory to be false the opponents propose a yet controversial (Sperry et al., 1996; Wei et al., 2000; Zimmermann et al., 2002b; Brooks, 2004) multiforce theory (Zimmermann et al., 1993; Zimmermann et al., 1994; Zimmermann et al., 2002a; Zimmermann et al., 2004) where the tension gradient is supported by forces such as tissue pressure (Canny, 1995) or gradients in the chemical activity of water (Plumb and Bridgman, 1972) and water absorption from the atmosphere adds to transpirational water uptake (Zimmermann et al., 2007). The basic pulling force resulting from transpiration implies that the water in xylem vessels is in a metastable state and thus vulnerable for cavitation which is the phase change from water to vapour. When the initially small bubble expands, blocking the ascent of water, the vessel is said to be embolized (Tyree and Sperry, 1989). Cavitation can be induced by drought or by freezing following two separate mechanisms (Cochard, 2006). Drought-induced cavitation takes place via intervessel pits, which are small canals in the vessel walls and vessel endings that allow transfer of water from one vessel to another (Fig. 1). By offering a way to circumvent gas-filled vessels they safeguard an efficient water flow but they are also the weak points where gas bubbles can nucleate.

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In this way intervessel pits at least partly determine the degree of compartmentalization of the water transport system that is supposed to be balanced against prevailing environmental conditions (Orians et al., 2004; Ellmore et al., 2006). The mechanism behind drought-induced cavitation is explained by the air-seeding theory (Sperry and Tyree, 1988; Oertli, 1993; Jarbeau et al., 1995; Salleo et al., 1996) that is based on the cohesion tension theory by putting a steep tension gradient, caused by soil water deficit or salinity, at its origin. In the middle of the pit chamber between two vessels, a more or less porous pit membrane is situated that is composed of remnants of the primary cell walls and the middle lamella (Fig. 1). According to the theory, an air bubble is seeded when the pressure gradient over the pit membrane surpasses a critical pressure, determined by the pit membrane pore size and the surface tension of the gas-water meniscus (Fig. 2a). The air is sucked in from neighbouring already air-filled vessels that are common in a plant (Tyree et al., 1994). Cycles of cavitation and refilling take place on a diurnal basis (Cochard et al., 2000; Melcher et al., 2001; Domec et al., 2006b; Woodruff et al., 2007), in parallel with variations in transpiration rate (Bucci et al., 2003) and also common processes like leaf abscission and herbivory cause cavitation. Moreover, although spontaneous air-seeding in xylem sap is extremely rare (Steudle, 2001), it is uncertain at this moment whether gas nucleation also happens via still other mechanisms than air-seeding at pit membranes and if yes what their contribution may be in the inactivation of parts of the sapwood (Tyree and Dixon, 1986; Tyree, 1997; Shen et al., 2002). Freeze-induced cavitation happens by air exsolving from the frozen water when melting. Intervessel pits are thus not involved in this mechanism (Hacke and Sperry, 2001).

Fig. 1 Intervessel pits and their dif-ferent parts. A Scanning electron micrograph of a vessel of Avicennia marina composed of vessel elements (VE) with intervessel pit fields (arrow heads). B Detail of a pit field with at the left intervessel pits with the pit membrane (PM) still attached and at the right several intervessel pits where the pit chamber (Pch), giving access to the pit canal (Pc) became visible after removal of the pit mem-brane. Vestures (v) stick out from the pit canal into the pit chamber. C Transmission electron micrograph from a transverse section of A. ma-rina vessels showing the intervessel pits as connecting pathways for the water transport between vessels. Pit membranes can be seen in line with the compound middle lamella (cml). D Detail of a sectioned intervessel pit with indication of the different parts.

D Air pockets in intervessel pits between a functional and a refilling vessel compartmentalize vessel refilling (white frame in C). The inclination of the pit chamber walls (2α) and the contact angle between water and ves-sel wall (θ) determine the strength of the air-water meniscus to disconnect the vessel, under positive pressure, from the suction force of transpiration until full embolism repair.

30 µm

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D

5 µm

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Embolisms are not necessarily permanent although refilling seems not to be a universal process (Hacke and Sperry, 2003) and the mechanism is still a matter of debate. The obscurity is in the process of pressurizing the air-filled vessel while neighbouring vessels are still under tension. In this respect the importance of vessel associated cells has been mentioned long before (Braun, 1984; Beeckman, 1999). Osmotic water shifting by secretion of sugars into vessels has even been suggested as a mechanism for water ascent alternating with transpiration. Although the permanent presence of positive pressures in the hydrosystem has not found wide support, it agrees with the currently suggested mechanism for embolism repair. Large osmotically active solutes are supposed to be transported from vessel associated cells to the embolized vessel. Since the solutes are too large to cross pit membranes water is pulled in from neighbouring active vessels or parenchyma cells to refill the blocked vessel (Fig. 2b) (Hacke and Sperry, 2003). In the case the solutes are small (Fig. 2c-d), air-pockets in the intervessel pits can disconnect the air-filled vessel from the transpiration stream as long as the vessel is not yet fully refilled (Holbrook and Zwieniecki, 1999). An alternative or perhaps additional mechanism is the hydrolysis of starch within vessel associated cells leading to pressure imbalances after water uptake driving water in the embolized vessel (Bucci et al., 2003; Salleo et al., 2004). The water flow could be directed towards the embolized vessel via activation of specific water channels (De Boer and

meniscus

embolized vesselfunctional vessel

pit membranepore

A

Pc PcPchPch

C

refilling vesselfunctional vessel

air-pocket

B

largesolutessolutetransportwater

air

2αΘ

D

Fig. 2 Drought-induced cavitation as explained by the air-seeding theory and mechanisms of embolism repair. A Air is seeded in from a pore in the pit membrane when a critical pressure difference, between the already embolized vessel and the functional vessel, is exceeded and the gas-water meniscus breaks. B One proposed mechanism to refill embolized vessels is based on the transport of large osmotically active solutes from living vessel associated cells. Because the solutes are too big to cross pit membranes water is attracted from both the transpiration stream and neighbouring cells. The inflowing water creates a positive pressure in the refilling vessel, forcing the air back into solution (Fig. adapted from Hacke and Sperry, 2003). C An alternative mecha-nism of embolism repair depends on small solutes that also draw in water from neighbouring vessels but not from adjacent functional vessels. Since these solutes can cross pit membranes the refilling vessel should be disconnected from the transpiration stream to create a positive pressure. (continued on p. 14)

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Volkov, 2003). The trigger for starch hydrolysis might be vibration of the vessel walls upon cavitation (Salleo et al., 2004; Salleo, 2006). Although at present there is no conclusive evidence for one or a combination of the presented strategies (Stiller et al., 2005), all mechanisms are conclusive about the metabolic control over the process of embolism repair. Living tissue such as parenchyma and phloem (Salleo et al., 1996; Salleo et al., 2004; Salleo, 2006) seem to be the basic requirement for refilling to take place, which can happen in a time range of minutes (Facette et al., 2001; Zwieniecki et al., 2001a; Salleo et al., 2004). Under natural drought stress conditions, however, it could take days (Hacke and Sperry, 2003).

Cycles of cavitation and repair can not go on infinitely since the refilling process slows down (Lee and Kim, 2007) and since cavitation fatigue takes place reducing cavitation resistance (Hacke et al., 2001b). This weakening is most likely caused by irreversible membrane damage after stretching during cavitation (Domec et al., 2006a). It could be minimized by fibers acting as buttresses (Jacobsen et al., 2005) although this could not be supported in a more recent study (Pratt et al., 2007). It is yet unknown whether it concerns a permanent state but in each case there is a moment embolism repair does no longer take place and vessels become permanently blocked where after they can be filled with gums, tyloses (Hargrave et al., 1994) or mucilages (Ewers et al., 2004). More and more studies, however, prove cavitation to be part of a hydraulic signal to regulate the water status of the tree rather than being an unavoidable evil, associated with the transport of water under tension. Cavitated vessels could function as water storage tissue (Stratton et al., 2000; Melcher et al., 2001). Older xylem vessels with a lower cavitation resistance might secure optimal functioning of the younger sapwood by acting as a water reservoir (Melcher et al., 2003). Spatial partitioning of xylem vulnerability to the most distal parts of the tree can have the advantage of sacrificing less important or more easily refilled parts of the tree, saving and mitigating tension in surviving more basally oriented branches (Hacke and Sperry, 2001; Choat et al., 2005b; Domec et al., 2006b; Woodruff et al., 2007). It follows that the hydraulic system is not simply generated to avoid gas nucleation but to reach the most beneficial activity state. Therefore, trees adapt both structurally and physiologically to the prevailing environmental conditions.

Approach: the added value of ecological wood anatomy in clarifying tree functioning

The adaptations of trees to the environment, with its spatial and temporal variations, are situated on three related levels: tree morphology, tree physiology and wood anatomy (Maseda and Fernández, 2006). To understand the findings of physiological studies a profound understanding of the hydraulic basis, the structure of the water transport system, is required (e.g. Meinzer, 2003; Choat et al., 2005a; Domec et al., 2007; Loepfe et al., 2007). The direct relationship between hydraulic structure and water transport is reflected in its adaptations to changing stress conditions within and between trees and species growing under different environmental conditions.

In the formation of their water transport system trees strive to an optimal compromise between efficiency and safety (Wheeler et al., 2005; Hacke et al., 2006; Sperry et al., 2008). The trade-off results from the fact that a high efficiency to transport water brings along a facilitated spread and expansion of gas bubbles, blocking the water flow in sometimes extended parts of the conduit network. Across distantly related species no or only a weak trade-off was observed (Maherali et al., 2004), which might be explained by ample strategies to balance the water status of the tree, next to structural adaptations of the hydrosystem (Loepfe et al., 2007). Tree morphology and tree physiology come also into play when extending to the whole-tree level (e.g. Melcher et al., 2001; Cruiziat et al., 2002; Kocacinar and Sage, 2004; Bréda et al., 2006; Junghans et al., 2006; Krauss et al., 2006b; Sobrado and Ewe, 2006). In addition, one should keep in mind that the hydraulic architecture is also

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determined by internal factors such as the genetic background of the tree (Baas et al., 1983; February and Manders, 1999; Reich et al., 2003; Dünisch et al., 2004) and its mechanical requirements (Taneda and Tateno, 2004) although there should not necessarily be a trade-off with transport needs (Woodrum et al., 2003; Jacobsen et al., 2005; Sperry et al., 2006).

Here, we will discuss how the hydraulic structure can be adjusted to meet the safety requirements for sap flow under water stress. Since no freezing occurs at the Kenyan coast, only adaptations to drought stress will be discussed and this from the tissue level, over the cellular level to the sub-cellular level of the intervessel pits.

The tissue level

Water storage tissue is an essential part of the hydraulic architecture when aiming at insight in the water transport of trees. It makes possible a flexible relationship between trees and environment in regulating sap flow (Sperry et al., 2008). In exchange with the transpiration stream, stored water replenishes water shortages on a daily (Goldstein et al., 1998; James et al., 2003; Meinzer et al., 2003) or seasonal basis (Chapotin et al., 2006). The water storage capacity of trees is determined by water stores of symplast (parenchyma) and apoplast (cavitated vessel lumina, intercellular spaces) and is especially important in the adaptation to drought stress (Borchert and Pockman, 2005; Lopez et al., 2005; Scholz et al., 2007). As mentioned in the section ‘Water transport and its limitations’ parenchyma cells and especially phloem cells may play an additional role in embolism repair as water or energy source (Salleo et al., 2004; Salleo, 2006). Therefore, the potential to refill embolized vessels and to store water, which are not linked as such (Chapotin et al., 2006), is considerably higher in trees showing secondary growth via successive cambia. While most trees produce wood via one vascular cambium just underneath the bark, a minority of tree species grows in an anomalous way (Carlquist, 2001). One type of cambial variant is a succession of cambia where the latest developed cambium adds to and finally replaces the wood production of the former cambium (Fig. 3a). Represented by the mangrove genus Avicennia (Chapman, 1947; Gill, 1971; Zamski, 1979), this growth form is characterized by an alternation of xylem tissue and phloem strands embedded in parenchyma tissue (Fig. 3a-b). On sanded stem discs (Fig. 3c) the regular development of a new vascular cambium (Fig. 3d) results in a pattern of light (phloem) and dark (xylem) coloured bands (Esau, 1969; Carlquist, 2001). Given that phloem tissue in regularly growing trees is restricted to a narrow layer at the outside of the stem, its proportion is relatively higher in trees with successive cambia. Among the group of woody species showing this anomalous growth pattern are several lianas (León-Gomez and Monroy-Ata, 2005), which is explained by the advantage for scandent growth of a flexible stem where the rigid wood tissue is interspersed with thin-walled phloem (Nair, 1993; Carlquist, 2001; Olson, 2003; Araújo and Costa, 2006). Until now, the advantage of successive cambia in enlarging the storage capacity of trees for water or photosynthates, facilitating embolism repair, has been proposed by several authors (Fahn and Shchori, 1967; van Veenendaal and den Outer, 1993; den Outer and van Veenendaal, 1995; Olson, 2003; Rajput et al., 2005; Araújo and Costa, 2006). The ecological and functional significance of secondary growth via successive cambia remains however speculative and is in need for further examination.

The cellular level

On the cellular scale, vessel characters can be adapted to reduce the impact of cavitation on sap flow by preventing gas expansion and by minimizing the loss of conductive area upon embolization. By studying the vessel anatomy of trees from a broad phylogenetic and geographic range, facing various degrees of water stress, some general trends between vessel geometry and water stress became clear (Baas et al., 1983; Carlquist and Hoekman, 1985; Lindorf, 1994; Carlquist, 2001). The multitude of

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small-scale studies underscore their findings and characterize a relatively safe hydraulic structure by vessels of a small diameter (Hargrave et al., 1994; Jarbeau et al., 1995; Villar-Salvador et al., 1997; Corcuera et al., 2004; Junghans et al., 2006; Sobrado, 2007), composed of short vessel elements (Sun and Lin, 1997; Villar-Salvador et al., 1997), joined together into short vessels (Sperry et al., 1988), occurring in high frequency (Choat et al., 2005a; Verheyden et al., 2005; Sobrado, 2007), in vessel groups rather than solitary (López et al., 2005) and with a thick vessel wall (Sperry et al., 1988).

The link between vessel diameter and cavitation resistance can be explained in two ways by the indirect influence, clarifying the flexibility of the correlation (Tyree et al., 1994; Mauseth and Plemons-Rodriguez, 1998; Hacke and Sperry, 2001; Sperry et al., 2006), of vessel size on the air-seeding process. First, small vessels have an equally small wall area, resulting in a relatively low pit area per vessel and thus cavitation risk (Choat et al., 2005b; Hacke et al., 2006). The correlation between the total pit membrane area and the cavitation vulnerability could be explained by the increased chance

X

X

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P

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SC

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D

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Ps XSCPA

PsPA

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Fig. 3 Secondary growth via successive cambia in Avicennia marina. A Transverse section of a portion of the outermost stem wood. Three cambia are simultaneously active (arrow heads), forming xylem (X) to the inside, sclereids (SC) and phloem (P) to the outside. At the barkside a new cambium has just developed. More inward, part of the xylem has not yet lignified. In the innermost growth layer, the parenchyma tissue (PA) in between the differentiating phloem strands (Ps) started lignification. Scale bar, 100 µm. B Transverse section of a seedling where the alternating xylem and phloem bands are clearly visible. The phloem bands consist of phloem strands embedded in a layer of parenchyma and delineated from the next growth layer by two to three cell layers of sclereids. Scale bar, 1000 µm. C Sanded stem disc with the phloem and xylem bands appearing as light and dark coloured bands, respectively. Scale bar, 1000 µm. D Detail of the outermost stem wood indi-cated by the frame in A. A new cambium has recently developed in the parenchyma layer to the outside of the sclereids. Scale bar, 100 µm. Double staining with safranin-fast green.

21


on an exceptionally large pit membrane pore, prone to air-seeding (Hargrave et al., 1994; Wheeler et al., 2005; Hacke et al., 2006). Second, vessels with a small diameter have a large surface-to-volume ratio which would aid in controlling the xylem sap composition (De Boer and Volkov, 2003). Given the ion regulation of the hydraulic conductance via hydrogels in pit membrane pores (see section ‘Approach’, ‘The sub-cellular level’) this could be of adaptive value under stress conditions. Recently, another positive effect of ions on sap flow was assigned to the prevention of coalescence rather than the development of air bubbles (Canny et al., 2007). The same authors presented a new theory saying that perforation plates of small vessels are more likely to trap cell wall residues from vessel element endings after hydrolysis that can act as gass nucleation sites. In this way small vessels could trap exsolved air, leaving degassed less cavitation-prone water for more efficient flow through wide vessels. This theory would only apply if cavitation triggered by constrictions along the flow path, occurs more easily than air seeding to which large vessels are more vulnerable. But in the first place, future research is challenged to underscore the formation of air bubbles via irregularities of the vessel wall next to air-seeding. This would have serious implications for the current views on structure-function relationships. Species with scalariform perforation plates, like Rhizophora, would inherently be more vulnerable to cavitation and we should have to revise our vision on the functional advantage of a bimodal distribution in vessel size, so often observed in arid environments (Baas et al., 1983).

In addition to reducing the air-seeding probability, small vessels mitigate the negative effect of it. Simply because of their small size, they maximize the proportion of active xylem upon hydraulic failure. The effect is enhanced by the correlation between vessel diameter and vessel length, across species (Sperry et al., 2005; Hacke et al., 2006) and within ring porous species when comparing latewood and earlywood (Zimmermann and Jeje, 1981). Reducing vessel element length (Baas et al., 1983), and especially total vessel length (Comstock and Sperry, 2000; Hacke and Sperry, 2001) hinders the spreading of embolisms by perforation plates or vessel endings, respectively. The parts of the conducting network that are blocked can be readily circumvented if vessel density and vessel grouping are high, providing more alternative routes to circumvent air bubbles (Villagra and Roig Juñent, 1997; Cruiziat et al., 2002; Kitin et al., 2004). Besides, the strategy of packing as many vessels as possible in the xylem guarantees a basic hydraulic conductance even after considerable losses to embolism (Hacke et al., 2006). The higher redundancy of the vessel network also implies a higher potential for air bubbles to spread to other vessels highlighting the complexity of studying the functional significance of the hydraulic structure (Loepfe et al., 2007). With respect to the connectivity dilemma, geometry of intervessel pits and wall chemistry should not be overlooked (Fig. 2d). The pressure needed to break the gas-water interface in the pit chamber between a functional and an embolized vessel depends on the contact angle between water and vessel wall, and the inclination of the pit chamber walls (Holbrook and Zwieniecki, 1999; Zwieniecki and Holbrook, 2000; Meyra et al., 2007). Gas crosses pit membranes thus less readily than water, making it easier to understand the advantages of a more integrated transport system. It provides not only the opportunity to bypass embolisms but also to exploit spatially localized soil resources (Orians et al., 2004; Ellmore et al., 2006), outweighing the potential disadvantage of an intense packing of vessels.

The increased xylem tension does not only lead to deactivation of vessels by embolization but also by vessel implosion. This can be hindered by more sturdy fibers to support vessels (Jacobsen et al., 2005; Cochard et al., 2007; Jacobsen et al., 2007b) or directly by increasing the ratio of the intervessel wall thickness to the vessel lumen diameter (Hacke et al., 2001a) that is related to wood density (Preston et al., 2006; Jacobsen et al., 2007a). It should be noted that between more related species the relationship between wood density and vulnerability to conductive failure seems to be missing (Cochard et al., 2007). Besides, the fact that a higher wood density results in a lower water storage capacity (Domec et al., 2006b) emphasizes the importance of integrative studies, looking at

22

General objective & hypotheses

the hydraulic architecture as a whole since it is the balance between all characteristics that determines the cavitation vulnerability of the tree.

The sub-cellular level

At the level of the intervessel pits we are at the origin of water stress, where gas bubbles seed in across the pit membrane and block the water stream. Studies on the structure of intervessel pits showed a remarkable variation across species (Sano, 2005; Choat et al., 2008) but, in contrast to studies dealing with vessel characters, ecological trends have rarely been investigated (Choat et al., 2003; Jansen et al., 2004). This is related to the relatively small number of studies dealing with pit geometry. The tiny size of pits requires electron microscopy analyses that need time-consuming sample preparation. Nevertheless, intervessel pit characteristics that generally show a higher cavitation resistance could be detected and are supposed to be beneficial in xeric environments: low pit membrane porosity, thick pit membranes and the presence of vestures (Choat et al., 2008).

The relationship between pit membrane structure and cavitation resistance shows some flexibility. A first explanation is given by the cavitation resistance that is determined by the maximum and not the average pore diameter of the pit membrane. If air seeding would take place at a random pore, pit membranes with a small mean pore size would have an increased cavitation resistance (Zimmermann, 1983; Sperry and Tyree, 1988). However, if air seeding would first take place at the largest pore per vessel, average pit membrane area per vessel would be the key factor determining cavitation resistance. The larger the pit area, the bigger the chance on an exceptional large pore increasing cavitation vulnerability (Wheeler et al., 2005; Hacke et al., 2006; Choat et al., 2008). This so-called pit area hypothesis is the most supported option at the moment. The size of the maximum pore is unrelated to the average pore diameter and is the result of pit membrane damage or stretching (Choat et al., 2003). The difficulty in observing these large, first air-seeding pores might be related to the temporary condition of their exceptional size (Choat et al., 2004). Alternatively, the large pore is in its natural state but rare (Wheeler et al., 2005). Next to the low frequency of these exceptionally large pores, their identification as the pores causing cavitation is hampered by the high risk for artefacts inherent to the delicate pit membranes (Choat et al., 2003; Choat et al., 2006). The stretching or even rupture of the pit membrane when aspirated by the pressure difference between a functional and an embolized vessel (Hacke et al., 2001b; Choat et al., 2003) gives a second clarification for the flexible correlation between pit membrane porosity and cavitation resistance. Several strategies have evolved to prevent deviation of the porosity from its pristine state. The variation observed between species in pit membrane thickness (Schmid and Machado, 1968; Sano, 2005; Choat et al., 2008) could translate in varying probability to stretch excessively and develop large pores. Next to changes of the pit membrane itself, vestures or appendices of the vessel wall in pit canals and sometimes also pit chambers (Fig. 4a), impede excessive stretching by supporting the pit membrane (Fig. 4b) and hence lowering the vulnerability for drought induced embolism (Zweypfenning, 1978; Jansen et al., 1998; Jansen et al., 2003; Choat et al., 2004; Jansen et al., 2004; Sperry and Hacke, 2004). Thirdly, next to structural adjustments, cavitation resistance can be affected by pit membrane chemistry influencing the air-water interface (see section ‘Approach’, ‘The cellular level’). Besides, pore size can be regulated by changes in the chemical composition of the xylem sap although its significance in planta remains to be demonstrated. Ion-regulation of pit membrane pore size is based on the hydrogel nature of pectins in the pit membrane (Tibbits et al., 1998; Ridley et al., 2001). Depending on the ionic composition of the xylem fluid, pectins swell under low ion content and shrink under high ion content when cross-linkages are being built. Adjusting hydraulic conductivity by changing ionic content provides an opportunity to compensate for the negative effect of embolisms (Zwieniecki et al., 2001b; Lopez-Portillo et al., 2005; van Ieperen, 2007). In this respect, less lignified vessels would offer the highest benefit. Because of the more intimate contact between pectins and ions, the hydraulic

23


v

v

Pch

PM

A B

10 µm

Fig. 4 The functional advantage of vestures in intervessel pits. A The intervessel pits of Sonneratia alba are characterized by extensive vestures (v), which are appendices of the vessel wall, projecting into the pit cham-ber (Pch). B Vestures can mitigate cavitation vulnerability by supporting the pit membrane (PM), hindering excessive stretching and the formation of large pores in the pit membrane. Figure B adapted from Choat et al. (2008).

response might increase (Boyce et al., 2004) as supported by the reduced lignin biosynthesis under water stress (Alvarez et al., 2008). Much more work is however needed to unravel the species-specific differences and temporal fluctuations in pectic composition, degree of methyl esterification of the pectic chains, ionic content of the xylem sap and its hydraulic effect as a combination of various ions (Schill et al., 1996; Gascó et al., 2006; Gascó et al., 2007; Nardini et al., 2007).

24


General objective & Hypotheses

In view of the extreme mangrove environment concerning stresses imposed on the water transport it is surprising that so little research has been done to gain knowledge about how mangroves adapt their hydraulic structure to their stressful and dynamic environment. This study is an endeavour to clarify the relationship between the hydraulic architecture of mangroves and their environment. To a broader extent, this study is intended to deepen our understanding in the water transport of trees, its threats and associated safety measures.

Hypothesis I: ecological plasticity within species

Although it is clear that a well-adapted hydrosystem, efficient in preventing and refilling cavitated conduits, plays a fundamental role in trees’ ecological success, mangrove wood anatomy has been studied scarcely in relation to its stressful environment. Nevertheless, mangrove species such as Rhizophora mucronata and Avicennia marina that grow under variable and partially overlapping environmental conditions, allow a study of the hydrosystem between but also within species, excluding the potential effect of genetic variability. The HYPOTHESIS tested is: “The hydraulic structure of mangrove trees and specifically the properties of their vessels and intervessel pits (with a risk of drought-induced cavitation), reflect the adaptation to secure the water transport under conditions of high temporal and spatial variability of the mangrove biotope.”

Hypothesis II: ecological significance of successive cambia

Despite the increasing support for the role of phloem tissue in the process of embolism repair, our understanding of trees with successive cambia is still limited to the anatomical description of their peculiarly composed wood. No conclusive information is available about the development of wood via successive cambia for an entire stem cross-section, nor about its relationship with environmental conditions or its putative difference in hydraulic demand. Several species with successive cambia grow under drought stress conditions. One of them is Avicennia marina, the mangrove species with the broadest latitudinal and local distribution, following from a high stress tolerance. Together with the expected role of phloem tissue in the refilling of embolized vessels, this raises the HYPOTHESIS that successive cambia offer an ecological benefit for trees growing under stressful conditions.

25


26

Part I

Hydraulic architecture of mangroves: ecological plasticity and functional significance

Part I

In wildness is preservation of the world.H.D. THOREAU

27

Part I

Summarizing abstractBackground and Aims Mangrove trees are subjected to exceptionally high and variable demands for the water transport, in time as well as space. The combination of saline water, periods of flooding alternating with periods of drought, and high temperature suggests an adaptive hydrosystem to control the impact of drought-induced cavitation. However, little is known about the ecological plasticity of the hydraulic architecture of mangrove trees and its functional significance to guarantee sap flow under all environmental conditions. In this first part of the thesis the variability of vessel structure and intervessel pit anatomy will be studied both along a salinity gradient and between seasons together with a first topochemical analysis of the intervessel pits and its intra-specific variation.

Methods Two species with a different ecological distribution, Rhizophora mucronata and Avicennia marina, have been studied in the mangrove forest of Gazi Bay, Kenya. Wood samples were collected at different sites characterized by different substrate salinity and inundation frequency. Several vessel and intervessel pit characteristics were quantitatively measured with Light microscopy, Scanning Electron Microscopy, Transmission Electron Microscopy and cellular UV Microspectrophotometry.

Key results Vessel density was shown to be a good proxy for soil water salinity while vessel diameter was remarkably constant with changing salinity levels. Also intervessel pit size did not vary significantly between sites. Pit membranes were slightly thicker in dry than in rainy season in R. mucronata. But, more remarkable was the seemingly random variation in electron density of the pit membranes and occurrence of deposits in pit canals. In comparison to R. mucronata the overall pit microstructure of A. marina might be interpreted as more resistant to drought-induced cavitation, which would then also fit the known ecological requirements of the species.

Conclusions The ecological plasticity in vessel density and pit membrane thickness can be related to a safe hydrosystem by increased redundancy in the conduit network and resistance of the pit membrane against air-seeding, respectively, with increasing substrate salinity. Vessel diameter and intervessel pit size, in contrast, seem to adopt a constant optimal value. The link between the constant vessel diameter, the seasonal changes in pit membrane thickness and the presence of deposits along vessel walls and in pit canals should be further investigated to explore their potential role in the regulation of the water transport in mangroves.

28

Chapter 1

...and they stand supported by their roots, like an octopus on its tentacles.THEOPHRASTUS

29

Chapter 1

Influence of a salinity gradient on the vessel characters of the mangrove speciesRhizophora mucronata

Vessel density as salinity proxy

Abstract

Background and aims Although mangroves are extensively studied, little is known about their ecological wood anatomy. This investigation examined the potential use of the vessel density as a proxy for soil water salinity in the mangrove species Rhizophora mucronata (Rhizophoraceae) from Kenya.

Methods In a time-standardized approach, 50 wood discs from trees growing in six salinity categories were investigated. Vessel densities, tangential and radial diameters of rainy and dry season wood of one distinct year, at three positions on the stem disks, were measured. A repeated measures ANOVA with the prevailing salinity was performed.

Key results Vessel density showed a significant increase with salinity supporting it as a prospective measure of salinity. Interestingly, the negative salinity response of the radial diameter of vessels was less striking, and tangential diameter was constant under the varying environmental conditions. An effect of age, growth rate or the presence of a vessel-dimorphism could be excluded as the cause of the lacking ecological trend.

Conclusions The clear trend in vessel density with salinity, together with the absence of a growth rate and age effect validates its potential as an environmental proxy. However, it can only be used as a relative measure of salinity since other environmental variables such as inundation frequency have an additional influence on the vessel density. In view of a reliable, absolute proxy future research should focus on finding wood anatomical features correlated exclusively with soil water salinity or inundation frequency. The plasticity in vessel density with differing salinity suggests a role in the establishment of a safe water transport system. To confirm this hypothesis the role of the inter-vessel pits, their relation to the rather constant vessel diameter and the underlying physiology and cell biology needs to be examined.

Key words: Rhizophora mucronata, mangrove, ecological wood anatomy, vessel density, vessel diameter, proxy, salinity, inundation frequency, Kenya, hydraulic architecture

Published in: Annals of Botany 98: 1321-1330

30

Chapter 1

Introduction

Mangroves are tropical and subtropical forests occurring in the intertidal areas of coastal shorelines protected from wave action. Mangrove forests provide a plethora of ecosystem services and products and play an important socio-economic as well as ecological role (Rönnbäck, 1999; Dahdouh-Guebas et al., 2000; Kairo et al., 2001; Moberg and Rönnbäck, 2003; Dahdouh-Guebas et al., 2005). World-wide disappearance of mangrove forests is undoubtedly mainly caused by large scale clear cutting and land conversion (Valiela et al., 2001; FAO, 2003). However, changing environmental conditions too, in particular salinity, can lead to mangrove degradation and die-off (Spalding et al., 1997; Kathiresan, 2002). Changes in soil water salinity can be influenced by climate (Drexler and Ewel, 2001) as well as by human impacts caused by the leakage from salt extraction pans or by the damming or redirection of rivers (Kovacs et al., 2001; Alongi, 2002). Nevertheless, relating mangrove degradation to changes in soil water salinity is still impeded by a lack of local, long-term environmental data (Kovacs et al., 2001). Performing salinity measurements on a long-term basis is impractical since the spatial and temporal variations in the mangrove habitat (c.f. Ridd and Renagi, 1996; Ball, 1998; Matthijs et al., 1999; Marchand et al., 2004) would require a high sampling intensity. Therefore, there is an urgent need for proxies of environmental conditions and in particular of salinity.

Recently, the presence of annual growth rings was discovered in the mangrove Rhizophora mucronata from Kenya (Verheyden et al., 2004) and R. mangle from North Brazil (Menezes et al., 2003). The annual rings are composed of a zone of low vessel density and a zone of high vessel density, which are produced during respectively, the rainy (earlywood) and dry season (latewood) (Verheyden et al., 2004). Verheyden et al. (2005) further suggested that wood anatomical features in this mangrove species could be a potential proxy for past environmental conditions. In particular, temporal changes in soil water salinity might be recorded in the vessel density (Verheyden et al., 2005) through the effect of salinity as a determining factor for the regulation of the water transport in mangroves (Naidoo, 1985, 1986; Clough and Sim, 1989; Lin and Sternberg, 1993; Zimmermann et al., 1994; Sobrado, 2001; Paliyavuth et al., 2004; Lopez-Portillo et al., 2005). However, precipitation which was taken as a measure of salinity exhibited the lowest correlation coefficient with the vessel density (Verheyden et al., 2005). The authors suggest that the low correlation might be caused due to environmental and climatic factors interfering with the salinity-rainfall relationship. During the rainy season, salt is not only flushed directly by the precipitation but also by land run-off, groundwater flow and river input, all related to the topography of the environment (Ewel et al., 1998; Hoguane et al., 1999). Other determinants of the salinity are the water retention capacity of the soil (Wiemann et al., 1998), the evaporation intensity (Naidoo, 1989) and the tidal inundation (Passioura et al., 1992; Lin and Sternberg, 1992, 1993). The effect of salinity per se on vascular features still needs to be demonstrated (Verheyden et al., 2005).

The present study investigated the potential of vessel features as a proxy for soil water salinity in the mangrove R. mucronata in Gazi Bay, Kenya. Time series of data records of the salinity per se are not available for the study area. Consequently, investigating the effect of temporal changes in salinity on vessel features is hindered. As an alternative, a time-standardized measuring approach is used to allow an accurate comparison between spatial differences in soil water salinity and vessel density and diameter. In this way, information will be indirectly gained about their proxy potential to trace temporal changes in salinity.

31


Materials and Methods

Study sites and sample collection

The study sites are located in the mangrove forest of Gazi Bay (39°30’E, 4°25’S), situated approximately 50 km south of Mombasa, Kenya. Two seasonal rivers discharge into the bay and provide a freshwater source for the mangroves: the Mkurumuji and the Kidogoweni (Kitheka, 1997).

Wood discs from the trunk of fifty trees (now part of the wood collection of the Royal Museum for Central Africa, Tervuren, Belgium, for accession numbers see Table 1) of R. mucronata were collected in Oct. 1999 and May 2002. Tree diameters at 1.3 m height varied between 2 and 10 cm and were associated with a cambial age in 1998 of 4 to 57 years (Table 1). The trees originate from eight sites (four to ten trees per site), selected for their differences in salinity and inundation class (Fig. 1; Table 1). Soil water was collected in 1998, 1999 and 2002 at 10 cm depth using a punctured plastic tube connected to a vacuum pump. At each site, one to three salinity measurements were carried out with a WTW P4 multiline conductivity meter (Wissenschaftlich-Technische Werkstätten GmbH, Weilheim, Germany). Within the studied mangrove stands, soil water salinity ranged from 26.4 ‰ to 49.2 ‰, which is the upper limit of distribution of R. mucronata in Gazi (data from Verheyden, 2004). Sites were divided into six salinity categories (SAL1-SAL6); site two and three as well as site four and five were combined (Table 1). This was justified since there was no statistical difference between the combined sites (ANOVA, Tukey’s Honest Significant Difference test). Depending on the topography and the tidal range, zones of different inundation classes can be defined. Inundation classes one, two, three and four correspond to an area being inundated by respectively 100-76, 75-51, 50-26 and 25-5 % of the high tides (cf. Tomlinson, 1994).

Site† n Tw number‡ Age (yr)§ Salinity (‰)*Salinity Inundation

category** class***1 10 55904 to -08, 56705 to -09 9-16 26.4 1 32 5 56710 to -14 17-37 31.9 2 33 4 55883 to -86 11-22 33.6 2 14 5 55872, -73, -76, -80, -81 17-33 35.2 3 25 5 55958, -75, -78, -90, -91 4-5 35.4 3 36 10 55890 to -94, 56725 to -29 11-24 38.2 4 27 7 55887 to -89, 56730, -33, -34, -36 25-57 42.7 5 48 4 56721 to -24 4-20 49.2 6 2

Table 1. Characteristics of the studied Rhizophora mucronata specimens and the different sampling sites

†See also Fig. 1; ‡Accession number of the samples in the Tervuren wood collection; §Cambial age in 1998; *Soil water salinity at 10 cm depth, data from Verheyden (2004), see also comment at the end of the chapter; **Salinity categories (see Materials and Methods); ***Inundation class according to Watson (1928) in Macnae (1968).

Climate description

The climate along the Kenyan coast is characterized by a bimodal distribution of the precipitation, which results in a long rainy season (Apr. – July), a short rainy season (Oct. – Nov.) and one distinct dry season (Jan. – Feb.) (Fig. 2). In accordance with earlywood formation (see Verheyden et al., 2004), the term “rainy season” will further be used to indicate the period of both long and short rainy season (Apr. –Nov.).

32

Sample preparation and wood anatomical measurements

Chapter 1

Fig. 1. Map of the study site, Gazi Bay (Kenya), indicating the different sites where wood discs were collected. Site four and eight are only 20 m from each other but site four is at the fringe and site eight in the middle of a basin forest (see also Table 1) (adapted from UNEP, 2001).

Fig. 2. Climate diagram of Mombasa (39°36’ E, 4°0’ S) adapted from Lieth (1999), showing the long (April - July) and short (October - November) rainy season and one distinct dry season (January

- February). Dotted area, dry season; hatched area, moist season. Precipitation scale is reduced to 1 / 10 above the horizontal line.

Fig. 3. Wood anatomical measurements were carried out in a time-standardized way. At three positions, chosen along a radius of high, moderate and slow growth rate two quadrats (size is exaggerated for clarity) covering rainy season wood (earlywood, E) and dry season wood (latewood, L) of the year 1998 were studied. Scale bar: 1 cm. Specimen number Tw56722, part of the Tervuren wood collection.

Wood samples were air-dried and sanded using a series of sandpaper from 100 to 1200 grit. Vessel features were measured directly on the sanded stem discs making use of digital image analysis software (AnalySIS Pro v.3, Soft Imaging System GmbH, Münster, Germany), at an optical magnification of 12.5 times. Vessel density (number of vessels per mm²), as well as average radial and tangential vessel diameter (µm) were measured at three positions on the wood disc. The size of the quadrats was chosen to include at least 20 vessels. Taking into account earlier findings on the temporal changes in vessel density in R. mucronata (Verheyden et al., 2005), the inter-annual variability was excluded by carrying out all measurements within the ring of the year 1998. The 1998 ring was the most recent ring that was fully developed in all samples (samples were collected in 1999 and 2002). In addition, intra-annual variation was investigated by measuring the wood produced during the rainy season

50

40

30

20

10

0

20

40

60

80

100

300

)C°( erutarep

meT )m

m ( n

o itati

pic e

rP

J A S O N D J F M A M J

33


(earlywood, see Verheyden et al., 2004) and the wood produced during the dry season (latewood) of the year 1998 separately (Fig. 3). However, for samples collected at site seven (corresponding to SAL5) earlywood and latewood could not be examined separately since growth rings were too narrow to allow differentiation. In order to include SAL5 in the statistical analysis and allow study of the variability of vessel features along the maximum salinity and inundation frequency gradient, annual averages were calculated for each site and included in the statistical analysis. Regarding asymmetrical wood discs, the three measuring positions were chosen along the longest, smallest and medium axis of the wood disc (see Fig. 3). Consequently, by comparing the three positions from each specimen with ring width, the within-tree growth rate effect on vessel features could be investigated. The between-tree correlation between growth rate and vessel diameter was investigated as well. For this purpose, growth rate data were used from a cambial marking experiment on 20 of the studied trees (Verheyden, 2004).

Statistical analysis

To trace trends in the three vessel characters considered, as a function of salinity and inundation class, a repeated measures ANOVA was carried out in STATISTICA 7.0 (StatSoft Inc., Tulsa, USA). Two separate analyses were performed, one with salinity and one with inundation class as a grouping factor. A combined analysis could not be performed since all salinity categories do not occur at each inundation class. Both an analysis based on annual averages and an analysis considering rainy and dry season wood separately (“season” as an additional categorical variable) were performed. Vessel density was inserted as a repeated measures factor with three levels, since measurements were carried out at three positions within each wood sample (Portney and Watkins, 2000). Post-hoc comparisons between group averages were made with “Tukey’s Honest Significant Difference test” (Tukey’s HSD) for unequal group sizes, as recommended by Quinn and Keough (2002). A student’s t-test for dependent samples was used to search for differences in vessel diameter and vessel density between dry and rainy season within sites. When the Levene’s test indicated heterogeneity of variances or the Shapiro-Wilk’s W test showed a non-normal distribution, a common logarithmic, natural logarithmic, square root or inverse transformation was executed to comply with the assumptions of an ANOVA / t-test. Finally, a Pearson-correlation coefficient was calculated between the log-transformed growth rate and the radial vessel diameter.

Results

The repeated-measures ANOVA did not show a within-tree positional effect for vessel density, tangential nor radial vessel diameter, for either salinity or inundation class (Table 2). Therefore, the vessel density obtained from the three positions on the stem disc was averaged and this average was used for all subsequent figures.

A significant positive relationship between vessel density and salinity was found for the rainy (earlywood) as well as for the dry season (latewood) (Fig. 4a, Table 2). This positive relationship was maintained when vessel densities were averaged (annual averages in order to include SAL5, see materials and methods) (Table 2). A distinctly lower vessel density was recorded at the sites with low salinity, relative to the sites with high salinity (SAL1 to SAL3 vs SAL5, P < 0.001, 0.01, 0.05) as shown by Tukey’s HSD-test .

Contrary to the vessel density, no significant effect of salinity on the radial and tangential diameter was found in either rainy or dry season wood (Table 2). The invariable nature of the tangential diameter is further expressed in the size distribution (Fig 5a). In contrast, a slight decline in radial diameter was

34

Chapter 1

EffectRainy season Dry season Annual average

df‡ MS§ F df MS F df MS FVessel density

Salinity 4(38) 2.78(sqrt) 3.44* 4(38) 0.47(ln) 3.24* 5(44) 0.14(log) 6.63***Position† 2 0.41(sqrt) 2.57 2 0.04(ln) 1.35 2 0.01(log) 2.13Inundation class 2(40) 0.89(ln) 7.92** 2(40) 1.02(ln) 7.51** 3(46) 1.54E-03(in) 10.07****Position 2 0.02(ln) 0.69 2 0.02(ln) 0.52 2 3.09E-05(in) 0.86

Tangential diameterSalinity 4(38) 28.22 0.41 4(38) 60.26 1.28 5(44) 80.30 1.46Position 2 1.84 0.12 2 33.72 2.06 2 22.46 1.46Inundation class 2(40) 58.04 0.89 2(40) 17.37 0.35 3(46) 68.36 1.20Position 2 1.57 0.10 2 23.48 1.37 2 12.32 0.76

Radial diameterSalinity 4(38) 147.47 1.11 4(38) 209.36 2.04 5(44) 392.78 3.36*Position 2 35.96 1.52 2 9.57 0.36 2 25.31 1.16Inundation class 2(40) 272.72 2.14 2(40) 158.30 1.43 3(46) 561.12 4.75**Position 2 46.84 1.87 2 2.07 0.08 2 33.90 1.49

Table 2. Repeated measures ANOVA’s of vessel density, radial and tangential diameter.

†Measurements were repeated at three positions on each stem disc (Fig. 3); ‡Error given in parentheses; §Mean Square (MS) with transformation type in parentheses: in = inverse, ln = natural logarithm, log = logarithm (common base 10), sqrt = square root; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

observed at increased salinity (Fig. 4b), which is supported by a significant relationship for the annual averages (Table 2) and a shift in the size distribution from SAL2 to SAL5 (Fig. 5b). Differences in vessel features between rainy season and dry season were significant for vessel density and radial diameter, but not for tangential diameter (paired t-test, Table 3).

Similar to the results obtained for salinity, a positive significant relationship was found between vessel density and inundation class and this for annual averages as well as for rainy and dry season measurements (Table 2). This relationship is most prominent when comparing class one vs. class four (P < 0.05, Tukey’s HSD-test). However, there is a considerably lower vessel density at inundation class three compared to inundation class two and four (df = 46, P < 0.01 and 0.002, Tukey’s HSD-test), which interrupts the positive trend. Concerning vessel diameter, a significant relationship with inundation class was only detected for the annually averaged radial diameter (Table 2) although a trend in the data could not be detected visually.

VariableRainy season Dry season

t-testMean ± s.d. (range) Mean ± s.d. (range)

Vessel density (# vessels mm-2) 23 ± 7 (11-57) 30 ± 9 (14-66) t = 13,31 P < 0,0001Radial diameter (µm) 81,26 ± 7,81(30.30-134.99) 79,87 ± 7,38(30.30-132.23) t = -2,50 P = 0,01

Tangential diameter (µm) 71,32 ± 5,73(33.06-115.7) 70,72 ± 5,36(35.81-115.7) t = 1,85 P = 0,07

Table 3. Vessel characteristics for rainy and dry season wood (values are means of all six salinity categories) and results for the t-test for dependent samples (n = 129).

35

Discussion

Vessel density increases in R. mucronata from low to high saline areas in the mangrove forest of Gazi (Fig. 4a), in accordance to the observations in the mangrove-associate Annona glabra (Yáñez-Espinosa and Terrazas, 2001). Similar to earlier findings (Verheyden et al., 2004; Verheyden et al., 2005), a higher vessel density was also found in the dry season as compared to the rainy season (Fig. 4a; Table 3), suggesting that vessel density can be used as an indicator for temporal changes in salinity. Indeed, seasonal fluctuations in vessel density can be partly attributed to an increase in salinity from rainy to dry season. Other seasonal factors possibly affecting vessel density are phenology (Slim et al., 1996; Drew, 1998; Salleo et al., 2003; Choat et al., 2005; Coupland et al., 2005), together with nutrients (February and Manders, 1999; Alongi et al., 2005), and water availability. A relatively higher vessel density is observed in the xylem of xerophytes compared to the vegetation of more mesic environments (Baas et al., 1983; Carlquist and Hoekman, 1985; Lindorf, 1994). Interestingly, Yáñez-Espinosa et al. (2001) found no differences in vessel number between different sites with presumed different soil water salinity (based on soil texture) in their study on Mexican R. mangle. The discrepancy between both studies can be due to small sample size (n = 12 vs 50) and a lacking control over inter- and intra-annual variability of the former study. Although taxonomic differences can not


Fig. 4. (A) Mean vessel density and (B) mean radial

Fig. 5. Frequency distributions (%) of the vessels, according to their (A) tangential and (B) radial vessel diameter (10 µm classes) for site seven and three (see also Table 1, Fig 1), representing contrasting salinity categories (SAL2 and SAL5) and inundation classes (one and four). Error bars correspond to standard er-rors.

0

10

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80 - 9090 - 1

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Tangential vessel diameter (10-µm classes)

Freq

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Radial vessel diameter (10-µm classes)

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ial v

esse

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vessel diameter in relation to salinity for both rainy season (dark bars) and dry season (light bars). Salinity category five represents the annual average value of the respective vessel feature since growth rings were too narrow to differentiate rainy season and dry season wood (see also Materials and Methods). Line, mean; Box, standard error; Whisker, standard deviation. SAL: Salinity category.

36

be omitted, we emphasize the importance of using a time-standardized measuring approach when comparing wood anatomical features between different sites in a dendroecological investigation. The developmental link between a rising vessel density with salinity can be explained by an adjustment in nutrient availability and/or auxin physiology with higher soil water salinity. Salt stress interferes with the uptake of nutrients such as Ca²+, Mg²+, K+ (Popp et al., 1985; Naidoo, 2006; Súarez and Medina, 2006) and is reported to decrease auxin concentrations in poplar, leading to a decrease in vessel size (Junghans et al., 2006). Although vessel density was not evaluated by Junghans et al. (2006), previous studies mention that also vessel density could be affected by auxin (Aloni, 2004).

The functional significance of the changes in vessel densities in trunks of R. mucronata are also considered. A high salinity creates an osmotic stress subjecting the xylem vessels to the risk of cavitation and subsequent embolism (Sperry and Tyree, 1988; Tyree and Sperry, 1989; Hacke and Sperry, 2001). The associated increase in vessel density may be interpreted as a strategy for conductive safety. The benefit is offered in two ways. First, when the same number of vessels is cavitated, a higher percentage of the transport system remains functional in high vessel density compared to low vessel density wood (Baas et al. 1983; Villar-Salvador et al. 1997; Mauseth and Plemons-Rodriguez, 1998). Second, because vessels are not linear features (Tyree and Zimmermann, 2002; Kitin et al. 2004), a high proportion of vessels are at least at one point along their length in contact with each other via inter-vessel pits. Therefore, embolized vessels can be circumvented by means of the high number of alternative routes for the water transport (Tyree et al., 1994; Carlquist, 2001).

The environmental responsiveness of the vessel diameter to the soil water salinity was found to be remarkably small, and therefore, its proxy potential is limited. Only a small tendency was shown towards smaller radial diameters at sites with a high salinity and during the dry season (Fig. 4b; Table 3). This finding does not correspond to an earlier study on R. mucronata from Kenya: a high-resolution time series analysis, but only considering one site, showed radial as well as tangential diameters were smaller in the dry than in the rainy season (Verheyden et al., 2005). A high-resolution approach might thus be required to reveal minor adjustments in tangential diameter (Table 2, 3). Similar to our results, Gillespie et al. (1998) during a study on Breonadia salicina, a tree of tropical, subtropical and semi-arid areas, also observed radial diameters to be more responsive to rainfall than tangential diameters. This finding is further supported by the strikingly similar size distributions of the tangential as opposed to the radial diameters at sites with contrasting soil water salinity and inundation frequency (Fig. 5a, b). The authors concluded that radial diameter measurements should not be neglected in favour of tangential diameters, which our study encourages.

Aside from a high vessel density, declining vessel dimensions are repeatedly mentioned in association with an increased conductive safety (Lo Gullo et al., 1995; Villagra and Roig Juñent, 1997; Arnold and Mauseth, 1999; Corcuera et al., 2004; Stevenson and Mauseth, 2004). The absence of a clear salinity effect on vessel diameters in this study (Table 2, Fig 4b) can be explained by one or a combination of the following aspects. First, the discrepancy between tangential and radial diameter does not result from a prospective relationship between radial diameter and growth rate, neither within (Table 2, Fig. 3) nor between trees (r² = 0.044, p = ns, n = 20). The reported growth rate effect on vessel diameters by Reich et al. (2003) in 17 oak species in Florida may thus be an effect of environmental factors, correlated with growth rate (e.g. salinity). Though, the incongruence may simply reflect inter-generic variation. Besides, an extensive analysis of the effect of growth rate on wood anatomy is needed to confirm our findings. Secondly, xylem vessels with a bimodal diameter distribution offer the advantage of an efficient (large vessels) and safe (small vessels) water transport system (Mauseth and Stevenson, 2004). The functional benefit of this vessel combination explains its frequent occurrence in the flora of arid regions (Baas et al., 1983; Baas and Schweingruber, 1987; Villagra and Roig Juñent, 1997). However, as in the mangrove Aegiceras corniculatum (Sun and Lin, 1997), no bi-modal vessel

Chapter 1

37


diameter distributions were detected in R. mucronata (Fig. 5). A third factor potentially interfering with an ecological trend in vessel diameter is age (Corcuera et al., 2004). In both radial (Verheyden et al., 2005) as well as tangential diameter (N Schmitz, VUB, Brussels, Belgium, unpubl. res.) an age-trend has sometimes been observed. To maintain a favourable water balance, when the tree is growing and increasing its leaf surface, trees usually produce longer and wider vessels in their stems when aging (Tyree and Ewers, 1991; Hudson et al., 1998; Cruiziat et al., 2002). The vessels in SAL4 and SAL6 are larger than expected if a negative trend with salinity would be present, but the impact of age can be excluded. The large vessels in both salinity categories are represented by young trees with a cambial age of respectively 11-24 and 4-20 years while the small vessels of SAL5 occurred in the oldest trees with a cambial age of 25-57 (Fig. 4b, Table 1). Alternatively, cell wall thickness and thus conduit reinforcement could be more important than vessel lumen area with respect to conductive safety (Hacke and Sperry, 2001; Hacke et al., 2001). The absence of a selective force might then explain the missing plasticity in vessel size. Also differences in nutrient availability between the study sites might have interfered with salinity. Lovelock et al. (2006) found an increase in vessel diameter in R. mangle in response to P addition while vessel density did not change considerably. Finally, future studies should address the role of the pit characteristics in the invariability of the vessel diameter with changing salinity conditions. According to the air-seeding hypothesis, tension-induced cavitation is the result of air being sucked in via the pores in the inter-vessel pit membranes (Tyree et al., 1994; Choat et al., 2003; Konrad and Roth-Nebelsick, 2003). The presence of small pit pores in small vessels would provide them with a higher cavitation resistance compared to large vessels (Tyree and Dixon 1986; Sperry and Tyree, 1988; Lo Gullo and Salleo 1991, 1993; Hargrave et al., 1994; Jarbeau et al., 1995). However, the relationship between vessel and pit pore diameter is still a subject of investigation. Small pit pore diameters (Tyree et al., 1994; Sperry and Hacke, 2004) and/or small surface area of the intervessel-pits (Orians et al., 2004; Wheeler et al., 2005; Ellmore et al., 2006; Hacke et al., 2006) have been reported to increase the cavitation resistance of the water transport system regardless of vessel diameter.

Similar to the vessel diameter, the vessel density is independent of both growth rate and age. Together with the clear trend of increased density with salinity, this validates the earlier suggestion of the vessel density as a salinity proxy (Verheyden et al., 2005). However, as indicated by the overlap in the data (Fig. 4), the application of the findings is restricted to comparative studies and no absolute salinity values can be predicted based on the vessel density. The merging data can at least partly be attributed to the interplay between salinity and inundation frequency in their influence on the mangrove trees’ vascular features. Next to a decrease in salinity a lower inundation class is also observed to result in the presence of relatively few vessels (George and Nielsen, 2000; Woodcock et al., 2000; Yáñez-Espinosa and Terrazas, 2001), as this study confirms (Table 2). Again, no such findings were reported by Yáñez-Espinosa et al. (2001) on R. mangle of Mexico. The interaction between inundation class and salinity as is reported here is a result of evaporation of the soil water when the tide is out, alternating with a regular or only an occasional flooding with sea water at respectively low and high inundation classes (Lin and Sternberg, 1992; Passioura et al., 1992; Lin and Sternberg, 1993). Aside from differences in tidal regime, the overlap in vessel density data can be caused by other factors making the salinity vary within and between sites. As mentioned before, these factors include land run-off, groundwater flow, river input, soil type and the degree of evaporation. In future studies, these factors have to be accounted for just as wood anatomical features have to be sought, that are exclusively related to salinity or inundation frequency. For example, ray height was found to give a good correlation with inundation frequency in some mangrove species of Mexico (Yáñez-Espinosa et al., 2001). A combination of different wood anatomical features might then result in a proxy that allows the reconstruction of absolute changes in salinity, rather than only relative changes when studying vessel density alone. The proxy will reach an intra-annual resolution since the salinity data are integrated over the rainy or dry season.

38

Conclusions and perspectives

In this study, a clear trend of increasing vessel density with rising soil water salinity was demonstrated. Although more information is needed, neither age nor growth rate were found to interfere with the relationship between vessel density and salinity. We can conclude that vessel density is a promising environmental proxy, in particular for tropical dendrochronology, since adequate proxies for tropical regions are limited (Robertson et al., 2004; Speer et al., 2004; Brienen and Zuidema, 2005; Heinrich and Banks, 2005). Vessel diameter was surprisingly less sensitive to changes in salinity. The radial diameter was slightly more responsive to environmental variations as opposed to the tangential diameter, which thus far could not be explained. In R. mucronata of Gazi Bay it is therefore a high vessel frequency and not a small vessel size that most likely represents a strategy for conductive safety. Our study contributed to elucidate the link between the hydraulic architecture of trees and their environment. But, to fully resolve this highly interesting research question insight has to be gained in the functional significance of the lacking plasticity in vessel diameter, the link with the intervessel pits and the underlying developmental and physiological processes.

Acknowledgements

The authors thank Dr. D.P. Gillikin for the helpful comments on an earlier version of the manuscript and assistance collecting the samples; the KMFRI staff for logistical help in the field; the two anonymous reviewers and Tim Colmer (Handling Editor) for their constructive comments and suggestions. This research was funded by a Ph.D. grant of the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen) and a project of the Belgian Federal Science Policy Office (MO/37/015).

Chapter 1

Comment

Soil water salinity in the mangrove forest is a highly dynamic environmental characteristic. Next to the spatial differences related to topography and tidal inundation, temporal differences can be large. The variability depends on the position along the coastal slope, the seasonal climate and the tidal regime with a daily, monthly and annual cycle. To document the entire salinity range, sampling should ideally be done: (1) before and after tidal inundation, which can take place two times a day or only few days in a month depending on the position along the coastal slope, (2) at the start and the end of the dry season, during the rainy season before and after a shower and at the end of the rainy season, (3) at highest spring tide during the equinoxes and at lowest neap tide. Since sampling of soil water and measurement of its salinity can only be done manually at the different positions in the forest this is an extensive job, not done yet thus far. Therefore, the salinity values given in table 1 that are based on one to six measurements carried out at one or two moments during the year give an idea of the difference in soil water salinity between the study sites. We can, however, not exclude that a full overview of the salinity variation will not influence interpretation of the data.

39


40

Chapter 2

41

Intervessel pits in two mangrove species

Chapter 2

Comparative anatomy of intervessel pits in two mangrove species growing along a natural salinity gradient in Gazi Bay, Kenya


Abstract

Background and Aims According to the air-seeding hypothesis, embolism vulnerability in xylem elements is linked directly to bordered pit structure and functioning. To elucidate the adaptive potential of intervessel pits towards fluctuating environmental conditions, we investigated two mangrove species with a distinct ecological distribution growing along a natural salinity gradient.

Methods Scanning and transmission electron microscopic observations were conducted to obtain qualitative and quantitative characteristics of alternate intervessel pits in Avicennia marina and scalariform intervessel pits in Rhizophora mucronata. Wood samples from three to six trees were collected at seven and five sites for A. marina and R. mucronata, respectively, with considerable differences between sites in soil water salinity.

Key Results Vestured pits without visible pores in the pit membrane were observed in A. marina, the mangrove species with the widest geographical distribution on global as well as local scale. Their thick pit membranes (on average 370 nm) and minute pit apertures may contribute to reduced vulnerability to cavitation of this highly salt tolerant species. The smaller ecological distribution of R. mucronata was in accordance with wider pit apertures and a slightly higher pitfield fraction (67 % vs. 60 % in A. marina). Nonetheless, its outer pit apertures were observed to be funnel-shaped shielding non-porous pit membranes. No trends in intervessel pit size were observed with increasing soil water salinity of the site.

Conclusions The contrasting ecological distribution of two mangrove species was reflected in the geometry and pit membrane characteristics of their intervessel pits. Within species, intervessel pit size seemed to be independent of spatial variations in environmental conditions and was only weakly correlated with vessel diameter. Further research on pit formation and function has to clarify the large variations in intervessel pit size within trees and even within single vessels.

Keywords: Rhizophora mucronata, Avicennia marina, intervessel pits, salinity, Kenya, pit membrane, vestures, ecological wood anatomy, cavitation vulnerability, xylem, field-emission SEM, TEM

42

Chapter 2

Introduction

Pits in xylem conductive elements fulfil an important role in vascular water transport in trees (Tyree and Zimmermann, 2002; Holbrook and Zwieniecki, 2005). Numerous investigations have evaluated the contribution of the structure of bordered pits and pit membranes to the efficiency and safety of sap ascent (e.g. Choat et al., 2003; Wheeler et al., 2005; Choat et al., 2006). However, detailed studies dealing with intra- and interspecific variation in intervessel pits, with respect to pit membrane as well as pit geometry, remain scarce (Sano, 2005; Domec et al., 2006).

Mangrove forests are an interesting habitat for studying intervessel pit characteristics along an ecological gradient because mangrove trees are subject to a salt stress that may change considerably even within a small area (e.g. Middelburg et al., 1996; Marchand et al., 2004). Mangrove trees growing at contrasting salinity levels have been shown to differ in cavitation vulnerability, suggesting a parallel variation in xylem structure (Melcher et al., 2001; Ewers et al., 2004), including both vessel and intervessel pit characteristics. A cavitation-resistant xylem structure is one of the strategies plants may use to safeguard their water transport (Tyree and Sperry, 1989; Tyree and Ewers, 1991; Reich et al., 2003). In particular, small conduit diameters are well known to cause a decrease in transport efficiency but also to provide greater hydraulic safety (Salleo and Lo Gullo, 1986; Mauseth and Plemons-Rodriguez, 1998; Corcuera et al., 2004; Mauseth and Stevenson, 2004). In the mangrove Rhizophora mucronata (Rhizophoraceae) from Kenya, vessels produced in the dry season are slightly smaller than those produced in the rainy season (Verheyden et al., 2004; Verheyden et al., 2005; Schmitz et al., 2006). Also in response to spatial differences in soil water salinity, vessel diameters only slightly varied (Schmitz et al., 2006). Regardless of the vessel diameter, small pit membrane pore diameters (Tyree et al., 1994; Jarbeau et al., 1995; Choat et al., 2003; Sperry and Hacke, 2004) and/or small surface area of the intervessel pits (Orians et al., 2004; Choat et al., 2005; Wheeler et al., 2005; Ellmore et al., 2006; Hacke et al., 2006) have been reported to increase the cavitation resistance of the water transport system. Therefore, we expect that variation in intervessel pit characteristics ensures cavitation resistance in R. mucronata. It is assumed that the functional significance of intervessel pits in this species is reflected in the ecological adaptation of its pit geometry.

The present study examines the ecological plasticity of intervessel pits in the mangrove species R. mucronata and Avicennia marina (Avicenniaceae) in Kenya. A. marina grows under the most extensive range of environmental conditions (Clough, 1984; Ball, 1988; Hegazy, 1998; Matthijs et al., 1999; Lopez-Portillo et al., 2005; Ye et al., 2005), both in terms of latitude and local distribution within mangrove forests (Duke, 1991; Duke et al., 1998; Dahdouh-Guebas et al., 2004). In contrast, the local distribution of R. mucronata is restricted to the seaward side of the forest and to riverine areas under moderate salinity (Table 1). The aim of this paper is to survey both qualitative and quantitative characteristics of intervessel pits, with electron microscopy techniques, in order to (1) compare intervessel pit anatomy between both species, and (2) to perform a within-species study, examining intervessel pits from sites differing in inundation class and salinity conditions. We tested three hypotheses with respect to ecological trends in pit morphology: (1) individual pit size and/or pitfield fraction (% pit membrane area / vessel wall area within a pitfield) is smaller in A. marina compared to R. mucronata, (2) the size and surface area of intervessel pits shows a negative trend within each species with increasing salinity, and (3) pit membrane size is not correlated with vessel diameter.

43


Materials and methods


The study sites are located in the mangrove forest of Gazi Bay (39°30’E, 4°25’S), situated approximately 50 km south of Mombasa, Kenya. Sampling was done in May 2005 at seven sites for A. marina and five sites for R. mucronata. Study sites were chosen to represent locations with different salinity and inundation frequency (Table 1). Soil water salinity data were available from about five (1-10) sampling dates in the rainy season (May 2005, June 2006) and for the A. marina sites also from the dry season (February 2006, except from site 5). At each site, the soil water was collected in triplicate at approximately 25 cm depth with a punctured plastic tube connected to a vacuum pump and measured with a hand-held refractometer (ATAGO, Tokyo, Japan). Depending on the topography and the tidal range, zones of different inundation classes can be defined. Inundation classes one, two, three and four correspond to an area being inundated by respectively 100-76 %, 75-51 %, 50-26 % and 25-5 % of the high tides (Tomlinson, 1994). Samples were excised at approximately 1.3 m height with a hollow puncher of 3 mm diameter for the R. mucronata trees and a hand saw for the A. marina trees. Three trees were sampled per site and per species, except from site four and five of R. mucronata, where five and six trees were sampled respectively. For both species, additional samples were collected from two trees at two sites. The samples were immediately stored in 30 % alcohol until analysis. The range of tree circumference (measured at the base of the tree) and tree height (calculated trigonometrically) was 4-135 cm and 1-7 m for A. marina and 12-33 cm and 3-7 m for R. mucronata respectively (Table 1).

SiteSalinity (‰)† Inundation Tree characters (range)

Min. Max. Range class‡ Circumference (cm)* Height (m)Avicennia marina

1 21 38 17 1 32-135 6-72 40 68 28 2 40-49 3-43 40 80 40 3 26-30 5-64 38 82 44 3 4-41 1-45 5 68 63 4 33-101 46 10 80 70 4 33-43 57 10 96 86 4 37-82 4-5

Rhizophora mucronata1 30 33 3 3 19-22 72 0 11 11 4 18-25 4-53 21 38 17 1 18-27 6-74 22 42 20 2 12-20 45 18 49 31 3 12-33 3-6

Table 1. Environmental and tree characteristics of the three Avicennia marina and Rhizophora mucronata trees sampled at each site in the mangrove forest of Gazi Bay (Kenya).

†Soil water salinity at 25 cm depth, representing spatial and temporal variations; ‡Inundation classes 1 to 4 correspond to an area being inundated by respectively 100-76 %, 75-51 %, 50-26 % and 25-5 % of the high tides (Tomlinson 1994); *Measured at the base of the tree.

Sample preparation and image analysis

Scanning electron microscopic (SEM) observations were carried out on three trees per site with a HITACHI cold field emission SEM S-4700 (Hitachi High Technologies Corp., Tokyo, Japan). Samples were trimmed into cubes of approximately 3 mm3 and split tangentially. The blocks were dehydrated

44

for five minutes in an ethanol series (50 %, 70 %, 90 %, 100 %) and air-dried. They were mounted on stubs with electron conductive carbon cement (Neubauer chemikaliën, Münster, Germany) and sputter coated with platinum using an EMITECH K550 sputter coater (Emitech Ltd., Ashford, U.K.). The remaining eight trees, two from site one and site three for A. marina, and two from site three and site five for R. mucronata were cut into blocks of about 2 mm³ for transmission electron microscopic (TEM) observations. The samples were dehydrated through a graded ethanol series (30 %, 50 %, 70 %, 90 %, 100 %). The ethanol was gradually replaced with LR WHITE resin (London Resin Co, Reading, U.K.) over several days. The resin was polymerized at 60 °C and 1,000 mm Hg for 18 to 24 hours. Embedded samples were trimmed and sectioned on an ultramicrotome (ULTRACUT, Reichert-Jung, Austria). Sections of 1 and 2 μm were cut with a glass knife, heat-fixed to glass slides and stained with 0.5 % toluidine blue O in 0.1 M phosphate buffer. Resin-embedded material was prepared for TEM-observations by cutting ultra-thin sections between 60 and 90 nm using a diamond knife. The sections were attached to FORMVAR grids and stained with uranyl acetate and lead citrate using a LKB 2168 ultrostainer (LKB-Produkter AB, Bromma, Sweden). Observations were carried out using a JEOL JEM-1210 TEM (Jeol, Tokyo, Japan) at 80 kV accelerating voltage and digital images were taken using a MegaView III camera (Soft Imaging System, Münster, Germany).

Anatomical measurements

Horizontal and vertical pit membrane diameters (Fig. 1D) of approximately 40 pits per vessel were measured on SEM images of A. marina. For images showing more than 40 pits a labelled grid was used to randomly select 40 pits. Similarly, about 20 pits per vessel were examined for R. mucronata (Fig. 1G and H). Measurements were carried out on three to seven vessels per tree, with a total number of three trees per study site in order to examine a total number of 600 and 300 pits per site for A. marina and R. mucronata, respectively. As for A. marina, pit density (number of pits per vessel wall area) was measured on the same images, in quadrats comprising approximately 40 pits per vessel. The shortest and longest axis of the pit apertures were measured in surface view on SEM images, at the broadest point including the vestures. For both outer and inner pit apertures, we measured three to seven random trees, including around 300 pits for A. marina and 150 pits for R. mucronata (Table 2). Measurements were carried out manually with the image analysis software AnalySIS 3.2 (Soft Imaging System GmbH, Münster, Germany). Pit membrane area was calculated via the formula of the area of an ellipse and together with the pit density they allowed us to calculate the percentage of pit membrane area per unit wall area in a pit field, hereafter referred to as pitfield fraction. With respect to R. mucronata, pit membrane areas were calculated via the formula of a rectangle and the sum was compared to the total wall area. SEM images of A. marina showing the full width of a vessel were used to determine the vessel diameter in comparison with the average pit membrane diameter. For R. mucronata, the horizontal pit membrane diameter was considered as similar to the vessel diameter. Consequently, vertical pit diameters were used to evaluate the intraspecific variation of R. mucronata instead of individual pit membrane areas as used for A. marina. Intervessel wall thickness, pit membrane thickness and pit chamber depth (see Fig. 2) were measured on TEM images from four A. marina trees and three R. mucronata trees (from one tree no measurements could be made).

Fig. 1. Scanning electron micrographs of intervessel pits of Avicennia marina (B-F) and Rhizophora mucronata (A,G,H) in surface view. (A) Vessel element of R. mucronata showing scalariform intervessel pitting and a scalariform perforation plate with five bars. (B) Vessel element of A. marina showing alternate intervessel pitting and a simple perforation plate. (C) Detail of intervessel pits of A. marina with detached pit membranes showing outer pit apertures, surrounded by lip-like vestures. Arrowheads indicate pits with intact pit membranes. (D) Lip-like vestures protruding from the outer aperture into the pit chamber. Dv, vertical pit diameter; Dh, horizontal pit diameter. (E) Vestures at the lumen side of the vessel, more or less extended to horizontal wall thickenings. Arrows indicate inner apertures.

Chapter 2

45

(continued from p. 42)(F) Vestures in their hypothesized role as supporters of the pit membrane (arrowheads), here only covering half of the pit chamber. (G) Part of a vessel element of R. mucronata showing scalariform pits. Dh, horizontal pit diameter. (H) Detail of scalariform pits with outer pit apertures partly covered with the pit membranes (arrowhead). Dv, vertical pit diameter.


46

Vessel grouping was measured in three A. marina trees of an additional site and five R. mucronata trees of site one (Table 1). The percentage of solitary vessels and the vessel grouping index were calculated. At both sites, average soil water salinity (32 ‰) and inundation class (class 3) were similar.


For statistical analyses raw data were used. One-way ANOVA analyses were performed to test the effect of different trees of the same site on individual pit size, pit membrane area in the case of A. marina and vertical pit membrane diameter in the case of R. mucronata. Since horizontal pit diameters of R. mucronata trees are related to the vessel diameters, pit areas are inappropriate for intraspecific comparison. Sites were ordered according to the salinity range of the site instead of the average salinity that is not experienced by the tree. Pearson and Spearman R correlation coefficients were calculated between the diameters of intervessel pits and xylem vessels of A. marina and R. mucronata respectively. T-tests for independent variables, with unpooled variances, were carried out to compare pit characteristics between the study species. When the assumption of normality was not met a Kolmogorov-Smirnov two-sample test was executed instead.

Intervessel Pit Characters A. marina n R. mucronata n P-value†

Pitfield fraction (%)‡ 60 ± 8 105 67 ± 4 79 < 0.0001*(39 - 91) (54 - 75)

Vertical pit diameter (µm) 3.0 ± 0.4 4359 3.4 ± 0.5 1919 < 0.0001*(1.5 - 5.2) (1.8 - 5.6)

Horizontal pit diameter (µm) 3.0 ± 0.5 4359 45 ± 17 1561 < 0.001(1.4 - 6.8) (4 - 85)

Pit membrane area (µm²) 7 ± 2 4359 136 ± 87 1827 < 0.001(2 - 15) (0 - 357)

Pit membrane thickness (µm) 0.37 ± 0.08 129 0.3 ± 0.1 83 < 0.001(0.23 - 0.61) (0.1 - 0.5)

Min. chamber depth (µm)§ 0.09 ± 0.05 55 0.15 ± 0.05 74 < 0.0001*(0 - 0.19) (0.07 - 0.30)

Max. chamber depth (µm)§ 0.28 ± 0.08 120 0.7 ± 0.1 53 < 0.001(0.16 - 0.57) (0.4 - 1)

Intervessel wall thickness (µm) 7 ± 2 108 9 ± 1 70 < 0.001(4 - 11) (7 - 11)

Shortest axis of inner aperture (µm) 0.6 ± 0.1 307 1.4 ± 0.3 135 < 0.001*(0.3 - 0.9) (0.7 - 2.4)

Longest axis of inner aperture (µm) 1.6 ± 0.3 307 36 ± 9 135 < 0.001*(0.9 - 2.9) (13 - 56)

Shortest axis of outer aperture (µm) 0.5 ± 0.2 301 0.9 ± 0.2 149 < 0.001(0.2 - 1.7) (0.3 - 1.7)

Longest axis of outer aperture (µm) 1.9 ± 0.4 301 46 ± 19 149 < 0.001(0.5 - 3.0) (7 - 68)

Table 2. Comparison of quantitative intervessel pit characteristics of Avicennia marina and Rhizophora mucro-nata. Values are means and standard deviations with the minimum and maximum values between brackets.

†Significance value of a t-test (*) or Kolmogorov-Smirnov test from independent samples depending on the normality of the data; ‡Percentage of the pit membrane area per vessel wall area within a pitfield; §Measured as illustrated in Fig. 2A,D.

Chapter 2

47

Fig. 2. Transmission electron micrographs of longitudinal sections of intervessel pits of Avicennia marina (A-C) and Rhizophora mucronata (D-F). (A) Intervessel pit of A. marina showing rudimentary vestures and pit membrane of low electron density. (B) Vestures extending from the outer apertures into the pit chamber and from the inner apertures into the vessel lumen. Note the transparent pit membranes. (C) Vestures in their hy-pothesized role as supporters of the pit membrane. (D) Intervessel pit of R. mucronata showing pit membrane and vessel wall lining of high electron density. (E-F) Overview of intervessel pits of R. mucronata with elec-tron dense pit membranes, a dark lining of the entire secondary wall, shallow pit chambers and a constriction of the pit channels near the outer apertures. Note the difference in pit membrane thickness between E and F. Arrowheads indicate pit membranes; arrows indicate inner apertures; circles indicate pit canal constrictions. *, vestures; a, minimal pit chamber depth; b, maximal pit chamber depth. w, intervessel wall thickness.


48

Results

SEM and TEM observations revealed the presence of intervessel pits with vestures in A. marina (Fig. 1C-F and 2A-C). The vestures were not extensively developed but they were consistently present in all intervessel pits. They adopted a lip-like, unbranched form although irregular forms were seen sporadically. Occasionally, vestures were observed at the lumen side of the vessel, sometimes extended as horizontal wall thickenings near inner pit apertures (Fig. 1E). Aspirated pits were seen in both SEM and TEM images, but with the pit chambers in both species relatively shallow (Table 2), it was difficult to determine whether pit membranes were truly aspirated or not (Fig. 1F and 2C). R. mucronata has non-vestured intervessel pits (Fig. 1G-H and 2D-F).

Our observation of 105 vessels in A. marina and 75 vessels in R. mucronata, which include around 4200 and 1500 intervessel pits respectively, showed that no pores were present in the pit membranes (Fig. 1E and H, 3A-C). Occasionally, small pores were observed in the pit membranes of A. marina but these were interpreted as artefacts due to sample preparation (Fig. 3D).

Chapter 2

Fig. 3. Scanning electron micrographs of intervessel pits of Avicennia marina (A, C-D) and Rhizophora mu-cronata (B) in surface view. Detail of non-porous pit membrane of (A) A. marina and (B) R. mucronata. (C) Porous pit membranes are completely absent in large pit fields of A. marina. (D) Artefactual pores occur in restricted pit field areas damaged by sample preparation.

Quantitative analysis of the pit geometry of both species, as based on SEM and TEM observations, demonstrated that the pitfield fraction, the vertical and horizontal pit diameter, the individual pit membrane area, the pit apertures, the pit chamber depth and the intervessel wall thickness are smaller in A. marina compared to R. mucronata (Table 2). The vessel diameter was only slightly correlated to the horizontal pit diameter in A. marina (Pearson: r² = 0.29, P < 0.01, n = 29) and to the vertical pit diameter in both A. marina (Pearson: r² = 0.13, P = 0.06, n = 29) and R. mucronata (Spearman R: r²

49


= 0.06, P < 0.0001, n = 996). The thickness of A. marina’s pit membranes showed a unimodal distribution and exceeded those of R. mucronata. A clear bimodal pattern was observed in the latter species (Fig. 2E-F and 4). Furthermore, the pit membrane was observed to be more electron dense in R. mucronata as opposed to A. marina (Fig. 2A and D). The pit chamber of both species studied was remarkably shallow, with a pit channel ending in a constriction in R. mucronata (Fig. 2E-F) or with vestures in A. marina. Consequently, one could distinguish a minimum and a maximum pit chamber depth (Fig. 2A and D; Table 2).

The distribution of the individual pit membrane area and vertical pit diameter in A. marina and R. mucronata, respectively, showed a wide range of variation (Fig. 5). Mean individual pit membrane area differed significantly between A. marina trees within a single site (Table 3, Fig. 5A). Within sites, mean vertical pit diameters also differed significantly between R. mucronata trees (Table 3, Fig. 5B).

Factor SiteA. marina R. mucronata

F-value n F-value n

Tree 1 95.83* 3 24.88* 3

2 122.41* 3 51.77* 3

3 75.82* 3 36.35* 3

4 39.14* 3 37.03* 5

5 195.41* 3 62.08* 6

6 181.33* 3

7 42.60* 3

Table 3. Results of one-way ANOVA’s, testing for differences in pit membrane area between Avicennia marina trees and vertical pit diameter between Rhi-zophora mucronata trees within the studied sites (see also Table 1). *P < 0.0001.

0.050.10

0.150.20

0.250.30

0.350.40

0.450.50

0.550.60

0.65

Pit membrane thickness (0.05 µm classes)

0

2

4

6

8

10

12

14

16

Num

ber o

f obs

erva

tions

in A

. mar

ina

0

5

10

15

20

25

30

35

40

45

Num

ber of observationsin R

. mucronata

Rhizophora mucronata Avicennia marina

Fig. 4. Distribution of intervessel pit membrane thick-ness in Rhizophora mucronata and Avicennia marina. Data are from 83 pits from three trees of R. mucronata and from 129 pits from four trees of A. marina.

Discussion

Vestured pits in Avicennia marina

We observed, as far as we know, for the first time vestured pits in A. marina. Due to the rudimentary nature of the vestures, it is not surprising that probably most previous studies overlooked the presence of this feature (Meylan and Butterfield, 1973; Matthew and Shah, 1983; Krishnamurthy and Sigamani, 1987; Sun and Suzuki, 2000). The vestures appeared as lip-like projections associated with the outer aperture and pointed into the pit chamber (Fig. 1D, 2A). At the lumen side of the vessel the vestures seemed to narrow down the inner pit apertures and to extend the pit canal (Fig. 1E and 2B). Moll and Janssonius (1920) reported that the numerous bordered pits in vessel walls of A. alba were needle-like and that the inner pit apertures showed a needle-like form. As Avicennia is a member of the Lamiales (Schwarzbach and McDade, 2002), the discovery of vestures in A. marina is especially noteworthy since this character is only known in some genera of the Oleaceae (Jansen et al., 2001). Mathew and Shah (1983) reported vestured pits in few genera of Verbenaceae, but not in A. marina. Their observations are, however, not convincing and most likely represent pseudo-vestures. SEM

50

0 10 20 30 40 50 60 70 80 90 1000

2

4

6

8

10

12

14

16

110

Vessel number

Pit m

embr

ane

area

(µm

²)

Site 1 Site 4 Site 7Site 6Site 5Site 3Site 2

Avicennia marinaA

0 10 20 30 40 50 60 70 80 90

Vessel number

2,0

2,5

3,0

3,5

4,0

4,5

5,0

5,5

Verti

cal p

it di

amet

er (µ

m)

Site 1 Site 5Site 4Site 3Site 2

Rhizophora mucronataB

Fig. 5. Distribution of intervessel pit size within ves-sels and trees along a natural salinity gradient. (A) Pit membrane area of the alternate pits of Avicennia marina in three trees for all seven sites with incre-asing salinity range (see Table 1). (B) Vertical pit diameter of Rhizophora mucronata in respectively three (Site 1-3), five (Site 4) and six (Site 5) trees for five sites with increasing salinity range (see Table 1). The horizontal diameter of the majority of the scalariform pits corresponds to the vessel diameter, making comparison of the pit membrane area inap-propriate. The different trees of each site are indica-ted by distinct point markers. On average 100 and 200 pits were measured per A. marina and R. mu-cronata tree, respectively, with 3-7 vessels studied per tree.

images of A. germinans from the Tervuren wood collection (RMCA Tervuren) and light microscopic observations of sections from the Jodrell slide collection (RBG Kew) suggested that vestured pits are present in other species of Avicennia. However, vestures are difficult to detect using a light microscope due to their small size and minute pit apertures. Therefore, SEM observations are required to confirm their occurrence.

The observation of vestures in A. marina is in accordance with the overall confinement of vestured pits to xeric or warm environments (Jansen et al., 2003; Jansen et al., 2004a). This may be related to the functional significance of vestures, at first formulated by Zweypfenning (1978) and later supported by ecological studies (Jansen et al., 2003; Jansen et al., 2004a). Inherently large pit membrane pores and especially an increased porosity or even rupture of the pit membrane has been suggested to be the cause of air-seeding (Hacke and Sperry, 2001; Choat et al., 2003; Sperry and Hacke, 2004; Wheeler et al., 2005). As an increased porosity may result from excessive stretching upon pit-aspiration, it is suggested that adaptations preventing the pit membrane from deflecting are extremely important in view of cavitation resistance. Zweypfenning’s hypothesis states that vestures could provide such advantage by offering mechanical support to stretched pit membranes. The funnel shaped pit channel could offer a similar advantage to the pit membranes of R. mucronata (Fig. 2E and F; Table 2). However, the shallow pit chamber and especially the thickness of the pit membrane itself could also play a substantial role in the prevention of excessive pit membrane stretching in both A. marina and R. mucronata (Fig. 2). Pit membranes are composed of a number of microfibrillar layers (Schmid and Machado, 1968; Sperry and Hacke, 2004; Sano, 2005) perforated by most likely tortuous rather than straight intervessel pathways (Choat et al., 2004). Since SEM images only show surface structures, the openings occasionally observed in pit membranes of A. marina are most likely artefacts resulting from sample preparation (Fig. 3D). Splitting of the wood samples might have removed one of the

Chapter 2

51

microfibrillar sheets of the pit membrane, rendering the membranes more sensitive to damage from preparative handlings such as dehydration. The artefactual nature of the pit membrane pores is supported by their irregular and inconsistent distribution: they are completely absent in many large pit fields (Fig. 3C) and restricted to particular areas (Fig. 3D). Furthermore, the finding of thick, non-porous pit membranes in the two species studied corresponds with previous observations. The thick pit membranes of Fraxinus are less likely to show pores than the thinner pit membranes of Betula, Salix and Ulmus (Sano, 2004, 2005; Choat et al., 2006). The main reason why pores in the pit membranes could not be seen with TEM is most likely due to the thickness of TEM-sections (60-90 nm), which is much larger than the majority of the pit membrane pores.

Intervessel pit morphology of two mangrove species

When comparing overall pit architecture of R. mucronata and A. marina the first conspicuous difference is their pit type (Fig. 1A and B). The minute alternate intervessel pits of A. marina, as opposed to R. mucronata’s scalariform pitting, resulted in a slightly smaller pitfield fraction in A. marina (Table 2). A small pitfield fraction implies a lower cavitation risk (Hargrave et al., 1994; Choat et al., 2003; Choat et al., 2004), since the occurrence and size of inherently large pit membrane pores is thought to increase with the total pit membrane area per vessel (Wheeler et al., 2005). However, because of the much higher percentage of solitary vessels in R. mucronata than in A. marina (79 ± 6 vs. 33 ± 24, t = -28.4, df = 127, P < 0.0001) and the lower vessel grouping index (1.25 ± 0.08 vs. 2.0 ± 0.6, t = 21.9, df = 359, P < 0.0001) R. mucronata’s overlapping pitfield area between neighbouring vessels may be smaller. It is thus possible that the intervessel pit membrane area of the entire vessel network is much higher in A. marina.

Structural differences in the pit micromorphology between both species could be interpreted as alternative solutions to cope with the saline mangrove environment. Inner and outer pit apertures were comparatively smaller in A. marina than in R. mucronata (Table 2). The minute pit apertures are related to the bordering vestures and result in an extended compartmentalization of the water transport system, minimizing conductivity loss from expanding embolisms (Ellmore et al., 2006). In addition, small intervessel pits reduce the actual sealing area of the pit membrane and thus increase the air seeding pressure (Wheeler et al., 2005). Pit membranes in A. marina were generally thicker, increasing on one side the hydraulic resistance (Choat et al., 2006), but on the other side decreasing the vulnerability to cycles of cavitation and refilling (Hacke et al., 2001). These are likely to occur in A. marina since vestured pits may help in embolism repair (Jansen et al., 2003), as do the abundant paratracheal parenchyma and the included phloem tissue (Holbrook and Zwieniecki, 1999; Tyree et al., 1999; Salleo et al., 2004; Stiller et al., 2005; Salleo, 2006). The thickness of R. mucronata’s pit membranes showed a bimodal distribution (Fig. 2E-F and 4), which could be caused by for instance wound induced depositions (Schmitt et al., 1997; Frankenstein et al., 2006) or by depositions due to seasonal variations (Wheeler, 1981; Sano, 2004). As far as we know, however, the wood samples collected were not from stems subject to any wounding. Seasonal changes are more likely. The average annual growth rate of R. mucronata in Gazi (Kenya) is 1.17 ± 0.73 mm/year (Verheyden et al., 2004). Given that the differences in the pit membrane thickness were found in radial sections of 2 mm², the sections possibly contained both wood formed during the dry season and the rainy season. Alternatively, the pit membrane thickness may well be related to the thickness of the secondary cell wall. Our observations showed that vessels with a narrow diameter have thinner cell walls than large vessels (results not shown), but further research is needed to test if this is also associated with a difference in pit membrane thickness. The electron density of pit membranes in R. mucronata contrasted strikingly with the more transparent pit membranes of A. marina (Fig. 2A and D). This could be due to a different chemical composition of the pit membrane in both species. An electron dense layer lining R. mucronata’s vessel walls (Fig. 2E and F) suggested that the wood samples of


52

this species are impregnated with substances characteristic of Rhizophora. Fresh material would be desired to see if the electron dense layer on the vessel walls is also present after fixation. Further TEM-observations would be interesting to examine the chemical composition of the pit membranes (Bauch and Berndt, 1973; Coleman et al., 2004).

As for many tropical trees, there is a lack of data on quantitative pit characters. In comparison to the few data on pit geometry in temperate trees, the intervessel pit anatomy of the two mangrove species studied suggests an increased hydraulic safety. The average pit chamber depth of the deciduous tree Sophora japonica is 0.84 µm and 0.2 µm, with and without inclusion of the vestures respectively. Fraxinus americana has pits with an average pit chamber of 0.61 µm deep (Choat et al., 2004). Compared to the mangrove species studied, these pit chambers are remarkably deep (Table 2). Schmid and Machado (1968) reported intervessel pit membranes of 0.25 - 0.35 µm thick in Leguminosae, with the membranes of air dried samples as thin as 100 - 200 nm. Pit membranes in vessels from temperate trees are generally less than 200 nm thick (SJ, unpublished data). The relatively thick pit membranes of R. mucronata and A. marina (Table 2) suggest a considerable impact of pit membrane thickness on the hydraulic resistance of a tree (Choat et al., 2006). When a high safety is not of prime importance the formation of thick pit membranes would be unfavourable. Furthermore, the shortest axis of the outer pit apertures was remarkably smaller in the mangrove species studied (Table 2) than the 0.8 – 1.89 µm sized apertures in Ulmus laciniata (Jansen et al., 2004b). The longest axis of the outer pit apertures, 1.64 – 3.29 µm in Ulmus laciniata (Jansen et al., 2004b), was wider in R. mucronata but generally shorter in A. marina (Table 2). Also, the pit aperture area of 2.3 - 3.9 µm² as reported for Acer and Betula species (Orians et al., 2004) is larger than the 0.7 µm² as calculated for A. marina (Table 2). The shallow pit chambers, thick pit membranes and small pit apertures could outweigh the negative effect on the hydraulic safety of both mangrove species’ comparatively large pitfield fraction (Table 2). The pitfield fraction of several temperate tree species (including evergreens) ranges from 9 % to 67 % (Orians et al., 2004; Choat et al., 2006; Ellmore et al., 2006; Hacke et al., 2006). These findings support the hypothesis that intervessel pit distribution is a compromise between hydraulic safety and efficiency (Sperry, 2003).

No intraspecific trends in intervessel pit size

The absence of an intraspecific trend in intervessel pit size with varying salinity conditions (Fig. 5) suggests that within species the ecological adaptability of the hydraulic architecture is restricted to vessel dimensions and vessel frequency. However, three trees per site are possibly insufficient to uncover a potential relationship between intervessel pit size and salinity because of the considerable variation within sites and trees. The potential difference in actual pit membrane pore sizes between and within the mangrove species studied could not be determined in this study. The natural porosity of the pit membranes remains to be verified since this character is closely related to cavitation vulnerability based on the air-seeding theory. Using fresh material, particle perfusion experiments should be performed in combination with air-seeding measurements to determine the size of the rare largest pores, which are responsible for cavitation (Choat et al., 2003; Choat et al., 2004; Wheeler et al., 2005).

Altogether, individual pit size and pit field fraction were smaller in A. marina than in R. mucronata and the diameter of the intervessel pits was only slightly correlated with the diameter of the corresponding vessels, as postulated in the introduction. The hypothesis of a decreasing trend in pit size with varying salinity was rejected for both species. It is proposed that intervessel pit size and geometry are mainly determined by genetic factors with the absence of a phenotypic plasticity related to the widely fluctuating environmental conditions of the mangrove habitat. The minor decrease in vessel diameter of R. mucronata with increasing substrate salinity (Schmitz et al., 2006), is thus not compensated for

Chapter 2

53

by a decrease in pit size offering a higher cavitation resistance. Therefore, the functional significance of the fluctuating vessel density should be addressed in future studies. Furthermore, there is need for additional comparative research, in combination with experimental tests, both between species and localities and within individual trees, to elucidate the adaptive and functional significance of the intervessel pits and their role in sap ascent.

Acknowledgements

This research was financially supported by the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen), the European Commission's Research Infrastructure Action via the SYNTHESYS Project, a travel grant from the National Fund for Scientific Research (FWO, Belgium) and the Schure-Beijerinck-Popping Fonds of the Royal Netherlands Academy of Arts and Sciences (KNAW, Amsterdam) and a project of the Belgian Federal Science Policy Office (MO/37/015). Financial support to SJ was provided by a grant from the Royal Society (2006/Rl) and NERC (NE/E001122/1). We thank two anonymous reviewers for their critical comments on an earlier version of this manuscript, Elisabeth Robert for the design of the photographs and Hamisi Ali Kirauni, the KMFRI staff in Gazi and all the people of Gazi for their invaluable assistance provided in the field and for their hospitality.


54

Chapter 3

Sea-water grows many living thingsof all sorts; and as it has its own kind of animal, so it has its own kind of plant.THEOPHRASTUS

55

Intervessel pit structure and histochemistry

Chapter 3

Intervessel pit structure and histochemistry of two mangrove species as revealed by cellular UV microspectrophotometry and electron microscopy: intraspecific variation and functional significance

Accepted for Microscopy and Microanalysis

Abstract

Background and aims Intervessel pits play a key-role in trees’ water transport, lying at the base of drought-induced cavitation and of the regulation of hydraulic conductivity via hydrogels bordering pit canals. Recently, their microstructure has been the focus of numerous studies but the considerable variation, even within species and the histochemistry of pit membranes remains largely unexplained.

Methods In the present study, intervessel pits of the outermost wood were examined for Avicennia marina, of dry and rainy season wood separately for Rhizophora mucronata. The thickness of the pit membranes was measured on Transmission Electron Micrographs while their topochemical nature was also analysed via cellular UV microspectrophotometry.

Key results Pit membranes of R. mucronata were slightly thicker in dry season than in rainy season wood but their spectra showed for both seasons a lignin and a yet unidentified higher wavelength absorbing component. It was suggested to be a derivative of the deposits, regularly filling pit canals. The vestures of A. marina chemically resembled pit membranes rather than cell walls.

Conclusions The hypothesis of seasonal changes causing differences in pit membrane thickness could only partly be accepted. The seasonal differences were weak and overshadowed by an enormous variability in electron density of the pit membranes. In addition, deposits were found filling pit canals and lining pit borders. But, they did not show a clear correlation with the variation of the pit membranes. The variability between as well as within trees should stimulate further research in the structure of intervessel pits, their chemical composition and ontogeny in order to come to a full understanding of their function in tree hydraulics.

Keywords: intervessel pit, pit membrane, cellular UV microspectrophotometry (UMSP), TEM, mangrove, vesture, lignin, Rhizophora mucronata, Avicennia marina, deposit

56

Chapter 3

Introduction

Intervessel pits are small openings in the vessel wall enabling communication between vessels. Instead of mere holes, porous intervessel pit membranes provide a way for a regulated sap flow (Zwieniecki et al., 2001; Gascó et al., 2006; van Ieperen, 2007). They play a central but dual role in the water transport. On the one side, they are the Achilles heel of the hydraulic structure where tiny air bubbles can be sucked through, that expand under the tension in the vessel (e.g. Sperry and Tyree, 1988; Sperry and Hacke, 2004; Wheeler et al., 2005; Domec et al., 2006). On the other side, they offer a way to circumvent these expanded air bubbles or drought-induced embolisms, that block the water transport (Orians et al., 2004; Ellmore et al., 2006; Hacke et al., 2006). Given their role in sap flow, the observation of a large variation in pit membrane thickness and porosity should not be surprising. Numerous recent studies focused on the microstructure of the intervessel pits and particularly their pit membranes (Singh et al., 1999; Pesacreta et al., 2005; Sano, 2005; Schmitz et al., 2007a). A tenfold variation in pit membrane thickness and a variation of even two orders of magnitude in maximum pit membrane porosity were demonstrated across fourteen hardwood species (Choat et al., 2008).

This structural variation of pit membranes can among others be related to different stages in pit development (Wardrop et al., 1963; Schmid and Machado, 1968) or to secondary deposits. Besides heartwood formation (Bonner and Thomas, 1972; Wheeler and Thomas, 1981; Wheeler, 1982; Sano and Fukuzawa, 1994; Streit and Fengel, 1994; Sano and Nakada, 1998; Koch et al., 2006), secondary deposits could be due to mechanical wounding (Morrow and Dute, 1999), biotic (Nemec, 1975; Hammerschmidt and Kuć, 1982; Street et al., 1986) or abiotic stresses (Robb et al., 1980; Robb and Busch, 1982) including seasonal changes (Yang, 1978; Wheeler, 1981; Sano et al., 1999). Under a seasonal tropical climate, trees experience an increased risk for drought-induced embolism during the dry season. In this case, thicker pit membranes are less likely to develop exceptionally large pores by excessive stretching when aspirated by the pressure difference between a functional and an embolized vessel (Hacke et al. 2001, Choat et al., 2003). It is this maximum pore size that is hypothesized to determine embolism vulnerability rather than the average porosity (Wheeler et al., 2005; Hacke et al., 2006; Choat et al., 2008). Next to changes of the pit membrane itself, vestures or appendices of the vessel wall in pit canals and/or pit chambers (Fig. 1a, l) can impede excessive stretching by supporting the pit membrane and hence lowering the vulnerability to embolism (Zweypfennig, 1978; Jansen et al., 1998; Jansen et al., 2003; Choat et al., 2004; Jansen et al., 2004; Sperry and Hacke, 2004).

The chemical nature of the intervessel pit membranes is just as variable and important for sap flow in trees as is its structure. Pit membrane porosity is not the sole determinant of the pressure that is needed between a functional and an air-filled vessel for air-seeding to occur. Also the contact angle between pit membrane and air-water interface, which is a function of the surface chemistry of the pit membrane, has an influence (Holbrook and Zwieniecki, 1999; Zwieniecki and Holbrook, 2000; Meyra et al., 2007). More hydrophobic substances, such as lignin, increase the contact angle and lower the air-seeding pressure. In addition, high lignin content has been assumed to hinder the

Fig. 1. Transmission electron micrographs of longitudinal sections of intervessel pits of Rhizophora mucro-nata (a-k) and Avicennia marina (l). (a-c) Intervessel pits with dark staining pit membranes and (d-f) nearly transparent pit membranes with pit annuli present (arrows). (g-h) Remarkably thick pit border linings at only one side of the vessel pair. (i-j) Membrane-like structures bordering inner and outer pit apertures, respectively (arrow heads). (k) Granular material lining pit borders. (l) Intervessel pits of A. marina with dark staining vestures (arrows), pit membranes of rather low electron density and a thin layer lining pit borders. Samples shown are 3878D, 3870D, 3884D, 3872D, 3873R, 3882D, 3876D, 3885R, 3879R, 3874D, 3871R and 3886 with D, dry season wood and R, rainy season wood (Table 1). Lv, vessel lining; Lpc, pit canal lining; PC, pit canal; Pch, pit chamber; PM, pit membrane; W, intervessel wall thickness.

57


58

Chapter 3

hydrogel activity of pectins in pit membranes (Tibbits et al., 1998; Ridley et al., 2001; Boyce et al., 2004). Pectic substances can swell and shrink in reaction to changes in the ionic composition of the xylem sap, altering pit membrane porosity. Lignin would hamper this adjustment and thus the regulation of the hydraulic conductance via hydrogels as found in some species (Zwieniecki et al., 2001; Gascó et al., 2006; van Ieperen, 2007). The functional advantage of pit membranes of low lignin content is supported by the reduced lignin biosynthesis under water stress (Donaldson, 2002; Alvarez et al., 2008).

Considering the significance of the chemical nature of pit membranes for water transport in trees, variation in the histochemistry of pit membranes is not unexpected. However, a conclusive explanation could not yet be given. Non-lignified pit membranes were observed, in both softwoods and hardwoods between rays and tracheary elements (Bamber, 1961; Chafe, 1974) and between fiber cells (Wardrop, 1957) using different staining methods or UV microscopy, respectively. Rudman (1965) expected lignin-free pit membranes between vessel cells too. In confirmation, little or no lignin was observed in juvenile wood of different hardwood species after diverse staining (O'Brien, 1970; Coleman et al., 2004) or immunolocalization techniques (Chaffey et al., 1997). It remains, however, unclear whether mature wood would have given the same results. Besides, the negative staining reaction for lignin in Salix (O'Brien, 1970) could be due to its extremely thin and porous pit membranes (Sano, 2005). These doubts are enforced by staining as well UV microspectrophotometry of mature wood samples showing lignified pit membranes between tracheary elements in different softwoods (Bauch and Berndt, 1973; Sano and Nakada, 1998; Donaldson, 2002). Atomic force microscopy of the hardwood Sapium sebiferum supported this finding (Pesacreta et al., 2005), as did a back-scattered electron microscopy analysis of vessel-fibre pit membranes in Fagus sylvatica (Fromm et al., 2003). Nevertheless, adding to the variation are the pit membranes of Pinus that only seemed to contain lignin in heartwood (Fengel and Wolfsgruber, 1971).

In this study, intervessel pit structure and chemistry of two Kenyan mangrove species will be examined. Rhizophora mucronata is restricted to the seaward side of the forest and areas influenced by fresh river water. Avicennia marina has a much wider distribution both globally and locally, where it occurs in a disjunct pattern at both seaward and landward side of the forest (Dahdouh-Guebas et al., 2004). The extreme mangrove environment concerning stresses imposed on the water transport makes them appropriate study species to assess the ecological plasticity of intervessel pit properties. Recently, a bimodal distribution of the pit membrane thickness was reported in R. mucronata but not in A. marina (Schmitz et al., 2007a) corresponding to their periodic (Verheyden et al., 2004) and patchy wood formation (Schmitz et al., 2008), respectively. In addition, the electron density of the pit membranes using Transmission Electron Microscopy (TEM) was considerably higher in R. mucronata compared to A. marina suggesting a different chemical composition (Schmitz et al., 2007a). The objectives of the study were approached via a combination of TEM and cellular UV microspectrophotometry (UMSP). The latter technique has proven its high value to visualize and quantify the chemical composition of cell wall structures and contents (Frankenstein et al., 2006; Koch et al., 2006). UV scans of semi-thin wood sections at a fixed wavelength, chosen at the absorbance maximum of the interested compound, inform about its absorbance intensity that is related to its concentration. UV spectra over a range of wavelengths but at a selected position of the section, inform about the chemical composition of the spot though without identifying observed absorbance peaks (Koch and Grünwald, 2004). First, the hypothesis will be tested that the dimorphism in the thickness of the intervessel pit membranes of R. mucronata is related to the seasonal climate at Gazi Bay. Second, we will explore the chemical composition of the intervessel pits of R. mucronata and A. marina. Pit membranes as well as pit chambers and pit canals will be addressed as a first step towards the clarification of intervessel pit chemistry and its internal variation, which to date remains largely unknown.

59



Study sites are located in the mangrove forest of Gazi Bay (39°30’E, 4°25’S), situated approx. 50 km south of Mombasa, Kenya. The climate along the Kenyan coast is characterized by a bimodal distribution of the precipitation. A distinct dry season (December - March) is followed by a long (April - July) and a short rainy season (October - November). During the wet season, the rivers Mkurumuji and Kidogoweni provide an important freshwater source for the mangroves of Gazi Bay (Kitheka, 1997). Samples were collected in February 2006 from two A. marina trees (Table 1, accession numbers 3886 and 3887) and in June 2006 from eight R. mucronata trees comprising two trees from four sites each (Table 1, accession numbers 3870-3885). Wedges of the outermost wood of about 10 x 3 x 4 cm³ were excised at approx. 1.3 m height with a handsaw and immediately stored in 50 % alcohol.


Species/LocationSample number C130

† Tree height Soil Water Salinity (‰) Inundation

Dry season Rainy season (cm) (m) Min. Max. class‡

Rhizophora mucronataSite 1 3878, -80 3879, -81 55 ± 12 8 ± 1 21 46 1Site 2 3870, -72 3871, -73 32 ± 7 7 ± 2 0 11 3Site 3 3874, -76 3875, -77 51 ± 12 9 ± 0 30 33 2Site 4 3882, -84 3881, -85 36 ± 9 6 ± 1 26 40 2

Avicennia marina

Site 5 3886-87* 25 ± 4 7 ± 1 5 68 3

Table 1. Environmental and tree characteristics of the eight Rhizophora mucronata trees and two Avicennia marina trees sampled. Consecutive sample numbers for R. mucronata are from one annual ring.

†Stem circumference at 130 cm height; ‡inundation classes 1 to 3 correspond to an area being inundated by respectively 100-76 %, 75-51 % and 50-26 % of the high tides (Tomlinson, 1994); *no distinction could be made between dry and rainy season wood (Schmitz et al., 2007b).

Sample preparation and analysis

From each R. mucronata wood sample a broad growth ring was chosen to separate the wood formed during the rainy season from the wood formed during the dry season (Table 1, pair and impair accession numbers, dry and rainy season respectively). The distinction between seasons is based on a corresponding change in vessel density (Verheyden et al., 2004; Schmitz et al., 2006). Wood samples were trimmed into cubes of 1 x 5 x 1 mm³, dehydrated in an acetone series (30 % - 100 %), infiltrated with Spurr’s epoxy resin through a series of propylene oxide / resin mixture and embedded at 70 ° C. Longitudinal sections of around 100 nm thickness were made with an ultramicrotome (Ultracut E, REICHERT - JUNG) using a diamond knife and stained with a 1 % potassium permanganate solution for TEM analysis with a Philips CM 12 at an accelerating voltage of 60 kV. Photographs were taken from one or two positions showing intervessel pits. Thickness of the pit membranes, of the vessel and pit canal linings (Fig. 1a, Lv and Lpc respectively) and of the intervessel walls (Fig. 1b, W) was measured with the image analysis software AnalySIS 3.2 (Soft Imaging System GmbH, Münster, Germany). Linings were defined as thin, electron dense layers that bordered vessel lumina including intervessel pits. They were measured for each vessel of the intervessel pit pair. T-tests for dependent samples and Pearson correlation analyses were performed in STATISTICA (StatSoft, Inc. 2006, data analysis software system, version 7.1., www.statsoft.com) after transforming the data with an inverse function, when data were not normally distributed. For the samples with two measuring positions, data were averaged per sample.

60

Chapter 3

For cellular UV microspectrophotometry, unstained sections of 1 µm thickness were transferred to quartz slides, immersed in a drop of non-UV absorbing glycerine and covered with a quartz cover slip. Scanning profiles were made with a ZEISS UMSP 80 at a constant wavelength of 280 nm (representing the absorbance maximum of lignin) using an ultrafluar 100:1 objective. Data were recorded and processed with the software programme APAMOS (Zeiss). For sample 3870 an additional scanning profile was made at 560 nm to obtain more details about deposits. The scan programme digitizes rectangular tissue portions with a local geometrical resolution of 0.25 µm² and a photometrical resolution of 4096 grey scales converted into 14 basic colours (Koch and Kleist, 2001). In addition, point measurements (diameter 1 µm) covering a wavelength range from 240 nm to 700 nm were made in 1 nm steps of pit membranes, pit canals, pit canal deposits, vessel walls and vestures using the programme LAMWIN (Zeiss). From the resulting spectra, mean curves were calculated after removing outliers from the dataset.

Results

Transmission Electron Microscopy

Intervessel pit membranes were thicker in the dry season wood compared to the rainy season wood in six of the eight studied R. mucronata trees (Table 2). The electron density of the pit membranes was similar in dry/rainy season wood (Fig. 1a/i, b/k, d/e) with the exception of three trees. Four out of five trees had pit membranes with a higher or similar electron density than the middle lamella in adjacent wall portions (Fig. 1a-b, g, i, k). The electron density was not strictly correlated with the average porosity of the pit membranes. While dark membranes were mostly opaque, some were very porous (Fig. 1c). The two A. marina trees had pit membranes with a similar or lower electron density than the middle lamella areas (Fig. 1l). Next to these general observations a large variability in intervessel pit properties was found between and even within trees. At one position of a dry season sample, several thin membrane-like layers were seen in the pit chamber (Fig. 1f). Two trees from which two positions were analysed for dry or rainy season wood, showed a considerable difference in pit membrane thickness between both positions with the average value being even larger in the rainy than in the dry season. Also the electron density, i.e. the intensity on a grey-scale, contrasted between two positions within the dry season wood of two trees. One tree had pit membranes that were nearly transparent although the pit annulus was highly electron dense (arrows, Fig. 1d-e).

In R. mucronata, vessel lumen, pit canal and pit chamber were lined by a dark staining substance (Fig. 1) with vessel linings somewhat thicker than linings of the corresponding pit border (Table 2). The electron density of the pit membranes (Fig. 1) was unrelated to the thickness of the corresponding linings. One sample had a relatively thicker lining (Fig. 1a) and very dark pit membranes. However, samples with a thin lining had pit membranes of variable electron density. Besides, three samples had a thin lining at one side of the intervessel pit and a thick lining at the other side (Fig. 1g-h). Next to the electron density of the pit membranes, pit membrane thickness was unrelated to thickness of vessel linings (r² = 0.12, p = ns, n = 19) or pit canal linings (r² = 0.19, p = ns, n = 19). On top of these linings, a membrane-like structure of similar electron density delineated the inner pit apertures in half of the samples (n = 16) with five samples being dry season wood (Fig. 1b). Not necessarily both sides of the vessel pair had their inner pit apertures lined by these membrane-like structures but if one aperture was lined, they all were lined at that side of the vessel pair (Fig. 1g, i, k). The same was true for a similar lining at the outer pit aperture that appeared in only one sample (Fig. 1j). Fuzzy, granular material of a similar electron density as the linings bordered the vessel wall from pit membranes to vessel lumen in four samples (Fig. 1b, h, k). No or only a very thin lining was observed in the two studied A. marina trees. The vestures were clearly more electron dense than the corresponding vessel wall (arrows, Fig.

61


Intervessel Pit Character†Median (range) SD‡ n P - value§

(µm)PM thickness D 0.5 (0.2 - 0.7) 0,1 10

< 0.05*PM thickness R 0.4 (0.2 - 0.7) 0,1 9

Vessel lining 0.1 (0.0 - 0.6) 0,1 38< 0.01

PC lining 0.06 (0.03 - 0.25) 0,05 38Intervessel wall thickness 11 (8 - 14) 2 19

Table 2. Quantitative description of the intervessel pits of Rhizophora mucronata wood sampled in the dry and rainy season as measured on longitudinal sections (Fig. 1a-b).

†Intervessel pit membrane thickness of wood formed in the dry (D) and rainy (R) season, thickness of the li-ning of vessel lumen and pit canal (PC) and wall thickness in between a vessel pair; ‡Standard Deviation; §sig-nificance value of a t-test for dependent samples. PM thicknesses were averaged per tree and per season, vessel lining and PC lining data were transformed via the inverse function to comply with the normality assumption; *n = 8, values of samples with more than one measurement were averaged.

1l). Deposits of variable electron density (Fig. 1) filled the pit canals homogenously in all trees of both species. The pit chamber was not filled or with a substance of lower electron density.

UV microspectrophotometry

The UV absorbance (280 nm) of the pit membranes of both species ranged only from about 0 to 0.2 in both R. mucronata (Fig. 2a, f, g) and A. marina (Fig. 2b-d) while the vessel walls were characterised by UV absorbance values from about 0.2 to 0.4 (Fig. 2). Only at one of eleven positions of one sample, pit membranes were of stronger absorbance than cell walls. Absorbance spectra of the pit membranes of both species showed a peak at around 280 nm and a second broad peak starting from about 580 nm up to 700 nm (Fig. 3a). Remarkably, these characteristic peaks were not found for one of the R. mucronata trees. No difference was observed between the spectral behaviour of the pit membranes of R. mucronata wood formed during the dry season or during the rainy season. The vestures in A. marina were not or only slightly absorbing at 280 nm as clearly seen in the scanning profile (arrows, Fig. 2d). The absorbance spectra of the vestures resembled more closely the spectra of the corresponding pit membranes than the vessel wall spectrum (Fig. 3b).

In the pit canals of both species deposits were regularly observed with a low absorbance at 280 nm and 560 nm (yellow arrow heads, Fig. 2a-b, d-f). The absorbance spectra of these low absorbent deposits showed a peak at 280 nm and a broad peak between 540 nm and 700 nm (pc, Fig. 3c). A second type of relatively high absorbent deposits was detected in the pit canals and/or pit chambers of R. mucronata sections analysed shortly after sectioning (black arrow heads, Fig. 2g). Their absorbance spectra showed two high peaks, one at 280 nm and one at around 540 nm or 620 nm (d, Fig. 3c). However, in sections studied later these highly absorbing deposits were only seen sporadically (black arrow heads, Fig. 2a, e-f) and in a time period of hours they had shrunk to the size of droplets and were located in the outer corners of the pit chambers, or in the inner aperture as observed from the vessel lumen in A. marina (Fig. 2h) or they had fully disappeared.

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Chapter 3

h

b

a

d

e

f

gc

Discussion

Remarkable variation of pit membranes within species

As was hypothesized, the seasonal climate in Kenya seemed to influence pit membrane thickness in R. mucronata with slightly thicker pit membranes in the dry season wood than in the rainy season wood (Table 2). Although this seasonal effect suggests a functional significance, its relationship with hydraulic conductance and embolism vulnerability needs to be tested. Moreover, the seasonal difference does not correspond to the bimodal distribution observed in an earlier study (peaks around 0.15 µm and 0.40 µm; Schmitz et al., 2007a), indicating that also other factors than season control pit membrane thickness. Tree size, linked with cambial age, seemed to be such an additional factor. While the mean pit membrane thickness recorded in the previous study, where younger trees were sampled, was 0.3 ± 0.1 µm (Schmitz et al., 2007a), it was 0.5 ± 0.2 µm in the present study. The formation of thicker pit membranes while growing taller might be an indication of the vessels’ requirements for mechanical strength next to conducting efficiency (Sperry et al., 2006).

63

Fig. 3. Mean curves of absorbance spectra of (a) intervessel pit membranes; (b) pit membranes, corresponding vestures and vessel cell wall of tree 3887; (c) high absorbent deposits of pit canals and pit chambers shrinking or even disappearing after some hours, and low absorbent pit canal deposits present for at least the entire study period of several days as seen in UV scanning profiles (Fig. 2). Ac-cession numbers 3870, -71, -73, Rhizophora mucro-nata; 3886-3887, Avicennia marina. In (c) values of 3870 and 3871 were averaged since it concerns dry and rainy season wood of the same tree respectively and curves did not differ considerably. n, number of pit membranes, vestures or deposits of which the ab-sorbance spectra were used for the average curve; SD, standard deviation.


Fig. 2. UV scanning profiles of intervessel pits of Rhizophora mucronata (a, e-g) and Avicen-nia marina in longitudinal view (b, d) and surface view (c, h). In (b) pit canals are not always crossed by sectioning (see also Fig. 3l). (c) External side of the vessel, (h) lumen side of the vessel. Different co-lours correspond to different UV absorbance values as represented in the colour legend. Images were ta-ken at a wavelength of 280 nm (a-d, f-h) for lignin abundance and at 560 nm for deposits (see Fig. 2e). Black arrows, pit membranes; grey arrows, vestures; black arrow heads, temporal high absorbent deposits in pit canals and pit chambers; yellow arrow heads, long term low absorbent pit canal deposits (see also Fig. 3c). Image magnification, 100x.

The differences in pit membrane thickness could not be strictly ascribed to the varying thickness of the deposits lining vessel lumina and pit borders. The coatings were unrelated to seasons, in contrast to former studies on different angiosperm tree species (Wardrop et al., 1963; Donaldson and Singh, 1990; Castro, 1991; Singh et al., 2002). All pits with an extremely dense and thick lining had an equally dense pit membrane. But, a thin vessel lining was commonly present pointing to an ontogenetic origin such as the tertiary wall or protoplast residues (Wardrop et al., 1963; Schmid, 1965). The extremely thick linings might result from heartwood formation as was found in Betula alleghaniensis (Yang, 1978), but also from reactions to past wounding. Although the age of heartwood formation in R. mucronata in Kenya is currently unknown, heartwood encrustations are unlikely because of three reasons. First, in R. mucronata from the Phillippines the sapwood extended over three to five centimetres (Panshin, 1932), which is the stem portion where the studied samples were taken. Second, the thickness of the linings of some vessel pairs was unequal (Fig. 1g-h) and third, a similar lining was found in Carya tomentosa surrounding pit chamber, pit canal and vessel lumen in both sapwood and heartwood (Thomas, 1976).

Independent of pit membrane thickness, differences in pit membrane porosity were observed in R. mucronata. In contrast to Schmid and Machado (1968), they do most probably not reflect different

64

Chapter 3

stages of pit development as samples were taken some distance from the cambium. More likely are (a)biotic stresses or increasing cambial age that could gradually lead to a degradation of the pit membranes via pectic enzymes secreted by xylem parenchyma (Barnett, 1981; Sperry et al., 1991; Choat et al., 2003). The remaining cellulose network, devoid of encrusting material (Sperry et al., 1991), would explain the increasing vulnerability to embolism with age (Melcher et al., 2003). While pit membranes could not be seen in 16 % (n = 19) of the studied vessel pairs, their pit annulus, assumed to be an accretion of encrusting material (Schmid and Machado, 1968; Sano et al., 1999), was clearly observed (Fig. 1d-e). Therefore, the apparently transparent pit membranes are almost certainly ripped off (Fig. 1e) or destroyed pit membranes as a preparation artefact. The appearance of dark dots in the pit membranes of one sample (Fig. 1d) and the loose membrane-like material in another (Fig. 1f) might be consistent with cracks in the pit membranes and layers peeling off from the pit membranes, respectively.

Exploration of intervessel pit chemistry

Pit membranes of both R. mucronata and A. marina were found to have low but not zero lignin content (Fig. 2). Their non-lignified appearance in surface view under UV light (Fig. 2c), resembling earlier observations in Aesculus hippocastanum (Chaffey et al., 1997), might be explained by the presence of a pectinaceous coating as was observed in the sapwood of Sapium sebiferum (Pesacreta et al., 2005). The authors suggested that the coating is very thin, not identifiable via scanning electron microscopy, and thus is different from the previously mentioned dense coatings covering heartwood pits (e.g. Bonner and Thomas, 1972; Kininmonth, 1972; Thomas, 1976; Wheeler and Thomas, 1981). These pectic substances (Schmid and Machado, 1968; Morrow and Dute, 1999) or other non-phenolic, potassium permanganate reactive components such as lipids (Nemec, 1975; Hoffmann and Parameswaran, 1976; Robb et al., 1980) might penetrate pit membranes. This would explain the mostly darker appearance of pit membranes than cell walls after staining with potassium permanganate but their lower UV absorbance (compare Fig. 1a,l with Fig. 2a-b). In A. marina, the vestures showed the same reaction to both methods (arrows, Fig. 1l vs. 2d) suggesting a mixed composition of lignin and polysaccharides. The lignin content (Fig. 3b), which is a possible reflection of their origin at the end of cell wall lignification (Parameswaran and Liese, 1977; Ohtani et al., 1984; Jansen et al., 1998) was in contrast to Ranjani and Krishnamurthy (1988) but in agreement with many other studies (Scurfield, 1970; Donaldson and Singh, 1990; Castro, 1991; Singh et al., 1999; Watanabe et al., 2006). The occasional observation of slightly stained pit membranes in R. mucronata (Fig. 1j) might be due to the activity of pectic enzymes with increasing cambial age. The second peak in the absorbance spectra at around 660 nm, consistent with some softwood species analysed by Bauch and Berndt (1973), could be a reflection of these encrustations (Fig. 3a).

In correspondence with this pectic coating, the deposits lining the pit borders in R. mucronata and less conspicuously also in A. marina could be polysaccharides (Fengel and Wolfsgruber, 1971; Castro, 1991). Although, the ability to resist bacterial degradation of a similar layer in Terminalia suggested a high lignin (Singh et al., 2002) and tannin content (Donaldson and Singh, 1990). Aside from these linings, pit canals were bordered in a few trees by granular material (Fig. 1b, h, k) as found before in the pit canals of parenchyma cells (Wheeler and Thomas, 1981), fibre tracheids and vessels (Yang, 1978). Lawn (1960) warned for granular precipitates as an artefact of potassium permanganate staining but they could also be remnants of plasmalemma, tonoplast or endoplasmatic reticulum (Scurfield, 1967). Of the same origin (Schmid, 1965; Scurfield, 1970) might be the membrane-like structures covering pit apertures (arrow heads, Fig. 1b, g, i-k), as found between two fibre tracheids of Paulownia tomentosa (Yang, 1986). Alternatively, it could be a layer of extractive material, protecting the tree against microbial attack and providing natural durability (Donaldson and Singh, 1990; Singh et al., 2002).

65


A similar function can be assigned to the deposits found to fill the pit canals in both studied species. The low absorbent deposits are most likely remnants of the highly absorbing ones after sample preparation given the more sporadic occurrence of the latter and the parallelism of their absorbance spectra (Fig. 3c). Next to protection against pathogens they could play a role in the water transport. Zimmermann et al. (1994; 2002) found mucopolysaccharides in xylem sap and attached to vessel walls in seedlings of Rhizophora mangle. Although they did not study intervessel pits and their findings are highly debated, these mucilages were proposed to play a major role in xylem sap flow by supporting the tension gradient in the water column. Whether the deposits found in R. mucronata and A. marina have a function in tree hydraulics or in contrast are an indication of non-active vessels remains to be clarified.

Conclusion and perspectives

Several authors noticed the large variation in pit membrane structure and chemistry within species, trees and even vessels (e.g. Bailey, 1957; Schmid and Machado, 1968; Bauch and Berndt, 1973; Sano and Nakada, 1998; Schmitz et al., 2007a). In this study, the hypothesis of seasonal changes causing differences in pit membrane thickness (Schmitz et al., 2007a) could only be partly accepted. The seasonal differences were weak and overshadowed by an enormous variability in electron density of the pit membranes. In addition, deposits were found filling pit canals and lining pit borders. But, they did not show a clear correlation with the variation of the pit membranes highlighting the importance of future research. Due to the time demanding preparation of electron microscopic and UV microscopic studies, sample sets are usually small relative to the heterogeneous pit membrane structure and composition. This limitation urges for numerous studies, considering an extended number of trees and several positions within individual trees to gather enough data in order to get an overview of the common and variable characteristics. Next to an extensive number of replicates, a wide ecological spectrum should be considered, as is the physiological status of the vessels. In this way, the potential role in tree hydraulics of the changes in pit membrane thickness and composition and of the deposits in pit canals can be elucidated. Besides the search for structure-function relationships, the resemblance in chemical composition of pit membranes and vestures as opposed to the cell wall added a new piece to the puzzle of their formation stimulating the expansion of the yet fragmentary knowledge.

Acknowledgements

We thank Tanja Potsch and Hamisi Ali Kirauni for their invaluable help during sample preparation and fieldwork respectively; the KMFRI staff in Gazi, especially Dr. J.G. Kairo, for the logistic support in Kenya and all people of Gazi and the vTI in Bergedorf for their assistance and hospitality. This research was financially supported by the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen), the COST Action E50 and travel grants from the National Fund for Scientific Research (FWO, Belgium), the Schure-Beijerinck-Popping Fonds (Koninklijke Nederlandse Akademie van Wetenschappen, Nederland) and the Flemish Interuniversity Council (VLIR).

66

Part II

Part II

Implications of successive cambiafor the hydraulic architecture of Avicennia marina

Nature does nothing uselessly.ARISTOTELES

67

Part II

Summarizing abstractBackground and Aims While most trees produce wood via a single cambium towards the outside of the stem, a select group of woody species develop secondary xylem via successive cambia. Although the formation of the resulting alternate bands of phloem and xylem tissue has been described anatomically for several species, the periodicity of the growth layer formation remains obscure. In this second part of the thesis secondary wood formation via successive cambia and the effect of soil water salinity on the differentiation of new ones, will be studied in the mangrove species Avicennia marina.

Methods Plantation trees of known age as well as trees that were marked, by wounding the cambium, and felled at a known moment in time were investigated. Studied trees were distributed over different sites in the mangrove forest of Gazi Bay (Kenya), characterized by different substrate salinity and inundation frequency. Growth layers were counted, the width of phloem and xylem tissue measured and related to several tree and environmental factors.

Key results Growth layer formation in A. marina was shown to be non-annual, closely related to radial increment and highly variable between trees and even within trees at different positions around the stem circumference. In contrast to the number of layers produced per time period, the width of the growth layers did clearly reduce with increasing soil water salinity of the site.

Conclusions A patchy growth mechanism was proposed with specific positions of optimal growth, changing the position of active growth zones with time. Such a growth strategy explains the observation of several growth layers forming at the time, even when the outermost layer had already been lignified. It could be an adaptation to optimize growth when environmental conditions are favourable. Besides, it provides the opportunity to create a hydraulic system of divergent structure in the different patches around the stem. In this way, the hydrosystem of the trees would under each condition be adapted to its environment in at least one part of the stem. The unpredictability of the environmental conditions in the habitat of A. marina could then be dealt with more effectively by this species. The development of a new cambium was indicated to be triggered endogenously as well as by salinity, supporting a potential role of phloem tissue in embolism repair.

68

Chapter 4

69

Chapter 4

Successive cambia development in Avicennia marina is not climatically driven in the seasonal climate at Gazi Bay, Kenya

Published in: Dendrochronologia 25(2): 87-96

Dendrochronological potential of Avicennia marina

Abstract

Background and aims The periodicity of growth ring formation and hence the dendrochronological potential remains elusive for many tropical tree species. This also applies to the most widely distributed mangrove species, Avicennia marina. This study is intended to provide early access to recent findings on the peculiar mode of secondary growth via successive cambia of A. marina in Kenya.

Methods To examine the periodicity of growth layer formation, growth layers were counted on three sanded stem discs from a cambial marking experiment and three from a plantation of known age. The effect of local site conditions, such as soil water salinity and inundation frequency, on growth layer development was examined by considering an extra 28 stem disks of trees from three different study sites.

Key results The respective number of growth layers produced during one year at the site of the cambial marking experiment and the plantation was on average a half and three. Growth layer development was shown to be strongly correlated with stem diameter. In addition, growth layer width decreased with increasing salinity and / or inundation class.

Conclusions Growth layer development in A. marina is not driven by the seasonal climate at Gazi Bay. Nevertheless, the effect of local sites conditions on growth layer width offers interesting perspectives. The larger proportion of xylem in comparison with phloem in trees with wide as opposed to narrow growth layers may provide extra mechanical strength. On the other hand, the larger fraction of phloem and parenchyma in trees with thin growth layers may be beneficial for the water balance of the tree. Next to the non-annual nature of the growth layers and their networking pattern, more than one cambium was found to be simultaneously active. We conclude that classical dendrochronological methods should not be applied to A. marina (from Kenya).

Key words: mangrove, tropical dendrochronology, cambial activity, inundation, salinity, tree ring

70

Chapter 4

Introduction

In contrast to trees of temperate regions, tropical trees are usually believed to show no growth rings. Regardless of a seasonal climate, growth rings are not always anatomically distinct in the tropics (Détienne, 1989; Jacoby, 1989; Sass et al., 1995; Worbes, 1995; Verheyden et al., 2004b) and if so, they do not necessarily imply an annual growth rhythm (Worbes, 1989; Stahle, 1999). However, these difficulties have not hindered applying dendrochronology to several tropical species (e.g., Gourlay, 1995; Pumijumnong et al., 1995; Eshete and Stahl, 1999; Worbes, 1999; Segala Alves and Angyalossy-Alfonso, 2000; Callado et al., 2001; Enquist and Leffler, 2001; Brienen and Zuidema, 2005; Heinrich and Banks, 2005; López et al., 2005) including the mangrove species Rhizophora mucronata (Verheyden et al., 2004a; Verheyden et al., 2004b; Verheyden et al., 2005) and Rhizophora mangle (Menezes et al., 2003) and a species of the genus Diospyros (Duke et al., 1981). Moreover, many other tropical tree species are still to be investigated (Worbes, 2002). The current study examines the growth layers of the most widely distributed mangrove, Avicennia marina (Tomlinson, 1994; Duke et al., 1998).

The mode of secondary growth in Avicennia is rather uncommon and occurs via successive cambia (Studholme and Philipson, 1966; Zamski, 1979; Carlquist 2001). They give rise to an alternating pattern of light coloured phloem and darker xylem, resulting in clearly visible growth layers (Fig. 1a). Nevertheless, no attempts have been made so far to unveil environmental factors affecting the development of a new cambium and only few investigations have dealt with the periodicity of growth layer formation (Chapman, 1944, 1947; Gill, 1971). Furthermore, these studies presented contradictory and inconclusive results. While Chapman (1944) assumed that the rings of A. nitida Jacq. were semi-annual, he later suggested that they could be annual (Chapman, 1947). This new assumption, however, was based on a single tree. Gill (1971) observed two to six ‘rings’ in one-year-old shoots, and showed a relationship between ‘ring’ numbers and stem diameter, but not age. He concluded that the development of successive cambia in A. germinans (L.) L. is probably endogenously controlled and is not environmentally influenced. However, firm conclusions can not be made since the study was partially based on branches that have been shown to behave differently in several species (Fegel, 1941; Zimmermann and Potter, 1982; Cherubini et al., 2003).

More than two decades after the study of Gill (1971), this paper is aimed at giving early access to our recent findings on secondary growth in A. marina of Gazi Bay (Kenya). The potential of the growth layers for age determination was evaluated by counting growth layers on stem disks of trees from a cambial marking experiment and of plantation trees of known age. In addition, the influence of local site conditions on growth layer formation was examined by using samples of the outermost wood as well as stem disks, from sites differing in salinity and inundation class.



Study sites are located in the mangrove forest of Gazi Bay (39°30’E, 4°25’S), which covers about 710 ha (UNEP, 2001) and is situated approximately 50 km south of Mombasa, Kenya. During the wet season, the rivers Mkurumuji and Kidogoweni provide an important freshwater source for the mangroves (Kitheka, 1997). Sampling was performed in nine sites, differing in salinity and inundation class (Fig. 2a, Table 1). Soil water salinity at approximately 20 cm depth ranged from 13.2 ‰ to 90 ‰ [data from this study and from Gillikin (2004), measurements carried out with a WTW P4 multiline conductivity meter, Wissenschaftlich-Technische Werkstätten GmbH, Weilheim, Germany]. Depending on the topography and the tidal range of on average 3.8 m and exceptionally 4.1 m (Kenya

71


Ports Authority tide tables for Kilindini, Mombasa), zones of different inundation classes can be defined. Inundation classes 1 to 4 correspond to an inundation frequency of 56-62, 45-56, 20-45 and 2-20 times a month respectively (Watson, 1928 in Macnae, 1968).

R

r

P

SCPA Ps

X

PA

BA

Fig. 1. (A) Asymmetric stem disc of an Avicennia marina tree showing the pattern of darker xylem bands and lighter coloured phloem bands formed by successive cambia. Scale bar = 1 cm. (B) Transverse section of the outer part of a wood sample showing the growth layers and their constituent tissues. Light microscope, scale bar = 1 mm. P phloem band, PA parenchyma, Ps phloem strand, r minimum radius, R maximum radius, SC sclereids, X xylem band. Brackets designate the growth layers.

Site† Salinity‡ (‰) Inundation n* Tw no.**

Average Min. Max. class§

1 28,5 20,9 34,6 1 8 (+ 8) 55924-26, 55928-322 29,3 26,6 34,0 4 10

3 32,4 28,1 34,2 3 (3) 57798-80***4 41,0 13,2 56,7 4 55 51,0 44,4 61,0 2 106 54,1 42,3 69,8 3 107 54,1 42,3 90,0 3 (4) 55933-368 58,1 40,0 68,2 4 10

9 61,5 46,5 79,9 3 10 + (3 + 8) 56737-39****, 55895, 55898, 55901-03

Table 1. Sample collection sites and corresponding environmental data

†See Fig. 2a; ‡Soil water salinity at approximately 20 cm depth; §Inundation class according to Watson (1928) in (Macnae, 1968); *Number of sampled trees with number of collected stem disks in parentheses; **Accession number in the Tervuren wood collection (Belgium) of the studied stem disks; ***Plantation trees; ****Trees from the cambial marking experiment.

Three trees from a plantation (site 3, Fig. 2a) established in February 1992 were sampled in May 2005. The planted trees were growing under natural conditions and were enclosed within a natural forest. Wood disks were sawn at the base of the stem and from two branches at a bifurcation to sample branches of the same age. Another three trees (site 9, Fig. 2a) were marked using the pinning technique in October 1999 with a surgical needle of 1.2 mm diameter (Verheyden et al., 2004b) and felled in May 2002. The pinning method has been proven to be very effective for examining radial growth in tropical trees (Shiokura, 1989). All wood samples are now part of the wood collection

72

of the Royal Museum for Central Africa, Tervuren, Belgium (for accession numbers see Table 1). From the xylarium an additional set of 28 samples from 20 trees was selected from site 1, 7 and 9 corresponding to a seaward site, a sand flat and a landward site. Finally, in May 2005 the outermost wood of 59 trees located at site 1, 2, 4, 5, 6, 8, 9 was collected. Samples (on average 3 x 1 x 1 cm) were taken at breast height with a handsaw and stored in FAA (Formalin-Acetic acid-Alcohol) to preserve the cambial cells.

Climate description

The climate along the Kenyan coast is characterized by a bimodal distribution of the precipitation. A distinct dry season (January - February) is followed by a long (April - July) and a short rainy season (October - November) (Fig. 2b). The average temperature at the Kenyan coast ranges from 22 to 30 °C, with a mean relative humidity of 65 % to 81 % (annual averages of minima and maxima for Mombasa for the period 1972 - 2001, data from the Kenyan Meteorological Department, Mombasa, Kenya).

50

40

30

20

10

0

20

40

60

80

100

300

)C°( erutarep

meT )m

m ( n

o itat i

pic e

rP


A B

Fig. 2. (A) Location of the nine study sites in Gazi Bay, Kenya. Zoom: Gazi is situated approximately 50 km south of Mombasa, adapted from Dahdouh-Guebas et al. (2002) (B) Climate diagram of Mombasa (39°36‘ E, 4°0‘ S) adapted from Lieth et al. (1999), showing the long (April - July) and short (October - November) rainy season and one distinct dry season (January - February). Precipitation scale is reduced to 1 / 10 above the horizontal line.

Sample preparation and microscopic analysis

In this study the following terms and definitions will be used (see also Fig. 1b): xylem band to designate the zone in between the phloem tissue and the sclereids. To the inside, this band also includes a few cell layers of parenchyma, which are the first derivatives of the new cambium. The term phloem band is used to designate a zone of phloem strands united in a band of parenchyma tissue and growth layer to designate one ontogenetic unit of parenchyma, xylem, phloem and sclereids-following the terminology of Parameswaran (1980).

Chapter 4

73

To investigate the annual nature of growth layer formation, growth layers were counted from three plantation trees of known age and from three trees from a cambial marking experiment. First, wood discs were air-dried and sanded using a series of sandpaper from 100 to 1200 grit, to make the phloem and xylem bands clearly visible (Fig. 1a). For the wood disks of site 1, 3, 7 and 9 the number of growth layers was then counted along the maximum as well as minimal radius making use of a magnifying glass. Because of the networking pattern of the growth layers (Fig. 1a) the convention was applied that a growth layer is counted if it crosses a pencil-drawn line from pith to bark along the maximum and minimum radius. For the calculation of the average number of growth layers formed per year, the measurement at the longer radius was used (see Worbes, 1989). The wound inflicted by the technique of cambial marking is visible as a datable scar, indicating the position of the cambium at the time of wounding. The number of rings from these cambial marks onwards was counted with the aid of a stereo microscope at 30 magnifications.

The samples preserved in FAA were cut into little blocks of approximately 8 mm sides with a scalpel, creating a transverse plane. Samples were washed in 50 % ethanol and dehydrated in an ethanol series (50, 75, 90, 96, 100 %) with the last step taking 48 h (Ruzin, 1999). Subsequently, they were soaked for 24 h with PEG 1500 (Pure, VWR International, Prolabo) at 60 °C and embedded with fresh PEG 1500 for another 24 h at room temperature. Samples were sectioned at 20 µm thickness with a sliding microtome (Microm), dehydrated and double stained with Safranin O (Merck) and Fast green FCF (C.I. 42053, Merck). Sections were mounted on slides with Canada balsam (Merck). The number of parenchyma, xylem, phloem and sclereid cells of the last growth layer were counted along three radial files per sample using digital image analysis software (AnalySIS Pro v.3, Soft Imaging System GmbH, Münster, Germany) with a microscope at a magnification of 125 times (Olympus). To standardize the measurement only xylem fibres were counted and neither vessels nor xylem parenchyma were considered. However, distinction between parenchyma and fibres was not always obvious. In addition, the width of the last as well as the previous growth layers was measured along three radial files at 25 magnifications from the beginning of the phloem band to the end of the xylem band, excluding the sclereids and the small parenchyma zone (Fig. 1b). This is justified since both the number of sclereids and parenchyma cells in each growth layer are not statistically different between sites (ANOVA test, F-values of 0.73 and 0.53 respectively, df = 6, p > 0.05). For comparison between and within trees, the width of the current growth layer was standardized since the stem of A. marina was often asymmetric (Fig. 1a), with narrower growth layers at the side of the smallest radius. The width of the last growth layer was divided by the average width of the preceding growth layers. Taking samples instead of disks obviously limited the data set. Between trees, variation could be considered but, within trees, variation could only be accounted for along the length of the 8 mm wide sample.


Simple linear regressions were performed to analyze the relationship between (i) number of growth layers and stem or branch diameters (all samples included in the analysis) and (ii) growth layer width and age (samples from plantation trees only). Time series of the growth layer width were compared to the growth layer number, as a measure of tree age. To test the difference between slopes we used a homogeneity-of-slopes model. Via a t-test for dependent samples the difference in growth layer width was tested between the small and large sides of the asymmetric stem disks. The assumption of homogeneity of variances was tested via Levene’s test. All statistics were executed in STATISTICA 7.0 (StatSoft Inc., Tulsa, USA).


74

Results

On average half a growth layer was formed per year in the trees from the cambial marking experiment of site nine and three growth layers in the trees of the 13.25 year old plantation (Fig. 2a, Table 1-2). From these plantation trees, growth layers were also counted in pairs of bifurcating branches. Along the maximum branch radius, two of the three branch pairs had an unequal number of growth layers. Along the minimum branch radius all three pairs of branches showed a different growth layer count (Table 2). The incongruence in growth layer formation both between and within trees was also pointed to by a microscopic analysis. The radial increment of the current growth layer of 59 trees of seven sites (Table 1, 1-2, 4-6 and 8-9) was measured. Using width instead of cell count was justified because of the high correlation between both characters (r² = 0.96, n = 189, p < 0.0001). The standardized width of the current growth layer (see Materials and methods) differed both within sites and trees (Fig. 3). The within-tree variation (Fig. 4a) explains the large standard deviation for some trees. Furthermore, the formation of a new growth layer can be started before completion of the previous one (Fig. 4b).

Tw number†Min. Radius Number of Max. radius* Number of Time period Number of

(cm) growth layers‡ (cm) growth layers‡ (yr) growth layers / yr§

Site 3, Plantation57798 3,7 37 6,2 50 13,25 3,8

branch1 1,2 16 1,6 21branch2 1 13 1,2 1457799 3 30 4,3 37 13,25 2,8

branch1 1,1 14 2,1 19branch2 1,2 16 2,4 2257800 2,9 27 4,7 42 13,25 3,2

branch1 1,1 17 1,5 19branch2 1,1 15 1,5 19

Site 9, Cambial marking56737 4.4 (0.19) 1,5 2,58 0,656738 2.7 (0.15) 1,0 2,58 0,456739 4.8 (0.10) 1,5 2,58 0,6

Table 2. Number of rings in three stem disks of known age collected from a plantation and in three trees from a cambial marking experiment.

Plantation: †Accession number in the Tervuren wood collection, samples were taken at the base of the stem and from a dichotomous branching; ‡Growth layers were counted as they crossed a drawn line from pith to bark at the max. and min. radius; §Calculated from the growth layers counted at the maximum radius. Cambial marking: ‡counted from the cambial mark (parenchyma band) to the bark. *Maximum radius of the stem disc, in parentheses the distance cambial mark - bark.

The number of growth layers was shown to be strongly correlated to the radius of the wood disks (Table 3). The larger the stem or branch, the more growth layers were counted from pith to bark (Table 2), despite the similar age of the three stem disks and of each pair of branches. Likewise, less growth layers were counted at the minimum than at the maximum radius of the wood disks (Fig. 1a, Table 2). This highly significant relationship between stem size and number of growth layers in the plantation trees was supported by analyzing an extra 28 wood disks from three other sites in the mangrove forest of Gazi Bay (Table 3, Fig. 5). Correlation coefficients between stem radius and growth layer count further increased when the data were grouped according to sampling site (Table 3). Furthermore, the slopes of the regression lines for the three sites separately were found to be significantly different (Table 3, Fig. 5). The associated growth layer width, ranged between 0.69 ± 0.09 mm (site 7) and 1.20 ± 0.34 mm (site 1) on average and did not show a correlation with age. No trend was observed from

Chapter 4

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juvenile to mature wood in none of the ring width chronologies of the three plantation trees (R²-values of respectively = 0.031, 0.11 and 0.000093, p ≥ 0.05). As shown by the three plantation trees and the 28 stem disks mentioned above, the width of the growth layers also differed within one tree. The width at the minimum branch radius was smaller than at the maximum branch radius in asymmetric stem disks (t = - 5.64, df = 30, p < 0.0001).

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

1 10 19 28 37 46 55

No. of trees

Av.

wid

th la

st g

row

th la

yer /

pr

evio

us g

row

th la

yers

Site 1 Site 2 Site 4 Site 5 Site 6 Site 8 Site 9

Fig. 3. The developmental stage of the last growth layer is illustrated for 63 trees of seven study sites. To exclude site-related differences the ratio was taken of the width of the last growth layer, averaged over three radial files, to the mean width of the previous growth layers. The different sites are ordered according to in-creasing salinity and separated by the use of alternating dark and white bars. Error bars correspond to standard deviations.

Discussion and conclusions

The growth layers in A. marina from Kenya were shown to be non-annual (Table 2). This finding indirectly demonstrates that the development of new cambia in A. marina is not controlled by the seasonal climate at the study site (Fig. 2b). In contrast, in the mangrove Rhizophora mucronata Lam., growing at Gazi Bay, the seasonality causes annual rings (Verheyden et al., 2004b; Verheyden et al., 2005). Also in other tropical trees with a single cambium, annual ring formation is primarily governed by rainfall seasonality or a period of inundation (Jacoby, 1989; Borchert, 1999). But, in A. marina the aclimatic growth rhythm is confirmed by the disparity in ring numbers in the even-aged pairs of branches (Table 2). Additional evidence is given by the widely fluctuating developmental stage of the

Site R²† df F p3, plantation 0,97 7 299,31 < 0.0001

1, 7, 9 0,83 26 136,12 < 0.00011, seaward 0,99 10 740,27 < 0.00017, sand flat 0,92 6 79,03 < 0.00019, landward 0,98 6 456,25 < 0.0001

Site*max radius‡ 22 54,72 < 0.0001

Table 3. Summary of the simple linear regressi-ons of the number of growth layers in function of the maximum stem radius.

†Adjusted R², ‡homogeneity-of-slopes model test.


76

current growth layer of 59 trees in Gazi (Fig. 3). If climate directed the production of new cambia, the last layer should have been in the same developmental stage in all trees. However, even along the circumference of a single stem this is not the case (Fig. 3, 4a), resulting in a growth-layer network (Fig. 1a). The cause may be an irregular distribution of assimilates and hormones as mentioned before in Dryobalanops sumatrensis (J.F.Gmelin) Kosterm. (Sass and Eckstein, 1995). This can be due to an irregular flowering in the branches of one tree (Clarke and Myerscough, 1991; Clarke, 1992). The differences within one site can thus be explained by the combination of an endogenous control on growth layer formation and an uneven-aged forest.

An internal control of cambial differentiation is in agreement with the observations by Gill (1971) and is supported by our study. The number of growth layers was found to be correlated to the diameter of the stem disc of both planted (Table 2) and naturally grown trees of three sites (Fig. 5). Moreover, the disappearance of growth layers at the slow growing side of asymmetric stems (Table 2) confirms the strong influence of the diameter on the production of new cambia and thus growth layers. An influence of endogenous factors on growth layer formation was reported before in Cupressus sempervirens L. (Liphschitz et al., 1981). In A. germinans growth control was suggested to be exclusively endogenous based on the finding of a common growth layer width of about 1.30 mm (Gill, 1971). On the contrary, this study on A. marina demonstrated a between-sites variation in the growth layer width (Fig. 5) and the number of yearly formed growth layers (Table 2). The incongruence between both studies can probably be attributed to the larger sample set of this investigation (n = 28 vs. 10), representing three study sites instead of one. The influencing local site conditions, presumably salinity and/or inundation frequency, may interact with phenological processes (Duke, 1990; Borchert, 1999; Ochieng and Erftemeijer, 2002). In an ongoing study on the phenology of A. marina in Gazi Bay, differences in leaf loss were found between sites (V.W. Wang’ondu, pers. comm.). A site dependent phenology, related to differences in the cambial activity and/or the active period of one cambium (Paliwal and Prasad, 1970; Borchert, 1999), might thus explain the discrepancy in width and number of yearly formed growth layers at different sites (Table 2, Fig. 5). Likewise, the regulation of the cambial activity in Azadirachta indica A. Juss. was mentioned to be determined by endogenous as well as some external factors like water supply and temperature (Rao and Rajput, 2001). On the contrary, an additional age effect on growth layer width could be excluded, in contradiction to earlier reports on other tropical tree species (Akachuku, 1984; Worbes et al., 2003). The inconsistency can be due to the internal control on growth layer formation. But, an extensive investigation, including trees of old age, is needed for confirmation.

A B

Fig. 4. Transverse sections of the outermost wood of two Avicennia marina trees. (A) A new cambium has been formed at the left side of the picture but not yet at the right side. (B) Three growth layers are being formed simultaneously implying a discontinuous time-axis. Light microscope, scale bar = 100 µm.

Chapter 4

77

Growth layers become narrower (steeper slopes in Fig. 5) with increasing soil water salinity, from the seaward (site 1), over the landward site (site 9) to the sand flat (site 7) (Table 1). Although the average soil water salinity at the sand flat is not so much higher than at the landward side, there are considerable differences in environmental conditions explaining the disparity in growth layer width. First, the sand flat shows an increased salinity fluctuation (Table 1) which imposes a larger stress on tree growth compared to a constant salinity (Lin and Sternberg, 1993). Second, in comparison to the sand flat the soil at the landward site has a higher loam fraction (Matthijs et al., 1999) making the substrate more effective in water storage. The functional significance of thin growth layers under high salinity conditions may be a higher proportion of living tissue, in this case phloem and parenchyma. Parenchyma sheaths may offer an improved regeneration capacity of the tree as do the phloem strands that are dispersed throughout the stem (Carlquist, 2001). They have been shown to serve as transporters of the auxin hormone that could promote vascular regeneration after wounding (Aloni, 2004). This is especially important for the A. marina trees of the more saline sites since die-back is a common phenomenon in trees subjected to drought-stress (Carlquist and Hoekman, 1985) that can be caused by a high salinity (Sperry et al., 1988; Tyree and Sperry, 1989; Hacke and Sperry, 2001). Additionally, the large proportion of phloem and parenchyma tissue may offer an advantage for water transport. That is because they have been put forward as actors in the process of embolism repair. Although the underlying mechanism is not yet clear, the refilling of embolized vessels may be accomplished by a water flow from living cells (Holbrook and Zwieniecki, 1999; Tyree et al., 1999; Stiller et al., 2005). Another benefit that is gained from the increased number of phloem bands per surface area involves the higher degree of compartmentalization. Sectoriality may diminish the spread of embolisms (Orians et al., 2004; Ellmore et al., 2006). In this study A. marina trees at the seaward fringe of the mangrove forest were found to have wider growth layers, implying a smaller proportion of parenchyma and phloem tissue and a larger fraction of fibres. It is likely that this provides the tree with an enhanced mechanical strength to withstand tidal force and strong winds (Sun and Suzuki, 2001). At the same time the increased fibre proportion could offer an alternative safety mechanism against cavitation (Jacobsen et al., 2005).

Finally, the finding that more than one cambium can be active simultaneously (Fig. 4b) implies that no dormancy takes place between the formation of two successive growth layers. This is consistent with the frequent observation of a continuous cambial activity in tropical trees (see Sass et al., 1995; Rao and Rajput, 2001; Verheyden et al., 2004b). Two active cambia at the same time were reported before in A. germinans and A. resinifera Forst. f. (Zamski, 1979). The resulting discontinuous time axis together with the network of non-annual growth layers impedes the application of conventional dendrochronological methods and simple age determinations on Avicennia marina from Kenya. The growth layers in this species turned out to be non-annual but related to stem size and local site conditions. More research is needed to confirm the hypothesis that the width and the yearly formed number of growth layers decrease with increasing salinity. The functional and/or mechanical

y = 10.80 + 4.72 xR² = 0.99

y = 2.41 + 8.35 x R² = 0.98

y = 3.55 + 12.45 xR² = 0.92

0 2 4 6 8 10 12Max. stem radius (cm)

No.

gro

wth

laye

rs

0

20

40

60

80

100

120Seaward Landward Sand flat Fig. 5. The number of growth layers as a function of

the maximum radius of the asymmetric stem disks for three sites differing in environmental conditi-ons. The three sites correspond to site 1 (seaward), 9 (landward) and 7 (sand flat) as given in Table 1.


78

significance of this trend present an extra research question. Finally, the annual variation in the yearly formed growth layers at a specific site has to be addressed to elucidate if growth layer count can still be used to give at least an estimation of tree age.

Acknowledgements

We are grateful to Hamisi Ali Kirauni and Nema Mwamoto Pashua of KMFRI Gazi and to Dr. D.P. Gillikin for their invaluable assistance provided in the field and to Bernard Kirui, Chomba Peter Kamanu, Judith Okello, Eric Okuku and all the people of Gazi for their hospitality. We thank the National Fund for Scientific Research (FWO, Belgium) and the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen) and the Belgian Federal Science Policy (project MO/37/015) for their financial support.

Chapter 4

79


80

Chapter 5

81

Tree growth via successive cambia

Chapter 5

A patchy growth via successive and simultaneous cambia: key to success of the most widespread mangrove species Avicennia marina?


Abstract

Background and aims Secondary growth via successive cambia has been intriguing researchers for decades. Insight in the mechanism of growth layer formation is, however, limited to the cellular level. The present study aims to clarify secondary growth via successive cambia in the mangrove species Avicennia marina on a macroscopic level, addressing the formation of the growth layer network as a whole. In addition, previously suggested effects of salinity on growth layer formation were reconsidered.

Methods A one year cambial marking experiment was performed on 80 trees from 8 sites in two mangrove forests in Kenya. Environmental (soil water salinity and nutrients, soil texture, inundation frequency) and tree characteristics (diameter, height, Leaf Area Index) were recorded for each site. Both groups of variables were analysed in relation to annual number of growth layers, annual radial increment and average growth layer width of stem disks.

Key results Between trees of the same site, the number of growth layers formed during the one year study period varied from only part of a growth layer up to four growth layers, and was highly correlated to the corresponding radial increment (0-5 mm/year), even along the different sides of asymmetric stem disks. The radial increment was unrelated to salinity but the growth layer width decreased with increasing salinity and decreasing tree height

Conclusions A patchy growth mechanism was proposed stating an optimal growth at distinct moments in time at different positions around the stem circumference. This strategy creates the opportunity to form several growth layers simultaneously, as observed in 14 % of the studied trees, which may optimize tree growth under favourable conditions. Strong evidence was given for a mainly endogenous trigger controlling cambium differentiation, with an additional influence of current environmental conditions in a trade-off between hydraulic efficiency and mechanical stability.

Key-words: Avicennia marina, cambial marking, mangrove, phloem, salinity, secondary growth, successive cambia, xylem

82

Chapter 5

Introduction

One of the curious mechanisms of plant growth is secondary wood formation via successive cambia (Wheeler et al., 1989). This means that each growth layer is formed by a new cambium and hence is composed of both xylem and phloem tissue. The cambial variant is especially present in lianas (eg., Nair, 1993; van Veenendaal and den Outer, 1993; Carlquist, 1996, 1999a; Jacques and De Franceschi, 2007) but is also reported in woody shrub and tree species with descriptions of the wood anatomy (McDonald, 1992; Carlquist and Gowans, 1995; Carlquist, 1996, 1999b, 2002, 2003a, b) and the wood formation (Wheat, 1977; Zamski, 1979, 1981; Fahn and Zimmermann, 1982; Nair and Mohan Ram, 1990; Terrazas, 1991; den Outer and van Veenendaal, 1995; Rajput and Rao, 1999; Carlquist, 2004; Schmitz et al., 2007). However, it remains unclear if the growth layers, originating from the combined action of the several cambia, are formed periodically or continuously and which factors trigger the formation of a new growth layer.

The mangrove genus Avicennia belongs to this exclusive group of trees showing secondary growth via successive cambia (Schmitz et al., 2007). Early studies speculate about the periodicity of the conspicuous pattern of light and dark coloured bands resulting from the alternating xylem and phloem tissue (Baker, 1915; Chapman, 1944, 1947; Gill, 1971). A preliminary study on six Avicennia marina trees in Gazi Bay, Kenya showed the non-annual nature of their growth layers (Schmitz et al., 2007). However, this study suggests that within one site a constant number of growth layers may form per year. While only half a growth layer was formed in a site of high salinity, an average of three growth layers was formed in a site with relatively lower salinity (Schmitz et al., 2007). Therefore, the formation of growth layers seems to be influenced by local environmental factors such as soil water salinity rather than by seasonal climate. In addition, the proportion of phloem tissue slightly increased in parallel with the salinity of the site (Schmitz et al., 2007). In agreement with these findings, Carlquist (2001) and Fahn and Shchori (1967) assume that dispersed phloem tissue over the entire stem offers a functional advantage to trees growing under xeric conditions.

Avicennia tree species tolerate a wide range of soil water salinity levels, related to different inundation frequencies and evaporation of the substrate (Matthijs et al., 1999; Dahdouh-Guebas et al., 2004; Ye et al., 2005; Naidoo, 2006; Sobrado and Ewe, 2006). To study the formation of growth layers, a technique with a cellular resolution is offered by mechanically wounding the cambium, leaving a datable scar (Kuroda and Shimaji, 1984). Thanks to the distinct time mark, the cambial mark, the presence of clear tree rings becomes superfluous in studies of wood formation, making the method especially useful for the study of tropical tree species (Détienne, 1989; Nobuchi et al., 1995; Sass et al., 1995; Jalil et al., 1998; Bauch and Dünisch, 2000; Ohashi et al., 2001; Heinrich and Banks, 2002), including mangrove trees (Shiokura, 1989; Verheyden et al., 2004; Schmitz et al., 2007).

In the present study the cambium of 80 A. marina trees was wounded. The trees were sampled from seven sites in Gazi Bay and one more distant location in Dabaso, both situated in Kenya. We aim to clarify the secondary growth mechanism of A. marina characterized by successive cambia. This study tested the hypotheses that (1) growth layer formation is periodic with the number of growth layers formed per year depending on the soil water salinity of the site, and (2) the width of the growth layers decreases with increasing soil water salinity.

83


Materials and methods

Study sites and Sample collection

Wood samples were collected in Gazi and Dabaso, two locations along the Kenyan coast. The mangrove forest of Gazi Bay (39°30’E, 4°25’S) covers about 710 ha (UNEP, 2001) and is situated approximately 50 km south of Mombasa. Sampling was performed in seven sites (Table 1), differing in local environmental conditions (Table 2). For comparison, an additional set of trees was sampled in Dabaso (39°21’E, 03°59’S), in the 1500 ha large mangrove forest of Mida Creek situated about 100 km north of Mombasa (Gang and Agatsiva, 1992). In the rainy season of 2005 and 2006 (May-June) and the dry season of 2006 and 2007 (February-March) soil water was collected at approximately 25 cm depth with a punctured plastic tube connected to a vacuum pump. At each site one to three salinity measurements were carried out with a hand-held refractometer (ATAGO, Tokyo, Japan). One day after spring tide in February 2007, soil water was collected in triplicate at the seven study sites in Gazi Bay. From these water samples NO3

-, NH4+ and P concentrations were measured by standard procedures

(APHA-AWWA-WEF, 1995) and expressed relative to soil water content, which was calculated from the fresh and oven dry weight. Soil texture was determined by standard field characterization methods (GLOBE, 2005). The height above datum was measured with tracing paper, placed at each site before high tide and the corresponding inundation classes were calculated. According to Tomlinson (1994) inundation classes one, two, three and four correspond to an area being inundated by respectively 100-76 %, 75-51 %, 50-26 % and 25-5 % of the high tides. The Leaf Area Index, integrated over the zenith angle 0 to 75°, was calculated from hemispherical images using the software program Gap Light Analyzer v. 2.0 (Simon Fraser University, British Columbia and the Institute of Ecosystem Studies, New York).

LocationAccession nr. Tree height R130

† Rbase‡

Discs Samples (m) (cm) (cm)Gazi

site 1 Tw58910-13,Tw58927-31 T72,T76,T85 11(7-14) 4(1-13) 43(20-100)site 2 Tw58905-09,Tw58926,Tw58988 T40-41 3(2-4) 2(1-3) 9(5-17)site 3 Tw58916-18,Tw58933-35 T26-28 2(2-4) 1(1-3) 7(3-11)site 4 Tw58943-48,Tw58987 T14-16 6(4-8) 3(2-5) 17(5-54)site 5 Tw58936-42 T45,T51,T56 6(6-6) 3(1-5) 7(3-10)site 6 Tw58900-04,Tw58932 T35,T37 4(2-5) 3(1-4) 6(6-14)site 7 Tw58920-25,Tw58989 T19-22 6(3-10) 4(1-9) 22(4-44)

Dabasosite 8 - Tw58950-61 10(6-14) 11(6-16) 36(16-100)

Table 1. Studied stem discs and outermost wood blocks of Avicennia marina with the corresponding tree cha-racteristics for the eight study sites at two locations in Kenya, Gazi and Dabaso. Values are means with ranges between brackets.

†Stem radius at 130 cm height; ‡stem radius at the base of the tree.

The cambial marking was performed with a surgical needle of 1.2 mm diameter (Verheyden et al., 2004) in May 2005 (wet season). In view of a potential time mark of the dry season by changes in wood anatomy, the trees were marked a second time in February 2006 (dry season). For two trees (Tw58955, -57) the cambium of both the stem and one branch was wounded. From these 80 trees, 49 were felled in June 2006 and samples are now part of the Xylarium of the Royal Museum for Central Africa, Tervuren, Belgium (discs, Table 1). From the remaining 31 trees wood samples were taken with a handsaw of the outermost wood comprising the cambial mark and 19 of them were subsequently stored in FAA (Formalin - Acetic Acid - Alcohol). Before felling or sampling of the

84

Chapter 5

trees, the stem circumference at 130 cm height and the stem diameter at the base were measured. The height was calculated trigonometrically.

Location Soil Water Nutrients (10-3 µmol/cm3 soil) LAI Soil textureSoil Water Salinity

(‰) Inundation

NO3- NH4

+ P min. max. classGazisite 1 0.5 (0.2 - 0.6) 3 (0 - 5) 1.4 (0.9 - 1.8) 1,42 silty clay 20,9 46,0 1site 2 0.3 (0.2 - 0.5) * 0 (0 - 0) 0,25 sandy loam (sl) 40,0 70,0 2site 3 0.15 (0.10 - 0.24) * 0.2 (0.1 - 0.3) 0,23 loamy sand (ls) 38,0 86,0 3site 4 0.3 (0.0 - 0.4) 1 (0 - 4) 2 (0 - 4) 1,29 ls - sl 5,0 68,2 3site 5 2 (0 - 6) 10 (7 - 14) 0.1 (0 - 0.3) 1,18 clay loam 40,0 79,9 3site 6 1 (0 - 4) 0.5 (0.1 - 1.1) 0.1 (0 - 0.3) 0,62 sandy loam 10,0 90,0 4site 7 23 (0 - 68) * 2 (0 - 4) 1,73 ls - sl 10,0 48,0 4

Dabasosite 8 * * * * * 33,0 72,2 3

Table 2. Stand characteristics of seven study sites in Gazi Bay and one study site in Dabaso, Kenya. Soil water for nutrient and salinity analyses was taken at about 25 cm depth. Inundation classes are according to Tomlin-son (1994). Values are mean with range between brackets

Climate description

The climate along the Kenyan coast is characterized by a bimodal distribution of the precipitation. A distinct dry season (January - February) is followed by a long (April - July) and a short rainy season (October - November) (Fig. 1). During the wet season, the rivers Mkurumuji and Kidogoweni provide an important freshwater source for the mangroves of Gazi Bay (Kitheka, 1997). The average temperature at the Kenyan coast ranges from 22 to 30 °C, with a mean relative humidity of 65 % to 81 % (annual averages of minima and maxima for Mombasa for the period 1972 - 2001, data from the Kenyan Meteorological Department, Mombasa, Kenya).

* No data records.

50

40

30

20

10

0

20

40

60

80

100

300

)C°( erutarep

meT )m

m ( n

o itati

pic e

rP


Fig. 1. Climate diagram of Mombasa (39°36’E, 4°0’S) adapted from Lieth et al. (1999), showing the long (April–July) and short (October–Novem-ber) rainy season and one distinct dry season (Ja-nuary–February). Dotted area, dry season; hatched area, wet season. The precipitation axis is redu-ced to one-tenth scale above the dotted horizontal line.

85


Sample preparation and microscopic analysis

In this study the following terms and definitions are used: phloem band to designate a zone of phloem strands united in a band of parenchyma tissue and bordered to the outside by a layer of sclereids; xylem band to designate the zone in between two phloem bands; growth layer to designate one ontogenetic unit of phloem and xylem (Fig. 2-3).

To investigate the periodicity of growth layer formation, the number of growth layers formed in the period from May 17th-26th 2005 to June 9th – July 9th 2006 was counted from 79 cambially marked trees. Wood discs and wood samples were prepared as described by Schmitz et al. (2007). In brief, wood discs were sanded with a series of sand paper from 100 to 1200 grit while wood samples were dehydrated in an ethanol series and embedded in PEG1500. Transverse sections were made with a sliding microtome and double stained with Safranin - Fast Green. The number of growth layers from the cambial mark onwards was counted using an Olympus BX 60 microscope. This was done at both sides of the wound and the growth layer count was averaged (Fig. 2B-C, 3A). The growth layer network of one stem, Tw57798 (see also Schmitz et al., 2007), was visualized with a CT-scan (Fig. 4; CT-scan Brilliance 64 slice, Philips, Nederland).

In addition, the increment during the period of the cambial marking experiment was measured with the image analysis software AnalySIS Pro 3.2 (Soft Imaging System GmbH, Münster, Germany) via a camera connected to a microscope (Olympus BX60). The increment was measured at about 2 mm distance from the abnormal growth at the wound, which is the furthest position showing a cambial mark. For trees showing a double wound, resulting from two simultaneously dividing cambia, the increment was corrected for the part of the xylem tissue of the outermost growth layer already formed at the time of pinning (Fig. 2C, 3A).

On each of 48 stem discs the width of the xylem and phloem bands of each growth layer was measured along three transects from pith to bark at the maximum, minimum and medium sized disc radius. Because of the networking pattern of the growth layers, a pencil line was drawn and all growth layers crossing the line were measured.

Statistics

To quantify relationships between different tree and growth layer characteristics Pearson correlation coefficients were calculated. Simple linear regressions were used to study relationships in growth increment with environmental and tree characteristics. One way ANOVAs were used to test (1) the difference in radial increment between the different study sites and (2) the relationship between the average ring width at different sides of asymmetric stem disks and corresponding radii. A t-test for independent variables was performed to compare the growth increment at opposite radii of asymmetric trees and between trees with and without signs of die-back (with separate variance estimates). To comply with the assumptions of a normal distribution of the data and homogenous variances data were transformed with either logarithmic or square root functions. If transformations failed, Spearman R correlation coefficients were calculated (STATISTICA, 2006).

Results

Wood anatomical description of the cambial mark

On a macroscopic level, the pin hole was regularly encircled by an extra periderm (Fig. 2A). Microscopically, the position of the cambium at the time of pinning could be determined from a

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Chapter 5

A

Pd

B

XX

X

P

P

CX

P

P

XP

Pd Pd

PdPd

X

Fig. 2. Macro- and microscopic characteristics of Avicennia marina stem wood after wounding the cambium with a needle of 1.2 mm diameter. (A) Pinhole encircled by wound periderm, scale bar = 1cm. (B-C) Detail of sanded stem discs showing (B) a decreased vessel density and vessel diameter in the zone around the pinhole, marking the time of cambial wounding and (C) a double cambial wound illustrating the simultaneous formati-on of the two growth layers. Scale bars = 1 mm. P, phloem band; Pd, periderm; X, xylem band. Arrows, radial increment from May 2005 to June 2006. Small arrows, part of the growth layer already formed at the time of cambial wounding. Asterisks, pinhole.

A

X

P

P

X

X

P

P

Pd

Pd Pd

BX

Ps PA

Sc

X

Fig. 3. Transverse microsections of the stem wood of Avicennia marina after wounding the cam-bium with a needle. (A) Microscopic wood structure after wounding of the two outermost cambia. Scale bar = 1 mm. (B) Detail of two phloem strands lo-cated inward to the pinning hole showing a coating of the cells and an induced meristem (arrowhead) in the right and left phloem strand, respectively. Scale bar = 500 µm. P, phloem band; PA, parenchyma; Pd, periderm; Ps, phloem strand; Sc, sclereids; X, xylem band. Arrows, radial increment from February 2006 to June 2006. Small arrows, part of the growth layer already formed at the time of cambial marking. As-terisks, pinhole.

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variable combination of wood anatomical changes (Fig. 2B-C, 3). In agreement with earlier reports, the time mark was formed by an initial parenchyma band in 53 % of the trees (n = 80) (Shiokura, 1989; Nobuchi et al., 1995; Ohashi et al., 2001; Stobbe et al., 2002) and relatively smaller vessels (Nobuchi et al., 1995; Schmitt et al., 2000; Ohashi et al., 2001) in a lower density (Carlquist, 2001) in an area of about 1 mm² at both sides of the pin hole. The pinning canal was closed by periderm (Carlquist, 2001; Stobbe et al., 2002), with an extra internal wound periderm in 14 % of the studied trees (n = 79) showing a double wound (Fig. 2C, 3A). Regularly, a coating that stained pink with safranin was observed in the phloem strands inward to the pinning canal (Fig. 3B). Suberin1 has been mentioned to coat parenchyma tissue of the reaction zone (Schmitt and Liese, 1993). It is known as a barrier forming agent protecting against invading pathogens (Franke and Schreiber, 2007) and necrosis of the deeper lying tissues (Schmitt and Liese, 1993). In addition, in 53 % (n = 19) of the microsections the phloem strands showed a meristematic zone (Fig. 3B), in correspondence with the finding in poplar of dedifferentiating secondary phloem cells into meristematic cambium cells (Frankenstein et al., 2005; Frankenstein, 2006). Finally, a dark zone of protection wood (Carlquist, 2001) was observed inward to the pinning canal (Fig. 2B-C) with partly occluded vessels and fibers (Schmitt et al., 2000).

Growth characteristic Mean ± SD Min Max1 Year study period

Growth rate (mm / yr) 1 ± 1 0 5

Included growth* (µm/yr) 249 ± 105 64 376Growth layers (nr / yr) 1.0 ± 0.7 0,1 4

Disc averageD(M-m) (nr) 4 ± 3 0 12Growth layer width (µm) 898 ± 137 402 1564

Xylem width 657 ± 107 257 1198Phloem width 241 ± 35 117 403

% Phloem / growth layer 28 ± 2 20 44

Factor F-value P-value n r2,*Environment

Min. Salinity 3.00 ns 73 0.03Max. Salinity 0.04 ns 73 -0.01Inundation class 1.48 ns 73 0.01

[NO3-] 1.11 ns 62 0.00

[NH4+] 0.02 ns 37 -0.03

[P] 1.09 ns 54 0.00LAI 6.05 <0.05 62 0.08

Tree

R130† 16.87 <0.001 73 0.18

Rb‡ 0.34 ns 73 -0.01

Height 9.13 <0.01 73 0.10

Table 3. Characteristics of the secondary growth of Avicennia marina averaged over the one-year study pe-riod between May 2005 and June 2006 (n = 80) or ave-raged over the entire stem disc (n = 49). D(M-m), diffe-rence in number of growth layers between the minimum and maximum disc radius; nr, number of growth layers; SD, standard deviation.

*Growth of the last but one growth layer (n = 10, see Fig. 2C, 3A)

Table 4. Results of simple linear regressions for the May 2005-June 2006 growth increment of Avicennia marina trees of different size and gro-wing under different environmental conditions (see Table 1). Data were transformed with a logarithmic function.

†Stem radius at 130 cm height; ‡stem radius at the base; *adjusted correlation coefficient.

Annual increment

Only a tenth of a growth layer up to four completed growth layers were formed in the period of the cambial marking experiment from May 2005 to June 2006 (Fig. 5, Table 3). The number of growth layers formed during the one year study period was highly correlated to the corresponding radial increment (r² = 0.73, n = 73, P < 0.0001). Also on the level of the entire stem disc, the number of growth layers showed a strong correlation with its radius (r² = 0.89, n = 141, P < 0.0001). In agreement with this high correlation, the difference between the maximum and minimum disc radius of asymmetrical stem discs was correlated with the corresponding difference in number of growth

1 See also comment at the end of the chapter.

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layers (r² = 0.37, n = 42, P < 0.0001). Growth layer width increased from minimum, over medium to maximum disc radius (F = 50.79, n = 2663, P < 0.0001; Linear trend analysis: F = 101.57, n = 2663, P < 0.0001).

No significant difference was observed in the number of growth layers formed in the one year study period between the eight study sites (Fig. 5B; F = 1.48, n = 79, P = ns). By analysing different environmental factors separately, characterizing the different study sites, no or only very weak relationships were found with the radial increment of the study period (Table 4). Also stem radius at the sampling height of the trees and tree height showed a weak but significant correlation with the radial increment measured from the cambial markings onwards (Table 4). The growth rate during the one year study period was significantly larger in trees showing a double cambial mark, resulting from the additional radial increment of the last but one growth layer (Fig. 2C, 3A; t = -3.2, n1 = 62 and n2 = 11, P < 0.01). The cambium of this inner growth layer produced on average 249 ± 105 µm (n = 10) of xylem tissue during the one year study period (Table 3). Within tree Tw58955 and Tw58957, radial growth was 370 % and 1065 % higher, respectively, in branches compared to stems. Trees without dead leaves or branches had a significant larger growth increment compared to trees with dead leaves and/or branches dispersed in the crown (t = -3.68, n1 = 15 and n2 = 57, P < 0.01). The growth from the cambial mark in February 2006, the middle of the dry season, until the felling in June 2006, the end of the long rainy season, was mostly small and restricted to the zone around the wound hindering relevant measurements and study of potential wood anatomical changes in the dry season.

Growth layer characteristics

The 2005-2006 growth increment was significantly correlated with the average growth layer width of a stem disc (r² = 0.31, t = 4.37, n = 44, P < 0.0001). The average growth layer width was strongly correlated with tree height (r² = 0.43, F = 36.35, n = 47, P < 0.0001) and weakly but significantly with soil water salinity, showing a higher correlation with maximum salinity (r² = 0.16, F = 9.60, n = 47, P < 0.01) than with minimum salinity (r² = 0.10, F = 5.97, n = 47, P < 0.05). No correlation was observed between growth layer width and age, represented by growth layer number, with the first growth layer at the pith having number one and the last growth layer at the bark having the highest number (Partial correlation, controlling for tree: r² = 0, t = 0.0067, n = 2663, P = ns). The width of a xylem band showed a strong positive correlation with the phloem band width of the same growth layer (Table 3, r² = 0.53, t = 54.78, n = 2663, P < 0.0001). However, the increase in phloem width was only 22 % of the corresponding xylem width resulting in a negative relationship between the percentage of phloem and the growth layer width (Spearman r² = 0.14, n = 2663, t = -21.26, P < 0.0001) with a lower limit at 20 % phloem per growth layer (Table 3).

Discussion

The patchy growth hypothesis

The first hypothesis of a periodic growth with the number of growth layers produced during the year depending on site conditions was rejected. Instead, we propose a patchy growth mechanism based on circumstantial evidence: (1) a network pattern of growth layers (Fig. 4), (2) two simultaneously forming growth layers without the occurrence of wood cracks or crushed phloem tissue indicating the absence of internal strains (Fig. 2C, 3A), (3) a remarkable variation in growth layer formation within the different study sites (Fig. 5B) and (4) an extensive variation of the radial increment, seemingly independent from any of the environmental factors considered (Table 3-4). The patchy growth hypothesis states a basic growth rate all around the tree with a more vigorous growth at one or several positions around the stem circumference. The side of active growth changes with time to

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Fig. 4. Networking pattern of the growth layers in (A) transverse and (B) longitudinal plane as visualized by a CT-scan of a tree stem portion of 7 cm diameter (Tw57798, Tervuren xylarium). Circles indicate positions of branching phloem bands.

Annual number of growth layers

Num

ber

of s

ampl

es

19%

31%

15%

26%

4% 4%1%

<0.5 0.5-1 1 1-2 2 2-3 40

5

10

15

20

25

30 AN

o. o

f gro

wth

laye

rs/y

r

Site

0

1

2

3

4

1 2 3 4 5 6 7 8

B

Fig. 5. Number of growth layers of 79 Avicennia marina trees formed in the period from May 2005 to June 2006 (A), distribution of the annual growth layer number over the eight study sites (B) (see also Table 2).

end up with a tree of regular circumference. A patchy growth would clarify the three-dimensional phloem network, first reconstructed from serial branch microsections by Zamski (1979) and here for the first time visualized in situ from a mature tree trunk. Not only around (Fig. 4A) but also along the stem (Fig. 4B), growth patches instead of rings were observed. Nair and Mohan Ram (1990) propose a similar explanation for the growth layer network of Dalbergia paniculata, resembling the one of A. marina. They suggest it could be due to the outermost parenchyma cells that do not invariably become meristematic. More recently, discontinuous new cambia have been observed in some species of Amaranthaceae (Rajput and Rao, 2000) and a liana species of the Menispermaceae (Jacques and De Franceschi, 2007).

The earlier suggestion of a simultaneous development of two growth layers (Schmitz et al., 2007) was illustrated by a double cambial mark (Fig. 2C, 3A). Cracks in the lignified part of the outermost growth layer can be overcome by collapse of phloem cells (Carlquist, 2007). Supporting the patchy growth hypothesis, this was not observed in the present study nor in A. resinifera and A. germinans which had at least the four outer phloem bands uncrushed (Zamski, 1979). Moreover, the finding of


90

14 % (n = 79) of the trees with more than one active cambium could be an underestimation. First, the depth of pinning was not standardized also because the depth needed to wound both cambia varies with the width of the outer growth layer. Recently, more than one active cambium was observed in the herbaceous and shrubby plants of Aizoaceae (Carlquist, 2007), questioning the existence of two mechanisms for successive cambia formation. Unlike Avicennia (Zamski, 1979) and some other species (Esau, 1969), Carlquist (Carlquist, 1999a; 1999b; 2003a; 2004) found in members of the Caryophyllales successive cambia originating from one lateral meristem in the cortex that also produces the rays and the tissue in between the phloem strands. This would be hindered by the partially formed outer growth layer in between the lateral meristem and the inner forming growth layer. Moreover, the patchy differentiation of new cambia makes the existence of one circular meristem, giving rise to the vascular cambia, unlikely in A. marina.

Secondary growth in discrete units would explain the variation in growth between A. marina trees (Fig. 5). On the side of wounding, the cambial activity and the duration of active growth during the study period would determine the number of growth layers produced and the annual radial increment. Therefore, radial increment was not a reflection of past environmental conditions (Table 4). However, the total increase in stem width is expected to be influenced by external factors such as soil water nutrients (Feller et al., 2003; Lovelock et al., 2004), salinity (Lin and Sternberg, 1992; Tuffers et al., 2001; Ball, 2002) and inundation periods (Worbes, 1989; Callado et al., 2001; Menezes et al., 2003; Verheyden, 2004). In favour of this idea, the average growth layer width of stem disks did show a decrease with increasing salinity. Despite the underestimation of total secondary growth, radial increment mirrored differences in leaf area related to crown die-back or to site specific LAI (Table 4). Although slightly, compared to tree species from Cameroon (Worbes et al., 2003), also tree size (Table 4) was reflected in A. marina’s growth rate for 2005-2006 as supported by the overall positive effect of tree height on mangrove biomass production (Saenger and Snedaker, 1993). The absence of a relationship between radial increment during the study period and basal diameter (Table 4) agrees with earlier findings of tree growth being determined by tree structural characters rather than by age (e.g., Pélissier and Pascal, 2000; Sterck et al., 2001; Worbes et al., 2003; King et al., 2005).

Additional support for the patchy growth is given by the slower radial growth, on average 0.57 mm/year, at site 5 during the previous study (Schmitz et al., 2007; here site 5 corresponds to site 9) compared to the present one. The longer study period of 2.5 years in combination with a cambial mark at a single position around the stem would have increased the underestimation of the total increase in stem width if growth takes place at discrete and changing positions around the stem. Therefore, when comparing the growth rate of A. marina with other tree species, one has to be cautious. The measured growth rate concerns the radial increment at the side of pinning. Depending on the number of positions around the stem circumference active during the study period, this value might underestimate the total increase in stem width. On the other side, growth layers growing at the time of pinning, have already produced (part of) their phloem band which might have resulted in an overestimation of the growth rate. Among mangrove species, our findings on radial growth corresponded quantitatively with the radial growth of Rhizophora mucronata growing in the same forest in Gazi Bay, <0.5 – 4.81 mm/yr (Verheyden, 2004), and of Heritiera fomes and Sonneratia apetala from Bangladesh, 1.4 ± 0.5 mm/yr and 2 ± 1 mm/yr respectively (Md Q Chowdhury, Wageningen University, Netherlands, unpubl. res.). Shiokura (1989) and Thampanya (2006) mention a higher growth rate of 3.8 – 5.7 mm/year for Avicennia species from Japan and Thailand. A soil water salinity level rarely exceeding 40 ‰ (see also Table 2) in these countries’ mangrove forests (Wakushima et al., 1994a; Wakushima et al., 1994b) might be responsible for the inconsistency. In India, three A. officinalis trees even had a diameter growth of 25.3 mm/year (Baker, 1915). The growth layers there are suggested to be monthly (Baker, 1915) and thus are approximately 1 mm wide, as those of A. marina (Table 3) and A. germinans from Japan (Sun and Suzuki, 2000).

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What triggers cambium differentiation?

The second hypothesis of the growth layer width being determined by soil water salinity was only partially accepted. The similar width of the growth layers of different Avicennia species together with the influence of radial growth on the formation of a new growth layer is in favour of a mainly endogenous control on cambium differentiation. An additional external influence of tree height and soil water salinity was recorded. Also in other species stem radius and number of growth layers are highly related (Ogden, 1981; Ruthrof et al., 2003) but in A. marina the relationship applies even along the different radii of a single asymmetric stem disc. Moreover, the radial increment of 2005-2006 was only slightly related to the width of the corresponding growth layers as compared to trees with one vascular cambium. A strictly controlled growth layer formation results in a regular dispersion of the phloem tissue over the entire stem disc. Within one growth layer the ratio of xylem to phloem tissue is lower compared to trees with conventional secondary growth (ratio: 2 - 9; Table 3) (Artschwager, 1950; Waisel et al., 1966; Rajput and Rao, 2000). Besides, they accumulate only a limited number of phloem increments making the total phloem fraction of an Avicennia stem considerably higher (Esau, 1969). The functional advantage of such a three-dimensional phloem network lies in its proposed role in embolism repair and water storage (Mauseth and Plemons-Rodriguez, 1997; Salleo et al., 2004; Salleo, 2006; Scholz et al., 2007), supported by the decreasing growth layer width with increasing soil water salinity. The smaller the growth layers are, the higher is the percentage of phloem tissue and the lower the fraction of thick-walled xylem fibers since the increase in phloem tissue was only 22 % of the xylem production. Therefore, the disappearance of growth layers at the smaller radius of asymmetric stems, instead of becoming smaller, might assure the mechanical stability of the tree. The finding of wider growth layers in larger trees confirms this idea though no age trend was observed. Nevertheless, the considerable variation in growth layer width within a tree, with an average range of 402 ± 92 µm - 1564 ± 354 µm (n = 47), underscored the existence of an external control on the development of a new cambium superimposed on a genetically defined basic growth layer width.

In conclusion, a patchy growth via successive cambia may offer a functional advantage under xeric and fluctuating environmental conditions as there are in the mangrove environment. We propose that growth layer width is partly predetermined by mature tree height and partly controlled by the micro-environmental conditions via a trade-off between hydraulic safety and mechanical stability. Optimal growth conditions could prematurely stimulate the differentiation of a new cambium resulting in two simultaneously forming growth layers. In this way beneficial growth conditions could be exploited, which is supported by the significantly larger increment of the trees showing a double wound. To test this hypothesis and the proposed patchy growth, future studies should focus on controlled eco-physiological experiments and address the cambial activity on an intra-annual level, considering intra-tree variations and the relationship with tree phenology. Further insight in the functioning of successive cambia in different tree species and the link with environmental conditions is needed to reveal this growth strategy as a functional adaptation or a mere structural oddity.

Acknowledgements

This research was financially supported by the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen), the Belgian Federal Science Policy (project MO/37/015), travel grants from the National Fund for Scientific Research (FWO, Belgium), the Schure-Beijerinck-Popping Fonds (Koninklijke Nederlandse Akademie van Wetenschappen, Nederland) and the Flemish Interuniversity Council (VLIR). We thank Dr. Samuel Teissier for assistance with the nutrient analyses and Dr. J. De Mey of the UZ-Brussels, Jette for the CT-scan photographs, Hassan Said Attuar and Hamisi Ali Kirauni for their invaluable help during the fieldwork and all people of Mwamba (A Rocha Kenya) and Gazi for their assistance and hospitality.


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Comment

Although suberin coatings have been reported before in reactions to wounding, a more detailed observation of the coating is needed for identification. While most previous studies used Sudan dyes to detect suberin (e.g. Pearce and Rutherford, 1981; Biggs, 1984; Pearce and Holloway, 1984; Hawkins and Boudet, 1996), Transmission Electron Microscopy (TEM) analyses are essen-tial. TEM micrographs allow the observation of suberin’s specific location inside of the primary cell wall close to the plasma membrane. In addition, they reveal the characteristic ultrastructure of suberin, which is an alternation of electron dense and more transparent lamellae (Nawrath, 2002).

Chapter 5

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94

General conclusion & perspectives

The tree which moves some to tears of joy is in the eyes of others only a green thing that stands in the way. Some see Nature all ridicule and deformity, and some scarce see Nature at all. But to the eyes of the man of imagination, Nature is Imagination itself.WILLIAM BLAKE

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General conclusion& perspectives

Growing on the edge

The transport of water is of vital importance for plants living on land (Sperry, 2003; Maseda and Fernández, 2006). Therefore, it should not be surprising that the hydrosystem of trees has been studied for a long time. A good job was done, describing the wood anatomy of a wide range of species from different ecosystems (Baas et al., 1983; Carlquist and Hoekman, 1985; Wheeler and Baas, 1991; Lindorf, 1994; Noshiro and Baas, 2000; Segala Alves and Angyalossy-Alfonso, 2000; Carlquist, 2001; Hacke et al., 2007; Wheeler et al., 2007) clarifying how trees can adapt their water transport system to the specific requirements of the environment. Noteworthy in this respect is the added value that studies on mangrove wood anatomy could offer.

Mangrove trees have to endure a peculiar combination of environmental demands. Growing on the edge between sea and land they are faced with salt water and a stunning dynamism inherent to the tidal regime, enforced or mitigated by topography. In addition, they are subjected at irregular intervals to short-term hydrology perturbations such as extreme floods and ENSO related climate changes, affecting inundation time and soil water salinity (Drexler and Ewel, 2001; Erftemeijer and Hamerlynck, 2005). It is however remarkable how little effort has been put in clarifying the structural adaptations facilitating the ascent of water in mangrove trees (Sperry et al., 1988; Sun and Lin, 1997; Yáñez-Espinosa et al., 2001; Sobrado, 2007). This study took up the challenge to explore the functional significance of the variability in hydraulic structure between and within mangrove trees and species. Often the value of descriptive studies is, totally undeserved, counted of inferior importance to functional ones. The current investigation, supported by a series of other recent studies (Domec et al., 2006a; Hacke et al., 2007; Sperry et al., 2007; Choat et al., 2008), dismisses this false idea and highlights the core importance of insight in the wood anatomical plasticity to deepen our understanding in the water transport of individual trees and even vegetations (Maherali et al., 2004; Hickler et al., 2006).

Mangrove hydraulic structure: flexibility meets stability

The value of redundancy in a dynamic environment

The hydrosystem of mangrove trees appeared to be highly responsive to their dynamic environment but, not in all vascular traits. While the vessel density of Rhizophora mucronata was sensitive to even small changes in soil water salinity, tangential vessel diameter and intervessel pit membrane size were unresponsive. The same contrast in sensitivity between vessel density and tangential vessel diameter was found in a recent study on Avicennia marina (Robert, 2007). Radial vessel diameter did show a minute decrease with higher salt levels in the soil suggesting a functional advantage of circular vessels under drought stress. The near constant vessel diameter was unexpected given the pit area hypothesis, stating that cavitation resistance depends on the total pit area per vessel (Wheeler et al., 2005). The finding raises questions about the role of vessel diameter in securing

96


hydraulic safety within species (Cochard et al., 2007). The pit area hypothesis fits in with previous studies that give the impression that vessel size is in general a major feature for a safe water transport system with decreasing vessel size under intensified stress conditions. Given the stunning diversity of trees’ hydraulic structure it should however not be surprising that one strategy does not fit all plants (Maherali et al., 2006; Preston et al., 2006). Packing of small vessels can mitigate the safety versus efficiency trade-off by compensating the hydraulically safe but inefficient tiny vessels with a large conductive area (Hacke et al., 2006; Arend and Fromm, 2007). But, no alternative for low vessel size has been given to provide safety. While the pit area hypothesis might still universally apply across species, the increased vessel density observed here seems to provide both efficiency and hydraulic safety as was recently observed for roots (Pratt et al., 2007). While in the mangrove species Aegiceras corniculatum, vessel diameter was similarly stable, vessel density showed the opposite trend with salinity (Sun and Lin, 1997). These findings underscore the need for studies integrating multiple variables, related to different plant parts and at all levels of organisation to fully understand the ecological adaptations of the trees’ hydrosystem (Reich et al., 2003; Maherali et al., 2004; Maherali et al., 2006). In the study species, a safe pit geometry and flexible pit membrane thickness might add to the explanation of a stable vessel diameter.

During periods of stress, smaller vessels are supposed to diminish the risk of cavitation because of their smaller pit area. The smaller volume per vessel available for flow also excludes the risk of a sudden drop in water transport. But, at the same time it impedes optimization of the water transport when conditions are favourable again. The higher vessel wall area per volume water transported increases the lumen resistance (Sperry et al., 2005). The nearly constant vessel diameter in both mangrove species could reflect an optimal value, equilibrating the overall safety and efficiency needs of the species’ habitat besides other adaptations to secure this safety. At locations or during periods of serious water stress, a higher number of transporting units could be an alternative to guarantee a minimal water supply (Ewers et al., 2007). In the following rainy season when conditions are favourable again, the high vessel density might offer the opportunity to optimize hydraulic conductivity by refilling embolized vessels. This hypothesis is supported by the indication that not only the last growth ring is functional in the studied species (unpublished results) and by the time-lag between the peak in precipitation and the decrease in vessel density (Verheyden et al., 2004). In contrast, the increase in vessel density starts several months before the onset of the driest months of the year. This might be explained by an endogenous trigger or rhythm such as an increase in leaf litter fall, found to parallel the periodicity in vessel density in R. mucronata (V.W. Wang’ondu, unpublished results). These phenological and developmental stages might also have a common cause, whether exogenous or endogenous. Nevertheless, the relationship between leaf loss and vessel density corresponds to earlier observations of the positive effect of defoliation on conductive area (Hilton et al., 1987; Salleo et al., 2003; Heinrich and Banks, 2006). The change in vessel density might thus partly be uncoupled from a direct functional adaptation to changing environmental conditions, clarifying the existence of annual changes in vessel density although more than one tree ring is conductive. Taking all together, the increased cavitation vulnerability of a more redundant hydraulic system, in contrast to a sectorial one with few connections between vessels, seems to be offset by its benefits for mangrove species growing in a variable environment both in time and space (Zanne et al., 2006).

Although the efficiency aspect seems to have won from the safety aspect in terms of pit area, other vessel characteristics might be responsible for a balance with hydraulic safety. Besides, an intensified integration of the hydraulic system would only be a threat for the water transport if cavitation takes place via air-seeding. Although this is the prevailing hypothesis, it remains elusive to which extent other mechanisms of bubble nucleation play a part in cavitation events (Cochard, 2006). But, bubble nucleation via air-seeding will most likely be the common cavitation mechanism as indicated by the ecological plasticity in intervessel pit structure.

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A dynamic role for the guardians of the transport network

Intervessel pits are often neglected in studies on ecological wood anatomy despite their fundamental but ambiguous role in the water transport (Choat et al., 2008). At the one hand they act as vessel crossings to keep a continuous water flow, offering a way to bypass air bubbles but on the other hand they create them by air-seeding via pores in the pit membranes. Compared to our knowledge about vessels in structure, chemistry, the variation herein, function and ecology, the understanding of intervessel pits is precious little. This study was amongst the first investigations (e.g. Mayr et al., 2002; Choat et al., 2003; Jansen et al., 2004) assessing the variability of the hydrosystem at the sub-cellular level in association with environmental changes. The sometimes slight structural differences emphasize the importance of quantification, large replication sizes and standardization of the measuring position (cf. Sano, 2005).

Despite the distinct arrangement, the geometry of the intervessel pits from A. marina and R. mucronata showed similarities, distinct from some temperate species pointing to a functional advantage under mangrove conditions. Vestures were discovered along the minute inner and outer pit apertures of A. marina, supposed to prevent membrane rupture. The funnelled pit canals in R. mucronata might offer the same functional advantage, as was the remarkable thickness of the pit membranes of both species. No pores were observed in the pit membranes, explained by their crooked route through the pit membrane although deposits could be involved too. Deposits, the origin and possible function of which need to be resolved, were observed in pit canals and along vessel walls of both studied mangrove species. Together with the discovery of a seasonal variation in the thickness of the pit membranes of R. mucronata and the high ionic strength of sea water, the potential of ionic regulation of the water transport deserves special attention in mangroves. While a kind of coarse regulation might take place at the level of the vessels by blocking and refilling (Domec et al., 2006b), fine-tuning could be handled by hydrogel swelling and shrinking in pit membrane pores in response to fluctuations in ionic content of the xylem sap (van Ieperen, 2007). To a certain degree both processes are interlinked. Following cavitation the ionic content of the xylem sap might increase, buffering the decline in hydraulic conductivity. In addition, the hydrogel effect might be regulated via seasonal fluctuations in the chemical nature of pectic substances (Nardini et al., 2007). This hypothesis got recent support from the finding of considerable changes in xylem sap composition under drought stress (Alvarez et al., 2008). The possibility of ion-mediated conductivity changes could be supported by the discovery in R. mucronata of a seasonal change in pit membrane thickness, if manifested in a changing pit membrane pore size. What’s more, the similar chemical composition of pit membranes and deposits in pit canals and along vessel walls could imply that not only pectic substances in but also on cell wall layers play a role in the regulation of the water transport.

Next to cavitation prevention an efficient embolism repair mechanism could compensate for the potentially higher cavitation risk related to the increased vessel density during the dry season. At least A. marina enjoys a privileged position in this respect because of the interspersed phloem tissue in the stem with an increasing proportion as soil water salinity increases.

The meaning of success in successive cambia

Avicennia marina occupies the most contrasting sites in the mangrove habitat and is the only mangrove species with the peculiar anatomical design caused by wood formation via successive cambia. This curious structure incited to detailed anatomical descriptions and ecological interpretations for species from various families (Carlquist, 2004, 2007b, a). Within species, however, the ecological significance of the alternation between phloem and xylem tissue and the developmental strategy at the tissue level instead of the cellular level had never been studied before. The clear pattern of xylem

98

and phloem bands should not be confused with annual tree rings. Stem diameter did not even grow in continuous circles but in patches, providing the tree with the opportunity to form several growth layers simultaneously. In the dynamic mangrove habitat a patchy growth could be beneficial to exploit favourable growth conditions whenever they occur. In addition, such a growth strategy results in a water transport system composed of various structures depending on the moment the patches were formed. In this way the tree would always be adapted to the ambient environmental conditions, at least in one sectional part of the stem.

Although growth layer formation was not controlled by climate, it was by local environmental conditions. The phloem fraction per stem increased with higher substrate salinity pointing to a relationship between this cambial variant and stress tolerance. It could underscore the suggested role of phloem tissue in embolism repair (Salleo, 2006) though two other factors could be involved too: (1) an increased sprouting capacity linked to regular activity of more than one cambium at the time but also the abundant phloem tissue could be beneficial in this respect (Aloni and Barnett, 1996), and (2) a higher water storage capacity thanks to the parenchyma tissue embedding the phloem strands (Carlquist, 2007a; Scholz et al., 2007). Given the association between vessel density and salinity in R. mucronata, a reduced wood density seems to be related to an increase in water stress in the studied mangrove species. The opposite finding across species (Preston et al., 2006; Jacobsen et al., 2007a; Jacobsen et al., 2007b) might indicate the benefit of an increased water storage capacity to deal with the comparatively small changes in water availability within species. While soil water salinity had a positive effect on the phloem-xylem ratio, the proportion of phloem tissue decreased with tree height. It illustrates the complexity of the relationships between hydraulic traits (Sperry et al., 2006; Pratt et al., 2007). The increase in the fraction of xylem with tree height does not necessarily support a trade-off between hydraulic efficiency and mechanical stability as reported earlier (McCulloh et al., 2004; Christensen-Dalsgaard et al., 2007). The interspersed phloem bands blur this relationship by affecting not only the mechanical strength of the tree but possibly also its hydraulic conductivity. To which extent secondary growth via successive cambia in A. marina represents a functional adaptation or a structural oddity is subject for future research.

Towards a functional understanding of the plasticity in hydraulic structure

Although water is a life saving resource of land plants, controversy still surrounds fundaments of sap flow such as the driving force and the mechanism(s) of cavitation. The high environmental stress mangroves have to endure together with the spatially heterogeneous habitat makes mangroves a worthy model system to clarify the regulation of the water transport. In an endeavour to reach this goal the functional significance of the anatomical variability of the conduit network has to be explored. This can be done by searching for the common structural and physiological characteristics of the water transport system within the phylogenetically diverse group of mangrove species. Only an integrative study of the ecological plasticity in hydraulic structure (Reich et al., 2003; Valladares et al., 2007), accounting for internal variations (McElrone et al., 2004; Burgess et al., 2006; Domec et al., 2006a; Petit et al., 2007; Pratt et al., 2007), in connection with its functional behaviour will clarify trees functioning (Maherali et al., 2006; Sperry et al., 2008). The occurrence of some mangrove species in a broad zone along the coastal slope enables studying the intraspecific variation in hydraulic structure in a gradient from land to sea. A comparison between mangrove species and terrestrial relatives will determine the specificity of the found characters for the mangrove habitat and its specific combination of stress factors. Vessel and intervessel pit structure should be studied on wood samples from root to canopy and from sap- to heartwood.


99

In view of the fluctuating thickness of the intervessel pit membranes, a challenge will be to elucidate the putative ion-regulation of the water transport via hydrogels in planta. The scanty knowledge about the remarkable variation in structure and chemistry of the intervessel pits should urgently be upgraded (Meyra et al., 2007). It will shed light on their dual role in cavitation events and regulatory processes of sap flow. Via a combination of Transmission Electron Microscopy and cellular UV Microspectrophotometry a good overview of the variation in the presence and chemistry of deposits on vessel walls, pit membranes and in pit canals can be gained. In a second step, pit membranes and deposits can be investigated more in detail by selective sampling with the technique of laser-microdissection (Angeles et al., 2006). The functional relevance of the variation in vessel and intervessel pit structure and chemistry can be clarified by combining measurements of hydraulic structure, hydraulic conductivity, osmotic potential and hydrostatic pressure of the xylem. Whether deposits play a role in sap flow or point to non-functional vessels can be answered by investigating in situ the presence of water, ions and deposits in vessels and intervessel pits via cryo-SEM (Scanning Electron Microscopy) fitted with EDAX (Energy Dispersive X-ray Analysis system). By investigating trees at several moments of the day and during different seasons, we can distinguish permanently blocked vessels from embolized vessels that are refilled afterwards.

In this respect, another challenge is to validate the role of phloem in embolism repair. The wood of A. marina formed by successive cambia provides an opportunity to test this hypothesis. Support is given when a relationship can be found between the activity of xylem bands in sap flow and the functionality of the corresponding phloem bands. Dysfunctional phloem bands can be recognized by collapsed phloem cells (Carlquist, 2007a). Finding out the trigger for the differentiation of successive cambia, determining the xylem-phloem ratio, and the link with stress conditions will be the ultimate obstacle to tackle. But it will also be rewarded with insight in the ontogeny and evolution of this puzzling phenomenon that is growth via successive cambia.


100

Mangroves, growing on the edge between sea and land

Dynamism of the intertidal environment�

Depends on:

▪ Tidal regime with a daily, monthly& annual cycle

▪ Topography▪ Seasons▪ Hydrology▪ Stochastic events (e.g. storms, ENSO)

�Fluctuating soil water salinity

�� Risk for drought-induced cavitation

Additional safety factor: possible role of phloem in embolism repair

Other factors:▪ Phenology▪ Mechanical strength

Efficiency-safety balance

vessels

pits

successive cambia

Dynamism in hydraulic structure�

Soil water salinity � results in:

� Redundancy of conduit networkStable conduit size

� Pit membrane thicknessSmall outer pit apertures

� Phloem-xylem ratio

Ecological plasticity in hydraulic structure is at least partly a response to the temporal and spatial variation in cavitation risk�

��


101


102

References

Alone with myselfThe trees bend to caress meThe shade hugs my heartCANDY POLGAR

103

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Mangrovebomen worden onderworpen aan uitzonderlijk hoge en variabele eisen voor het watertransport zowel in tijd als ruimte. De combinatie van zout water, periodes van overstroming afgewisseld door periodes van droogte, en hoge temperaturen suggereren dat een adaptief hydrosysteem de impact van droogte-geïnduceerde cavitatie reguleert. Er is echter weinig geweten over de ecologische plasticiteit van de hydraulische architectuur van mangrovebomen en zijn functionele betekenis voor het verzekeren van het watertransport. In het EERSTE DEEL VAN DE THESIS wordt de variabiliteit van de vatstructuur en de anatomie van de stippels tussen de vaten bestudeerd langsheen een gradiënt in saliniteit, zowel als tussen de seizoenen. Daarnaast wordt een eerste topochemische analyse uitgevoerd van de stippels tussen de vaten en hun intraspecifieke variatie onderzocht. In het TWEEDE DEEL VAN DE THESIS wordt het effect behandeld van bodemwatersaliniteit op de vorming van successieve cambia in Avicennia marina. Hoewel het merendeel van de boomsoorten hout vormt via één cambium gelegen aan de buitenkant van de stam, is er een beperkte groep van houtige soorten die hout vormen via opeenvolgende, nieuw gevormde cambia. Hoe dit gebeurt werd reeds beschreven op cellulair niveau voor verschillende soorten. De periodiciteit van de vorming van de opeenvolgende banden van floëem- en xyleemweefsel die zo ontstaan bleef tot op heden echter onduidelijk.

Twee soorten, die sterk verschillen in hun ecologische verspreiding over het mangrovewoud in Gazi Bay (Kenia), en eveneens biogeografisch belangrijke areaalverschillen tonen m.b.t. klimaatgebieden, werden bestudeerd: Rhizophora mucronata en Avicennia marina. Houtstalen werden verzameld op verschillende locaties gekenmerkt door uiteenlopende saliniteit van het bodemwater en overstromingsfrequentie. Diverse kenmerken van de vaten en de stippels daartussen werden gekwantificeerd met behulp van lichtmicroscopie, Scanning Elektronen Microscopie, Transmissie Elektronen Microscopie en cellulaire UV Microspectrofotometrie. De houtstalen van A. marina voor de analyse van de secundaire diktegroei werden genomen van bomen waarop een tijdsmarkering werd aangebracht door het cambium te verwonden, waarna ze op gekende datum geveld werden. Daarnaast werd er gebruik gemaakt van bomen van een aanplanting van gekende leeftijd. Groeiringen werden geteld, de breedte van de floëem- en xyleembanden gemeten en gerelateerd aan de verschillende omgevings- en boomkenmerken.

De bevindingen duiden de vatendichtheid aan als een goede proxy voor de saliniteit van het bodemwater terwijl vatdiameter eerder constant bleef tussen de sites. Ook de diameter van de stippels tussen de vaten vertoonde geen opmerkelijke verschillen tussen de sites. De stippelmembranen waren wel lichtjes dikker in het droogseizoen dan in het regenseizoen in R. mucronata. Maar meer opvallend was de variatie in elektronendichtheid van de stippelmembranen en het voorkomen van afzettingen in de stippelkanalen. In vergelijking met R. mucronata, kon de algemene anatomie van de stippels van A. marina geïnterpreteerd worden als meer resistent tegen droogte-geïnduceerde cavitatie. Ook de speciale groeistrategie kan hiertoe bijdragen. De vorming van de opeenvolgende xyleem-floëemlagen vertoonde geen jaarlijks karakter maar wel een sterke correlatie met de radiale diametergroei. Bovendien was de diktegroei zeer variabel zowel tussen als binnen bomen aan verschillende zijden van de stamomtrek. In contrast tot het aantal, daalde de breedte van de gevormde banden duidelijk met de bodemwatersaliniteit van de site.

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Als besluit kunnen we stellen dat de ecologische plasticiteit in vatendichtheid en dikte van de stippelmembranen kan gerelateerd worden aan een veilig hydrosysteem. De redundantie van het vatennetwerk en de resistentie van de stippelmembranen tegen cavitatie, respectievelijk, nam toe in parallel met stijgende saliniteit. De diameter van de vaten en de stippels ertussen lijken daarentegen een stabiele, optimale waarde aan te nemen. Het verband tussen de statische vatdiameter, de seizoenale veranderingen in de dikte van de stippelmembranen en de mogelijke afzettingen langsheen vatwanden en in stippelkanalen vraagt om verder onderzoek om de mogelijke rol van deze eigenschappen te onderzoeken in de regulatie van het watertransport in mangroven. Ook de gesegmenteerde groeistrategie van A. marina zou hierin een rol kunnen spelen. Optimale groei vindt enkel plaats op specifieke posities rondom de stam, die elkaar afwisselen in de tijd. Dit zou de waarneming kunnen verklaren van de simultane vorming van meerdere xyleem-floëem lagen, zelfs wanneer de buitenste laag al gelignificeerd is. Het kan een adaptatie zijn om de groei te optimaliseren onder gunstige omgevingsomstandigheden. Daarnaast geeft het de mogelijkheid een hydraulisch systeem te creëren bestaande uit segmentjes van verschillende anatomische opbouw. Op deze manier, zou het watertransportsysteem altijd aangepast zijn aan de omgeving, aan ten minste één zijde van de stamomtrek. De vorming van een nieuw cambium lijkt gecontroleerd te worden door een endogene factor zowel als door de saliniteit van het bodemwater dat de potentiële rol van floëemweefsel in het herstellen van embolismen ondersteunt. De uitdaging voor de toekomst ligt in het ophelderen van de functionele betekenis van successieve cambia, naast hun bijzonderheid als houtanatomische structuur.

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Acknowledgements

Enjoy the little things,for one day you may look backand discoverthey were the big things

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Acknowledgements

This study was inspired by the results of my Master‘s thesis, set up by Anouk Verheyden and came to a favourable conclusion via the scientific, technical and moral support of my promoters Nico Koe-dam, Hans Beeckman and James Gitundu Kairo; my supervisors on missions abroad: Steven Jansen of the Jodrell Laboratory (Royal Botanic Gardens Kew), Tanja Potsch, Gerald Koch and Uwe Schmitt of the Johann Heinrich von Thünen-Institut (vTI) (Institute for Wood Technology and Wood Biology, Hamburg); my field assistants in Kenya: Hamissi Ali Kirauni and Hassan Said Attuar; my colleagues at the Royal Museum of Central Africa, the Vrije Universiteit Brussel and at the Gazi field station and last but not least my parents, Veerle, Goele and Tom who could always cheer me up and put issues into perspective.

Acknowledgements

Thanks

Thank you, Thanks for looking after meThanks for having someone to look upto and admireThanks for giving me timeThanks for giving me spaceThanks for giving me adviceThanks for making me laughThanks for making me cryThanks for making me singThanks for making me sighThanks for giving me everything you canThank you

LUCY VERNEZZE

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Curriculum vitae

Use what talent you possess:the woods would be very silentif no birds sangexcept those that sang best.HENRY VAN DYKE

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Curriculum vitae

Curriculum vitae

Nele Schmitz°Leuven, 2nd of February 1982

Started a Master in Biology at the Vrije Universiteit Brussel in September 2000 and graduated summa cum laude on the 17th of September 2004. During her master thesis she got inspired by the research of mangrove wood. In September 2004 she successfully applied for a scholarship of the “The Institute for the Promotion of Innovation by Science and Technology in Flanders” (IWT), who was the major funding organisation of this PhD thesis.

Professional experiences & scholarships

PhD▪ Transitional benefit Vrije Universiteit Brussel (VUB): 1 Oct 2004 – 1 Jan 2005▪ Grant of The Institute for the Promotion of Innovation by Science and Technology in Flanders (IWT): 1 Jan 2005 – 31 Dec 2008.

Professional experiences abroad▪ Jodrell Laboratory, Royal Botanic Gardens Kew, UK: analysis of intervessel pits of mangrove wood with SEM and TEM (see publications). Funded by a Synthesys grant, 26 April – 24 May 2006.▪ Institute for Wood Biology and Wood Protection, Federal Research Centre for Forestry and Forest Products Hamburg, Germany: analysis of intervessel pits of mangrove wood with UMSP and TEM (see publications). Funded by a Short Term Scientific Mission within the COST E50 action, 16 April – 28 May 2007.

Field work expeditions▪ Kenya: May-June 2005, February 2006, June-July 2006, February-March 2007. Funded by the Research Foundation – Flanders (FWO), The Royal Netherlands Academy of Arts and Sciences (KNAW), the Flemish Interuniversity Council (VLIR).▪ Democratic Republic Congo: August-September 2007. Funded by the World Wide Fund (WWF).

MeetingsFunding of Vrije Universiteit Brussel (VUB), Research Foundation – Flanders (FWO), COST Action E50 to attend meetings in Belgium and abroad (see publications).

Publications

In peer reviewed international journals: 5As co-auteur in peer reviewed international journals: 3Non-peer reviewed scientific publications: 4Oral presentations at international scientific meetings: 6Posters presented at international scientific meetings: 7Supervised master theses: 2Reviews performed for international scientific journals: 7

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Peer reviewed papers

Beelaerts V, De Ridder F, Schmitz N, Bauwens M, Dehairs F, Schoukens J, Pintelon R, Submitted. On the elimination of bias averaging-errors in proxy records. Mathematical Geology.

Chowdhury MQ, Schmitz N, Verheyden A, Sass-Klaassen U, Koedam N, Beeckman H, accepted. Nature and periodicity of growth rings in two Bangladeshi mangrove species. IAWA Journal.

Schmitz N, Koch G, Schmitt U, Koedam N, Beeckman H, accepted. Intervessel pit structure and histochemistry of two mangrove species as revealed by cellular UV microspectrophotometry and electron microscopy: intraspecific variation and functional significance. Microscopy and Microanalysis.

Schmitz N, Robert EMR, Verheyden A, Kairo JG, Beeckman H, Koedam N, 2008. A patchy growth via successive and simultaneous cambia: key to success of the most widespread mangrove species Avicennia marina? Annals of Botany 101, 49-58.

Schmitz N, Verheyden A, Kairo JG, Beeckman H, Koedam N, 2007b. Successive cambia development in Avicennia marina (Forssk.) Vierh. is not climatically driven in the seasonal climate at Gazi Bay, Kenya. Dendrochronologia 25(2), 87-96.

Schmitz N, Jansen S, Verheyden A, Kairo JG, Beeckman H, Koedam N, 2007a. Comparative anatomy of intervessel pits in two mangrove species growing along a natural salinity gradient in Gazi Bay, Kenya. Annals of Botany 100, 271-281.

Schmitz N, Verheyden A, Beeckman H, Kairo JG, Koedam N, 2006. Influence of a salinity gradient on the vessel characters of the mangrove species Rhizophora mucronata Lam. Annals of Botany 98, 1321-1330.

Verheyden A, De Ridder F, Schmitz N, Beeckman H, Koedam N, 2005. High-resolution time series of vessel density in Kenyan mangrove trees reveal a link with climate. New Phytologist 167, 425-435.

Non-peer reviewed papers

Beeckman H, Boeren I, Couralet C, De Ridder M, Schmitz N, Tavernier W, Toirambe B, Verheyden A, 2007. De zwarte doos van Afrikaans hout. Het Afrodendro-project van het KMMA. Science connection 18, 10-14 (in Dutch and French).

Schmitz N, Verheyden A, De Ridder F, Beeckman H, Koedam N, 2006. Hydraulic architecture of the mangrove Rhizophora mucronata under different salinity and flooding conditions. Proceedings Tree Rings in Archaeology, Climatology and Ecology April 21-23 2005, Fribourg, Switzerland.

Schmitz N, 2004. Houtanatomie van de mangrove Rhizophora mucronata Lamk. en de relatie met de omgeving. De betekenis van de vatendichtheid voor de veiligheid van het watertransportsysteem. Biology Msc thesis, Vrije Universiteit Brussel.

Verheyden A, Schmitz N, De Ridder F, Beeckman H, Koedam N, 2004. Potential of high-resolution wood anatomy and stable isotope measurements for tropical dendrochronology: a case study on a Kenyan mangrove tree species. Tropical forests in a changing global context November 8-9 2004, Brussels, Belgium.

Oral presentations at scientific meetings

Beelaerts V, De Ridder F, Schmitz N, Bauwens M, Pintelon R, 2008. Identification of a harmonic signal in the presence of additive noise, an unknown time base distortion, and an averaging effect. International Instrumentation and Measurement Technology Conference May 12-15 2008, British Columbia, Canada.

Schmitz N, Verheyden A, Kairo JG, Beeckman H, Koedam N, 2007. Growth characteristics of the mangrove species Avicennia marina as revealed by the pinning technique. Workshop Quantitative Wood Anatomy May 29-31 2007, Birmensdorf, Switzerland.

Schmitz N, Verheyden A, Kairo JG, Beeckman H, Koedam N, 2006. Growth layer formation in Avicennia marina: the story beyond. Tree Rings in Archaeology, Climatology and Ecology April

Curriculum vitae

133

21-23 2006, Tervuren, Belgium.Schmitz N, Verheyden A, Beeckman H, Koedam N, 2005. Periodicity in phloem rings of Avicennia

marina? Workshop Intra-annual analysis of wood formation October 2-5 2005, San Vito di Cadore, Belluno, Italy.

Schmitz N, Verheyden A, De Ridder F, Beeckman H, Koedam N, 2005. Hydraulic architecture of Rhizophora mucronata under different salinity and flooding conditions. Tree Rings in Archaeology, Climatology and Ecology April 21-23 2005, Fribourg, Switzerland.

Schmitz N, Verheyden A, De Ridder F, Beeckman H, Koedam N, 2004. Wood anatomy of the mangrove Rhizophora mucronata Lamk. and the relationship with the environment: the significance of the vessel density for the safety of the water transport system. Young Botanist Day November 18 2004, Brussels, Belgium.

Posters presented at scientific meetings

Schmitz N, Jansen S, Koch G, Schmitt U, Koedam N, Beeckman H, 2008. Intervessel pit anatomy in two mangrove species and its ecological significance. EuroDendro Conference of the European Working Group for Dendrochronology May 28-June 1 2008, Hallstatt, Austria.

Schmitz N, Kairo JG, Beeckman H, Koedam N, 2008. Wound response in the mangrove species Avicennia marina. COST E-50 CEMARE Conference Wound reactions in trees and wood quality April 11-12 2008, Ljubljana, Slovenia.

Schmitz N, Verheyden A, Kairo JG, Beeckman H, Koedam N, 2007. Houtvorming via successieve cambia in de mangrove Avicennia marina. Starters in het Bosonderzoek 22 maart 2007, Brussels, Belgium.

Schmitz N, Verheyden A, Kairo JG, Beeckman H, Koedam N, 2006. What controls successive cambia formation in the mangrove species Avicennia marina (Forssk.) Vierh. in Kenya? COST E-50 CEMARE Conference October 19-22 2006, Warsaw, Poland.

Chowdhury MQ, Schmitz N, Verheyden A, Sass-Klaassen U, Koedam N, Beeckman H, 2006. Nature and periodicity of growth rings in Sonneratia apetala Buch.-Ham., a Bangladesh mangrove species. Tree Rings in Archaeology, Climatology and Ecology April 21-23 2006, Tervuren, Belgium.

Schmitz N, Verheyden A, De Ridder F, Beeckman H, Koedam N, 2004. Vessel density as a potential proxy for environmental parameters in the mangrove Rhizophora mucronata. NecoV Symposium Exploitation and restoration of tropical aquatic ecosystems October 7 2004, Brussels, Belgium.

Schmitz N, Verheyden A, De Ridder F, Beeckman H, Koedam N, 2004. Vessel density as a potential proxy for environmental parameters in the mangrove Rhizophora mucronata Lamk. Third International Postgraduate Symposium The Quaternary Research Association September 14-17 2004, Brussels, Belgium.

Supervised theses

Robert E, 2006-2007. Wood anatomy of Avicennia marina along an ecological gradient in Gazi Bay, Kenya. Biology Msc thesis, Vrije Universiteit Brussel.

Okello J, 2007-2008. Dendrochronological potential of six mangrove species in Gazi Bay, Kenya. Ecological Marine Management Msc thesis, Vrije Universiteit Brussel.

Reviews performed for scientific journals

Australian Journal of Botany, short communication, 26/12/2006Forest Ecology and Management, original article 17/08/2007Functional Plant Biology, research paper 15/10/2007New Phytologist, regular manuscript 3/12/2007Dendrochronologia, original article 21/12/2007Hydrobiologia, primary research paper 22/2/2008Plant Biology, Research paper 15/03/2008

Curriculum vitae

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