Gregory R. Moyer

Permanent Genetic Resources added to Molecular Ecology Resources Database 1 August 2009-30 September 2009

This article documents the addition of 229 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Acacia auriculiformis × Acacia mangium hybrid, Alabama argillacea, Anoplopoma fimbria, Aplochiton zebra, Brevicoryne brassicae, Bruguiera gymnorhiza, Bucorvus leadbeateri, Delphacodes detecta, Tumidagena minuta, Dictyostelium giganteum, Echinogammarus berilloni, Epimedium sagittatum, Fraxinus excelsior, Labeo chrysophekadion, Oncorhynchus clarki lewisi, Paratrechina longicornis, Phaeocystis antarctica, Pinus roxburghii and Potamilus capax. These loci were cross‐tested on the following species: Acacia peregrinalis, Acacia crassicarpa, Bruguiera cylindrica, Delphacodes detecta, Tumidagena minuta, Dictyostelium macrocephalum, Dictyostelium discoideum, Dictyostelium purpureum, Dictyostelium mucoroides, Dictyostelium rosarium, Polysphondylium pallidum, Epimedium brevicornum, Epimedium koreanum, Epimedium pubescens, Epimedium wushanese and Fraxinus angustifolia.

Assessing Environmental DNA Detection in Controlled Lentic Systems

Little consideration has been given to environmental DNA (eDNA) sampling strategies for rare species. The certainty of species detection relies on understanding false positive and false negative error rates. We used artificial ponds together with logistic regression models to assess the detection of African jewelfish eDNA at varying fish densities (0, 0.32, 1.75, and 5.25 fish/m3). Our objectives were to determine the most effective water stratum for eDNA detection, estimate true and false positive eDNA detection rates, and assess the number of water samples necessary to minimize the risk of false negatives. There were 28 eDNA detections in 324, 1-L, water samples collected from four experimental ponds. The best-approximating model indicated that the per-L-sample probability of eDNA detection was 4.86 times more likely for every 2.53 fish/m3 (1 SD) increase in fish density and 1.67 times less likely for every 1.02 C (1 SD) increase in water temperature. The best section of the water column to detect eDNA was the surface and to a lesser extent the bottom. Although no false positives were detected, the estimated likely number of false positives in samples from ponds that contained fish averaged 3.62. At high densities of African jewelfish, 3–5 L of water provided a >95% probability for the presence/absence of its eDNA. Conversely, at moderate and low densities, the number of water samples necessary to achieve a >95% probability of eDNA detection approximated 42–73 and >100 L, respectively. Potential biases associated with incomplete detection of eDNA could be alleviated via formal estimation of eDNA detection probabilities under an occupancy modeling framework; alternatively, the filtration of hundreds of liters of water may be required to achieve a high (e.g., 95%) level of certainty that African jewelfish eDNA will be detected at low densities (i.e., <0.32 fish/m3 or 1.75 g/m3).

Linkage disequilibrium and effective population size when generations overlap

Estimates of effective population size are critical for species of conservation concern. Genetic datasets can be used to provide robust estimates of this important parameter. However, the methods used to obtain these estimates assume that generations are discrete. We used simulated data to assess the influences of overlapping generations on the estimates of effective size provided by the linkage disequilibrium (LD) method. Our simulations focus on two factors: the degree of reproductive skew exhibited by the focal species and the generation time, without considering sample size or the level of polymorphism at marker loci. In situations where a majority of reproduction is achieved by a small fraction of the population, the effective number of breeders can be much smaller than the per‐generation effective population size. The LD in samples of newborns can provide estimates of the former size, while our results indicate that the latter size is best estimated using random samples of reproductively mature adults. Using samples of adults, the downwards bias was less than approximately 15% across our simulated life histories. As noted in previous assessments, precision of the estimate depends on the magnitude of effective size itself, with greater precision achieved for small populations.

Rapid development of molecular resources for a freshwater mussel, Villosa lienosa (Bivalvia:Unionidae), using an RNA-seq-based approach

Abstract.  Freshwater mussels (Unionidae) are among the most endangered groups of organisms in the world, and their conservation and recovery are priorities throughout North America, especially the southeastern USA. We used a ribonucleic acid sequencing (RNA-seq)-based approach to develop molecular resources for Villosa lienosa, the little spectaclecase. We sequenced barcoded samples (Illumina HiSeq 2000 platform) and assembled (Trans-ABySS) 778,234 contigs (average length  =  707.5 base pairs [bp]) from 162 million filtered reads. We identified 23,742 unigene hits against the National Center for Biotechnology Information nonredundant database and 36,582 microsatellites with sufficient flanking sequence for primer design. Microsatellite validation indicated a 36% polymorphic rate (16/44 tested markers) in the tested population (26 individuals; mean  =  5 alleles/marker). Analysis of differentially expressed genes between heat-stressed and untreated controls enabled us to identify 604 genes involved in stress-response pathways. Real-time polymerase chain reaction validation of gene-expression results using individual samples confirmed RNA-seq patterns (r  =  0.847, p < 0.001). RNA-seq is a powerful tool for rapid development of molecular resources in nonmodel species, and our study is the first large-scale transcriptome project in freshwater mussels. The validated microsatellite set and stress-associated genes are being used in parentage analysis and health-assessment surveys to support mussel conservation.

Isolation and characterization of microsatellite loci for alligator gar (Atractosteus spatula) and their variability in two other species (Lepisosteus oculatus and L-osseus) of Lepisosteidae

We report on the isolation of 17 polymorphic microsatellite loci from alligator gar (Atractosteus spatula), a large‐bodied species that has experienced population declines across much of its range. These loci possessed 2–19 alleles and observed heterozygosities of 0–0.974. All loci conformed to Hardy–Weinberg equilibrium expectations, and none exhibited linkage disequilibrium. Nine and eight of these loci were found to be polymorphic in the related species Lepisosteus oculatus and L. osseus, respectively. These microsatellite loci should prove useful in conservation efforts of A. spatula through the study of population structure and hatchery broodstock management.

Population structure and genetic diversity in the endangered bluemask darter (Etheostoma akatulo)

Bluemask darters (Etheostoma akatulo) were sampled from the four drainages where extant populations of this narrowly endemic freshwater fish are known to exist. Population genetic diversity and structure were assessed at 10 microsatellite loci. All populations exhibited low levels of genetic variation, with expected heterozygosity ranging from 0.2 to 0.35. Significant population subdivision was found among most tributaries, and genetic divergence was strongly correlated with geographic distance. Bayesian population assignment and pairwise population differentiation measures both identified a lack of differentiation between E. akatulo populations inhabiting Cane Creek and the Caney Fork. This observation reduced the number of distinct breeding populations of this species to three. We also used approximate Bayesian computation to compare three models of demographic history in this species. A constant population size model was favored over models that included historic or recent population reductions. Our results suggest that impoundment of the Caney Fork and its tributaries, by completion of Great Falls Dam in 1916, was not responsible for the reduced genetic diversity in the sampled populations. Given the low levels of genetic diversity within populations and the limited geographic distribution, future conservation efforts should seek to maximize available habitat while simultaneously limiting the influences of anthropogenic stressors in the system.

Genetic Evaluation of a Conservation Hatchery Program for Reintroduction of Sicklefin Redhorse Moxostoma sp. in the Tuckasegee River, North Carolina

Abstract Restoration and reintroduction efforts for the sicklefin redhorse Moxostoma sp. have been initiated by state, tribal, and federal agencies owing to the limited geographic distribution of this species and threats associated with the physical alteration of its habitat. A critical component of a successful reintroduction is that the source and recipient populations have similar genetic resources and life history patterns. We used 10 microsatellite loci to estimate and compare indices of genetic diversity between the Little Tennessee River population of wild adults and the hatchery broodstock being used for initial reintroduction efforts. We also compared relatedness values for the broodstock used for restoration efforts. There were no significant differences between hatchery broodstock and wild adults with respect to average gene diversity, but the average number of alleles for each brood year was significantly less than that for wild adults. While this trend persisted when the 2007 and 2008 brood y...

Isolation and characterization of microsatellite loci in the federally endangered fat threeridge mussel (Amblema neislerii)

We isolated and characterize 16 microsatellite loci from Amblema neislerii, a federally endangered freshwater mussel. Fifteen of 16 loci were polymorphic with an average of 11.62 alleles per locus and an average observed and expected heterozygosity of 0.63 and 0.75, respectively. Fourteen of 16 loci conformed to Hardy–Weinberg expectations and all pairs of loci showed no significant genotypic disequilibrium. These microsatellite loci will serve as important tools in ongoing conservation efforts of A. neislerii.

Identification of endangered Alabama lampmussel (Lampsilis virescens) specimens collected in the Emory river, Tennessee, USA via DNA barcoding

The federally endangered Alabama lampmussel (Lampsilis virescens) has been presumed extirpated in the Emory river, TN for almost four decades. In the fall of 2011, three specimens that were morphologically identified as L. virescens (based on external shell morphology) were collected from the Emory river. The significance of such a find led biologists to take a noninvasive tissue sample from each individual for genetic confirmation. Approximately 400 nucleotides of the mtDNA COI gene were sequenced for each individual along with two samples of L. virescens from the Paint Rock river. DNA sequence data was also obtained from Genbank for other Lampsilis sp. Maximum parsimony and Bayesian phylogenetic methods revealed strong support for a clade consisting of putative Emory river L. virescens and known L. virescens (Paint Rock river origin) that was sister to all other taxa. These results indicated that the individuals collected from the Emory river were indeed L. virescens.

Isolation and characterization of microsatellite loci for the oval pigtoe mussel, Pleurobema pyriforme (Lea 1857)

We isolated nine microsatellite loci from Pleurobema pyriforme, a freshwater mussel species currently protected under the Endangered Species Act. Eight loci were polymorphic with 2–15 alleles and observed heterozygosities of 0.130–0.957. All loci conformed to Hardy–Weinberg expectations and all pairs of loci showed no significant genotypic disequilibrium. These are the first isolated microsatellite loci for this species and will serve in ongoing conservation efforts of P. pyriforme.