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Breeding Chronology, Sexual Dimorphism, and Genetic Diversity ofCongeneric Ambystomatid Salamanders

ROD N. WILLIAMS,1 DAVID GOPURENKO, KEVIN R. KEMP, BROOKE WILLIAMS, AND

J. ANDREW DEWOODY

Department of Forestry and Natural Resources, Purdue University, West Lafayette, Indiana 47907 USA

ABSTRACT.—Many amphibians are explosive breeders, but the key factors that influence migrations to (and

from) breeding sites are not well understood for all species. We used a comparative approach to compare and

contrast the complex relationships among breeding chronology, environmental conditions, sexual

dimorphism/body size, sex ratio, and genetic variation in Small-mouthed (Ambystoma texanum) and Eastern

Tiger Salamanders (Ambystoma tigrinum tigrinum). We intercepted 171 A. texanum and 330 adult A. t.

tigrinum on their annual migration to breeding ponds over two consecutive breeding seasons (2003–04). Both

species immigrated over a short period of time (4–17 days) and displayed no clear pattern regarding whether

males or females typically arrived first at breeding ponds. Sex ratios were skewed toward males in both

species but varied between years. Consistent with their expected sexual selection regimes, intraspecific

sexual dimorphism was subtle in A. texanum but pronounced in A. t. tigrinum. There was no size-ordered

immigration or emigration for either species examined; migration events were triggered by temperature and

precipitation. We genotyped four hypervariable microsatellite loci and found no gross difference in the

overall level of genetic variation between species but document that our study populations are genetically

diverse (mean of .18 alleles per locus and heterozygosities .0.75 in each species), presumably as a result of

historically large effective population sizes.

In a diverse array of animal taxa, males andfemales arrive at breeding areas according todifferent schedules (Morbey and Ydenberg,2001). The timing of arrival can vary bothintrasexually, when variation occurs becauseof individual quality (Hardy and Raymond,1980; Briggler et al., 2004), and intersexually,when there is a reproductive advantage (Lichtand Bogart, 1990; Krenz and Sever, 1995).Understanding the factors affecting variationin animal movements during breeding events isa crucial component to understanding theselective pressures driving the evolution ofbreeding strategies. Some research on aggregatebreeding amphibians has emphasized the im-portance of temperature and precipitation, butlittle attention has been directed toward adultbody size and how that corresponds to thetiming of arrival to the breeding ponds (Sem-litsch, 1983; Sexton et al., 1990; Palis, 1997).Fewer studies have focused on the inherentvariation (i.e., sexual dimorphisms, sex ratios,etc.) found in salamander breeding aggrega-tions and how this variation may influencepopulation demography, particularly geneticvariation. Herein, we capitalize on the differ-ences in breeding biology of Small-mouthedSalamanders (Ambystoma texanum) and EasternTiger Salamanders (Ambystoma tigrinum tigri-

num) to compare breeding chronology, mor-phology, sex ratios, and genetic diversity.

Ambystomatid salamanders are typicallyspring breeders with migrations to breedingsites occurring at night during rainy weather.Breeding is usually confined to one to threebrief bouts during or immediately followingrainfall events (Petranka, 1998). Males canarrive anywhere from two days to severalweeks ahead of females at breeding sites wherethey may outnumber females. Ambystomatidsalamanders exhibit diverse breeding behaviorsand strategies that present abundant opportu-nities for comparative study.

Courtship and breeding strategies vary in A.texanum (Wyman 1971; Trauth, 2005), but mostevidence suggests that, upon arrival at thewetland, adults gather in groups and engagein mass breeding with little courtship; afterencountering and nudging a potential mate, amale soon moves away and makes limitedcontact with the female (Garton, 1972; Petranka,1982; Licht and Bogart, 1990; McWilliams, 1992).Furthermore, A. texanum is one of only fourspecies of Ambystoma that lacks the classic‘‘cloacal nudging walk’’ where females nudgethe male’s cloaca, likely receiving chemical cuesfrom him (Houck and Arnold, 2003). Notsurprisingly, female mate choice is not apparentin A. texanum (Gopurenko et al., 2007). Directmale-male competition is also limited, but malesfrequently participate in sexual interference via1 Corresponding Author. E-mail: [email protected]

Journal of Herpetology, Vol. 43, No. 3, pp. 438–449, 2009Copyright 2009 Society for the Study of Amphibians and Reptiles

sperm capping (Garton, 1972; Licht and Bogart,1990). While at breeding ponds, the reported sexratios of A. texanum adults range from an even1:1 (Plummer, 1977; Parmelee, 1993) to moremale biased ratios of 2.4:1 (Gopurenko et al.,2007).

The Tiger Salamander (A. tigrinum) is themost widely distributed salamander in NorthAmerica (Petranka, 1998), and, as such, indi-viduals found across this expansive geographicdistribution vary considerably with respect tocolor pattern, behavior, habitat, and life-historypatterns (Lannoo and Phillips, 2005). In EasternTiger Salamanders (A. t. tigrinum), males com-pete for access to females who then choosewhether to mate with a particular male (Kumpf,1934). Unlike A. texanum, A. t. tigrinum femalesemploy the ‘‘cloacal nudging walk.’’ When afemale nudges a male’s cloaca, he leads theprospective mate forward, keeping physicalcontact with her by tapping his tail on herdorsum while she follows and continues push-ing on his cloaca with her snout (Arnold, 1976;Houck and Arnold, 2003). Upon spermataphoredeposition by the male, a female quicklydecides whether to induct (Kumpf, 1934).Breeding males compete in several ways,including overt pushing/shoving, sperm cap-ping, and female mimicry (Arnold, 1976; Houckand Arnold, 2003). Males typically outnumberfemales at breeding ponds with reported sexratios of 1:1, 3.2:1, and 5.3:1 in Indiana (Peckhamand Dineen, 1954; Sever and Dineen, 1978) and2:1 in South Carolina (Semlitsch, 1983).

These differences in breeding behaviors be-tween A. texanum and A. t. tigrinum lead toseveral general predictions regarding morphol-ogy, migratory behavior, and genetics. Forexample, the ardent male-male competitionand subsequent female mate choice in A. t.tigrinum would suggest that intraspecific sexualdimorphisms should be more pronounced thanin A. texanum. Additionally, reproductive skewleads to a reduction in effective population size,which can impact genetic variation (Sugg andChesser, 1994; Hedrick, 2005). Thus, one mightexpect that the strongly male-biased sex ratiosin A. t. tigrinum could lead to reduced levels ofgenetic variation relative to the near-lotterysystem of A. texanum.

Our data address these and other predictionsin a natural history framework that considersthe substantial influence of environmentalconditions. For each species, the specific goalsof the study were to (1) document sex ratios andsexual dimorphisms among breeding popula-tions of A. texanum and A. t. tigrinum; (2)investigate the relative importance of body sizeand environmental factors on breeding migra-tions; and (3) compare levels of microsatellite

diversity among species to investigate thepotential genetic effects of differential sexualselection between species.

MATERIALS AND METHODS

Study Areas and Breeding Season.—Breeding A.texanum and A. t. tigrinum populations weremonitored at two ephemeral wetlands in Tip-pecanoe County, Indiana, during 2003 and 2004.The South River Road (SRR) field site is anisolated ephemeral wetland approximately 2 hain size located along the floodplains of theWabash River. Water depths are maintained atapproximately 40 cm in years with averagelevels of precipitation; however, the wetland isgenerally dry by June. Ambystoma tigrinumtigrinum have been captured at the SRR wet-land, but they are much less common than A.texanum.

The Purdue Wildlife Area (PWA) consists of apermanent wetland complex extending overabout 100 ha. A small ephemeral wetlandlocated within this complex (‘‘the swale’’) waschosen based on its location and manageablesize of approximately 0.8 ha. The swale main-tains a water depth of about 85 cm during mostof the spring and early summer months and isusually dry by late July. Both A. texanum and A.t. tigrinum are present at PWA; however, A. t.tigrinum are far more numerous at this site. ThePWA field site is located approximately 12 kmnorthwest of the SRR field site and is not part ofthe contemporary Wabash River floodplain.

Sample Collection/Analyses.—Approximately850 and 400 m of aluminum flashing drift fencewere used to encircle the SRR and PWAwetlands, respectively. Drift fences were placed1–5 m from the edge of each breeding pool.Pitfall traps (15 cm in diameter 3 17 cm deep)were buried on both the inside and the outsideof the enclosure fences at 10-m intervals toallow for the capture of individuals immigrat-ing to or emigrating from the breeding wet-lands. In 2003, all traps were opened andchecked twice daily (morning and evening)from 9 February through 15 June at both fieldsites. At the conclusion of the 2003 breedingseason, the SRR enclosure was dismantled, andno further trapping occurred. Trapping at thePWA field site continued into 2004, where trapswere checked twice daily from 15 Februarythrough 17 June.

Upon initial capture, each individual sala-mander was uniquely toe clipped for individualidentification (Twitty, 1966) and to providetissue for subsequent DNA extraction andgenetic analyses. In addition, we recorded thedates of immigration and emigration, traplocation, sex, mass, total body length, tail

AMBYSTOMATID BREEDING BIOLOGY 439

height, and snout–vent length before placing theindividual on the opposite side of the fence.Snout–vent lengths were measured from theanterior end of the snout to the anterior end ofthe cloacal opening. Captured salamanderswere sexed by presence or absence of cloacalswellings, where cloacal swelling indicated abreeding male. All measurements were to thenearest 0.5 mm as measured with a Vernierhand caliper and mass to the nearest 0.5 g witha Pesola hand-held spring scale. Precipitationand air temperatures were recorded fromnearby weather stations.

We evaluated the influence of morphologyand weather cues on immigration and emigra-tion dates using multiple linear regression (SASInstitute, Cary, NC, 2003). The body sizecharacteristics used in the model buildingprocedure of SAS were total body length (TL),snout–vent length (SVL), tail length (TAL), tailheight (TH), and mass (M). The weathervariables used in regression analyses weremaximum daily temperature (uC) and dailyprecipitation (mm). Correlations among vari-ables were evaluated using Spearman’s rankcorrelation. Total body length, tail length, andtail height were often correlated; thus, weremoved the effects of body length on each tailmeasure by obtaining residual values andregressing each variable against body length(as in Howard et al., 1997). We also used acondition index as a measure of body conditionby obtaining residuals after regressing the cuberoot of mass against body length (as in Baker,1992). Chi-square tests were used to determinewhether overall sex ratios differed from parity.We evaluated differences in male-female bodymeasurements using multivariate analyses ofvariance (MANOVA). Effects found to besignificant were subsequently analyzed usingunivariate ANOVAs. Means are given 6 1 SD.All statistical tests were considered significantat the a 5 0.05 level.

Genetic Analyses.—We obtained 122 adult A.texanum toe clips from the SRR site and 49 fromwithin the PWA site. Ambystoma t. tigrinumsamples (N 5 330) were collected only from thePWA site. All tissue samples were stored inlysis buffer at room temperature until genomicDNA was extracted using a standard proteinaseK/phenol-chloroform procedure (Sambrookand Russell, 2001). We used four microsatelliteloci for A. t. tigrinum: Atex 65 (Williams andDeWoody, 2004), At-5–7 (Mech et al., 2003),52.34 (Parra-Olea et al., 2007), and 60.9 (K.Zamudio, pers. comm.), using the conditionsdescribed in Gopurenko et al. (2006). Also, weused four microsatellite loci (Atex 49, 65, 102,and 141) for A. texanum using the conditions asdescribed in Gopurenko et al. (2007). Amplicons

from each individual were scored for size viaelectrophoresis on an ABI 377 sequencer (Ap-plied Biosystems) using associated GeneScan3.1.2 and Genotyper 2.5.2 software.

We estimated both expected and observedlevels of heterozygosity as well as allelicdiversity for all populations of each species.Genetic structure among the populations of A.texanum at SRR and PWA field sites wasassessed using F-statistics, calculated as Weirand Cockerhams’ h, using the software packageGDA v 1.0 (Lewis and Zaykin, 2001).

RESULTS

Sex Ratios and Breeding Chronology.—We cap-tured 122 adult A. texanum and four A. t. tigrinummigrating to and from the SRR breeding site in2003. Of the 122 A. texanum captures, only thoseindividuals captured immigrating into the wet-land (N 5 90) were used for t-test and chi-squareanalyses. The 32 A. texanum not included in theanalysis were initially captured from within theenclosure (i.e., they overwintered in the wetlandand no migration occurred). The sex ratio of A.texanum (58:32 M:F) was significantly skewedtoward males (x2

1 5 4.72, P 5 0.03); the A. t.tigrinum sample size precluded any meaningfultest of parity. The species composition wasreversed at the PWA field site and resulted in110 A. t. tigrinum and seven A. texanum in 2003.Again, of the 110 A. t. tigrinum caught, only thosecaptured immigrating into the wetland (N 5 63)were used for t-test and chi-squared analyses. In2003 at the PWA field site, the A. t. tigrinum sexratio (38:25 M:F) was statistically indistinguish-able from 1:1 (x2

1 5 2.33, P 5 0.13). In 2004, wecollected 220 adult A. t. tigrinum (of these, lessthan 3% were recaptures from 2003, and all weremale) and 49 A. texanum at the PWA field site.The sex ratio for A. t. tigrinum (175:45 M:F) wassignificantly skewed toward males (x2

1 , 0.001,P , 0.001), whereas A. texanum (29:20 M:F)showed no significant deviation from parity (x2

1

5 1.65, P 5 0.20).In both species, the annual migration to

breeding ponds occurred over a short timeperiod. In 2003, A. texanum males immigratedduring an eight-day period from 13–21 March,whereas females immigrated over a four-dayperiod (17–21 March; Fig. 1A). In A. t. tigrinum,both sexes immigrated over a four-day periodfrom 17–21 March (Fig. 1B). In 2004, A. texanummales immigrated over a 10-day period (24February to 5 March), whereas females immi-grated over a 14-day period (21 February to 6March; Fig. 2A). Ambystoma t. tigrinum malesimmigrated over a 17-day period (23 Februaryto 11 March) and females over a 10-day period(1–11 March; Fig. 2B).

440 R. N. WILLIAMS ET AL.

In 2003, the 23 A. texanum males and 22females with known immigration and emigra-tion dates (i.e., toe-clipped recaptures) spent anaverage of 73 6 30.05 and 74 6 26.4 days in theSRR enclosure, respectively (t43 5 0.171, P 5

0.86). The 37 A. t. tigrinum males and 22 femaleswith known immigration and emigration datesat the PWA field site spent an average of 32 6

25.4 and 29 6 29.5 days in the enclosure,respectively (t57 5 0.515, P 5 0.61). In 2004,

the 35 A. texanum males and 14 females withknown immigration and emigration dates spentan average of 88 6 31.0 and 95 6 22.3 days inthe PWA enclosure, respectively (t47 5 0.781, P5 0.44). The 97 A. t. tigrinum males and 42females with known immigration and emigra-tion dates spent an average of 72 6 40.6 and 756 39.4 days within the PWA enclosure, respec-tively (t137 5 0.514, P 5 0.61). Ambystoma t.tigrinum spent significantly longer periods of

FIG. 1. The number of male and female salamanders immigrating to breeding ponds during 2003. The left y-axis shows the number of immigrants: the black bars represent the number of females whereas white barsrepresent the number of males. The right y-axis shows the range of temperature and precipitation: solid linesrepresent daily high temperatures (uC), whereas dotted lines represent daily rainfall (mm). (A) Ambystomatexanum immigrating to the SRR field site. (B) A. t. tigrinum immigrating to the PWA field site.


time within the enclosure in 2004 (wet) than in2003 (dry) for both males (t132 5 5.481, P ,0.001) and females (t63 5 4.896, P , 0.001).Sample sizes for A. texanum in 2003 precludedany statistical tests between wet and dry years.

Sexual Dimorphism.—Body size differed signif-icantly among males and females (Table 1). Bothspecies showed some evidence of sexual sizedimorphism among the five metrics, between thetwo field sites, and across sampling years(Table 1). Female A. texanum had a greater bodymass in both years and a greater SVL in 2004(Table 1), but no other intraspecific metricsdiffered significantly. However, A. t. tigrinum

displayed sexual size dimorphism for four of thefive metrics (Table 1). In general, our resultsindicate that migrating females are significantlyheavier (prior to oviposition) than males in bothspecies and that A. texanum display very littlesexual size dimorphism at either field site. Sexualsize dimorphism was more evident in A. t.tigrinum, where males typically have significantlylonger and taller tails, but females are longer(SVL). The varying levels of sexual size dimor-phism between species is not entirely unexpectedgiven their differences in courtship and naturalhistory (Kumpf 1934; Petranka 1998), whichsuggest sexual selection is more extreme in A. t.

FIG. 2. The number of male and female salamanders immigrating to breeding ponds during 2004. The left y-axis shows the number of immigrants: the black bars represent the number of females, whereas white barsrepresent the number of males. The right y-axis shows the range of temperature and precipitation: solid linesrepresent daily high temperatures (uC), whereas dotted lines represent daily rainfall (mm). (A) Ambystomatexanum immigrating to PWA field site. (B) A. t. tigrinum immigrating to PWA field site.


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tigrinum but is difficult to reconcile with geneticparentage data (Gopurenko et al., 2007; Williamsand DeWoody, 2009; see Discussion).

Size and Breeding Migrations.—In general, wefound that precipitation and temperature werethe best predictors of immigration date (Ta-ble 2, Figs. 1A, B), whereas morphologicalparameters were generally poor predictors.Tail height and body condition were correlatedwith immigration date in A. texanum, but inboth cases, the parameters explained very littlevariation (Table 2). Likewise, we found that tailheight and body condition were correlatedwith female A. t. tigrinum immigration, butagain the metrics explained very little variationin the model (Table 2). The general pattern wasreversed in 2004, with temperature, not precip-itation, serving as the best predictor of immi-gration date (Table 2; Figs. 2A, B). We found norelationship between the date of immigrationand snout–vent length, tail length, or mass,suggesting that gross morphology has littlebearing on when salamanders migrate tobreeding ponds.

In most cases, we found a significant correla-tion between the number of individuals leavingthe wetland in a 24-h period and both theduration of wetland stay (i.e., departure date)and daily temperature (Table 3). Daily rainfalland mass were correlated with the time femalesspent within breeding ponds, but both explainedlittle of the overall variation. The number of A. t.tigrinum emigrating on a given day was correlat-ed to mass and daily rainfall (males) and rainfall

(females), but daily temperature explained mostof the variation in each of those cases.

Genetic Variation and Structure.—Genetic var-iability was high in both species and at bothsampling sites (Table 4). In A. t. tigrinum, themean number of alleles per locus was 18.5,mean observed heterozygosity was 0.75, andtests for significant differences in allele frequen-cies between years (2003 and 2004) werenegative (h 5 0.004; P . 0.05). Ambystomatexanum from the PWA site had a mean of 14.8alleles per locus, with a mean observed hetero-zygosity of 0.78, compared to 19.8 alleles perlocus and a mean observed heterozygosity of0.90 in A. texanum at the SRR site. There wassignificant genetic differentiation between thetwo A. texanum sampling sites (h5 0.05; P ,0.05). This differentiation is highlighted byprivate alleles; over all four loci there were 29unique A. texanum alleles in the SRR populationand 8 in the PWA population, suggesting thathistorical gene flow has been limited (as in otherurodeles; Storfer, 1999; Jones et al., 2001).

DISCUSSION

We compare morphology, breeding chronol-ogy, and genetics in two salamander specieswith very different mating behaviors that arelikely shaped by sexual selection. Recall that A.texanum populations typically consist of evensex ratios, and courtship is simple with littledirect male/male competition, and femalechoice is not apparent (Petranka, 1998; Gopur-

TABLE 2. Linear regression analysis of the total number of immigrating Ambystoma texanum and A. t. tigrinum

and both environmental cues and body size measurements. Coefficients of partial determination (partial r2),coefficients of determination (r2), and probabilities are presented. Sample sizes are given in parentheses. SRR 5

South River Road, PWA 5 Purdue Wildlife Area.

Species Year Field site Sex Significant model parameters Partial r2 r2 P

A. texanum 2003 SRR Female (N 5 23) Rain 0.9337 0.9337 ,0.0001Rain + Temperature 20.0414 0.9751 ,0.0001

Male (N 5 56) Rain 0.4793 0.4793 ,0.0001Rain + Temperature 0.2973 0.7766 ,0.0001Rain + Temperature + Tail Height 20.0230 0.7996 0.0179Rain + Temperature + Body

Condition0.0092 0.9844 0.003

A. texanum 2004 PWA Female (N 5 20) Temperature 0.5771 0.5771 0.001Male (N 5 29) Temperature 0.7203 0.7203 ,0.0001

A. tigrinum 2003 PWA Female (N 5 25) Rain 0.7864 0.7864 ,0.0001Rain + Temperature 20.1540 0.9404 ,0.0001Rain + Temperature + Body

Condition0.0149 0.9553 0.0150

Male (N 5 35) Rain 0.7504 0.7504 ,0.0001Rain + Temperature 20.1845 0.9349 ,0.0001

A. tigrinum 2004 PWA Female (N 5 45) Rain 0.1676 0.1676 0.0052Male (N 5 175) Temperature 0.0525 0.0525 0.0023

* Model parameters include total length, tail length, snout-vent length, tail height, mass, body condition, temperature,precipitation.


enko et al., 2007). However, breeding popula-tions of A. t. tigrinum have strongly male-biasedsex ratios and complex courtship, with malescompeting for females who ultimately choosetheir mates based on these courtship activities(Arnold, 1976; Semlitsch, 1983; Howard et al.,1997). Here, we summarize key similarities anddifferences between the two species.

Breeding Chronology.—We found that annualA. texanum migrations to breeding pondsoccurred over relatively short periods of time(4–14 days) and varied slightly from year toyear (Figs. 1A and 2A). Many authors haveindicated that male salamanders appear at

breeding sites much earlier than females (Hillis,1977; Douglas, 1979; Hardy and Raymond, 1980;Semlitsch, 1985), but at our sites in Indiana, noclear pattern emerged regarding whether malesor females typically arrive first at breedingponds. Furthermore, both sexes stayed withinthe wetland nearly twice as long as previouslyreported (Brown et al., 1983). Thus, at our studysites, both A. texanum sexes migrate more or lesssimultaneously and remain within the wetlandlong after reproduction has occurred.

Ambystoma t. tigrinum migration to breedingponds occurred over timescales (4–17 days)nearly identical to that of A. texanum (Figs. 1B

TABLE 3. Linear regression analysis of the total number of emigrating Ambystoma texanum and A. t. tigrinum

and both environmental cues and body size measurements. Coefficients of partial determination (partial r2),coefficients of determination (r2), and probabilities are presented. Sample sizes are given in parentheses. SRR 5

South River Road, PWA 5 Purdue Wildlife Area.

Species Year Field site Sex Significant model parameters Partial r2 r2 P

A. texanum 2003 SRR Female (N 5 23) Temperature 0.9080 0.9080 ,0.0001Temperature + Rain 20.0612 0.9693 ,0.0001Temperature + Rain + Tail

Length20.0065 0.9758 0.0357

Male (N 5 23) Temperature 0.8007 0.8007 ,0.0001Temperature + Rain 20.1453 0.9460 ,0.0001

A. texanum 2004 PWA Female (N 5 20) Temperature 0.4909 0.4909 0.0006Male (N 5 29) Temperature 0.6899 0.6899 ,0.0001

A. t. tigrinum 2003 PWA Female (N 5 21) Temperature 20.3432 0.3432 0.0053Temperature + Tail Height 20.1612 0.5044 0.0263

Male (N 5 34) None — — —A. t. tigrinum 2004 PWA Female (N 5 43) Temperature 0.5499 0.5499 ,0.0001

Temperature + Rain 0.0487 0.5986 0.0334Male (N 5 96) Temperature 0.4939 0.4939 ,0.0001

Temperature + Rain 0.0693 0.5632 0.0002Temperature + Rain + Body

Condition20.0252 0.5884 0.0196

* Model parameters include total length, tail length, snout–vent length, tail height, mass, body condition, temperature,precipitation.

TABLE 4. Genetic variability results for Ambystoma texanum and A. t. tigrinum. A 5 number of alleles perlocus, He 5 expected heterozygosity, Ho 5 observed heterozygosity.

Species Field site Locus Sample size A He Ho

A. texanum South River Road Atex 141 119 24 0.92 0.90Atex 102 118 17 0.91 0.91Atex 65 120 22 0.93 0.91Atex 49 121 16 0.91 0.87mean 120 19.8 0.92 0.90

A. texanum Purdue Wildlife Area Atex 141 61 16 0.90 0.97Atex 102 59 12 0.85 0.78Atex 65 61 20 0.93 0.92Atex 49 60 11 0.71 0.47mean 60 14.8 0.85 0.78

A. t. tigrinum Purdue Wildlife Area 52.34 245 21 0.88 0.89Atex 65 242 32 0.95 0.935–7 243 4 0.60 0.5560.9 232 17 0.83 0.64mean 241 18.5 0.81 0.75


and 2B). Again, no clear pattern emergedregarding whether A. t. tigrinum males or femalestypically arrive first at breeding ponds. Thelength of time spent within the wetland variednot between the sexes but between samplingyears. The PWA swale was dry in 2003, and notonly was the absolute number of migratingsalamanders much smaller but the mean timemales and females spent within the wetland wassignificantly reduced (30 days in 2003 comparedto 72 days in 2004). Thus, A. t. tigrinum males andfemales at our study sites migrated in similartime frames, and both remained in the wetlandlong after reproduction had occurred. Theamount of time the sexes spent within thewetland differed across years and with environ-mental conditions (as in Semlitsch, 1983).

Sexual Dimorphism.—Sexual dimorphisms aremost pronounced in species that are understrong sexual selection either by mate choice ormate competition (Andersson, 1994). There is noevidence for mate choice in A. texanum (seeGopurenko et al., 2007), and because their lifehistory strategy includes no parental care and ashort breeding season, there is little a priorireason to suspect strong female choice (Trivers,1972; Verrell, 1989). However, sexual selectionin the form of mate competition (e.g., sexualinterference via sperm capping) is well docu-mented in A. texanum (McWilliams, 1992),although this may not result in selection formorphological differences. We determined thatgravid A. texanum females were consistentlyheavier than males (similar to Finkler andCullum, 2002), and in the PWA population,females had larger snout–vent lengths, a similarfinding to Plummer (1977). However, no othermorphological differences were detected, andthe overall lack of sexual dimorphism isconsistent with the weak intensity of sexualselection predicted in A. texanum based on itsavailable natural history.

Sexual dimorphism has been detected insome A. t. tigrinum populations (Howard etal., 1997) but not in others (Semlitsch, 1983).Semlitsch (1983) found no sexual dimorphism inSVL between male and females from SouthCarolina, whereas Howard et al. (1997) foundthat in Indiana males tend to have longer tails.In fact, Howard et al. (1997) found that femaleA. t. tigrinum preferred mates with longer tailsand, thus, concluded that larger males (i.e.,those with longer tails) have a mating advan-tage over smaller males. This should result inlarge variances in male reproductive success,which can lead to positive feedback (runawayselection). Like Howard et al. (1997), we alsofound large differences in sexual dimorphismamong males and females in A. t. tigrinum;males typically had significantly greater tail

lengths and tail heights, whereas females hadsignificantly greater SVL.

Genetic parentage data are largely consistentwith a random union of gametes in thesespecies (Gopurenko et al. 2007; Williams andDeWoody, 2009). This begs the question ofwhether sexual dimorphisms are driven bysexual selection or by other factors. It is entirelypossible that the sexual dimorphisms weobserved are a by-product of life-historypatterns (e.g., gravid females are older thanmales) or by sex-specific differences (e.g.,females may have longer SVL to store moreova). Unfortunately, we cannot distinguishbetween these (or other) hypotheses with ourexisting data sets.

Body Size and Migration.—The large differenc-es in A. t. tigrinum sexual dimorphism (and, toa lesser extent, in A. texanum) suggested thatbody size could influence immigration, emi-gration, or duration of stay in the breedingwetland. The influence of body size on migra-tion is often confounded by weather (Douglas,1979; Semlitsch, 1985; Sexton et al., 1990; Palis,1997). Weather notwithstanding, Briggler et al.(2004) found that in the autumn breeding A.annulatum, larger individuals of both sexesmigrated to breeding ponds earlier than small-er individuals. However, Hardy and Raymond(1980) found that, in the spring breeding A.talpoideum, smaller (and presumably younger)males immigrated before larger males. Theysuggested the earlier arrival of smaller males atthe pond provides them a competitive fitnessadvantage over the older males (which arrivelater). Thus, one might suspect that conditionsfavorable for migration may differ not onlybetween the sexes but also between individualsof the same sex. For example, male Ambystomacould migrate earlier than females because oflower environmental thresholds (Douglas,1979; Sexton et al., 1990) or to increase theprobability of finding a receptive mate (Krenzand Sever, 1995). Conversely, larger femalesproduce a greater number of ova and, hence,have a greater investment in reproduction(Plummer, 1977; Licht and Bogart, 1990).Therefore, larger females might be expected toarrive later to ensure environmental conditionsare favorable or that high-quality males arepresent (Douglas, 1979). Within this context,the potential exists for a size-dependent ‘‘or-der’’ in which individuals migrate to and frombreeding sites, which could ultimately influ-ence their reproductive success (i.e., individu-als that arrive first and leave last increaseaccess to mates).

Our data not only show that males andfemales arrive at approximately the same timein both species but also that neither sex exhibits


a size-ordered migration (i.e., only temperatureand precipitation were correlated with salaman-der migrations). Thus, environmental factorsare the most important factor in predictingmigration events in Indiana. The number ofimmigrating adults was consistently correlatedwith the total amount of precipitation, a findingsimilar to Semlitsch (1983). We did not detect acorrelation between the duration of stay andany morphological measurement, suggestingthat once environmental conditions reach somecritical threshold, individuals of all sizes emi-grate from the wetland.

Sex Ratios.—Male-biased sex ratios have longbeen reported in Ambystoma salamanders (Peck-ham and Dineen, 1954; Sever and Dineen, 1978;Semlitsch, 1983; Williams and MacGowan,2004). Our current data (Table 1) support thisnotion in both A. texanum and A. t. tigrinum, butthe magnitude of sex ratio bias is nearly twiceas strong in A. t. tigrinum than in A. texanum(1.7:1 vs. 3:1, averaged within each species oversites across years). One possible factor drivingsex-ratio bias in A.t. tigrinum was suggested byAnderson et al. (1971), who argued thatreproductive cycles were biennial for femalesand annual for males. This argument waslargely refuted by Sever and Dineen’s (1978)evidence of breeding female recaptures oversuccessive years, suggesting that a large pro-portion of females in the population wereannual breeders. Our 2003–04 recapture dataon A. t. tigrinum indicated that not only didfemales not return to the same breeding pond insuccessive years but that the recapture rate formales was very low as well (,3%). Sever andDineen (1978) argued that three factors may bedriving sex-ratio bias in A. t. tigrinum: sex ratioat birth, differential mortality, and differentialmaturation rate between sexes. With our data,we can only address differential maturation.Biased sex ratios among breeding adults mightbe driven by age and size structure, where onlyfemales of a particular minimum size (andpresumably age) breed, whereas nearly allmales try to mate. If so, then we expectbreeding sex ratios to be weighted againstfemales and the average body size (as well asage) of females to be larger (older) than males.This is exactly the pattern shown by our data,where females were generally larger than malesin both species. We assume in this discussionthat age and size are tightly correlated, but thecritical tests required of this assumption (e.g.,skeletochronology) were not conducted in thisstudy.

Genetic Diversity.—Species that have exagger-ated sex ratios or show evidence of extensivereproductive variance in one sex may have smalleffective population size (Ne) relative to their

absolute census population (Nunney, 1993; Suggand Chesser, 1994). Subsequently, loss of allelicdiversity via genetic drift is exaggerated inspecies that have consistently low Ne (Hedrick,2005). We predicted that allelic diversity wouldbe less in A. t. tigrinum than in A. texanum becauseof more pronounced male-biased sex ratios andcomplex courtship behaviors that may lead togreater reproductive variance among individu-als. Nevertheless, our data do not reveal anysubstantial difference in the level of geneticvariation between species (Table 4).

The data reveal very high levels of geneticvariation in both species, much higher thanfound in most animals (DeWoody and Avise,2000). Indeed, levels of neutral genetic variationin these salamanders are roughly twice thatfound in most terrestrial animal populationsand rival those of marine fishes, which oftenhave huge effective population sizes. In A. t.tigrinum, the microsatellite data are buttressedby substantial levels of nonneutral variation inthat species (Bos and DeWoody, 2005; Bos et al.,2008).

Genetic diversity may be similar in A.texanum and A. t. tigrinum if their sexualselection regimes are similar. Gopurenko et al.(2006) reported that variance in reproductivesuccess of female A. t. tigrinum was roughlyhalf that found in males and suggest that themore elaborate courtship behaviors (e.g., malecoercion or female mate choice) could reducethe need for genetic bet hedging. Conversely,the variance in reproductive success is not onlymuch higher in A. texanum, it is much higher infemales than in males (Gopurenko et al., 2007).These findings support the idea that, in specieswith simple courtship behaviors like A. tex-anum, male mating success is more likelydriven by the number of males involved inmate competition via spermatophore deposi-tion rather than by female mate choice or malecoercion.

Nunney (1993) investigated Ne for specieswith lottery versus polygynous breeding strat-egies, and his theoretical work suggests thathigh variance in reproductive success results inlow Ne. However, this is the case only inpopulations with nonoverlapping generationsand when generation time is short. In highlypolygynous species with overlapping genera-tions and long generation times (like molesalamanders), Ne can approach one-half theharmonic census population size. In A. texanumand A. t. tigrinum, the high estimates of Ne

suggested by measures of genetic diversity maybe a function of their overlapping generations,annual breeding events over a relatively longlifetime, and large population sizes.


Summary.—The disparities in the breedingbiology of A. texanum and A. t. tigrinum providea rich theoretical framework for studying theevolution of their morphology, natural history,and genes. We document some striking simi-larities between species (breeding chronologies,genetic diversity) and some pronounced differ-ences (sex ratios, sexual dimorphisms). Also,our data show that abiotic factors seem totrigger breeding immigrations in both species.Although the data presented herein highlightmany behavioral similarities and differencesbetween these two congeners, they also raisesome interesting evolutionary questions per-taining to the role of morphology in mating andreproductive success.

Acknowledgments.—We thank J. Barany forpermission to conduct research at the SouthRiver Road field site and S. Baker, L. Sheets, S.Hecht, and numerous other technicians for helpin collecting salamanders. We also thank mem-bers of the DeWoody lab for helpful commentson earlier drafts of this manuscript. Animalswere collected under permits issued by theIndiana Department of Natural Resources andthe Purdue University animal care and usecommittee. Financial support was provided byPurdue University and the National ScienceFoundation (DEB-0514815). This is AgriculturalResearch Programs contribution 2006-17882from Purdue University.

LITERATURE CITED

ANDERSON, J. D., D. D. HASSINGER, AND G. H. DARYMPLE.1971. Natural mortality of eggs and larvae ofAmbystoma t. tigrinum. Ecology 52:1107–1112.

ANDERSSON, M. 1994. Sexual Selection. PrincetonUniversity Press, Princeton, NJ.

ARNOLD, S. J. 1976. Sexual behavior, sexual interfer-ence, and sexual defense in the salamandersAmbystoma maculatum, Ambystoma tigrinum, andPlethodonjordani. Zeitschrift fur Tierpsychologie 42:247–300.

BAKER, J. M. R. 1992. Body condition and tail height inGreat Crested Newts, Triturus cristatus. AnimalBehaviour 43:157–159.

BOS, D. H., AND J. A. DEWOODY. 2005. Molecularcharacterization of major histocompatibilitycomplex class II alleles in wild Tiger Salaman-ders (Ambystoma tigrinum). Immunogenetics 57:775–781.

BOS, D. H., D. GOPURENKO, R. N. WILLIAMS, AND J. A.DEWOODY. 2008. Inferring population history anddemography using microsatellites, mitrochondrialDNA, and major histocompatibility complex(MHC) genes. Evolution 62:1458–1568.

BRIGGLER, J. T., J. E. JOHNSON, AND D. D. RAMBO. 2004.Demographics of a Ringed Salamander (Ambysto-ma annulatum) breeding migration. SouthwesternNaturalist 49:209–217.

BROWN, B. A., W. W. CUDMORE, AND J. O. WHITAKER JR.1983. Miscellaneous notes on Ambystoma texanumin Vigo County, Indiana. Proceedings of theIndiana Academy of Science 92:473–478.

DEWOODY, J. A., AND J. C. AVISE. 2000. Microsatellitevariation in marine, freshwater and anadromousfishes compared with other animals. Journal ofFish Biology 56:461–473.

DOUGLAS, M. E. 1979. Migration and sexual selection inAmbystoma jeffersonianum. Canadian Journal ofZoology 57:2303–2310.

FINKLER, M. S., AND K. A. CULLUM. 2002. Sex-relateddifferences in metabolic rate and energy reservesin spring-breeding Small-mouthed Salamanders(Ambystoma texanum). Copeia 2002:824–829.

GARTON, J. S. 1972. Courtship of the Small-mouthedSalamander, Ambystoma texanum, in southernIllinois. Herpetologica 28:41–45.

GOPURENKO, D., R. N. WILLIAMS, C. R. MCCORMICK, AND

J. A. DEWOODY. 2006. Insights into the matinghabits of the Tiger Salamander (Ambystoma t.tigrinum) as revealed by genetic parentage analy-ses. Molecular Ecology 15:1917–1928.

GOPURENKO, D., R. N. WILLIAMS, AND J. A. DEWOODY.2007. Reproductive and mating success in theSmall-mouthed Salamander (Ambystoma texanum)estimated via microsatellite parentage analysis.Evolutionary Biology 34:130–139.

HARDY, L. M., AND L. R. RAYMOND. 1980. The breedingmigration of the mole salamander, Ambystomatalpoideum, in Louisiana. Journal of Herpetology14:327–335.

HEDRICK, P. 2005. Large variance in reproductivesuccess and the Ne/N ratio. Evolution 59:1596–1599.

HILLIS, D. M. 1977. Sex ratio, mortality rate, andbreeding stimuli in a Maryland population ofAmbystoma maculatum. Bulletin Maryland Herpe-tological Society 13:84–91.

HOUCK, L. D., AND S. J. ARNOLD. 2003. Courtship andmating behavior. In D. M. Sever (ed.), Reproduc-tive Biology and Phylogeny of Urodela,pp. 383–424. Science Publishers, Inc., Enfield, NH.

HOWARD, R. D., R. S. MOORMAN, AND H. H. WHITEMAN.1997. Differential effects of mate competition andmate choice on Eastern Tiger Salamanders. AnimalBehaviour 53:1345–1356.

JONES, A. G., M. S. BLOUIN, AND S. J. ARNOLD. 2001.Genetic variation in two populations of the Rough-Skinned Newt (Taricha granulosa) assessed usingnovel tetranucleotide microsatellite loci. MolecularEcology Notes 1:293–296.

KRENZ, J. D., AND D. M. SEVER. 1995. Mating andoviposition in paedomorphic Ambystoma talpoi-deum precedes the arrival of terrestrial males.Herpetologica 51:387–393.

KUMPF, K. F. 1934. The courtship of Ambystomatigrinum. Copeia 1934:7–10.

LANNOO, M. J., AND C. A. PHILLIPS. 2005. Ambystomatigrinum. In M. J. Lannoo (ed.), AmphibianDeclines: The Conservation Status of United StatesSpecies, pp. 636–639. University of CaliforniaPress, Berkeley, CA.

LEWIS, P. O., AND D. ZAYKIN. 2001. Genetic dataanalysis: computer program for the analysis ofallelic data. Version 1.0 (d16c). Free program


distributed by the authors from http://lewis.eeb.uconn.edu/lewishome/software.html

LICHT, L. E., AND J. P. BOGART. 1990. Courtship behaviorof Ambystoma texanum on Pelee Island, Ontario.Journal of Herpetology 24:450–452.

MCWILLIAMS, S. R. 1992. Courtship behavior of theSmall-mouthed Salamander (Ambystoma texanum):the effects of conspecific males on male matingtactics. Behavior 121:3–19.

MECH, S. G., A. STORFER, J. A. ERNST, M. W. REUDINK,AND S. D. MALONEY. 2003. Polymorphic microsatel-lite loci for Tiger Salamanders, Ambystoma tigri-num. Molecular Ecology Notes 3:79–81.

MORBEY, Y. E., AND R. C. YDENBERG. 2001. Protandrousarrival timing to breeding areas: a review. EcologyLetters 4:663–673.

NUNNEY, L. 1993. The influence of mating system andoverlapping generations on effective populationsize. Evolution 47:1329–1341.

PALIS, J. G. 1997. Breeding migration of Ambystomacingulatum in Florida. Journal of Herpetology31:71–78.

PARMELEE, J. R. 1993. Microhabitat segregation andspatial relationships among four species of molesalamander (genus Ambystoma). Occasional Papersof the Museum of Natural History, University ofKansas 160:1–33.

PARRA-OLEA, G., E. RECUERO, AND K. R. ZAMUDIO. 2007.Polymorphic microsatellite markers for Mexicansalamanders of the genus Ambystoma. MolecularEcology Notes 7:818–820.

PECKHAM, R. S., AND C. F. DINEEN. 1954. Springmigrations of salamanders. Proceedings of theIndiana Academy of Science 64:278–280.

PETRANKA, J. W. 1982. Courtship behavior of the Small-mouthed Salamander, (Ambystoma texanum) incentral Kentucky. Herpetologica 38:333–336.

———. 1998. Salamanders of the United States andCanada. Smithsonian Institution, Washington, DC.

PLUMMER, M. V. 1977. Observations on breedingmigrations of Ambystoma texanum. HerpetologicalReview 8:79–80.

SAMBROOK, J., AND D. W. RUSSELL. 2001. MolecularCloning: A Laboratory Manual. 3rd ed. ColdSprings. Harbor Laboratory Press, Cold SpringsHarbor, NY.

SEMLITSCH, R. D. 1983. Structure and dynamics of twobreeding populations of the Eastern Tiger Sala-mander, Ambystoma tigrinum. Copeia 1983:608–616.

———. 1985. Analysis of climatic factors influencingmigrations of the salamander Ambystoma talpoi-deum. Copeia 1985:477–489.

SEVER, D. M., AND C. F. DINEEN. 1978. Reproductiveecology of the Tiger Salamander, Ambystomatigrinum, in northern Indiana. Proceedings of theIndiana Academy of Science 87:189–203.

SEXTON, O. J., C. PHILLIPS, AND J. E. BRAMBLE. 1990. Theeffects of temperature and precipitation on themigration of the Spotted Salamander (Ambystomamaculatum). Copeia 1990:781–787.

STORFER, A. 1999. Gene flow and population subdivi-sion in the Streamside Salamander, Ambystomabarbouri. Copeia 1999:174–181.

SUGG, D. W., AND R. K. CHESSER. 1994. Effectivepopulation sizes with multiple paternity. Genetics137:1147–1155.

TRAUTH, S. E. 2005. Ambystoma texanum. In M. J.Lannoo (ed.), Amphibian Declines: The Conserva-tion Status of United States Species, pp. 634–636.University of California Press, Berkeley.

TRIVERS, R. L. 1972. Parental investment and sexualselection. In B. Campbell (ed.), Sexual Selectionand the Descent of Man, 1871–1971, pp. 136–179.Heinemann, London.

TWITTY, V. C. 1966. Of Scientists and Salamanders.Freeman, San Francisco, CA.

VERRELL, P. A. 1989. The sexual strategies of naturalpopulations of newts and salamanders. Herpeto-logica 45:265–281.

WILLIAMS, R. N., AND J. A. DEWOODY. 2004. FluorescentdUTP helps characterize 10 novel tetranucleotidemicrosatellites from an enriched salamander (Am-bystoma texanum) genomic library. Molecular Ecol-ogy Notes 4:17–19.

———. 2009. Reproductive success and sexualselection in wild Tiger Salamanders (Ambystomatigrinum tigrinum). Evolutionary Biology 36:201–213.

WILLIAMS, R. N., AND B. J. MACGOWAN. 2004. Naturalhistory data on the mole salamander in Indiana.Proceedings of the Indiana Academy of Science113:147–150.

WYMAN, R. L. 1971. The courtship behavior of theSmall-mouthed Salamander, Ambystoma texanum.Herpetologica 27:491–498.

Accepted: 18 November 2008.


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