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At least 199 records · Page 11Linked to original sources

Environmental, morphological, and molecular data reveal a new species of freshwater mussel, Strophitus howellsi, endemic to the Edwards Plateau in Texas

Freshwater mussels are considered the most imperiled group of organisms in North America and systematics research has played an integral role in the development and implementation of their conservation. Despite the importance of systematics in conservation planning, the evolutionary relationships between many mussel taxa remain poorly explored, clearly illustrated by Strophitus undulatus . This species is wide-ranging, occurring in streams across the United States and Canada with a disjunct population in the Colorado River drainage in central Texas. The widespread distribution of S. undulatus , as well as high intraspecific morphological variation, has led previous authors to doubt the taxon is representative of a single species. In this study, we set out to investigate species boundaries in S. undulatus by integrating environmental, molecular, and morphological datasets. Molecular and morphological data supported S. undulatus from the Colorado River as distinct, which was supplemented by a species distribution modeling approach, suggesting potential adaptation to Edwards Plateau streams has contributed to speciation. Given our findings, we formally describe a new species of freshwater mussel, Strophitus howellsi , endemic to streams along the Edwards Plateau in the Colorado River drainage. A conservation assessment of S. howellsi suggests the species is extremely rare within a highly restricted distribution and may warrant future recovery actions. Our findings build on a growing body of literature highlighting aquatic endemism along the Edwards Plateau and have significant conservation implications for freshwater mussels in Texas.

Conservation Genetics

Development and characterization of 17 polymorphic microsatellite loci in the faucet snail, Bithynia tentaculata (Gastroposa: Caenogastropoda; Bithyniidae)

Bithynia tentaculata (Linnaeus, 1758), a snail native to Europe, was introduced into the US Great Lakes in the 1870's and has spread to rivers throughout the Northeastern US and Upper Mississippi River (UMR). Trematode parasites, for which B. tentaculata is a host, have also been introduced and are causing widespread waterfowl mortality in the UMR. Waterfowl mortality is caused by ingestion of trematode-infected B. tentaculata or insects infected with parasites released from the snails. We isolated and characterized 17 microsatellite loci from the invasive faucet snail, B. tentaculata (Gastropoda: Caenogastropoda: Bithyniidae). Loci were screened in 24 individuals of B. tentaculata. The number of alleles per locus ranged from 2 to 6, observed heterozygosity ranged from 0.050 to 0.783, and the probability of identity values ranged from 0.10 to 0.91. These new loci provide tools for examining the origin and spread of invasive populations in the US and management activities to prevent waterfowl mortality.

Conservation Genetics Resources

Tools for assessing kinship, population structure, phylogeography, and interspecific hybridization in Asian carps invasive to the Mississippi River, USA: isolation and characterization of novel tetranucleotide microsatellite DNA loci in silver carp Hypophthalmichthys molitrix

We document the isolation and characterization of novel tetranucleotide microsatellite DNA markers for the invasive silver carp Hypophthalmichthys molitrix and provide the results of cross-species amplification for three additional invasive carp species: bighead (H. nobilis), grass (Ctenopharyngodon idella) and black (Mylopharyngodon piceus). In the target species these markers yielded levels of allelic diversity (average 4.4 alleles/locus) and heterozygosity (average 54.7%) sufficient to: (1) provide unique multilocus genotypes; (2) delineate kinship relationships; (3) differentiate populations/species; (4) estimate effective population sizes; and (5) provide unique demographic perspectives for control or eradication. Currently these markers are being utilized to determine the degree of introgressive hybridization between H. molitrix and H. nobilis, to quantify gene flow between different sub-basins established in the central United States, and to assess the demographic status of sub-basin groups. This information will be critically important in the management/control of these invasive species.

Mississippi River

Isolation of microsatellite loci from the lesser long-nosed bat (Leptonycteris yerbuenae)

Leptonycteris yerbabuenae is a nectarivore (subfamily: Glossophaginae, family: Phyllostomidae), is found from southern Arizona/southwestern New Mexico to southern Mexico including the Baja California peninsula (Ceballos et al.1997; Cockrum 1991). Leptonycteris yerbabuenae is listed as endangered in the United States (Shull 1988) and threatened in Mexico (SEMARNAT 2002). They migrate up to 1,800 km between wintering and breeding grounds (Fleming and Eby 2001). Females mate in the winter in southern Mexico, and migrate to maternity roosts in northern Mexico/southern Arizona to give birth in late spring-early summer (Ceballos et al.1997; Cockrum 1991). Wilkinson and Fleming (1996) have suggested two separate migration corridors where bats occupy southeastern Arizona and southwestern Arizona arrive and leave through separate corridors. We isolated 12 microsatellites loci to estimate gene flow between southwestern and southeastern Arizona roosts, as well as Sonora, Baja California, and Jalisco, Mexico. Genomic DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen). Approximately 5 μg of genomic DNA was digested with RsaI (NEB) and fragments were ligated to double-stranded SuperSNX-24 linkers to construct an enriched genomic DNA library using a modified version of Glenn and Schable (2005). Linker-ligated fragments ranging from 300–1400 bp were recovered using the polymerase chain reaction (PCR) with a SuperSNX-24 forward primer and Platinum high-fidelity Taq DNA polymerase (Invitrogen), and were hybridized to 5′-biotinylated microsatellite oligonucleotide probes (GT) 15 , (CT) 15 , (GATA) 10 and (GACA) 8 . Hybridized fragments were captured on streptavidin-coated paramagnetic beads (Dynal) and then recovered by PCR. These fragments were ligated into the vector PCR4-TOPO (Invitrogen), and transformed into TOP10 chemically competent E. coli cells (Invitrogen) following the manufacturer’s protocol.

Conservation Genetics Resources

New microsatellite loci isolated via next-generation sequencing for two endangered pronghorn from the Sonoran Desert

We isolated 16 novel microsatellite loci in two subspecies of endangered desert pronghorns (Antilocapra americana sonoriensis and Antilocapra americana peninsularis) using a shotgun pyrosequencing approach. All and 87.5 % of the loci were polymorphic within each subspecies, respectively. The mean number of alleles per locus was 4.86 (range 2–8) and 2.5 alleles per locus (range 1–4 alleles), and observed heterozygosity ranged from 0.13 to 0.78 (mean 0.48) and 0.00 to 0.61 (mean 0.31), respectively. We did not find significant linkage disequilibrium among loci pairs and only one locus deviated significantly from Hardy–Weinberg equilibrium in peninsularis.

Conservation Genetics Resources

Development and characterization of 21 polymorphic microsatellite markers for the barren-ground shrew, Sorex ugyunak (Mammalia: Sorcidae), through next-generation sequencing, and cross-species amplification in the masked shrew, S. cinereus

We used next generation shotgun sequencing to develop 21 novel microsatellite markers for the barren-ground shrew ( Sorex ugyunak ), which were polymorphic among individuals from northern Alaska. The loci displayed moderate allelic diversity (averaging 6.81 alleles per locus) and heterozygosity (averaging 70 %). Two loci deviated from Hardy–Weinberg equilibrium (HWE) due to heterozygote deficiency. While the population did not deviate from HWE overall, it showed significant linkage disequilibrium suggesting this population is not in mutation-drift equilibrium. Nineteen of 21 loci were polymorphic in masked shrews ( S. cinereus ) from interior Alaska and exhibited linkage equilibrium and HWE overall. All loci yielded sufficient variability for use in population studies.

Conservation Genetics Resources

Characterization of microsatellite loci from two-spotted octopus Octopus bimaculatus Verrill 1883 from pyrosequencing reads

We characterized 22 novel microsatellite loci in the two-spotted octopus Octopus bimaculatus using 454 pyrosequencing reads. All loci were polymorphic and will be used in studies of marine connectivity aimed at increasing sustainability of the resource. The mean number alleles per locus was 13.09 (range 7–19) and observed heterozygosities ranged from 0.50 to 1.00. Four loci pairs were linked and three deviated from Hardy–Weinberg equilibrium. Eighteen and 12 loci were polymorphic in Octopus bimaculoides and Octopus hubbsorum , respectively.

Conservation Genetics Resources

Development and characterization of polymorphic microsatellitemarkers for the crested caracara, Caracara cheriway

We isolated novel microsatellites from the crested caracara ( Caracara cheriway ) with a shotgun pyrosequencing approach. We tested 80 loci for polymorphism among 20 individuals from the threatened Florida population. Fourteen loci were polymorphic. The mean number of alleles was 2.21 (range 2–3) and the mean observed heterozygosity was 0.41 (range 0.15–0.65). None of the 14 polymorphic loci exhibited significant linkage disequilibrium nor did they deviate significantly from Hardy–Weinberg expectations. We also report 16 monomorphic loci.

Florida

Development of 20 TaqMan assays differentiating the endangered shortnose and Lost River suckers

Accurate species identification is vital to conservation and management of species at risk. Species identification is challenging when taxa express similar phenotypic characters and form hybrids, for example the endangered shortnose sucker ( Chasmistes brevirostris ) and Lost River sucker ( Deltistes luxatus ). Here, we developed 20 Taqman assays that differentiate these species (19 nuclear DNA and one mitochondrial DNA). Assays were evaluated in 160 young-of-the-year identified to species using meristic counts. Alleles were not fixed between species, but species were highly differentiated ( F ST = 0.753, P < 0.001). The assays developed herein will be a valuable tool for resource managers.

Conservation Genetics Resources

A probe-based quantitative PCR assay for detecting Tetracapsuloides bryosalmonae in fish tissue and environmental DNA water samples

A probe-based quantitative real-time PCR assay was developed to detect Tetracapsuloides bryosalmonae , which causes proliferative kidney disease in salmonid fish, in kidney tissue and environmental DNA (eDNA) water samples. The limits of detection and quantification were 7 and 100 DNA copies for calibration standards and T. bryosalmonae was reliably detected down to 100 copies in tissue and eDNA samples. The assay presented here is a highly sensitive and quantitative tool for detecting T. bryosalmonae with potential applications for tissue diagnostics and environmental detection.

Conservation Genetics Resources

Ethanol and sodium acetate as a preservation method to delay degradation of environmental DNA

Environmental DNA (eDNA) samples that are collected from remote locations depend on rapid stabilization of the DNA. The degradation of eDNA in water samples is minimized when samples are stored at ≤ 4 °C. Developing a preservation technique to maintain eDNA integrity at room temperature would allow a wider range of locations to be sampled. We evaluated an ethanol and sodium acetate solution to maintain the integrity of the DNA samples for the time between collection and lab testing. For this evaluation, replicate water samples taken from a tank housing Asian carp were placed on ice or held at room temperature. At both temperatures, water samples were left untreated or were preserved with an ethanol and sodium acetate solution (EtOH–NaAc). Every day for 6 days following collection, a subset of the samples was removed from each preservation method and DNA was extracted and nuclear and mitochondrial markers were assayed with qPCR. Results showed comparable persistence of DNA between iced samples without the EtOH–NaAc treatment and samples that received EtOH–NaAc treatment that were kept at room temperature. We found that DNA can be amplified from preserved samples using an EtOH–NaAc solution after up to 7 days at room temperature.

Conservation Genetics Resources

Using redundant primer sets to detect multiple native Alaskan fish species from environmental DNA

Accurate and timely data regarding freshwater fish communities is important for informed decision-making by local, state, tribal, and federal land and resource managers; however, conducting traditional gear-based fish surveys can be an expensive and time-consuming process, particularly in remote areas, like those that characterize much of Alaska. To help address this challenge, we developed and tested five multi-species environmental DNA (eDNA) primer sets for the simultaneous detection of up to 37 target fish species in a single sample. Using these primer sets can reduce the cost and time needed to perform future studies of fish communities. Our results comparing multiple samples from multiple lakes and streams using multiple next-generation sequencing runs show the efficacy and reproducibility of these primers.

Alaska

Development of two quantitative PCR assays for detection of several Cottus species from environmental DNA in Pacific coast watersheds of North America

We developed two quantitative PCR assays for use with environmental DNA (eDNA) to detect numerous species in the genus Cottus that are indigenous to the Pacific coast watersheds of North America. We conducted in vitro assay validations on ten Cottus species and 32 potentially co-occurring non- Cottus species. We demonstrate the efficacy of these assays by field testing eDNA samples collected from streams inhabited by Cottus . These assays will be particularly useful for detecting Cottus in habitats where one (or more) of several Cottus species could be present.

Pacific Northwest

Development of a quantitative PCR assay for detecting Egeria densa in environmental DNA samples

Brazilian elodea ( Egeria densa ) is an invasive freshwater plant that demonstrates widespread ecological impacts in freshwater ecosystems and causes substantial economic damage. Here, we developed an environmental DNA assay for detection of E. densa to provide resource managers with a tool for early detection, identification, and monitoring of invasive populations.

Conservation Genetics Resources

A conservation-oriented SNP panel for Smallmouth Bass (Micropterus dolomieu), with emphasis on Interior Highlands lineages

The Smallmouth Bass ( Micropterus dolomieu ; SMB) is a widely distributed black bass species, but the southwestern edge of the species range within the Interior Highlands contains some of the most divergent ecotypes. The Neosho subspecies ( M. d. velox ) inhabits tributaries of the Arkansas River within the Ozark Mountains and a second lineage is reported from drainages of the Ouachita Mountains. We sought to develop a single nucleotide polymorphism (SNP) panel to (1) diagnose hybridization with sympatric Spotted Bass ( Micropterus punctulatus ; SPB) and non-native Northern SMB ( M. d. dolomieu ) stocked in the region, and (2) delineate population structure within the ranges of the Neosho and Ouachita SMB lineages. We obtained 76 individual SMB samples from across their range but concentrated within the Interior Highlands ( n = 50). We also included 3 SPB to allow for hybrid detection and 3 Shoal Bass ( Micropterus cataractae ) as an outgroup. Phylogenetic trees constructed with the generated SNP data corroborated the existence of at least three major lineages of SMB (Northern, Neosho, and Ouachita), each containing varying degrees of differentiation among major drainages. Simulation analyses revealed that chosen SNPs had high power (> 0.9) to assign SMB × SPB hybrid categories and similarly high power (> 0.8) for Northern SMB × Interior Highlands SMB hybrids. Clustering methods delineated major inter-basin population structure within the native ranges of Neosho and Ouachita SMB with chosen SNPs. Anticipated uses of the resulting 192-loci SNP panel include conservation planning, fisheries management assessments, and ecological investigations of the Neosho and Ouachita SMB lineages.

Arkansas, Missouri, Oklahoma

Development of a ddPCR assay for the detection of the Smoky Madtom (Noturus baileyi) from eDNA in stream water samples

The Smoky Madtom Noturus baileyi is a federally endangered species, whose native distribution includes lower Abrams Creek in Great Smoky Mountains National Park (GRSM) and Citico Creek in nearby Cherokee National Forest. Due to challenges for bio-monitoring posed by its nocturnality and cryptic life history, an environmental DNA (eDNA)-based approach for detection would be useful to complement existing electrofishing and seining efforts to better understand the distribution of this species. We developed a probe-based droplet digital PCR (ddPCR) assay to detect Smoky Madtoms from non-invasively collected water samples. The assay was specific to N. baileyi and did not amplify concentrated genomic DNA of 16 co-occurring or regional fish species, including the yellowfin madtom N. flavipinnis and stonecat N. flavus . The assay limit of detection (LOD) was determined to be 4.18 copies (95% CI: 3.95, 4.41). Several 2 L water samples collected from throughout various streams in GRSM in 2016 and 2017 were tested for the presence of N. baileyi using the ddPCR assay. N. baileyi was detected at two different sites in 2016 and 2017 within Abrams Creek previously known to contain N. baileyi , but no novel detections in other sampled streams were observed. This assay should prove useful for continued surveys of N. baileyi in GRSM.

Great Smoky Mountains National Park

Validation of a species-specific probe-based qPCR assay for the threatened meltwater stonefly, Lednia tumana, in environmental samples

A probe-based quantitative real-time PCR assay was developed to detect meltwater stonefly ( Lednia tumana ) environmental (e)DNA in water samples. The limits of detection and quantification, respectively, were 12.1 and 58.4 gene copies for calibration standards and these values were similarly low in a relevant environmental sample matrix (8.6 and 174.2, respectively). The assay’s utility was demonstrated in situ on water samples with concomitant manual invertebrate surveys from a wide range of alpine streams across L. tumana ’s native range.

Conservation Genetics Resources

Development of a quantitative PCR assay for detecting northwest salamander (ambystoma gracile) in environmental DNA samples

We developed a primer and probe based quantitative PCR assay for use with environmental DNA to detect Northwest salamander ( Ambystoma gracile ), a species endemic to the temperate Pacific coastal region of North America. The assay targets a region in the mitochondrial DNA (mtDNA) D-loop gene. Tests of the assay were performed in silico (using the NCBI BLAST tool), in vitro (using DNA extracted from A. gracile and related species) and in situ (water samples collected from a lake with a known population of A. gracile ). This assay will be useful in efforts to monitor the distribution and occupancy of this species.

Conservation Genetics Resources