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Life History and Skeletal Adaptations in the Galapagos Marine Iguana(Amblyrhynchus cristatus) as Reconstructed with Bone Histological Data—AComparative Study of IguaninesAuthor(s): Jasmina Hugi and Marcelo R. Sánchez-VillagraSource: Journal of Herpetology, 46(3):312-324. 2012.Published By: The Society for the Study of Amphibians and ReptilesDOI: http://dx.doi.org/10.1670/11-071URL: http://www.bioone.org/doi/full/10.1670/11-071

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Journal of Herpetology, Vol. 46, No. 3, 312–324, 2012Copyright 2012 Society for the Study of Amphibians and Reptiles

Life History and Skeletal Adaptations in the Galapagos Marine Iguana (Amblyrhynchuscristatus) as Reconstructed with Bone Histological Data—A Comparative Study of Iguanines

JASMINA HUGI1

AND MARCELO R. SANCHEZ-VILLAGRA

Palaontologisches Institut und Museum, Universitat Zurich, Karl-Schmid-Strabe 4, 8006 Zurich, Switzerland

ABSTRACT.—The skeletal adaptations of Amblyrhynchus cristatus (Marine Iguanas) are of particular interest based on their amphibiouslifestyle, which is unique among living lacertilian squamates. The well-known ecological data are applied to new bone histological findings,

which revealed expected and unexpected congruencies. The cortical bone matrix consists of avascular lamellar-zonal bone tissue type. The

geometrical disposition of the growth marks (i.e., their spacing) shows an unusual pattern for lizards: the growth cycles maintain a constant

thickness until the growth is terminated, which is marked by the development of the external fundamental system (efs). Minor resorptionprocesses within the inner periosteal cortical region and the occurrence of these thick growth cycles in A. cristatus result in high mean bone

compactness values. The reported life-history data from ecological studies and the hypothesized annuality of the growth cycles indicate that this

first decline in annual bone deposition rate is not congruent with the attainment of sexual maturity. In contrast, this event might be indicated byother histological changes in the growth record of A. cristatus, which they share exclusively with their sister group, Conolophus subcristatus(Land Iguana). The bone matrix of the growth zones and annuli differ in their thickness, their color in polarized light, and vary slightly in the

amount and shape of osteocyte lacunae in both A. cristatus and C. subcristatus. These well-recognizable growth zones and annuli of the growth

cycles change their thickness abruptly within the reported time frame of the attainment of sexual maturity in A. cristatus.

Amblyrhynchus cristatus Bell, 1825 (Reptilia: Iguanidae) aremedium-sized lizards (200–340 mm, adult snout–vent length)distributed on the islands of the Galapagos Archipelago(Boersma, 1983). They show a unique lifestyle among all lizardsby foraging exclusively in the cold sea (Trillmich and Trillmich,1986; Laurie and Brown, 1990). Their habitat and specializeddiet have led to many studies on their physiology with focus onthe thermoregulatory behavior (Bartholomew, 1966; Bennett etal., 1975; Bartholomew et al., 1976; Boersma, 1983), as well as ontheir ecology (Trillmich and Trillmich, 1984; Laurie and Brown,1990; Wikelski and Hau, 1995; Wikelski and Trillmich, 1997).The climate of the Galapagos Archipelago is characterized bytwo seasons, a warm and wet season that lasts from January toJune and a dry and cold season from July to December(Colinvaux, 1972). The seasons are regular except for thereturning El Nino rainfalls in every fourth or fifth year(Colinvaux, 1972). These two distinct seasons favor or decreasethe growth of macrophytic algae, the main food supply of A.cristatus (Laurie and Brown, 1990; Wikelski et al., 1993, 1997),and as a result of this high dependence, reproduction in theMarine Iguana starts during the cold and dry season (Trillmichand Trillmich, 1984).

Is the unique lifestyle of A. cristatus coupled with changes inthe bone microstructure compared to terrestrial iguanidrelatives? Little information exists on the bone histology of A.cristatus, and available data concern only bone compactness ofthe humerus, radius, or the tibia (Germain and Laurin, 2005;Kriloff et al., 2008; Canoville and Laurin, 2010; Houssaye et al.,2010). These studies show that A. cristatus generally have highercompactness values in these limb bones but not in theirvertebrae in comparison to terrestrial squamate relatives. Theterm pachyostosis sensu lato (i.e., hyperplasy of the cortexwhich results in a swollen appearance from external view;Buffrenil and Rage, 1993) has not been observed in any bone ofA. cristatus (Houssaye, 2009).

The long bones of vertebrates are generally informativestructures to study life histories using histological data (e.g.,Castanet et al., 1993). These data can provide information on

rate and duration of growth (Francillon-Vieillot et al., 1990),whereas counting of growth marks (skeletochronology) allowsestimation of the age at sexual maturity and the longevity of anindividual (Castanet et al., 1993). The bone tissue types and thedegree of remodeling processes result in varying compactnessvalues that can be, for example, evaluated with the programBone Profiler (Girondot and Laurin, 2003). An annuallydeposited growth cycle is composed of a growth zone, anannulus, which is occasionally but not always associated with aline of arrested growth (lag) (Castanet et al., 1993). Theannuality of growth units have been supported by studies onrecent reptiles of known age (Castanet and Naulleau, 1985;Castanet et al. 1993; Erickson et al., 2001). Although lags aremost obvious in taxa living in high latitudes/altitudes (areaswith marked seasonality), their occurrence has also beenreported in the bones of taxa living in aseasonal environments(Patnaik and Behera, 1981; Castanet and Gasc, 1986). Castanet etal. (1993) proposed that lags arise as a result of an endogenousbiological rhythm that may be synchronized by external factorssuch as food availability. Such external and internal factors areoften preserved in the histological record by remodelingprocesses (e.g., for the production of eggs), a change in bonetissue type, as well as a decrease in the spacing between the lags(e.g., sexual maturity, termination of growth). The terminationof growth is expressed histologically by the development of anexternal fundamental system (efs after Horner et al., 2001; orOCL: outer circumferential layer after Chinsamy-Turan, 2005).

The aim of this study is to assess the well-known life historyof A. cristatus, in comparison to its terrestrial iguanid relativesusing rare data on the microstructure of limb bones and tocompare them with known life-history data. The questions weaddress here are: (1) whether bone histological data correspondto data on the plasticity of the reproduction cycle, longevity, aswell as the age of sexual maturity (Tables 1, 2), and (2) whetherthe bone histology of A. cristatus is different in comparison to itsterrestrial relatives based on its derived lifestyle. This study (1)describes the bone histology of A. cristatus and (2) applies thewell-known ecological data on the new bone histological datafrom A. cristatus.

1Corresponding Author. E-mail: jasmina.hugi@gmail.comDOI: 10.1670/11-071

TABLE 1. General information about the studied iguanine lizards regarding their home range, the climate they live in, the age of reaching sexualmaturity, the longevity, the adult snout–tail length. Abbreviations: S: summer; sm-t.: age at sexual maturity; svl: snout–vent length; W: winter.

General information

Species Species range Climate Seasons

Amblyrhynchus cristatus Galapagos Archipelago "S’’: warm, rainy; ‘‘W’’: cold,dry

warm season (January–June), dry season(July–December)

Brachylophus fasciatus Fiji Islands S: hot, humid; W: warm, dry S: November–February; W: April–SeptemberConolophus subcristatus Galapagos Archipelago "S’’: warm, rainy; ‘‘W’’: cold,

drywarm season (January–June), dry season

(July–December)Ctenosaura similis Mexico to Panama S: rainy; W: dry S: May–October, W: November–AprilCyclura cornuta Hispaniola (Dominican

Republic)S: hot, rainy; W: warm, dry S: May–October, W: November–April

Dipsosaurus dorsalis SW U.S./NW Mexico S and W: rainfall; arid, hot arid/hot, two rainfall seasonsS: July–September; W: October–March

(hibernation)Iguana iguana Mexico to South

America; West IndiesS: rainfall; W: dry S: May–October, W: November–April

Sauromalus obesus SW U.S./NW Mexico S and W: rainfall; arid andhot

arid/hot, two rainfall seasonsS: July–September; W: October–March,

hibernation

Species sm-t/longevity* SVL [in mm] References

Amblyrhynchus cristatus female: 3–5, male: 6–8/ca. up to 20–30 years

200–340 Etheridge, 1982; Laurie, 1990; Wikelski et al.,1993

Brachylophus fasciatus 2.5 years (ca. 16months)/ca. up to atleast 20 years

185–220 Etheridge, 1982; Iverson, 1982; Gibbons,1984

Conolophus subcristatus female: 7–10, male: 11–16/ca. up to 20–30years

380–490 Etheridge, 1982; Werner, 1983; Christian etal., 1984

Ctenosaura similis 2 years/ca. up to 13years

200–500 Fitch and Henderson, 1977; Etheridge, 1982;Iverson, 1982

Cyclura cornuta female: 12 years, male:? years, in captivity:female: 6–7 years,male: 4–5 years/ca.at least up to 20–30years

-750 Etheridge, 1982; Iverson, 1982; Perez-Buitrago et al., 2008

Dipsosaurus dorsalis 2 years (up to 30months)/ca. up to 14years

100–122 Moberly, 1963; Etheridge, 1982; Iverson,1982; Mautz and Nagy, 1987

Iguana iguana 18–24 months/ca. up to20–30 years

390–450 Etheridge, 1982; Iverson, 1982; Troyer, 1984;Zug and Rand, 1987

Sauromalus obesus females: 2 years, males:2–3 years/ca. up to14 years

175–200 Nagy, 1973; Etheridge, 1982; Abts, 1987

Sampled animals

Species Collection number Sex Lived in . . .

A. c. venustissimus NKMB-30260 Adult male Natural habitat, SLV: 390 mm. Weight: 2570g, male, Espanola (former Hood Island).Capture date: 10 January 1973

A. c. cristatus ZFMK-uncataloged Adult male Natural habitat, FernandinaA. c. cristatus ZFMK-uncataloged Adult female Natural habitat, FernandinaA. c. cristatus ZFMK-uncataloged Subadult male Natural habitat, FernandinaB. fasciatus SMF-72758 Adult male Natural habitatC. subcristatus CAS-10475 Adult male Natural habitat, SLV: 480 mm, male, South

Seymour Islands. Capture date: 21November 1905 by J. R. Slevin

C. similis SMF-52071 Adult male Natural habitatC. cornuta ZFMK-5223 Adult male Natural habitatD. dorsalis SMF-71284 Subadult female Kept in captivityI. iguana NMB-5743 Adult male Kept in captivityS. obesus NMB-13801 Adult female Kept in captivity

BONE HISTOLOGY OF THE GALAPAGOS MARINE IGUANA 313

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314 J. HUGI AND M. R. SANCHEZ-VILLAGRA

MATERIALS AND METHODS

The stylo- and zeugopodial bones of the forelimbs (humerus,radius, ulna) and hind limbs (femur, tibia, fibula) of four A.cristatus from two different islands (Amblyrhynchus cristatusvenustissimus Eibl-Eibesfeldt, 1962: NKMB-30260, Espanola;Amblyrhynchus cristatus cristatus Bell, 1825: ZFMK-uncatalogedFernandina, Table 1), as well as specimens of seven terrestrialiguanines, including Conolophus subcristatus (Galapagos LandIguana), were investigated (Fig. 1; Table 1). The museumspecimens used in this study were critical because these lizardsin some cases hold endangered status. Further development ofimaging techniques, which enable the study of skeletochrono-logical data without being invasive, may be used in the future tofurther test the observations we discuss here with ecologicaldata.

Males were preferred as samples because the long bones ofthe females usually serve as calcium storage during reproduc-tion destroying the inner growth record (Buffrenil et al., 2010).Mid-shaft diaphyseal thin sections were prepared and docu-mented following standard petrographic preparation tech-niques (c.f. Scheyer and Sanchez-Villagra, 2007). Bonecompactness profiles and values were quantified (Table 2) usingthe program Bone Profiler 3.20 (Girondot and Laurin, 2003). Thebone histological terminology follows Francillon-Vieillot et al.(1990). Student’s paired t-tests were performed to report theprobability (P) of the following null hypotheses: (1) there is nosignificant difference in the mean compactness values of thelimb bones between the female and the male A. cristatus; (2)there are no significant differences in the mean compactnessvalues of the limb bones between A. cristatus and the otheriguanid taxa; (3) there is no significant difference in the meancompactness vales of the forelimbs and hind limbs in A.cristatus; (4) there are no significant differences in the meancompactness values of the forelimbs and hind limbs in theterrestrial iguanid taxa. A rejection of the second, third, andfourth null hypothesis was expected based on previous studyresults on bone compactness values (Germain and Laurin, 2005;Kriloff et al., 2008; Canoville and Laurin, 2010; Houssaye et al.,2010). The histology was assessed qualitatively (Table 3).Several long bones which are pictured appear blurred, whichis a result of the great amount of lipid deposits in the cortex.Lipid deposits could not be completely removed chemicallyalthough the long bones were treated for almost half a year withmethylene chloride.

RESULTS

Bone Compactness, Histology, and Skeletochronology of TerrestrialIguanines.—All terrestrial iguanines share several bone histolog-ical features in their limb bones. They all show avascularlamellar-zonal bone tissue type (Figs. 2–4). The mean compact-ness values of the forelimbs are significantly higher thanequivalent means of the hind limbs (paired t-test: t = 2.02, P <0.05). Erosion cavities scattered in the cortex are rarely preservedin any terrestrial iguanine, contrary to the frequent resorptionprocesses in the inner cortical region that surrounds themedullary cavity. Larger terrestrial iguanine species (large size:Cyclura cornuta, Conolophus subcristatus, Iguana iguana, Brachylo-phus fasciatus, Ctenosaura similis, Figs. 3, 6; Table 1) show a higherloss of the growth record of the inner cortical region by resorptionprocesses than smaller-bodied species (Dipsosaurus dorsalis,Sauromalus obesus; Table 1; Fig. 2). Therefore, bone samples of

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BONE HISTOLOGY OF THE GALAPAGOS MARINE IGUANA 315

iguanines of smaller adult size often still display parts of theembryonic bone as innermost cortical growth cycle (Fig. 2).However, all terrestrial iguanines show a thin layer of endosteallamellar bone that is deposited around the medullary cavity(Figs. 2, 3A,C–F, 6C–F).

All terrestrial iguanines were sexually mature. The age of theattainment of sexual maturity is marked by the first steep andabrupt decline in growth cycle thickness. The subsequentgrowth cycles instead slightly increase their thickness for oneor two growth cycles again before continuously decreasing theirthicknesses annually (Figs. 2, 3C–F). Therefore, the lagsconverge continuously before they cannot be distinguishedany more from each other in the outermost cortical region (i.e.,efs). Iguanines of smaller-bodied adult size show a shorter timeperiod between the attainment of sexual maturity and thedevelopment of the efs as measured by the counting of thegrowth cycles (Fig. 2). The majority of growth is reached beforesexual maturity in small iguanines. In iguanines of larger adultbody size, in contrast, sexual maturity occurs well beforegrowth is terminated (Fig. 3).

The growth zones and annuli are placed randomly within thegrowth cycles in all terrestrial iguanines except for C.subcristatus, the sister group of A. cristatus. Conolophus sub-cristatus shows alternating and regular distribution of thegrowth zones and annuli within the growth cycles.

In the limb bone samples of C. subcristatus a maximum of 13growth cycles are preserved (Fig. 6C–D). The first abruptdecrease in growth cycle thickness, which indicates sexualmaturity, is still preserved although large parts of the innerperiosteal growth record are destroyed by resorption (Fig. 6C–D). The growth cycles of C. subcristatus also increase againslightly in thickness after the attainment of sexual maturity, butcontrary to the data of other terrestrial iguanines, the growthcycle thicknesses then remain rather constant, but of smallerthickness than before the abrupt decline, until the efs isdeveloped (Fig. 6C–D). Therefore, C. subcristatus shows amixture between the growth patterns of A. cristatus and theother terrestrial iguanines. In addition, the growth zones andannuli show a similar color pattern in polarized light like A.cristatus. The collagenous fibers of each growth zone are all

FIG. 1. Phylogeny of the studied iguanines and summary of results. Left column shows the phylogeny of the studied iguanines, which is modifiedfrom Wiens and Hollingsworth (2000). The results are summarized on the right column of the figure.

TABLE 3. Specific skeletochronological and histological information for the studied Marine Iguanas. The number of the lags varies only in the femurand the tibia, which show higher resorption of the inner wall of the cortex. Abbreviations: an: annulus, efs: external fundamental system, gz: growthzone, sm-t: growth cycle when sexual maturation is reached. *: Fernandina specimens are generally larger than Espanola specimens. **: this individualis morphologically regarded as a subadult because of smaller size compared to the adults from Fernandina.

Specimen Sex

Length

humerus [mm]

Preserved growth

cycle number

Age at change

of light pattern Development of efs

NKMB 30260 Adult male 63.2 17 sixth year 13. to 20. growth cycleZFMK (uncataloged) Adult male* 75.0 10 sixth year not yet an efsZFMK (uncataloged) Adult female* 62.9 13 third year 7. to 9. growth cycleZFMK (uncataloged) Subadult male** 64.5 9 seventh year not yet an efs

316 J. HUGI AND M. R. SANCHEZ-VILLAGRA

oriented equally throughout the growth record until the efs isdeveloped. The collagenous fibers of the annuli are all equallyoriented but in another direction than the ones of the growthzones, which results in a constant pattern of alternating bands oftwo colors. This specific color pattern is maintained until the efsis developed (Fig. 6D,F). At sexual maturity, the growth zonesand annuli equally decrease their thickness within the corre-sponding growth cycle. The growth zones and annuli are ofequal thickness within the growth cycles after sexual maturity isreached and remain similarly distributed until the efs isdeveloped (Fig. 6E–F).

Bone Compactness and Histology of Amblyrhynchus cristatus.—Alllong bones of the limbs show high compactness values, except forthe femur and tibia (Table 2; Fig. 1). Therefore, the meancompactness values of the forelimbs of A. cristatus weresignificantly higher than the equivalent data of the hind limbs(paired t-test: t = 2.1, P < 0.05). The mean compactness values ofall limb bones of A. cristatus were significantly higher than theequivalent data of the terrestrial iguanines (paired t-test: t =1.998, P < 0.05). The humerus, radius, ulna, and fibula of A.cristatus show the highest cortico-diaphyseal index or CDI(thickness of the cortex of the bone divided by the radius of thebone, Castanet et al., 2000) with even less resorption of the innerregion of the cortex than the femur and tibia. In addition, smallcircular layers of endosteally deposited lamellar bone arepreserved surrounding the medullary cavity (Figs. 4C,D, 5B,D).The increase of bone compactness values of all limb bones isreached by a continuous and regular accretion of primary,centrifugally deposited, periosteal bone along the outer corticalregion combined with minor remodeling processes of the innercortical region. The medullary cavity is free of endosteal bonedeposits in the single female but not in all the males. The humeriof the two male specimens from Fernandina show trabeculae,which are not only preserved in the medullary cavity of theepiphyseal and metaphyseal region but also in the diaphysis (Fig.5G,H). In all the males, the occurrence of resorption processes isrestricted to the inner cortical region, whereas one limb bone ofthe female, the tibia, also shows various erosion cavities scatteredin the cortex (ZFMK-uncataloged female) (Fig. 5A,B). However,this difference did not affect the mean compactness values of thelimb bones of the female specimen compared to equivalent dataof the male A. cristatus (paired t-test: t = 2.2, P < 0.05).

The cortices of the limb bones are of avascular lamellar-zonal

bone tissue type (Figs. 4, 5). The limb bones, which show least

remodeling processes (i.e., humerus, radius, ulna, and fibula),

show one innermost periosteal bone layer of a matrix of less

organized woven-fibered bone grading into parallel-fibered bone

(wp; Figs. 4C,D, 5D) that is identified as embryonic bone

(Francillon-Vieillot et al., 1990). The wp layer is bordered by a lag

that represents the hatching line (Fig. 5D; Pilorge and Castanet,

1981; Nouira et al., 1982). The bone matrix of the embryonic bone

differs from the other periosteal growth cycles in being opaque or

monofringent in polarized light based on the high amount of

unorganized collagenous fibers. This innermost periosteal layer

further displays a high number of roundish osteocyte lacunae

(Figs. 4G–H, 5E–F). The osteocyte lacunae of the other,

subsequent growth cycles, in contrast, are scattered and roundish

to flattened and the bone matrix is birefringent in polarized light.

The growth cycles are of constant thickness up to the

outermost cortical region where the distribution of lags abruptly

becomes closely spaced (efs; Figs. 4C–H, 5D–H; Table 3). All

growth cycles, besides the innermost wp layer and the

outermost ones that belong to the efs, are divided clearly into

a growth zone and an annulus. The growth zone and annuli

vary in the shape of the osteocyte lacunae as well as in the color

of the bone matrix visible in polarized light (Glimcher and Muir,

1984). Each growth cycle consists of these two well-separated

subunits that are visible as bands of two alternating colors in the

cortex in polarized light in all males and the one female. The

thicknesses of the two subunits distinctly change one time

during the ontogeny: at a constant time frame in both the males

(sixth to seventh year, Table 3) and in the female (third year,

Table 3). Before this change, the growth zones are thick, whereas

the annuli are thin (Figs. 4F,H, 5F). This distinct change is

accompanied further by the higher presence of supernumerary

lags. The change in the characteristics of the color pattern and

the presence of supernumerary lags occur during the sixth and

seventh growth cycle in the male specimens (Figs. 4C–H, 5G–H;

Table 3) and during the third to fourth year in the female

specimen (Fig. 5D–F; Table 3). The peculiarity of this event is

less developed in the sampled female: growth zones are thicker

than the annuli before this event but of almost equal thickness

after it (Fig. 4E–H).

FIG. 2. Microstructure of the long bones of Dipsosaurus dorsalis. Diaphyseal transverse sections of the humerus of an adult Desert Iguana (SMF71284) as an example for the spacing pattern of small-bodied lizards and random color pattern within the growth cycles in terrestrial iguanines, withConolophus subcristatus as an exception. Image A and B in normal transmitted light. Image C in polarized light with Lambda compensator. In allimages A, B, and C, the specimen shows five lags (arrow heads) and, therefore, was at its sixth year at death. Image A shows the steep decrease in thethickness at sexual maturity (number 2). The subsequent growth cycles increase in thickness after this event before decreasing again until the efs isdeveloped. Abbreviations: hl: hatching line; el: endosteally deposited lamellar bone that surrounds the medullary cavity; wp: woven-fibered bonegrading into parallel-fibered bone.

BONE HISTOLOGY OF THE GALAPAGOS MARINE IGUANA 317

DISCUSSION

The limb bones of A. cristatus differ in mean bone

compactness values, as well as in the growth pattern of the

cortex (i.e., constant growth cycle thicknesses up to the efs, well-

separated growth zones and annuli up to the efs) in comparison

with terrestrial iguanid relatives. We discuss the bone histology

in the light of ecological findings for A. cristatus that report

prolonged longevity, delayed sexual maturity, and climate-

dependent reproductive seasons.

Are Thick Cortices a Result of Foraging Strategies?—The mean

compactness values of the limb bones of A. cristatus were

significantly higher than the equivalent data for terrestrial

iguanid relatives and other lizards (Kriloff et al., 2008; Canoville

and Laurin, 2010; this study). All iguanines have forelimbs with

higher compactness values than the hind limbs, but the deviation

was more distinct in A. cristatus, which also showed higher mean

compactness values (Table 2). Therefore, the limb bones of A.

cristatus might be used for buoyancy control while exclusively

feeding on intertidal and subtidal macrophytic algae (e.g.,

Trillmich and Trillmich, 1986). Heavy bones act as ballast

allowing a hydrostatic control of body trim in water (Hoffstetter,

1955; Taylor, 2000). Increase in bone compactness, especially in

the forelimbs, counteracts lung buoyancy and, therefore, facili-

tates diving and long-lasting underwater stays (Kaiser, 1966;

Ricqles and Buffrenil, 2001). Moreover, the increase in body

density would counteract the action of waves and improve

stability in rough water (Ricqles and Buffrenil, 2001). The limb

bone sample of the female A. cristatus was not significantly

FIG. 3. Microstructure of the long bones of Cyclura cornuta. Diaphyseal transverse section of the tibia (image A to F) of an adult male RhinocerosIguana (ZFMK 5223) as an example of the spacing pattern in terrestrial iguanines, with Conolophus subcristatus as an exception. The growth cyclescontinuously decrease in thickness after the onset of sexual maturity (grey arrow head nr. 3*), which is marked by a steep decrease in the growth cyclethickness. The growth cycle thicknesses continuously decrease until the efs is developed (grey arrow heads in the outermost region of the cortex, nr. 7to 8). Arrows mark supernumerary lags. Images A, C, E in normal transmitted light, whereas image B, D, F in polarized light. Image C and D show aminimum number of eight lags. The more detailed view of images E and F do not reveal a distinct color pattern at least not after sexual maturity isreached. Growth cycles are randomly composed of lighter and darker colored bone layers in polarized light. Abbreviation: res.: resorption processes inthe inner cortical region. For additional abbreviations see Figure 2.

318 J. HUGI AND M. R. SANCHEZ-VILLAGRA

FIG. 4. Microstructure of the long bones of Amblyrhynchus cristatus (I). Diaphyseal transverse sections of the radius of the adult male A. cristatusfrom Espanola (NKMB 30260). Images A, C, E, G in normal transmitted light, whereas images B, D, F, H in polarized light. Images C and D show thecomplete growth record with up to 17 growth cycles (numbered arrow heads). The growth cycles only decrease in thickness after the age of 10 yearswhen the efs is developed (grey arrow heads in the outermost region of the cortex). Arrows mark supernumerary lags. Images E to H aremagnifications of the cortical region where the thicknesses of the growth zones (gz) and annuli (an) change. Abbreviations: efs: external fundamentalsystem; f. ost.: flattened osteocyte lacunae; r. ost.: round osteocyte lacunae; sm-t: bone histological transition of sexual maturity. For additionalabbreviations, see Figure 2.

BONE HISTOLOGY OF THE GALAPAGOS MARINE IGUANA 319

FIG. 5. Microstructure of the long bones of Amblyrhynchus cristatus (II). Images A and B show the diaphyseal transverse sections of the tibia of theadult female A. cristatus from Fernandina (ZFMK-uncataloged) in normal transmitted light. The tibia shows high resorption processes of the innercortical region (res.), as well as secondary erosion cavities (e.c) in the cortical region. Images C, D, E (normal transmitted light), and F (polarized lightwith Lambda compensator) show the diaphyseal transverse section of the humerus of the female A. cristatus. The sampled female was in its 10th yearof life with nine preserved lags (arrow heads). Images E and F show a magnification of the area at which the characteristics of the growth zoneschange. Image G show the exact transverse sections of the mid-shaft of the humerus of the adult male A. cristatus from Fernandina (ZFMK-uncataloged), whereas image H shows the subadult male specimen from the same island. Both males exhibit trabeculae (p. trab.) within the medullarycavity. For abbreviations see Figure 2–4.

320 J. HUGI AND M. R. SANCHEZ-VILLAGRA

different from the bone compactness values of the males,although the female specimen shows erosion cavities in thecortex of the tibia, which are not present in any male specimen.The exclusive presence of erosion cavities in the tibia of thefemale reflects other data of reabsorption of calcium duringreproduction in lizards (e.g., Varanus niloticus, see Buffrenil andFrancillon-Vieillot, 2001).

The high mean compactness values of the limb bones of thefemale A. cristatus was not expected, because females feedexclusively in the intertidal zones (Bartholomew, 1966; Trillmichand Trillmich, 1986) where diving is not necessary. Therefore,limb bones of higher compactness values might also be usefulfor counterbalancing high muscular forces that are developedfor providing a strong hold to the shore while feeding inintertidal zones. Another explanation could be that a shortage offood forces A. cristatus use different feeding grounds (juvenilesand adult females in intertidal zones, adult males in subtidalzones; e.g., Bartholomew, 1966) and that the high compactnessvalues are an exaptation in females. The bone compactnessvalues of A. cristatus, therefore, show no deviation within adults

of different body size (females vs. males). The variouspopulations of A. cristatus on different islands differ amongeach other in body size. Our sample contains animals from twodifferent islands for which this size dimorphism is reported: A.cristatus from Fernandina are generally larger in body size thanthe ones from Espanola (Wikelski and Trillmich, 1997; Eibl-Eibesfeldt, 2001; Wikelski, 2005), but they show the same meancompactness values as the males of smaller adult size fromEspanola. The males from Fernandina further show trabeculaein the mid-shaft region of the medullary cavity of exclusive thehumerus (Fig. 5G,H).

Constant Thickness of the Growth Cycles up to efs.—Thethicknesses of the growth cycles of A. cristatus remain constantuntil the efs is developed (Fig. 7B). This annual bone growthperiod, which produces constant growth cycle thicknesses, is anunusual pattern for lizards and has, at least to our knowledge,not been reported in any other lizard in such a regular mannerfrom all bones of several specimens. One lizard, with a similarbut less extreme distribution of growth cycles of equalthicknesses, is the Nile monitor, Varanus niloticus. However, the

FIG. 6. Microstructure of the long bones of Conolophus subcristatus. Diaphyseal transverse sections of the fibula of an adult male Galapagos LandIguana (CAS 10475). Images A, C, and D in normal transmitted light, image B, D, and F in polarized light. Images C and D show the region with themost complete growth record, where 13 growth cycles are preserved (arrow heads). Although the growth cycles decrease in thickness at the sm-t (greyarrow head numbered with *5), they are of small but rather equal thickness until the efs is developed (grey arrow heads in the outermost region of thecortex). The higher magnification of this transition is shown in images E and F. For abbreviations see Figures 2–4.

BONE HISTOLOGY OF THE GALAPAGOS MARINE IGUANA 321

occurrence of this pattern varies (fig. 4 in Buffrenil and Castanet,2000). The combination of such a regular annual growthdeposition until the efs is developed and minor resorption ofthe inner cortical bone material produces these high compactnessvalues in the limb bones of A. cristatus.

Wikelski and Thom (2000) examined the growth of A. cristatusduring El Nino rainfalls and reported that A. cristatus ‘‘shrink’’(reduction in their body length) during these re-occurringrainfalls, most probably attributable to energetic stress and lowfood availability. An abrupt decrease in the growth cyclethickness is only preserved when the efs is developed. In thosespecimens for which data are available (Table 1), no severe ElNino rainfall was documented during their life spans.

Bone Histological Indicators for Embryonic Bone and SexualMaturity.—The bone matrix of the innermost periosteal growthcycle of the limb bones with the most complete growth record(humerus, radius, ulna, fibula) is composed of woven-fiberedbone grading into parallel-fibered bone. This bone tissue,classically considered to result from a high bone deposition rate,is characterized by its monorefringent reaction to polarized lightand a high density of osteocyte lacunae with numerous canaliculioriginating in these lacunae (Francillon-Vieillot et al., 1990; Figs.4C–D, 5D).

The attainment of sexual maturity of A. cristatus is notindicated by an abrupt decrease in the growth cycle thickness asin other lizards (e.g., Castanet, 1985; Castanet et al., 1993; Fig.7A) but might instead be indicated by a distinct change in thecolor pattern of the cortex visible in polarized light (Figs. 4C–H;5D–F). Each growth cycle of the cortex of the A. cristatus iscomposed of a well-separated growth zone and an annulus. Thegrowth zones and annuli differ slightly from each other in thebone tissue based on different bone deposition rates duringfavored and nonfavored seasons (Castanet et al., 1993). In A.cristatus, the thickness of these two subunits, but not amountand shape of the osteocyte lacunae and the color in polarizedlight, change once in the growth record within a similar timeframe in the males but earlier in the only female (Table 3). Theages that can be inferred from counting the annually depositedgrowth cycles coincides with the reported age when A. cristatusreach sexual maturity (Trillmich and Trillmich, 1984). Wehypothesize that the change of this color pattern indicates theattainment of sexual maturity in A. cristatus.

Neither is the development of the efs taken as indicator thatsexual maturity is reached; instead, closely aligned lags of theefs mark the termination of the growth of an animal (Horner etal., 2001). The first steep decrease in growth rate is equivalentto the reported ages when sexual maturity is reached (e.g., Zugand Rand, 1987; Castanet et al., 1988; Castanet and Baez, 1991).It is well known that small-bodied lizards show shorter termlife histories compared to larger relatives, although inter- andintraspecific variation occurs (Andrews, 1982; Shine andCharnov, 1992). The variation between the size differencesbetween small and large taxa is mainly a result of thedifferences in the maintenance of growth after sexual maturityis reached: large taxa reach sexual maturity well before thetermination of growth, whereas small-bodied lizards areskeletally mature shortly after this event (Andrews, 1982;Shine and Charnov, 1992; e.g., Cyclura cornuta vs. Dipsosaurusdorsalis: this study). There is no exception to this rule for thedata from A. cristatus. This consistency would support theecological evidence that A. cristatus reaches sexual maturitywell before the attainment of full adult size as in other middle-sized or large iguanines, which is the plesiomorphic reptiliancondition (Erickson et al., 2007).

The change in the thickness of the growth zones and theannuli could be a result of the beginning of mating behavior,which is sex dependent (Trillmich and Trillmich, 1984). Themating cycles of A. cristatus are annual and dependent on twodistinct seasons (Christian and Tracy, 1982; Werner, 1982).Conolophus subcristatus, which shows a similar color pattern,exhibits the same conserved reproductive cycle as the otherterrestrial iguanines (e.g., Wiewandt, 1982; Table 1), startingthe mating season at the beginning of the warm and wetseason. The reproductive season of A. cristatus, in contrast,starts in the cold and dry season, which is introduced by theoccurrence of the cold and nutrient-rich Humboldt currentswhich, in turn, strongly influence the reproduction and growthof the algae. Therefore, A. cristatus serves as example ofplasticity in the reproductive cycle of lizards, being dependentmainly on the food availability. Bone histological data appearto be congruent with this observation. First, the histologicalchange in the color pattern of the growth cycles occurs duringthe time when females and males start to reproduce (Tables 1,3). In A. cristatus, this hypothesis is supported by slightdifferences of the color pattern between male and femalespecimens as well as by the timing when the pattern changes.

FIG. 7. The spatial distribution of the lags and the color pattern interrestrial iguanines in comparison to Amblyrhynchus cristatus with itspossible link to the reproduction cycle. (A) The general growth patternof terrestrial iguanines. (1) The deposition of bone (lzb) in the outercortical region and the resorption processes of the inner cortical region(res.) are more or less ‘‘balanced,’’ (2) one innermost periosteal bonelayer of woven-fibered bone grading into parallel-fibered bone (wp) isdeposited during the early ontogeny, (3) continuous decrease of growthcycle thicknesses before the efs and an abrupt decrease in growth cyclethickness when sexual maturity is reached (sm-t). (B) General growthpattern of A. cristatus. (1) The deposition of bone (lzb) in the outercortical region and the resorption processes of the inner cortical region(res.) show an ‘‘imbalance,’’ (2) one innermost periosteal bone layer ofwoven-fibered bone grading into parallel-fibered bone (wp) is depositedduring the early ontogeny, (3) growth cycle thicknesses remain constantuntil the efs is developed and no or only a slight decrease in thicknessoccurs when sexual maturity is reached (sm-t?).

322 J. HUGI AND M. R. SANCHEZ-VILLAGRA

Ecological studies of A. cristatus report sexual maturity at threeto five years in females and six to eight years for males (Laurie,1990), which corresponds to the growth cycle number at whichthe first change is observed (Figs. 4, 5C–F; Table 3).

Second, there is a slight histological variation between malesand females (Figs. 4, 5C–F). In females, the thicknesses of thegrowth zones and the annuli are more or less equal after sexualmaturity is reached, whereas they show other thicknesses atsexual maturity (one subunit becoming thinner and the otherone becoming thicker), which is maintained until the efs isdeveloped. This could be explained as follows: A. cristatus,which are not yet sexually mature, spend all their time baskingand feeding on algae. After they reach sexual maturity, thepremating and mating period takes most of the dry season orannulus season (Trillmich and Trillmich, 1984). Males do notfeed or feed little during the period of premating and mating,because they start to be highly territorial, showing malecompetition to monopolize access to females as soon as theyarrive at mating sites (Trillmich and Trillmich, 1984). In contrast,females continue to forage during the premating and matingperiod and only stop feeding when aggregating to the nestingground and while defending their nests for several weeks to amonth. The relatively long period of fasting of the malescompared to females could be reflected in the more drasticchange in the characteristics of the growth zone and annulus atand after sexual maturity is reached. Male C. subcristatus, incontrast, are not territorial and, therefore, still feed, althoughless, during the reproductive season (Werner, 1982, 1983), whichcould explain the less drastic change in the characteristics of thegrowth zones and annuli at sexual maturity compared to maleA. cristatus. Another explanation could be the relatively shorterreproduction cycle relative to that of A. cristatus.

The life history of A. cristatus reconstructed with bonehistology appears to be strikingly congruent with observationsprovided by ecological studies (Table 1, Fig. 7). Bone histologicalfeatures appear to coincide with the ecological data on theplasticity of the reproduction cycle, longevity, as well as the agewhen sexual maturity is reached. Specifically, single life stagesof A. cristatus appear to be reflected in the growth record of thelimb bones by the presence of wp (embryonic bone), the changein the thickness of alternating colored bands (sexual maturity),as well as the development of the efs (termination of growth). Incontrast to A. cristatus, life-history data based on bone histologyfor other iguanines is often more or less obscured as a result ofhigher remodeling processes, which often destroy the innermostgrowth record of the cortex (Fig. 3). Only the long bones of thesmall-sized iguanines, Dipsosaurus dorsalis (Fig. 2) and Sauroma-lus obesus (Table 1) reveal the complete growth record in thecortices by showing minor remodeling processes that only affectparts of the inner cortical region. These data on skeletochronol-ogy are congruent with previously published demographic andecological data (Table 1).

Acknowledgments.—We would like to thank T. Scheyer, J.Neenan, U. Koller, and B. Hausler for their invaluable help.Samples were kindly made available for the study by A. E.Leviton (San Francisco), G. Kohler (Frankfurt), R. Winkler (Basel),W. Bohme (Bonn), F. Zachos and A. Honnen (formerly Kiel). Weare thankful to N. Klein and A. Houssaye and several anonymousreviewers for providing very useful suggestions on earlierversions of the manuscript. The Swiss National Science Founda-tion supported this project (grant 31003A-133032/1 to MRS-V).

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Accepted: 12 October 2011.

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