Microsatellite markers for the endangered Roanoke logperch, Percina rex (Percidae) and their potential utility for other darter species

The Roanoke logperch (Percina rex Jordan and Evermann), an endangered fish, occurs in only six watersheds in the Roanoke and Chowan river drainages of Virginia, USA. The species’ population genetic structure is poorly known. We developed 16 microsatellite markers that were reliably scorable and polymorphic P. rex. Markers were also screened in seven other darter species of the genus Percina. Most markers exhibited successful amplification and polymorphism in several species. These markers may therefore prove useful for population genetic studies in other darters, a diverse but highly imperiled group.     

Comparison of visual survey and seining methods for estimating abundance of an endangered,benthic stream fish

We compared visual survey and seining methods for estimating abundance of endangered Okaloosa darters, Etheostoma okaloosae, in 12 replicate stream reaches during August 2001. For each 20-m stream reach, two divers systematically located and marked the position of darters and then a second crew of three to five people came through with a small-mesh seine and exhaustively sampled the same area. Visual surveys required little extra time to complete. Visual counts (24.2 ± 12.0; mean ± one SD) considerably exceeded seine captures (7.4 ± 4.8), and counts from the two methods were uncorrelated. Visual surveys, but not seines, detected the presence of Okaloosa darters at one site with low population densities. In 2003, we performed a depletion removal study in 10 replicate stream reaches to assess the accuracy of the visual survey method. Visual surveys detected 59% of Okaloosa darters present, and visual counts and removal estimates were positively correlated. Taken together, our comparisons indicate that visual surveys more accurately and precisely estimate abundance of Okaloosa darters than seining and more reliably detect presence at low population densities. We recommend evaluation of visual survey methods when designing programs to monitor abundance of benthic fishes in clear streams, especially for threatened and endangered species that may be sensitive to handling and habitat disturbance.     

Microsatellite DNA markers for the rainbow darter,Etheostoma caeruleum (Percidae), and their potential utility for other darter species

The rainbow darter Etheostoma caeruleum is a small fish in the perch family (Percidae) that is adapted to fast‐flowing streams in eastern North America. It is relatively sensitive to habitat degradation and is widely used as a sentinel of stream condition. To provide a complementary tool for assessing the integrity of stream ecosystems, 16 highly polymorphic tetranucleotide microsatellite markers were identified for these darters. Between four and 16 loci were found to be useful in five congeneric species. These markers will be useful for characterizing population genetic structure and diversity of rainbow darters and related fishes.     

Role of drainage and barriers in the genetic structuring of a tessellated darter population

While population genetic structuring is easily identified, the causes of the structure can be difficult to determine. Habitat fragmentation in aquatic systems has often been identified as a major source of increased population structure and decreased genetic diversity in fish, including benthic resident species such as darters. However, these findings are often not replicated across natural and manmade barriers and come from endangered or threatened populations where the genetic structure is likely already compromised due to small population size. To evaluate the factors involved in structuring a healthy darter population, we genotyped 506 tessellated darters from 18 sites in three different river drainages and one large lake. Sites were all in the same watershed but separated from one another by one or more of three different types of barriers: dams, natural fall lines and causeways. We found that while diversity and allele frequency varied largely by drainage, within drainage variation was minimal even across multiple barriers. No single barrier type appeared to be more formidable than any other. Our results indicate that healthy populations of darters may naturally be structured by drainage, but likely disperse across barriers enough to retain drainage-wide homogeneity.     

Genetic diversity and divergence in the fountain darter (<Emphasis Type="Italic">Etheostoma fonticola</Emphasis>): implications for conservation of an endangered species

The endangered fountain darter Etheostoma fonticola is found only in the Comal and San Marcos rivers in the Guadalupe River basin in central Texas, USA. Comal River fountain darters were believed to be extirpated following a severe drought in the 1950s and were reintroduced in the early 1970s using 457 darters from the San Marcos River. In this study we used 23 microsatellite loci to describe and evaluate the genetic diversity, population structure and effective population size (N _e) of fountain darters. We also evaluated the genetic effect of the Comal River reintroduction and the influence of low-head dams (dams) on dispersal in both rivers. Bayesian analysis of individual genotypes and Analysis of Molecular Variation supported two distinct populations concordant with the two rivers. Estimates of N _e were much smaller (<10 %) than census size (N _c) in both rivers but did not indicate the populations are at risk of an immediate and rapid loss of genetic diversity. Coalescent-based estimates of the genetically effective number of founders (Nf) for the Comal River averaged about 49 darters and, together with the indices of genetic diversity and the bottleneck test (heterozygosity excess) results, were consistent with a founder event following the reintroduction in the Comal River. Finally, our results regarding the influence of dams on fountain darter dispersal were equivocal and did not support a conclusion. We recommend this issue be examined further as part of the fountain darter recovery program.     

Isolation and identification of eight microsatellite loci in the Cherokee darter (Etheostoma scotti) and their variability in other members of the genera Etheostoma, Ammocrypta, and Percina

The Cherokee darter Etheostoma scotti is a federally threatened fish endemic to the Etowah River system of northwest Georgia. In order to analyse the population structure and genetic diversity of this fish, eight tetranucleotide microsatellite genetic markers were developed. The marker set was applied to 13 additional darter species to test cross-species amplification and polymorphism. Successful amplification was obtained for all eight loci in each of the 13 other species of darters, with between seven and eight polymorphic loci per species.     

Regional population structure of the endangered Bridle Shiner (<Emphasis Type="Italic">Notropis bifrenatus</Emphasis>)

In the last 100 years, the Bridle Shiner has declined over significant parts of its range. We used mitochondrial and nuclear microsatellite markers to investigate population structure of this species. Tissue samples were collected from populations in six drainages in PA, NJ, NY and CT. One predominant haplotype was observed in the Delaware, Housatonic, Passaic, and Raritan specimens. Specimens from the Hudson and St. Lawrence drainages had a separate unique haplotype. Microsatellite loci revealed low within-population genetic diversity and pairwise population comparisons of allelic divergence showed significant genetic differences among most drainages. Population structure analysis of microsatellite loci recover the same population clusters inferred using mtDNA. Within the Delaware drainage, there was significant differentiation among areas separated by 128 km. Several alternate scenarios of population divergence and population bottlenecks were investigated using approximate Bayesian computing. These supported a scenario with a bottleneck occurring in the ancestral population of Bridle Shiner followed by recent divergence of Northern and Southern drainages.     

Microhabitat estimation of an imperiled headwater fish,the Yazoo darter (<Emphasis Type="Italic">Etheostoma raneyi</Emphasis>), in coastal plain streams

Headwater fishes in the southeastern United States make up much of the fish biodiversity of the region yet many are imperiled. Despite this, the specific habitat requirements of imperiled headwater fishes in lowland Coastal Plain streams have rarely been quantified. Using data collected over three years of seasonal sampling we provide estimates of the microhabitat requirements of the imperiled Yazoo darter (Etheostoma raneyi Suttkus and Bart), a small benthic insectivore. Our results indicate that the species is a microhabitat specialist and that optimum microhabitat within degraded contemporary streams consists of a narrow range of water depths (about 20–30 cm), current velocity ≥ 0.25 m·s^?1, complex stable debris piles, rooted macrophytes, and likely coarse substrate. No pronounced or generalized seasonal shifts in microhabitat use occurs, and no evidence exists for intraspecific partitioning of microhabitat. Though stable and complex instream cover is one of the most important variables explaining variation in microhabitat use by Yazoo darters, such cover is rare in the degraded streams within the range of the species. Current conservation classifications of the Yazoo darter by governmental agencies and nongovernmental organizations as well as associated management plans that are based on the assumption that Yazoo darters are habitat generalists should be reviewed in recognition of the increased risk of decline because Yazoo darters are microhabitat specialists. These considerations should also be extended to other closely related imperiled species of snubnose darters.     

PERMANENT GENETIC RESOURCES: Microsatellite DNA primers for the candy darter, Etheostoma osburni and variegate darter, Etheostoma variatum, and cross-species amplification in other darters (Percidae)

In order to investigate a potential hybrid zone between the candy darter, Etheostoma osburni, and variegate darter, Etheostoma variatum, and examine population variation within E. osburni, a suite of primers for 15 polymorphic microsatellite loci were developed. The average number of alleles per locus was 5.5 in E. osburni and 7.6 in E. variatum, and the average observed heterozygosities were 62.5% and 71.4%, respectively. There were no deviations from Hardy-Weinberg equilibrium and no observed linkage disequilibrium after Bonferroni correction. The utility of these primers was also tested in 11 species of darters representing all four genera of darters. Success of cross-species amplification was largely consistent with phylogenetic relationships of darters.     

Phylogeography of the cyprinid Squalius aradensis and implications for conservation of the endemic freshwater fauna of southern Portugal

The Iberian cyprinid fauna, characterized by the presence of numerous endemic species, has suffered from significant habitat degradation. The critically endangered Squalius aradensis is restricted to small drainages of southern Portugal, habitats that typically exhibit a characteristic Mediterranean-type heterogeneous hydrological system throughout the year, including alternation of flooding events during winter and complete drought in large river sections during summer. To assess the effect of historical and recent processes on genetic diversity in S. aradensis we examined within- and among-population variability in cytochrome b and six polymorphic microsatellite loci. Estimates of genetic diversity in time and space through the combined use of traditional Phi-/F-statistics, phylogenetic trees, ordination methods and nested clade analysis indicated significant and congruent structuring among populations. Data suggest that the Arade drainage represent the evolutionary centre of the species, with subsequent allopatric fragmentation across drainages. Factors other than isolation by distance strongly affected the within-drainage genetic differentiation observed in these Mediterranean-type drainages, including recent population expansion from a bottleneck event and restricted gene flow imposed by a long-term barrier (brackish water area). Significant correlation was found between S. aradensis allelic diversity and upstream drainage area. The relevance of findings for conservation issues is discussed in relation to local intermittent hydrological conditions, the highly restricted distribution and the critically endangered status of the species.     

Life history of the gulf darter,Etheostoma swaini (Pisces: Percidae)

Synopsis Etheostoma swaini, the gulf darter, was collected from the Black Creek drainage in southern Mississippi (February 1978 – April 1979). The gulf darter generally inhabits small- to moderate-size creeks and occurs over a sand or sandy mud bottom, often in association with aquatic vegetation or a layer of organic debris. Larval dipterans were the most important food items, both numerically and volumetrically. Chironomids were found in 71–100% of the stomachs in all except the unusual March 16 collection. The length frequency distribution and the scale annuli analysis indicated there were three year-classes present in the population at any one time. Fifty-one percent of the specimens taken were less than 12 months old. During the mid-February to late March spawning season gulf darters were most often collected over clean gravel or gravelsand substrates. Laboratory observations suggest that the female burrows into the gravel where the demersal, adhesive eggs are deposited. Female gulf darters significantly outnumbered males at a ratio of 59:41.     

Characterization of eight polymorphic microsatellite DNA markers for the greenside darter,Etheostoma blennioides (Percidae)

The greenside darter, Etheostoma blennioides is a small benthic fish found in fast‐flowing streams in eastern North America. In Canada, this species is native to three, and introduced into one, Great Lakes tributaries in southwestern Ontario. It is currently listed as a species of Special Concern. To characterize population genetic structure and diversity in the Canadian populations of greenside darter, eight polymorphic microsatellite markers were developed for the species. The polymerase chain reaction primers were tested between 32 and 60 individuals from the Sydenham River and yielded a high number of alleles (four to 42 per locus), and observed heterozygosities ranging from 0.14 to 0.82.     

Spatial extent of analysis influences observed patterns of population genetic structure in a widespread darter species (Percidae)

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Population genetic characterization of the endangered silver skipper,Leptalina unicolor (Lepidoptera: Hesperiidae), using novel microsatellite markers and mitochondrial DNA gene sequences

The silver stripped skipper, Leptalina unicolor, is listed as an endangered species with a paucity of leftover populations in Korea. Prior population genetic analysis is essential for the establishment of effective conservation strategies. In the present study, we investigated the genetic diversity and the relationships of L. unicolor populations in Korea using 12 newly developed L. unicolor-specific microsatellite markers and two mitochondrial DNA (mtDNA) sequences (a total of 1283 bp from COI and CytB). Due to the endangered status and rarity, a total of 33 individuals from two localities in Gangwon-do Province (Seohwa and Girin) and one in Gyeongsangnam-do Province (Danjang) were sampled. The concatenated mtDNA sequences revealed very low genetic diversity in each population, with only one to three independent haplotypes per population. F_ST, principal coordinate analysis, and independent structural analysis of the concatenated mtDNA sequences and the 12 microsatellite loci developed in this study showed that each L. unicolor population in Korea is nearly completely isolated, although inbreeding has not yet occurred. Long-term conservation habitat recovery that increases the available population appears to be important in the prevention of genetic drift and inbreeding, which occur in small isolated populations and reduce the viability of populations under fluctuating environmental conditions.     

Microsatellite DNA primers for the endangered vermilion darter,Etheostoma chermocki,and cross‐species amplification in other darters (Percidae: Etheostoma)

The endangered vermilion darter (Etheostoma chermocki) is endemic to the Black Warrior River system in the Mobile Basin in Alabama. Restoration and conservation of this species require an understanding of its population genetic structure, which can be characterized using microsatellite DNA. Nine microsatellite loci were developed; eight loci were polymorphic. Although observed heterozygosity was lower than expected heterozygosity in most polymorphic loci, only one locus showed significant deviation from Hardy–Weinberg equilibrium. These nine markers were tested in an additional 24 species of Etheostoma and appear to have sufficient allelic variation to be useful in studies of population genetic structure.     

Molecular phylogeny of the snubnose darters, subgenus Ulocentra (genus Etheostoma, family Percidae)

Snubnose darters comprise one of the largest subgenera of the percid genus Etheostoma. Many species are described based on differences in male breeding coloration. Few morphological synapomorphies have been proposed for the subgenus and their relatives, making it difficult to delineate monophyletic clades. The phylogenetic relationships of the 20 snubnose darter species of the subgenus Ulocentra and 11 members of its proposed sister subgenus Etheostoma were investigated with partial mitochondrial DNA sequences including 1033 bp encompassing the entire mitochondrial control region, the tRNA-Phe gene, and part of the 12S rRNA gene. Two hypotheses on the relationship and monophyly of the two subgenera were evaluated. Both maximum-parsimony and neighbor-joining analyses supported monophyly of the subgenus Ulocentra and resolved some species-level relationships. The banded darter, E. zonale, and its sister taxon, E. lynceum, were not closely related to the snubnose darters and appear to be diverged from the other members of the subgenus Etheostoma, fitting their former distinction as the recognized subgenus Nanostoma. The sister group to Ulocentra appears to be a restricted species assemblage within the subgenus Etheostoma containing E. blennioides, E. rupestre, E. blennius, and the E. thalassinum species group. The placement of the harlequin darter, E. histrio, is problematic, and it may represent a basal member of Ulocentra or of the restricted subgenus Etheostoma. Despite recent estimates of divergence times between nominal Ulocentra taxa, each species exhibits its own unique set of mtDNA haplotypes, providing no direct evidence for current genetic exchange between species. The nominal taxa of snubnose darters thus appear to be evolving independently from each other and therefore constitute valid species under the Phylogenetic Species Concept.     

Unique and isolated: population structure has implications for management of the endangered New Zealand sea lion

Female otariids (eared seals) frequently display strong levels of philopatry, a behaviour that has the potential to influence population structure, particularly at the mitochondrial level. Conversely, male otariids often move between breeding colonies, likely facilitating nuclear gene flow between colonies. Such gender-specific movements have the potential to influence species population structure. Here we investigate the genetic population structure of the endangered New Zealand (NZ) sea lion, using nuclear (microsatellite) and mitochondrial molecular markers, with the intention to better inform conservation through identification of management units for the species. The strong levels of female philopatry in this species have potential to lead to population structure at the mitochondrial loci. In contrast, weak or no population structure is expected across nuclear loci. NZ sea lions were sampled from the main breeding areas across the species’ current distribution (three Auckland Islands sites, two Campbell Island sites, one Stewart Island site and one Otago Peninsula site). Individuals were screened for microsatellite (n?=?271; 16 loci) and mitochondrial (n?=?56; 1027 bp D-loop and 1189 bp cytb). Despite a small (c. 9880 individuals) population size, moderate levels of microsatellite variation are observed in the NZ sea lions, in contrast to low levels of mitochondrial genetic variation. Results from mitochondrial DNA analyses revealed no population structure, suggesting that the strong level of female philopatry in NZ sea lions alone is not sufficient to maintain genetic population structure. Due to the frequent male movements between breeding colonies, no population structure was detected across the nuclear loci either. The absence of genetic structure suggests that, from a genetic perspective, NZ sea lions can be considered to be a single population. Despite this, the differing impacts of threats (e.g. fisheries by-catch) to each individual breeding colony must also be taken into consideration when defining management units for this endangered species.     

Population structure and conservation genetic assessment of the endangered Pugnose Shiner,Notropis anogenus

The Pugnose Shiner is a small minnow with a fragmented distribution across the Great Lakes and Upper Mississippi River in North America. The species is listed federally as endangered in Canada, and in the United States its status varies by state, from Special Concern to Endangered (as well as Extirpated). We conducted a thorough genetic assessment of the Pugnose Shiner using both microsatellite loci and mitochondrial DNA collected for samples across the species range. Our results indicate high levels of population differentiation suggesting restricted dispersal, in some cases at very small geographical scales. We also found strong evidence of small effective population sizes and one case of a genetic bottleneck. Although significant range-wide genetic variation was observed in both microsatellite loci and mitochondrial DNA, the species is best characterized as a single evolutionarily significant unit for conservation purposes.     

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.     

Water availability strongly impacts population genetic patterns of an imperiled Great Plains endemic fish

Genetic, demographic, and environmental processes affect natural populations synergistically, and understanding their interplay is crucial for the conservation of biodiversity. Stream fishes in metapopulations are particularly sensitive to habitat fragmentation because persistence depends on dispersal and colonization of new habitat but dispersal is constrained to stream networks. Great Plains streams are increasingly fragmented by water diversion and climate change, threatening connectivity of fish populations in this ecosystem. We used seven microsatellite loci to describe population and landscape genetic patterns across 614 individuals from 12 remaining populations of Arkansas darter (Etheostoma cragini) in Colorado, a candidate species for listing under the U.S. Endangered Species Act. We found small effective population sizes, low levels of genetic diversity within populations, and high levels of genetic structure, especially among basins. Both at- and between-site landscape features were associated with genetic diversity and connectivity, respectively. Available stream habitat and amount of continuous wetted area were positively associated with genetic diversity within a site, while stream distance and intermittency were the best predictors of genetic divergence among sites. We found little genetic contribution from historic supplementation efforts, and we provide a set of management recommendations for this species that incorporate a conservation genetics perspective.