Rod N. Williams

An eDNA approach to detect eastern hellbenders (Cryptobranchus a. alleganiensis) using samples of water

Abstract Context. Environmental DNA, or eDNA, methods are a novel application of non-invasive genetic sampling in which DNA from organisms is detected via sampling of water or soil, typically for the purposes of determining the presence or absence of an organism. eDNA methods have the potential to revolutionise the study of rare or endangered taxa. Aims. We evaluated the efficacy of eDNA sampling to detect populations of an amphibian of conservation concern, the eastern hellbender (Cryptobranchus a. alleganiensis), indirectly from their aquatic environments. Methods. We developed species-specific primers, validated their specificity and sensitivity, and assessed the utility of our methods in silico and in laboratory trials. In the field, we collected water samples from three sites with known densities of hellbenders, and from one site where hellbenders do not occur. We filtered water samples, extracted DNA from filters, and assayed the extraction products for hellbender DNA by using polymerase chain reaction (PCR) and gel electrophoresis. Key results. Our methods detected hellbenders at densities approaching the lowest of reported natural densities. The low-density site (0.16 hellbenders per 100 m2) yielded two positive amplifications, the medium-density site (0.38 hellbenders per 100 m2) yielded eight positive amplifications, and the high-density site (0.88 hellbenders per 100 m2) yielded 10 positive amplifications. The apparent relationship between density and detection was obfuscated when river discharge was considered. There was no amplification in any negative control. Conclusion. eDNA methods may represent a cost-effective means by which to establish broad-scale patterns of occupancy for hellbenders. Implications. eDNA can be considered a valuable tool for detecting many species that are otherwise difficult to study.

INFERRING POPULATION HISTORY AND DEMOGRAPHY USING MICROSATELLITES, MITOCHONDRIAL DNA, AND MAJOR HISTOCOMPATIBILITY COMPLEX (MHC) GENES

Abstract Microsatellites and mitochondrial DNA (mtDNA) have traditionally been used in population genetics because of their variability and presumed neutrality, whereas genes of the major histocompatibility complex (MHC) are increasingly of interest because strong selective pressures shape their standing variation. Despite the potential for MHC genes, microsatellites, and mtDNA sequences to complement one another in deciphering population history and demography, the three are rarely used in tandem. Here we report on MHC, microsatellite, and mtDNA variability in a single large population of the eastern tiger salamander (Ambystoma tigrinum tigrinum). We use the mtDNA mismatch distribution and, on microsatellite data, the imbalance index and bottleneck tests to infer aspects of population history and demography. Haplotype and allelic variation was high at all loci surveyed, and heterozygosity was high at the nuclear loci. We find concordance among neutral molecular markers that suggests our study population originated from post-Pleistocene expansions of multiple, fragmented sources that shared few migrants. Differences in Ne estimates derived from haploid and diploid genetic markers are potentially attributable to secondary contact among source populations that experienced rapid mtDNA divergence and comparatively low levels of nuclear DNA divergence. We find strong evidence of natural selection acting on MHC genes and estimate long-term effective population sizes (Ne) that are very large, making small selection intensities significant evolutionary forces in this population.

Condition-dependent mate choice and a reproductive disadvantage for MHC-divergent male tiger salamanders.

Major histocompatibility complex (MHC) alleles likely have adaptive value because of overdominance, in which case MHC heterozygous individuals have increased fitness relative to homozygotes. Because of this potential benefit, the evolution of sexual reproduction between MHC‐divergent individuals (i.e. negative assortative mating, NAM) may be favoured. However, the strongest evidence for MHC‐based NAM comes from inbred animals, and context‐dependent mating preferences have rarely been evaluated although they often occur in nature. We assessed the extent MHC‐based mating preferences among wild tiger salamanders (Ambystoma tigrinum) using multiple molecular approaches. We genotyped 102 adults and 864 larvae from 36 breeding trials at both microsatellite and MHC loci. Parentage analysis revealed that reproductive success among males was positively associated with increased tail length and that with respect to the focal female, MHC‐similar males sired a significantly higher number of offspring than more dissimilar males. This trend was consistent, even under context‐dependent scenarios that favour traditional MHC‐based NAM. These results suggest that the most MHC‐divergent males may be at a reproductive disadvantage in pairwise breeding trials. Our data add to a growing body of evidence that suggests where it exists, MHC‐based choice is probably dynamic and mediated by many factors that vary in the wild, notably signals from other indicator traits and by the quality and quantity of potential mates.

ASSESSMENT OF GENETIC VARIANCE AMONG SOURCE AND REINTRODUCED FISHER POPULATIONS

Abstract A series of reintroduction programs have reestablished fisher (Martes pennanti) populations to a large part of their former range. Horizontal starch-gel electrophoresis of 20 presumptive gene loci was used from 1996 to 1998 to investigate gene dynamics in 4 remnant fisher populations (sources) and 4 reintroduced fisher populations from the northeastern and Great Lakes regions of the United States. Mean multilocus heterozygosities, mean number of alleles per locus, and percentage polymorphic loci were 0.027–0.090, 1.2–1.6, and 10.0–30.0, respectively, in the 8 populations surveyed. Significant allelic frequency differentiation was detected among the 4 source populations and among the 4 reintroduced populations, but a significant proportion of the genetic variance was partitioned only among the 4 reintroduced populations. Pairwise comparisons between each source and its associated reintroduced population indicated that only older reintroductions have attained significant differentiation of allelic frequency from their sources. Significant heterozygotic deficiencies were detected for statewide populations and regional populations within states, suggesting that breeding biology of the fisher, presumably among females, is creating levels of fine-scale genetic structure within populations.

Reproductive and Mating Success in the Small-Mouthed Salamander (Ambystoma texanum) Estimated via Microsatellite Parentage Analysis

Bateman’s principles of sexual selection predict that the sex with “cheaper” gametes may maximize reproductive efforts by mating multiply and so display greater positive covariance between reproductive and mating success. We conducted a semi-controlled breeding experiment to genetically quantify sexual selection in adult Ambystoma texanum, a sexually monomorphic salamander with simple courtship behaviors. We used four polymorphic microsatellite loci to genotype 57 adults enclosed in a breeding wetland and compared their multilocus profiles to that of 862 embryos collected from the enclosure. The molecular data were used to assign parentage, investigate the mating system, and measure sexual selection intensity. Parentage analyses indicated 36% of dams and 93% of sires were genetically sampled via their gametes but physically unsampled, suggesting that a large number of breeders over-wintered within the enclosure and/or some females released into the enclosure were already inseminated. We used the genetic data to generate estimates of individual reproductive and mating success and we interpret these in light of salamander behavior and sexual selection theory. The incidence of multiple mating in females (86%) was considerably higher than in males (32%) and the standardized variance in mating success was significantly greater in females. The correlations between reproductive and mating success were significant and of similar magnitude between the sexes, indicating that both sexes increased reproductive success through increased mating success. This pattern may be a function of differential opportunities for mating success between the sexes.

Insights into the mating habits of the tiger salamander (Ambystoma tigrinum tigrinum) as revealed by genetic parentage analyses.

Among urodeles, ambystomatid salamanders are particularly amenable to genetic parentage analyses because they are explosive aggregate breeders that typically have large progeny arrays. Such analyses can lead to direct inferences about otherwise cryptic aspects of salamander natural history, including the rate of multiple mating, individual reproductive success, and the spatial distribution of clutches. In 2002, we collected eastern tiger salamander (Ambystoma tigrinum tigrinum) egg masses (> 1000 embryos) from a ~80 m linear transect in Indiana, USA. Embryos were genotyped at four variable microsatellite loci and the resulting progeny array data were used to reconstruct multilocus genotypes of the parental dams and sires for each egg mass. UPGMA analysis of genetic distances among embryos resolved four instances of egg mass admixture, where two or more females had oviposited at exactly the same site resulting in the mixing of independent cohorts. In total, 41 discrete egg masses were available for parentage analyses. Twenty‐three egg masses (56%) consisted exclusively of full‐siblings (i.e. were singly sired) and 18 (44%) were multiply sired (mean 2.6 males/clutch). Parentage could be genetically assigned to one of 17 distinct parent pairs involving at least 15 females and 14 different males. Reproductive skew was evident among males who sired multiply sired clutches. Additional evidence of the effects of sexual selection on male reproductive success was apparent via significant positive correlations between male mating and reproductive success. Females frequently partitioned their clutches into multiple discrete egg masses that were separated from one another by as many as 43 m. Collectively, these data provide the first direct evidence for polygynandry in a wild population of tiger salamanders.

Reproductive Success and Sexual Selection in Wild Eastern Tiger Salamanders (Ambystoma t. tigrinum)

Variation in reproductive success is most pronounced in species with strongly biased operational sex ratios, prominent sexual dimorphisms, and where mate competition and choice are likely. We studied sexual selection in eastern tiger salamanders (Ambystoma t. tigrinum) and examined the role of body size on reproductive success. We genotyped 155 adults and 1,341 larvae from 90 egg masses at six microsatellite loci. Parentage analyses revealed both sexes engaged in multiple matings, but was more common among females (64%) than males (27%). However, the standardized variance in mating and reproductive success was higher in males. Bateman gradients were significant and nearly identical in both sexes, suggesting that sexual selection was roughly equal between sexes. Body size was not correlated with mating or reproductive success in either sex. The apparent lack of sexual selection on body size may be a result of sperm storage, sperm competition, alternative mating tactics, and/or random induction of spermatophores.

Amphibian malformations and inbreeding.

Inbreeding may lead to morphological malformations in a wide variety of taxa. We used genetic markers to evaluate whether malformed urodeles were more inbred and/or had less genetic diversity than normal salamanders. We captured 687 adult and 1259 larval tiger salamanders (Ambystoma tigrinum tigrinum), assessed each individual for gross malformations, and surveyed genetic variation among malformed and normal individuals using both cytoplasmic and nuclear markers. The most common malformations in both adults and larvae were brachydactyly, ectrodactyly and polyphalangy. The overall frequency of adults with malformations was 0.078 compared to 0.081 in larval samples. Genetic diversity was high in both normal and malformed salamanders, and there were no significant difference in measures of inbreeding (f and F), allele frequencies, mean individual heterozygosity or mean internal relatedness. Environmental contaminants or other extrinsic factors may lead to genome alternations that ultimately cause malformations, but our data indicate that inbreeding is not a causal mechanism.

Short-Term Forest Management Effects on a Long-Lived Ectotherm

Timber harvesting has been shown to have both positive and negative effects on forest dwelling species. We examined the immediate effects of timber harvests (clearcuts and group selection openings) on ectotherm behavior, using the eastern box turtle as a model. We monitored the movement and thermal ecology of 50 adult box turtles using radiotelemetry from May–October for two years prior to, and two years following scheduled timber harvests in the Central Hardwoods Region of the U.S. Annual home ranges (7.45 ha, 100% MCP) did not differ in any year or in response to timber harvests, but were 33% larger than previous estimates (range 0.47–187.67 ha). Distance of daily movements decreased post-harvest (from 22 m±1.2 m to 15 m±0.9 m) whereas thermal optima increased (from 23±1°C to 25±1°C). Microclimatic conditions varied by habitat type, but monthly average temperatures were warmer in harvested areas by as much as 13°C. Animals that used harvest openings were exposed to extreme monthly average temperatures (∼40°C). As a result, the animals made shorter and more frequent movements in and out of the harvest areas while maintaining 9% higher body temperatures. This experimental design coupled with radiotelemetry and behavioral observation of a wild ectotherm population prior to and in response to anthropogenic habitat alteration is the first of its kind. Our results indicate that even in a relatively contiguous forested landscape with small-scale timber harvests, there are local effects on the thermal ecology of ectotherms. Ultimately, the results of this research can benefit the conservation and management of temperature-dependent species by informing effects of timber management across landscapes amid changing climates.

Temporal variance in hematologic and plasma biochemical reference intervals for free-ranging eastern box turtles (Terrapene carolina carolina).

Eastern box turtle (Terrapene carolina carolina) populations are in decline, likely due to anthropogenic forces and disease, necessitating hematologic and biochemical data from healthy individuals for evaluation of wild populations. We repeatedly sampled 21 free-ranging eastern box turtles from May to September 2009 in the spring, summer, and fall to establish temporal hematologic and biochemical reference intervals. Packed cell volume, aspartate aminotransferase, and potassium levels declined significantly as the active season progressed. High levels of albumin, globulin, and calcium coincided with the presence of eggs in females. These reference intervals should provide baseline data for the clinical evaluation of wild box turtles presented for veterinary care or for studies of wild populations.