Schyler O. Nunziata

32 species validation of a new Illumina paired-end approach for the development of microsatellites

Development and optimization of novel species-specific microsatellites, or simple sequence repeats (SSRs) remains an important step for studies in ecology, evolution, and behavior. Numerous approaches exist for identifying new SSRs that vary widely in terms of both time and cost investments. A recent approach of using paired-end Illumina sequence data in conjunction with the bioinformatics pipeline, PAL_FINDER, has the potential to substantially reduce the cost and labor investment while also improving efficiency. However, it does not appear that the approach has been widely adopted, perhaps due to concerns over its broad applicability across taxa. Therefore, to validate the utility of the approach we developed SSRs for 32 species representing 30 families, 25 orders, 11 classes, and six phyla and optimized SSRs for 13 of the species. Overall the IPE method worked extremely well and we identified 1000s of SSRs for all species (mean = 128,485), with 17% of loci being potentially amplifiable loci, and 25% of these met our most stringent criteria designed to that avoid SSRs associated with repetitive elements. Approximately 61% of screened primers yielded strong amplification of a single locus.

Habitat structure and colony structure constrain extrapair paternity in a colonial bird

Individual variation in sexual fidelity and extrapair paternity (EPP) is widely attributed to environmental heterogeneity, but the only variables known to be influential are food abundance and density of conspecific breeders (potential extrapair partners). Habitat structure is thought to impact EPP but is rarely measured and, when considered, is usually confounded with food abundance and predation pressure. To sidestep these confounds, we tested whether EPP is associated with habitat structure variables and with local conspecific density in a species whose nesting habitat is not used for feeding and lacks predators. In a blue-footed booby, Sula nebouxii, colony, the probability of EPP in a females nest was highest in parts of the study plot where there were few obstacles to locomotion, and was quadratically related to local density of sexually active males, even though local males did not sire the EP chicks. The probability of a male breeder siring EP (extrapair) chicks elsewhere was quadratically related to local density of sexually active males around his nest. From these patterns we infer that both sexes may foray for EP interactions, that males and females nesting at intermediate density are most likely to be accessed by forayers, and that obstacles in the vicinity of a females nest constrain access of foraying males. To our knowledge, this is the first demonstration that individual variation in EPP is associated with habitat structure in the absence of confounding variation in food availability, predation pressure or breeder quality, and the first evidence that EPP opportunities of female and male breeders are reduced by high

Development and characterization of twenty-three microsatellite markers for the freshwater minnow Santa Ana speckled dace (Rhinichthys osculus spp., Cyprinidae) using paired-end Illumina shotgun sequencing

We isolated and characterized a total of 23 microsatellite loci from the Santa Ana speckled dace, Rhinichthys osculus spp., a freshwater minnow restricted to southern California. Loci were screened in 24 individuals from five watersheds. The number of alleles per locus ranged from 7 to 25, observed heterozygosity ranged from 0.409 to 0.875, and the probability of identity values ranged from 0.005 to 0.081. These new loci will provide tools for examining taxonomic status, population structure, and management strategies.

Characterization of 42 polymorphic microsatellite loci in Mimulus ringens (Phrymaceae) using Illumina sequencing

UNLABELLED PREMISE OF THE STUDY Microsatellite markers were isolated and characterized in Mimulus ringens (Phrymaceae), a herbaceous wetland perennial, to facilitate studies of mating patterns and population genetic structure. • METHODS AND RESULTS A total of 42 polymorphic loci were identified from a sample of 24 individuals from a single population in Ohio, USA. The number of alleles per locus ranged from two to nine, and median observed heterozygosity was 0.435. • CONCLUSIONS This large number of polymorphic loci will enable researchers to quantify male fitness, patterns of multiple paternity, selfing, and biparental inbreeding in large natural populations of this species. These markers will also permit detailed study of fine-scale patterns of genetic structure.

Population and Conservation Genetics of Crawfish Frogs, Lithobates areolatus, at Their Northeastern Range Limit

Abstract Crawfish Frogs (Lithobates areolatus) are a North American ranid, considered near threatened globally with populations in decline throughout their range. We studied populations of Crawfish Frogs on local and regional scales at their northeastern range limit to (1) assess the level of genetic diversity within populations, (2) estimate fine-scale genetic structure, and (3) estimate genetic differentiation between populations at the regional level. We used 10 microsatellite loci to genotype frogs collected from three regional sites in Indiana separated by 50–172 km and at one of these sites within a network of three breeding ponds <1 km apart. Heterozygosity estimates revealed high levels of diversity within these populations (mean HO: 0.54–0.67 per site), which is encouraging for future management. The degree of population subdivision was low at the regional level (FST = 0.071 for sites within 172 km). Genetic differentiation was related to geographic distance between sampling sites, as predicted by an isolation-by-distance model. We observed no genetic differentiation between individuals sampled from ponds approximately 250 m apart and slight divergence of individuals from a pond approximately 750 m away. This suggests ponds within 1 km form a genetically distinct single breeding unit composed of multiple subpopulations. Finally, we observed high genetic differentiation between southwest and southeast Indiana sites indicating historical (rather than recent) isolation of these sites. These data will be applied to a regional management plan in an attempt to recover Crawfish Frogs along the northeastern extreme of their range.

Development of 31 polymorphic microsatellite markers for the mole salamander (Ambystoma talpoideum) using Illumina paired-end sequencing

We isolated and characterized a total of 31 microsatellite loci from the mole salamander, Ambystoma talpoideum. Loci were screened in 20 individuals from a single location in Aiken, South Carolina. The number of alleles per locus ranged from 3 to 11, observed heterozygosity ranged from 0.000 to 0.700, and the probability of identity values ranged from 0.031 to 0.400. These new loci will provide tools for examining the genetic diversity, structure, mating system, and adult morph determination of A. talpoideum.

Development and characterization of microsatellite markers for Berberis thunbergii (Berberidaceae).

PREMISE OF THE STUDY Microsatellite markers were isolated and characterized in Berberis thunbergii, an invasive and ornamental shrub in the eastern United States, to assess genetic diversity among populations and potentially identify horticultural cultivars. METHODS AND RESULTS A total of 12 loci were identified for the species. Eight of the loci were polymorphic and were screened in 24 individuals from two native (Tochigi and Ibaraki prefectures, Japan) and one invasive (Connecticut, USA) population and 21 horticultural cultivars. The number of alleles per locus ranged from three to seven, and observed heterozygosity ranged from 0.048 to 0.636. CONCLUSIONS These new markers will provide tools for examining genetic relatedness of B. thunbergii plants in the native and invasive range, including phylogeographic studies and assessment of rapid evolution in the invasive range. These markers may also provide tools for examining hybridization with other related species in the invasive range.

Cryptic Diversity and Conservation of Gopher Frogs across the Southeastern United States

Identifying cryptic biodiversity is fundamental to evolutionary biology and to conservation efforts. This study investigated range-wide genetic diversity of Gopher Frogs, Lithobates capito, across the southeastern United States coastal plain to determine implications for taxonomy and conservation. We collected data for two mtDNA regions in 21 populations to identify genetic structure across the geographic distribution of the species. Based on population genetic, phylogenetic, and genealogical analyses, we recovered three reciprocally monophyletic mtDNA lineages corresponding to mainland coastal plain populations and two lineages within peninsular Florida. Breakpoints for these lineages did not occur in previously identified hotspots of amphibian phylogeographic breaks and did not follow currently recognized subspecies designations. We recommend these lineages be recognized as separate distinct population segments and be considered separately by the U.S. Fish and Wildlife Service for listing under the Endangered Species Act. Additionally, we propose an evolutionary hotspot for amphibians that deserves further attention.

Fourteen novel microsatellite markers for the gopher frog, Lithobates capito (Amphibia: Ranidae)

We isolated and characterized a total of 14 microsatellite loci from gopher frogs, Lithobates capito. This species is of conservation concern because most populations have gone locally extinct across the geographic distribution. Loci were screened for 21 individuals from a single population in Florida. The number of alleles per locus ranged from 7 to 17, observed heterozygosity ranged from 0.667 to 0.947, and the probability of identity values ranged from 0.011 to 0.077. These new loci provide tools for examining the genetic diversity and population structure of L. capito populations and addressing factors associated with their decline.

Patterns of amphibian infection prevalence across wetlands on the Savannah River Site, South Carolina, USA.

Amphibian diseases, such as chytridiomycosis caused by Batrachochytrium dendrobatidis (Bd) and ranaviral disease caused by ranaviruses, are often linked to global amphibian population declines, yet the ecological dynamics of both pathogens are poorly understood. The goal of our study was to determine the baseline prevalence, pathogen loads, and co-infection rate of Bd and ranavirus across the Savannah River Site (SRS) in South Carolina, USA, a region with rich amphibian diversity and a history of amphibian-based research. We tested over 1000 individuals, encompassing 21 amphibian species from 11 wetlands for both Bd and ranavirus. The prevalence of Bd across individuals was 9.7%. Using wetland means, the mean (±SE) Bd prevalence was 7.9 ± 2.9%. Among toad species, Anaxyrus terrestris had 95 and 380% greater odds of being infected with Bd than Scaphiopus holbrookii and Gastrophryne carolinensis, respectively. Odds of Bd infection in adult A. terrestris and Lithobates sphenocephalus were 75 to 77% greater in metal-contaminated sites. The prevalence of ranavirus infections across all individuals was 37.4%. Mean wetland ranavirus prevalence was 29.8 ± 8.8% and was higher in post-metamorphic individuals than in aquatic larvae. Ambystoma tigrinum had 83 to 85% higher odds of ranavirus infection than A. opacum and A. talpoideum. We detected a 4.8% co-infection rate, with individuals positive for ranavirus having a 5% higher occurrence of Bd. In adult Anaxyrus terrestris, odds of Bd infection were 13% higher in ranavirus-positive animals and odds of co-infection were 23% higher in contaminated wetlands. Overall, we found the pathogen prevalence varied by wetland, species, and life stage.