Jennifer A. Dever

Low levels of nucleotide diversity in Crocodylus moreletii and evidence of hybridization with C. acutus

Examinations of both population genetic structure and the processes that lead to such structure in crocodilians have been initiated in several species in response to a call by the IUCN Crocodile Specialist Group. A recent study used microsatellite markers to characterize Morelets crocodile (Crocodylus moreletii) populations in north-central Belize and presented evidence for isolation by distance. To further investigate this hypothesis, we sequenced a portion of the mitochondrial control region for representative animals after including samples from additional locales in Belize, Guatemala and Mexico. While there is limited evidence of subdivision involving other locales, we found that most of the differentiation among populations of C. moreletiican be attributed to animals collected from a single locale in Belize, Banana Bank Lagoon. Furthermore, mitochondrial DNA sequence analysis showed that animals from this and certain other locales display a haplotype characteristic of the American crocodile, C. acutus, rather than C. moreletii. We interpret this as evidence of hybridization between the two species and comment on how these new data have influenced our interpretation of previous findings. We also find very low levels of nucleotide diversity in C. moreletiihaplotypes and provide evidence for a low rate of substitution in the crocodilian mitochondrial control region. Finally, the conservation implications of these findings are discussed.

Genetic diversity, population subdivision, and gene flow in Morelet's crocodile (Crocodylus moreletii) from Belize, central America

Abstract The lack of information surrounding natural history and ecology of the endangered Morelets crocodile (Crocodylus moreletii) has prompted a baseline study of the population genetics for this species. Nine microsatellite loci have been used to estimate genetic structure within and gene flow patterns among crocodiles (using a recently described maximum likelihood approach) from seven localities in north-central Belize. Individuals from the seven localities grouped into four apparent populations. Within localities, a high degree of genetic heterogeneity was observed. Among all localities, some subdivision was present (FST = 0.062; RST = 0.100). Furthermore, among the apparent populations, we found a significant correlation between geographic distance and genetic subdivision. Our findings suggest a relatively high level of migration among populations (Nm = 5.15) and are consistent with an isolation-by-distance model of gene flow. Two contiguous subpopulations in particular, New River and New River Lagoon, may form an important source for genetic variation for smaller populations throughout the region. These data will allow us to test hypotheses of relatedness among C. moreletii for other drainages in Belize and will be useful in optimizing future management programs for C. moreletii.

Microsatellites in Morelet's Crocodile (Crocodylus moreletii) and Their Utility in Addressing Crocodilian Population Genetics Questions

Acknowledgments.-We thank K. Oseen, T. Jockel, and I. Rot-Nikcevic for help with animal care. M. Yamashita provided us with the Bombina for this study. Helpful comments on the manuscript were provided by K. Oseen, Y. Malashichev, and M. Yamashita. This research was supported by the Natural Sciences and Engineering Research Council of Canada and the Admissions Office of the Faculty of Medicine at Dalhousie University.

ORGANOCHLORINE PESTICIDES IN CHORIOALLANTOIC MEMBRANES OF MORELET'S CROCODILE EGGS FROM BELIZE

Recent studies examined the utility of the chorioallantoic membrane (CAM) as a nonlethal, noninvasive indicator of environmental contaminant exposure in oviparous wildlife. The CAM is a highly vascularized extraembryonic membrane that functions as a site for respiration, nutrient transport, and waste storage during embryonic development. After hatching, the CAM is usually discarded with the eggshell and can be used for chemical residue analysis. Chorioallantoic membranes have been used successfully to examine contaminant exposure and predict chemical concentrations in multiple species of birds and reptiles. In this study, we examined organochlorine (OC) pesticide concentrations in CAMs from eggs of Morelets crocodiles (Crocodylus moreletii) from northern Belize. Multiple OCs were detected in crocodile CAMs, including aldrin, dieldrin, endrin, dichlorodiphenyltrichloroethane, dichlorodiphenyldichloroethane, dichlorodiphenyldichloroethylene (DDE), heptachlor, lindane, and methoxychlor. Number and concentrations of OC compounds in CAMs were variable. The most prevalent contaminant detected was DDE, which occurred in 69% of CAMs, with concentrations ranging from 0.3 parts per billion (ppb) to 17.0 ppb. The OC burdens in crocodile CAMs confirm contamination of eggs and suggest exposure in embryos and maternal females. These results further support the use of CAMs as qualitative indicators of OC exposure in oviparous wildlife. The efficacy of this sampling technique in the field will depend on the logistics and cost associated with CAM collection and the specific life history traits of the wildlife species.

Evidence of multiple paternity in Morelet's Crocodile (Crocodylus moreletii) in Belize, CA, inferred from microsatellite markers

Microsatellite data were generated from hatchlings collected from ten nests of Morelets Crocodile (Crocodylus moreletii) from New River Lagoon and Gold Button Lagoon in Belize to test for evidence of multiple paternity. Nine microsatellite loci were genotyped for 188 individuals from the 10 nests, alongside 42 nonhatchlings from Gold Button Lagoon. Then mitochondrial control region sequences were generated for the nonhatchlings and for one individual from each nest to test for presence of C. acutus-like haplotypes. Analyses of five of the nine microsatellite loci revealed evidence that progeny from five of the ten nests were sired by at least two males. These data suggest the presence of multiple paternity as a mating strategy in the true crocodiles. This information may be useful in the application of conservation and management techniques to the 12 species in this genus, most of which are threatened or endangered.

Fine-Scale Genetic Structure in the Threatened Foothill Yellow-Legged Frog (Rana boylii)

Abstract The Foothill Yellow-Legged Frog (Rana boylii) is a threatened river-dwelling amphibian endemic to California and Oregon. Determining the genetic structure of populations that have not yet declined is an important tool for their conservation. In this study, molecular markers were used to asses the genetic structure of R. boylii. The ND2 region of the mitochondrial genome and Random Amplified Polymorphic DNA (RAPD) markers were examined amongst 51 individuals collected from seven relatively pristine tributaries branching off the Eel River in Northern California. Both markers exhibited significant genetic differentiation among tributaries; however, the RAPD markers revealed a positive correlation between geographic distance and genetic distance. Cluster analysis illustrated a distinct separation between northern and southern tributaries within the study site. In contrast, relatively little geographic structure was apparent when mtDNA haplotypes were examined. Discordance may be caused by the number of loci examined in the mitochondrial and nuclear genomes, recent divergence, and sex-biased dispersal.

Cryptic Torrent Frogs of Myanmar: An Examination of the Amolops marmoratus Species Complex with the Resurrection of Amolops afghanus and the Identification of a New Species

We investigated diversity in the Amolops marmoratus species complex within Myanmar using both molecular and morphological characters from recently collected specimens. Based on congruence between multivariate analyses of quantitative morphological characters and phylogenetic analyses of nucleotide variation in the 16S ribosomal gene conducted on 43 out of 182 frogs examined, we recognize A. marmoratus for specimens from the states of Mon and Shan and northern Tanintharyi Division and designate a neotype for this species; resurrect A. afghanus (Günther, 1858) from synonymy with A. marmoratus for specimens from the northern state of Kachin and designate a lectotype for this species; recognize A. panhai for specimens from Tanintharyi, a new country record; and describe a new species for specimens from the western states of Chin and Rakhine, and Sagaing Division.

Sibling relatedness and social play in juvenile ground squirrels

We described the relationship between relatedness as full or maternal half siblings and expression of social play and other social behaviors in juvenile Belding’s ground squirrel (Urocitellus beldingi) litters and evaluated the possible role of play in establishing social bonds between juvenile females. We used microsatellite analysis to determine relatedness. Juvenile females did not interact preferentially with full over half siblings, suggesting that they may form bonds equally with full and half sisters. The probability that females will have a surviving full sister beyond the juvenile period may be low in U. beldingi, and establishing a cooperative relationship with a half sister may sometimes be the best available option in adulthood. As the proportion of females within litters increased, rates of play decreased, suggesting that low social play may be adequate for social bonding among females. Among juvenile male U. beldingi, play bouts lasted longer between full than half brothers; however, juvenile males did not interact preferentially with full brothers in play or other social interactions. Body mass differences were smaller between full than half brothers, and in both full and half brother pairings, play bouts lasted longest when body mass differences were small. Because male U. beldingi do not ordinarily interact with littermate siblings after emigrating from the natal area, it is unlikely that play behavior functions to establish long-term social bonds between full brothers. Rather, young males may favor play interactions with phenotypically similar partners who can provide optimal challenges in interactions that promote motor development.

Rediscovery and Redescription of Theloderma phrynoderma (Ahl, 1927) (Anura: Rhacophoridae) from Myanmar

Theloderma is a widely distributed yet little-known genus of camouflaged tree frogs found throughout Southeast Asia. One member, T. phrynoderma, known only from the moist evergreen forest of the Karen Hills of Myanmar, is redescribed from two recently collected specimens and examination of type specimens. To date the only information available about T. phrynoderma is Boulenger’s brief 1893 description of two type specimens collected in 1888, and phylogenetic analyses to test its placement among other species of Theloderma is lacking due to an absence of specimens. In the present study, we compared two individuals collected in 2009 and 2010 from the Tanintharyi Nature Reserve to the type specimens of T. phrynoderma and proposed that they are also members of this species. We then used two mitochondrial genes (12S and 16S rRNA) and two nuclear genes (rhodopsin and tyrosinase) to infer the phylogenetic relationship of the putative T. phrynoderma to other members of Rhacophoridae, with a special emphasis on Theloderma. The recently collected individuals are of the same species within Theloderma but distinct from all other DNA sequenced congeners. The species redescription is based on a comparison of the newly found reference specimens with the lectotype and paralectotype. In addition, using a combination of morphological characters we provide a more complete diagnosis. The species is distinct from other congeners by a combination of the following characters: a mid-body size (female 44 mm SVL; male mean 41.3 mm SVL); tympanum diameter to eye diameter (70%); partial webbing between fingers; rugose skin with clumped, white-tipped calcified tubercles throughout the dorsal surface; webbing between fingers; distinct darker brown inverted V-marking between its shoulders; absence of vomerine teeth; and absence of vocal sacs.

A New Cryptic Species of the Theloderma asperum Complex (Anura: Rhacophoridae) from Myanmar

Abstract I describe a new species of Theloderma from two regions in Myanmar (Chin State in western Myanmar and Kachin State in northern Myanmar). Highly similar in appearance and size to Theloderma albopunctatum and Theloderma asperum, the new species differs by the presence of small, bilateral vocal sac openings absent in T. albopunctatum and T. asperum. Molecular phylogenetic analysis from two mitochondrial and four nuclear gene fragments infers that individuals are members of a unique genetic lineage within the T. asperum Complex.