Reproductive strategies of Huperzia

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GOLA E.M. 2008. Reproductive strategies of Huperzia. � In: E. Szczê�niak, E. Gola (eds),Club mosses, horsetails and ferns in Poland � resources and protection. � Institute of PlantBiology, University of Wroc³aw, Wroc³aw, p. 5�14.

Reproductive strategies of Huperzia

EDYTA M. GOLA

Institute of Plant Biology, University of Wroc³aw, Kanonia 6/8,PL-50-328 Wroc³aw, Poland; e-mail: [email protected]

ABSTRACT: The genus Huperzia has the ability to reproduce vegetatively due to thepresence of special vegetative propagules called bulbils. This ability makes a relativelyquick propagation and dispersal of a plant possible and it is particularly importantfor the species having long-lasting life-cycles such as Huperzia. The generative andvegetative reproduction occurs alternately during the vegetative season in Huperziaspecies. Interestingly, the populations differ in their strategies of balancing thegenerative and vegetative reproduction with the extensive bulbil production in themountain locations in opposition to the average amount of propagules produced bythe lowland plants. In this paper, different anatomical aspects of the vegetative andgenerative reproduction as well as developmental changes in the bulbil structureassociated with the process of a bulbil detachment are briefly given and discussed.

ABSTRAKT: Przedstawiciele rodzaju Huperzia posiadaj¹ zdolno�æ do rozmna¿aniawegetatywnego dziêki obecno�ci specyficznych rozmnó¿ek. Zdolno�æ ta umo¿liwiaro�linom wzglêdnie szybkie rozmna¿anie i rozprzestrzenianie, co jest szczególniekorzystn¹ cech¹ dla ro�lin o d³ugim cyklu ¿yciowym, takich jak np. Huperzia.Rozmna¿anie generatywne i wegetatywne wystêpuje u tego gatunku naprzemienniew trakcie sezonu wegetacyjnego. W zale¿no�ci od warunków �rodowiskowychpopulacje Huperzia przyjmuj¹ ró¿ne strategie rozmna¿ania: w populacjach górskichintensywnie produkowane s¹ rozmnó¿ki wegetatywne w przeciwieñstwie do populacjini¿owych, w których liczba inicjowanych rozmnó¿ek jest stosunkowo niewielka.W komunikacie przedstawione i przedyskutowane s¹ wybrane anatomiczne aspektyrozmna¿ania wegetatywnego i generatywnego oraz zmiany rozwojowe w budowierozmnó¿ek, zwi¹zane z procesem ich od³¹czania i rozprzestrzeniania.

KEY WORDS: bulbil, bulbil detachment, Huperzia, sporangium development, sporegermination, vegetative propagule

Introduction

Periodic formation of the specific vegetative propagules at the shoot apex,which are called bulbils or gemmae, is a unique feature of some species ofHuperzia (Øllgaard 1987). Bulbils are fully pre-formed plantlets, which are

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able to develop rapidly just after detachment from the mother plant. They playan important role in a plant dispersal and propagation, since the duration ofthe entire life-cycle in Huperzia can take several years in natural conditions(Reutter 1987; Whittier 1998). Previous experimental approaches to inducespore germination and gametophyte development of different representativesof the Lycopodiaceae s. l. in axenic culture succeeded only in part (Whittier1998), suggesting that the delay in spore germination and the slow pace ofgametophyte and young sporophyte growth may be under the genetic and/orphysiological control. Considering the Huperzia ability to produce bulbils, itbecomes interesting to analyze the reproductive strategies, which may reflecta plant adaptation to specific environmental conditions. The obvious advan-tage of the gene exchange during the generative reproduction seems to becounteracted by the extremely extended life-cycle in Huperzia. On the otherhand, the vegetative propagation is a quick process, and resultant clonal plantletshave the genotype that has already been environmentally tested. In order toestablish the strategies of reproduction in representatives of the genus Huperzia,some aspects of the H. lucidula and H. selago anatomy and developmentwere studied and presented in this communication.

1. Materials and methods

The Huperzia lucidula (Michx.) Trevisan plants came from the commer-cial supplier, the Carolina Biological Supply, NC, USA. Shoots of H. selago(L.) Schrank et Mart. were collected in Finland near Lammi, in the KarkonoskiNational Park, and in the Wigierski National Park. For the spore culture, sporesof H. lucidula were pre-soaked overnight in distilled water or in mixture of100 µM IAA and 50 µM kinetin, sterilized in 70% ethanol and 5% sodiumhypochlorite, and then, the spore-coat was digested with 1% (v/w) cellulaseand 0.5% (v/v) pectinase. Spores were sown on the plates containing a stan-dard Murashige-Skoog medium (Gamborg 1991) of half-strength with 1% sucrose,solidified with 0.8% agar.

To analyze the anatomical structure of developing sporangia and bulbils,shoots of H. lucidula were collected. Plant material fixed in the formyl-ace-tic acid-alcohol mixture (FAA) was proceeded according to the standard pro-tocols for paraffin sectioning (O�Brien, McCully 1981; Ruzin 1999). Anatomicalsections were observed and documented in the bright field microscope, andsome properties of the cell walls were additionally analyzed in the fluorescentmicroscope (Olympus Optical Co., Poland).

Bulbils produced by a specimen in the last season and, based on the stalkremnants, in the previous years were counted, and their total amount wascalculated per one functioning apex.


2. Results and Discussion

Development of sporangia is similar in both species studied. They are ini-tiated separately in the microphyll axils in May/June. In the central part ofa growing primordium, the mass of sporogenic tissue is formed, which multi-plies by mitotic divisions (Fig. 1A). By the end of September, the sporogenic cellscease divisions and prior to meiosis become the spore mother cells (Fig. 1B).

Fig. 1. Sporangium and gametophyte development in Huperzia lucidula. A. In the youngsporangium (sg) the sporogenic tissue (spt) is formed, which upon maturation (B) becomesthe spore mother cells (smc) and undergoes meiosis, giving rise to the tetrads of spores(sp; C). The internal layer of the sporangium wall forms a tapetum (t), which providesthe nutrition for developing spores (sp). D. Germinating spore � an arrow points the siteof the fracture in the spore-coat releasing the spore. E. Young gametophytes (g) clusteredtogether. F. Top view of the mature gametophyte with archegonia (marked with asterisks;phot. E. Gola)

Ryc. 1. Rozwój zarodni i gametofitów u Huperzia lucidula. A. Powstaj¹ca zarodnia (sg)z widoczn¹ tkank¹ sporogenn¹ (spt). B. Stadium komórek macierzystych (smc), które pomejozie tworz¹ tetrady spor (sp; C). Wewnêtrzna warstwa �ciany zarodni wykszta³ca siêjako tapetum (t), od¿ywiaj¹ce rozwijaj¹ce siê zarodniki (sp). D. Kie³kuj¹cy zarodnik �miejsce nadtrawienia �ciany zarodnika i uwolnienia spory wskazane jest strza³k¹. E. M³odegametofity. F. Dojrza³y gametofit (widok z góry) z kana³ami rodni (zaznaczonymi gwiazdkami;fot. E. Gola)

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Further spore development is continued in the next season. The tetrads of sporesformed by the meiotic divisions are visible in sporangia in following April.Correspondingly to maturation of the sporogenic tissue, the inner layer of thesporangium wall forms a tapetum, which plays a role in nutrition of the devel-oping spores (Figs 1A�C). The outer, epidermal layer becomes thick-walled,protecting the sporangium and facilitating its dehiscence (Fig. 1C). By this time,spores are ripe (Fig. 1C) but sporangia do not dehisce until the end of follow-ing July. Thus, the complete development of spores and sporangia finishedby their dehiscence is accomplished in two seasons. Such a developmentalsequence was characteristic of both studied species, Huperzia lucidula andH. selago, and corresponded well with the data on H. selago (Case 1943).

Spores of H. lucidula, in natural conditions, germinate approximately af-ter three to five years, in axenic culture this process may be hastened, althoughit still takes two or more years to germinate (Whittier, Webster 1986; Whittier1998). The employed in this study spore pre-treatment resulted in induction ofthe spore germination and emergence of gametophytes already four monthsafter sowing. The recent studies on angiosperms showed that the seed dor-mancy and germination depends on the levels of the endogenous abscisic acid(ABA) and gibberellins (GAs; Grappin et al. 2000; Gubler et al. 2005). Likely,the spore dormancy may be controlled in the similar way. Thus, the appliedspore pre-soaking in the hormone solution could possibly change the hormonebalance in a dormant spore triggering its germination, while the enzymatic digestionof the spore-coat facilitated the spore release (Fig. 1D). Subsequent divisionsof the germinated spore resulted in formation of a gametophyte that initiallywas composed of few cells (Fig. 1E). The growth of young gametophytes wasaccomplished by two months, when gametangia developed on the upper sur-face of a gametophyte (Fig. 1F). Further stages of the Huperzia development,i.e., fertilization and embryo formation, were not observed in the course ofthis study. Despite the results presented in this communication are preliminaryand the gametophyte development was induced only in about 1% of all plates,it may be supposed that the spore experimental pre-treatment with differenthormone solutions may break the spore dormancy increasing the amount ofgrowing gametophytes in culture. Further studies are, however, necessary toestablish the optimal experimental conditions for the Huperzia spore germi-nation and the growth of gametophytes.

The vegetative propagules are periodically produced at the apex of H. lucidulaand H. selago in a microphyll leaf position (Fig. 2A). The basal part of a develop-ing bulbil forms a short stalk at which the bulbil proper is placed (Figs 2B�C).The stalk and its microphyllous leaves form an apparatus involved in a bulbildetachment and dispersal (Smith 1920; Stevenson 1976). Specifically, a bigsupportive microphyll on the abaxial side of the stalk helps in throwing out the


bulbil proper at some distance (Smith 1920; Figs 2B�C). The bulbil detach-ment from the mother plant is not a random process. Smith (1920) suggestedthat the cells in the narrow region between the bulbil proper and the stalkunderwent a process of gelatinization prior to detachment. Recent experimentswith immunolabeling of the pectin epitopes confirmed ontogenetic modifica-tions of the cell wall properties and suggested that the unspecialized abscis-sion zone was formed during the bulbil development (Figs 2D�E; Gola, Jernstedt2003). Axially elongated cells, which occur in the region between the bulbilproper and the stalk, upon maturation of the abscission zone start dividingperpendicularly to the bulbil long axis (Fig. 2E). Since the walls of these cellsafter division are located at the similar level the unspecialized abscission layeremerges. The cell wall composition and metabolism change in cells belongingto this layer, making possible to hydrolyze their middle lamellas what in turnfacilitates the separation of the neighbouring cells (Gola, Jernstedt 2003). Suchanatomical and physiological changes, including additional cell divisions andloosening of the cell wall structure, are well recognized in the abscission zonesof several seed plants (Sexton, Roberts 1982) and the features of the detach-ment process in Huperzia comply well with them. The surface of the stalk,exposed after bulbil detachment (Fig. 2C), undergoes additional changes de-veloping the protective layer. First, the callose is rapidly synthesized just afterbulbil detachment, blocking plasmodesmata and restricting symplasmic trans-port in the subsuperficial cells of the stalk (Fig. 2F). The rapid callose synthe-sis and deposition is a well-known response to wounding or pathogenic infec-tion, regulating the transport through plasmodesmata (Aloni et al. 1990; Radfordet al. 1998; Roberts 2005) and it seems to be a part of the mechanismprotecting the exposed stalk surface after bulbil detachment in Huperzia. Theformation of the protective layer accomplishes when the cell walls becomeimpregnated with the lignin and/or suberin, visualized by autofluorescence(Fig. 2F), additionally defending the stalk surface against the pathogens(Fig. 2F; Poovaiah 1974; Sexton, Roberts 1982). Taken together anatomical,developmental and physiological properties of the region between the bulbilproper and the stalk it may be concluded that the morphologically unspecializedbut functionally fully effective abscission zone develops in Huperzia. The for-mation of the abscission zone in lycophytes appears to be an evolutionary novelty,which recruited and specified, temporally as well as spatially, the already knownin pteridophytes mechanism of the hydrolysis of the cell wall components togain the new quality � precisely controlled detachment of a plant organ.

The comparison of the H. selago specimens coming from different popu-lations showed differences in the number of bulbils produced and in distribu-tion of sporangia vs. bulbils on the shoot (Figs 3A�B; Tab. 1).

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Fig. 3. Huperzia selago shoots coming from the lowland (A) and the mountain (B�C)populations. The difference in the number of bulbils produced is distinct. The overlappinggenerative and vegetative zones are particularly well-seen in the C (phot. E. Gola)

Ryc. 3. Porównanie pêdów Huperzia selago pochodz¹cych z populacji ni¿owych (A) orazgórskiej (B�C). Zaznacza siê ró¿nica w liczbie tworzonych rozmnó¿ek oraz wystêpowanieobok siebie na pêdzie strefy zarodniono�nej i strefy rozmna¿ania wegetatywnego u ro�linpochodz¹cych z górskiej populacji (C; fot. E. Gola)


Tab. 1. The number of bulbils produced in lowland vs. mountain populationsTab. 1. Porównanie badanych populacji pod wzglêdem liczby inicjowanych rozmnó¿ek

KNP � Karkonoski National Park; WNP � Wigierski National Park; segment* � thefunctional unit of the dichotomizing shoot formed by the activity of a single apex. As theindependent segments the twin axes after dichotomy as well as a shoot fragment beforebifurcation are counted.

KNP � Karkonoski Park Narodowy; WNP � Wigierski Park Narodowy; segment* � jednostkafunkcjonalna dychotomizuj¹cego pêdu, tworzona przez aktywno�æ pojedynczego wierzcho³ka.Niezale¿nymi segmentami s¹ osie potomne po dychotomii oraz fragment pêdu przedrozga³êzieniem.

No. of bulbils per an apex (Liczba rozmnó¿ek na pêdzie)

Species/population (Gatunek/populacja)

No. of segments* examined

(Liczba zbadanych segmentów) mean (�rednia) min.–max.

H. lucidula 395 4.1 0–23

H. selago, KNP 56 8.7 2–23

H. selago, Finland 49 0.2 0–3

H. selago, WNP 33 0 0

Fig. 2. The bulbil structure. A. Side-view of the H. lucidula shoot presenting distributionof bulbils (bl) which are exclusive to sporangia (sg). B�C. Close-ups of the bulbil proper(bl) and its stalk (st) with a distinctive supportive leaf (smi) at the abaxial side of the stalk.In the picture C, the exposed surface of the stalk (st) after bulbil detachment is visible(marked with asterisks). D�E. The longitudinal section of the narrow region between thebulbil proper (bl) and the stalk (st) where the detachment process takes place. The arrowsindicate the recent cell divisions resulting in formation of the unspecialized abscission zone.F. The exposed surface of the stalk (st) protected by lignin impregnating the cell walls.Yellow autofluorescence of lignin is distinct in superficial cells (phot. E. Gola)

Ryc. 2. Struktura rozmnó¿ek wegetatywnych. A. Pêd Huperzia lucidula widziany z boku,prezentuj¹cy rozmieszczenie zarodni (sg) i rozmnó¿ek (bl) wzd³u¿ osi. B�C. Strukturarozmnó¿ki: rozmnó¿ka w³a�ciwa (bl) osadzona jest na krótkim trzonku (st), który postronie odosiowej posiada wyra�ny li�æ wspieraj¹cy (smi), wchodz¹cy w sk³ad aparatuwyrzucaj¹cego rozmnó¿kê. Powierzchnia trzonka, eksponowana po od³¹czeniu rozmnó¿ki,zaznaczona jest gwiazdk¹ na zdjêciu C. D�E. Przekrój pod³u¿ny przez obszar przewê¿eniamiêdzy rozmnó¿k¹ w³a�ciw¹ (bl) a trzonkiem (st), w którym nastêpuje od³¹czanie rozmnó¿ki.�ciany komórek powsta³ych w wyniku podzia³ów formatywnych, tworz¹cych warstwêodcinaj¹c¹, wskazane s¹ strza³kami. F. Powierzchnia trzonka po od³¹czeniu rozmnó¿kichroniona przez wysycenie �cian komórkowych lignin¹ (wyra�na jasno¿ó³ta autofluorescencjaligniny; (fot. E. Gola)

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Plants growing in the lowlands produced a few or even no bulbils at thespecific apex during a season. Moreover, the relatively distinct vegetative andgenerative zones altered regularly along the axis (Fig. 3A). Oppositely, themountain population was characterized by the presence of numerous vegeta-tive propagules (Figs 3B�C). Also, the vegetative/generative zones in H. selagoplants from the mountain location overlapped along the shoot axis forming themosaic pattern. Nevertheless, the presence of a sporangium in the microphyllaxil excluded initiation of a bulbil in the same position and vice versa (Fig. 3C).These findings suggest that the frequency of the bulbil occurrence may pos-sibly be controlled by the environmental factors, such as e.g., temperature,altitude, insolation, etc. Likely, these factors may switch the genes that areresponsible for the transition between vegetative and generative developmentalphases, resulting in production either of sporangia or vegetative propagules atthe apex. Recently, it has been shown that the changes in the temporal andspatial expression of the gene LAMB1 are associated with the sporangial tis-sue development in Lycopodium annotinum (Svensson et al. 2000). It can-not be ruled out that the similar control, differing however in the spatial pat-tern of the homologous gene expression, exists in Huperzia. It would be in-teresting to test if plants originally coming from the lowland population andre-planted in the mountains start generating numerous vegetative propagules,or if this ability to increase the bulbil production is independent of the epigenomicinfluences.

References

ALONI R., BAUM S.F., PETERSON C.A. 1990. The role of cytokinin in sieve tuberegeneration and callose production in wounded Coleus internodes. � PlantPhysiology 93: 982�989.

CASE I.M. 1943. Periodicity in the development of fertile and sterile zones inLycopodium selago. � New Phytologist 42: 93�96.

GAMBORG O.L. 1991. Media preparation. � In: Plant tissue culture manual A1:1�24. � Kluwer Academic Publishers, the Netherlands.

GOLA E.M., JERNSTEDT J.A. 2003. Detachment of Huperzia propagules: Letting goand leaving home. � In: Botany 2003. Aquatic and Wetland Plants: Wet & Wild,July 26�31, 2003, Mobile, AL, USA.

GRAPPIN P., BOUINOT D., SOTTA B., MIGINIAC E., JULLIEN M. 2000. Control of seeddormancy in Nicotiana plumbaginifolia: post-imbibition abscisic acid synthesisimposes dormancy maintenance. � Planta 210: 279�285.

GUBLER F., MILLAR A.A., JACOBSEN J.V. 2005. Dormancy release, ABA and pre-harvest sprouting. � Current Opinion in Plant Biology 8: 183�187.


O�BRIEN T.P., MCCULLY M.E. 1981. The study of plant structure principles andselected methods. � Termarcarphi Pty. Ltd., Melbourne, Australia.

ØLLGA ARD B. 1987. A revised classification of the Lycopodiaceae s. lat. � OperaBotanica 92: 153�178.

POOVAIAH B.W. 1974. Formation of callose and lignin during leaf abscission. �American Journal of Botany 61: 829�834.

RADFORD J.E., VESK M., OVERALL R.L. 1998. Callose deposition at plasmodesmata.� Protoplasma 201: 30�37.

REUTTER U. 1987. Growth patterns of gemmlings of Lycopodium lucidulum. �American Fern Journal 77(2): 50�57.

ROBERTS A.G. 2005. Plasmodesmatal structure and development. � In: OPARKA K.J.(ed.), Plasmodesmata. � Blackwell Publishing Ltd., p. 1�32.

RUZIN S.E. 1999. Plant microtechnique and microscopy. � Oxford UniversityPress, New York, Oxford.

SEXTON R., ROBERTS J.A. 1982. Cell biology of abscission. � Annual Review ofPlant Physiology 33: 133�162.

SMITH R.W. 1920. Bulbils of Lycopodium lucidulum. � Botanical Gazette 69: 426�437.

STEVENSON D.W. 1976. Observations on phyllotaxis, stelar morphology, the shootapex and gemmae of Lycopodium lucidulum Michaux (Lycopodiaceae). �Botanical Journal of the Linnean Society 72: 81�100.

SVENSSON M.E., JOHANNESSON H., ENGSTRÖM P. 2000. The LAMB1 gene from theclubmoss, Lycopodium annotinum, is a divergent MADS-box gene, expressedspecifically in sporogenic structures. � Gene 253: 31�43.

WHITTIER D.P. 1998. Germination of spores of the Lycopodiaceae in axenic culture.� American Fern Journal 88(3): 106�113.

WHITTIER D.P., WEBSTER T.R. 1986. Gametophytes of Lycopodium lucidulum fromaxenic culture. � American Fern Journal 76: 48�55.

Strategie rozmna¿ania Huperzia

U badanych gatunków Huperzia lucidula i H. selago zarodnie inicjowanes¹ pojedynczo w pachwinach mikrofili na prze³omie maja i czerwca. Rozwójtkanki sporogennej i zarodni obserwowany u obu badanych gatunków zgodnyjest z opisem Case (1943) dla H. selago. Kie³kowanie zarodników trwa od3 do 5 lat w warunkach naturalnych i tylko nieznacznie mo¿e byæ skróconew hodowli in vitro (Whittier, Webster 1986; Whittier 1998). W prowadzonychbadaniach uzyskano dojrza³e gametofity ju¿ po sze�ciu miesi¹cach dziêkizastosowaniu kombinacji hormonów do zniesienia spoczynku spor i przyspieszeniaich kie³kowania. Wydaje siê, ¿e spoczynek zarodników mo¿e byæ kontrolowanyprzez poziom endogennych hormonów, podobnie jak ma to miejsce w nasionachro�lin okrytozal¹¿kowych (Grappin i in. 2000; Gubler i in. 2005). Traktowanie

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zarodników roztworami hormonów mog³o wiêc zmieniæ równowagê hormonaln¹skutkuj¹c przyspieszeniem procesu kie³kowania. Okre�lenie optymalnychwarunków hodowli spor i gametofitów H. lucidula wymaga jednak dalszychbadañ.

D³ugi cykl rozwoju generatywnego Huperzia jest kompensowany przeztworzenie rozmnó¿ek wegetatywnych. W³a�ciwa rozmnó¿ka osadzona jest nakrótkim trzonku, który wraz ze zmodyfikowanymi li�æmi tworzy specjalny aparatwyrzucaj¹cy (Smith 1920; Stevenson 1976). Od³¹czanie rozmnó¿ek mo¿liwejest dziêki wykszta³ceniu specyficznej strefy odcinania w przewê¿eniu pomiêdzyw³a�ciw¹ rozmnó¿k¹ i trzonkiem (Gola, Jernstedt 2003). Strefa odcinania tworzysiê u Huperzia przez ukierunkowane podzia³y komórek w miejscu przewê¿enia.W tym obszarze zmienia siê te¿ stopieñ metylestryfikacji pektyn i sk³ad �cianykomórkowej, najprawdopodobniej w wyniku hydrolizy jej komponentów. �cianykomórek na powierzchni trzonka ulegaj¹ silnej lignifikacji i tworz¹ pok³adzabezpieczaj¹cy. Zmiany anatomiczne i fizjologiczne prowadz¹ce do powstanianiewyspecjalizowanej strefy odcinania u Huperzia przypominaj¹ proces abscyzjiu ro�lin nasiennych (Sexton, Roberts 1982). Modyfikacje sk³adników �cianykomórkowej, znane jak dot¹d z procesu otwierania sporangiów u paprotników,u Huperzia po raz pierwszy w ewolucji s¹ wykorzystane do kontrolowanegood³¹czania organów.

Porównanie populacji H. selago pochodz¹cych z obszarów górskich i ni¿owychwykaza³o ró¿nice w liczbie tworzonych rozmnó¿ek. Wydaje siê, ¿e czynniki�rodowiskowe mog¹ zmieniaæ ekspresjê genów decyduj¹cych o to¿samo�ciprodukowanych struktur: rozmnó¿ek lub sporangiów.

Reproductive strategies of Huperzia

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