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Molecular genetics at the Fort Collins Science Center

The Fort Collins Science Center operates a molecular genetic and systematics research facility (FORT Molecular Ecology Laboratory) that uses molecular genetic tools to provide genetic information needed to inform natural resource management decisions. For many wildlife species, the data generated have become increasingly important in the development of their long-term management strategies, leading to a better understanding of species diversity, population dynamics and ecology, and future conservation and management needs. The Molecular Ecology Lab serves Federal research and resource management agencies by developing scientifically rigorous research programs using nuclear, mitochondrial and chloroplast DNA to help address many of today's conservation biology and natural resource management issues.

Fact Sheet

Using Molecular Genetic Markers to Resolve a Subspecies Boundary: The Northern Boundary of the Southwestern Willow Flycatcher in the Four-Corner States

*Executive Summary* The northern boundary of the endangered Southwestern Willow Flycatcher (Empidonax traillii extimus) is currently approximated as running through southern Colorado and Utah, but the exact placement is uncertain because this subspecies shares a border with the more northern and non-endangered E. t. adastus. To help resolve this issue, we evaluated the geographic distribution of mitochondrial and nuclear DNA by sampling breeding sites across the four-corner states (Arizona, Colorado, New Mexico, and Utah). We found that breeding sites clustered into two major groups generally consistent with the currently designated boundary, with the exception of three sites situated along the current boundary. However, delineating a precise boundary that would separate the two subspecies is made difficult because (1) we found evidence for a region of intergradation along the boundary area, suggesting the boundary is not discreet, and (2) the boundary region is sparsely populated, with too few extant breeding populations to precisely locate a boundary. The boundary region encompasses an area where elevation changes markedly over relatively short distances, with low elevation deserts to the south and more mesic, higher elevation habitats to the north. We hypothesized that latitudinal and elevational differences and their concomitant ecological effects could form an ecological barrier that inhibited gene flow between the subspecies, forming the basis for the subspecies boundary. We modeled changes in geographic patterns of genetic markers as a function of latitude and elevation finding significant support for this relationship. The model was brought into a GIS environment to create multiple subspecies boundaries, with the strength of each predicted boundary evaluated on the basis of how much genetic variation it explained. The candidate boundary that accounted for the most genetic variation was situated generally near the currently recognized subspecies boundary, but should be more biologically meaningful because it incorporates the landscape features that may be driving separation of the subspecies. Even so, we caution that using any narrow boundary line as an indicator of subspecies identity could be misleading because biologically the boundary is a region of intergradation rather than a discrete line. Designating, a boundary ultimately becomes a regulatory and management decision based on how much of the genetic variation unique to a subspecies should be protected. We discuss how the results of this study can help guide this decision process by wildlife policy makers.

Open-File Report

Hair of the dog: obtaining samples from coyotes and wolves noninvasively

Canids can be difficult to detect and their populations difficult to monitor. We tested whether hair samples could be collected from coyotes (Canis latrans) in Texas, USA and gray wolves (C. lupus) in Montana, USA using lure to elicit rubbing behavior at both man-made and natural collection devices. We used mitochondrial and nuclear DNA to determine whether collected hair samples were from coyote, wolf, or nontarget species. Both coyotes and wolves rubbed on man-made barbed surfaces but coyotes in Texas seldom rubbed on hanging barbed surfaces. Wolves in Montana showed a tendency to rub at stations where natural-material collection devices (sticks and debris) were present. Time to detection was relatively short (5 nights and 4 nights for coyotes and wolves, respectively) with nontarget and unknown species comprising approximately 26% of the detections in both locations. Eliciting rubbing behavior from coyotes and wolves using lures has advantages over opportunistic genetic sampling methods (e.g., scat transects) because it elicits a behavior that deposits a hair sample at a fixed sampling location, thereby increasing the efficiency of sampling for these canids. Hair samples from rub stations could be used to provide estimates of abundance, measures of genetic diversity and health, and detection-nondetection data useful for cost-effective population monitoring.

Texas;Montana

Freshwater mussels (Unionidae): Central and West Texas Final Report

The goal of this study was two-fold: (1) assess the taxonomic identity and phylogenetic placement of Quadrula aurea (Golden Orb) [Federal Candidate], Quadrula houstonensis (Smooth Pimpleback) [Federal Candidate], and Quadrula petrina (Texas Pimpleback) [Federal Candidate] through mitochondrial and nuclear DNA sequencing; and (2) provided additional information on the distribution and abundance for mussel species petitioned for listing under ESA through field surveys in portions of several major rivers in Central and West Texas. The final report is organized by 4 research tasks as per contract (see Appendix A) and submitted proposals. Below is an outline of goal-oriented tasks for this project: Task 1 – Conduct comprehensive surveys of portions of the Brazos, Colorado, and Guadalupe River basins Task 2 – Conduct comprehensive surveys of portions of the Rio Grande Basin Task 3 – Develop conservation status assessment maps for 9 state-threatened mussel species in Texas Task 4 – Delineate species boundaries, test for cryptic species, and assess phylogenetic relationships for threatened Texas mussel species in the genus Quadrula Note the following 2 tasks were either not funded in the current contract or was a modification of the contract and added as a project deliverable: Task 5 – Delineate species boundaries, test for cryptic species, and assess phylogenetic relationships for east Texas mussel species in the genus Fusconaia (Not Funded) Task 6 – Evaluate the conservation status of Texas hornshell and other mussels in the Pecos and Devils Rivers (Contract amendment, results combined with Task 2). Detailed descriptions of the research tasks and findings are found within each chapter of the report. Here, we outline and summarize project deliverables and major findings for each task.

Texas

Evidence for a duplicated mitochondrial region in Audubon’s shearwater based on MinION sequencing

Mitochondrial genetic markers have been extensively used to study the phylogenetics and phylogeography of many birds, including seabirds of the order Procellariiformes. Evidence suggests that part of the mitochondrial genome of Procellariiformes, especially albatrosses, is duplicated, but no DNA fragment covering the entire duplication has been sequenced. We sequenced the complete mitochondrial genome of a non-albatross species of Procellariiformes, Puffinus lherminieri (Audubon’s shearwater) using the long-read MinION (ONT) technology. Two mitogenomes were assembled from the same individual, differing by 52 SNPs and in length. The shorter was 19 kb long while the longer was 21 kb, due to the presence of two identical copies of nad6 , three tRNA, and two dissimilar copies of the control region (CR). Contrary to albatrosses, cob was not duplicated. We further detected a complex repeated region of undetermined length between the CR and 12S. Long-read sequencing suggests heteroplasmy and a novel arrangement within the duplicated region, indicating a complex evolution of the mitogenome in Procellariiformes.

Mitochondrial DNA Part A

Environmental DNA metabarcoding as a tool for biodiversity assessment and monitoring: Reconstructing established fish communities of north-temperate lakes and rivers

Aim To evaluate the ability of precipitation-based environmental DNA (eDNA) sample collection and mitochondrial 12S metabarcoding sequencing to reconstruct well-studied fish communities in lakes and rivers. Specific objectives were to 1) determine correlations between eDNA species detections and known community composition based on conventional field sampling, 2) compare efficiency of eDNA to detect fish biodiversity among systems with variable morphologies and trophic states, and 3) determine if species habitat preferences predict eDNA detection. Location Upper Great Lakes Region, North America. Methods Fish community composition was estimated for seven lakes and two Mississippi River navigation pools using sequence data from the mitochondrial 12S gene amplified from 10 to 50 water samples per waterbody collected in 50-mL centrifuge tubes at a single time point. Environmental DNA (eDNA) was concentrated without filtration by centrifuging samples to reduce per-sample handling time. Taxonomic detections from eDNA were compared to established community monitoring databases containing up to 40 years of sampling and a detailed habitat/substrate preference matrix to identify patterns of bias. Results Mitochondrial 12S gene metabarcoding detected 15%–47% of the known species at each waterbody and 30%–76% of known genera. Non-metric multidimensional scaling (NMDS) assessment of the community structure indicated that eDNA-detected communities grouped in a similar pattern as known communities. Discriminant analysis of principal components indicated that there was a high degree of overlap in habitat/substrate preference of eDNA-detected and eDNA-undetected species suggesting limited habitat bias for eDNA sampling. Main conclusions Large numbers of small volume samples sequenced at the mitochondrial 12S gene can describe the coarse community structure of freshwater systems. However, additional conventional sampling and environmental DNA sampling may be necessary for a complete diversity census.

Illinois, Iowa, Wisconsin

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

Arrested development of the myxozoan parasite, Myxobolus cerebralis, in certain populations of mitochondrial 16S lineage III Tubifex tubifex

Laboratory populations of Tubifex tubifex from mitochondrial (mt)16S ribosomal DNA (rDNA) lineage III were generated from single cocoons of adult worms releasing the triactinomyxon stages (TAMs) of the myxozoan parasite, Myxobolus cerebralis. Subsequent worm populations from these cocoons, referred to as clonal lines, were tested for susceptibility to infection with the myxospore stages of M. cerebralis. Development and release of TAMs occurred in five clonal lines, while four clonal lines showed immature parasitic forms that were not expelled from the worm (non-TAM producers). Oligochaetes from TAM- and non-TAM-producing clonal lines were confirmed as lineage III based on mt16S rDNA and internal transcribed spacer region 1 (ITS1) sequences, but these genes did not differentiate these phenotypes. In contrast, random amplified polymorphic DNA analyses of genomic DNA demonstrated unique banding patterns that distinguished the phenotypes. Cohabitation of parasite-exposed TAM- and non-TAM-producing phenotypes showed an overall decrease in expected TAM production compared to the same exposure dose of the TAM-producing phenotype without cohabitation. These studies suggest that differences in susceptibility to parasite infection can occur in genetically similar T. tubifex populations, and their coexistence may affect overall M. cerebralis production, a factor that may influence the severity of whirling disease in wild trout populations. ?? 2007 Springer-Verlag.

Parasitology Research

Assessing reproductive and endocrine parameters in male largescale suckers ( Catostomus macrocheilus ) along a contaminant gradient in the lower Columbia River, USA

Persistent organochlorine pollutants such as polychlorinated biphenyls (PCBs), dichlorodiphenyldichloroethylene (p,p′-DDE), and polybrominated diphenyl ethers (PBDEs) are stable, bioaccumulative, and widely found in the environment, wildlife, and the human population. To explore the hypothesis that reproduction in male fish is associated with environmental exposures in the lower Columbia River (LCR), reproductive and endocrine parameters were studied in male resident, non-anadromous largescale sucker (Catostomus macrocheilus) (LSS) in the same habitats as anadromous salmonids having conservation status. Testes, thyroid tissue and plasma collected in 2010 from Longview (LV), Columbia City (CC), and Skamania (SK; reference) were studied. Sperm morphologies and thyrocyte heights were measured by light microscopy, sperm motilities by computer-assisted sperm motion analysis, sperm adenosine triphosphate (ATP) with luciferase, and plasma vitellogenin (VTG), thyroxine (T4), and triiodothyronine (T3) by immunoassay. Sperm apoptosis, viability, mitochondrial membrane potential, nuclear DNA fragmentation, and reproductive stage were measured by flow cytometry. Sperm quality parameters (except counts) and VTG were significantly different among sites, with correlations between VTG and 7 sperm parameters. Thyrocyte heights, T4, T3, gonadosomatic index and Fulton's condition factor differed among sites, but not significantly. Sperm quality was significantly lower and VTG higher where liver contaminants and water estrogen equivalents were highest (LV site). Total PCBs (specifically PCB-138, -146, -151, -170, -174, -177, -180, -183, -187, -194, and -206) and total PBDEs (specifically BDE-47, -100, -153, and -154) were negatively correlated with sperm motility. PCB-206 and BDE-154 were positively correlated with DNA fragmentation, and pentachloroanisole and VTG were positively correlated with sperm apoptosis and negatively correlated with ATP. BDE-99 was positively correlated with sperm counts and motility; T4 was negatively correlated with counts and positively correlated with motility, thus indicating possible androgenic mechanisms and thyroid endocrine disruption. Male LSS proved to be an informative model for studying reproductive and endocrine biomarkers in the LCR.

Oregon;Washington

Recurrent hybridization and recent origin obscure phylogenetic relationships within the ‘white-headed’ gull (Larus sp.) complex

Species complexes that have undergone recent radiations are often characterized by extensive allele sharing due to recent ancestry and (or) introgressive hybridization. This can result in discordant evolutionary histories of genes and heterogeneous genomes, making delineating species limits difficult. Here we examine the phylogenetic relationships among a complex group of birds, the white-headed gulls (Aves: Laridae), which offer a unique window into the speciation process due to their recent evolutionary history and propensity to hybridize. Relationships were examined among 17 species (61 populations) using a multilocus approach, including mitochondrial and nuclear intron DNA sequences and microsatellite genotype information. Analyses of microsatellite and intron data resulted in some species-based groupings, although most species were not represented by a single cluster. Considerable allele and haplotype sharing among white-headed gull species was observed; no locus contained a species-specific clade. Despite this, our multilocus approach provided better resolution among some species than previous studies. Interestingly, most clades appear to correspond to geographic locality: our BEAST analysis recovered strong support for a northern European/Icelandic clade, a southern European/Russian clade, and a western North American/ canus clade, with weak evidence for a high latitude clade spanning North America and northwestern Europe. This geographical structuring is concordant with behavioral observations of pervasive hybridization in areas of secondary contact. The extent of allele and haplotype sharing indicates that ecological and sexual selection are likely not strong enough to complete reproductive isolation within several species in the white-headed gull complex. This suggests that just a few genes are driving the speciation process.

Molecular Phylogenetics and Evolution

Population connectivity of endangered Ozark big-eared bats ( Corynorhinus townsendii ingens )

The endangered Ozark big-eared bat ( Corynorhinus townsendii ingens ) is restricted to eastern Oklahoma and western and north-central Arkansas, where populations may be susceptible to losses of genetic variation due to patchy distribution of colonies and potentially small effective population sizes. We used mitochondrial D-loop DNA sequences and 15 nuclear microsatellite loci to determine population connectivity among Ozark big-eared bat caves. Assessment of 7 caves revealed a haplotype not detected in a previous study (2002–2003) and gene flow among colonies in eastern Oklahoma. Our data suggest genetic mixing of individuals, which may be occurring at nearby swarming sites in the autumn. Further evidence of limited gene flow between caves in Oklahoma with a cave in Arkansas highlights the importance of including samples from geographically widespread caves to fully understand gene flow in this subspecies. It appears autumn swarming sites and winter hibernacula play an important role in providing opportunities for mating; therefore, we suggest protection of these sites, maternity caves, and surrounding habitat to facilitate gene flow among populations of Ozark big-eared bats.

Arkansas, Oklahoma

Influence of volcanic activity on the population genetic structure of Hawaiian Tetragnatha spiders: Fragmentation, rapid population growth and the potential for accelerated evolution

Volcanic activity on the island of Hawaii results in a cyclical pattern of habitat destruction and fragmentation by lava, followed by habitat regeneration on newly formed substrates. While this pattern has been hypothesized to promote the diversification of Hawaiian lineages, there have been few attempts to link geological processes to measurable changes in population structure. We investigated the genetic structure of three species of Hawaiian spiders in forests fragmented by a 150-year-old lava flow on Mauna Loa Volcano, island of Hawaii: Tetragnatha quasimodo (forest and lava flow generalist), T. anuenue and T. brevignatha (forest specialists). To estimate fragmentation effects on population subdivision in each species, we examined variation in mitochondrial and nuclear genomes (DNA sequences and allozymes, respectively). Population subdivision was higher for forest specialists than for the generalist in fragments separated by lava. Patterns of mtDNA sequence evolution also revealed that forest specialists have undergone rapid expansion, while the generalist has experienced more gradual population growth. Results confirm that patterns of neutral genetic variation reflect patterns of volcanic activity in some Tetragnatha species. Our study further suggests that population subdivision and expansion can occur across small spatial and temporal scales, which may facilitate the rapid spread of new character states, leading to speciation as hypothesized by H. L. Carson 30 years ago.

Molecular Ecology

Polyphyly of Campylorhamphus , and description of a new genus for C. pucherani (Dendrocolaptinae)

We investigated the phylogenetic relationships of Campylorhamphus pucherani using DNA sequences from three mitochondrial genes and a nuclear intron, as well as 84 morphological characters from the skeleton, the integument, and the musculature. The molecular phylogeny indicated that C. pucherani is not part of Campylorhamphus ; instead, it is the sister species to Drymornis bridgesii , in a clade that also contains Lepidocolaptes . The morphological phylogeny also placed C. pucherani in a clade that contains Drymornis and Lepidocolaptes . Using a morphometric analysis of size and shape diversity, we demonstrated that the inclusion of C. pucherani in Drymornis would create an excessively heterogeneous genus compared with other dendrocolaptine genera. Because no generic name is available for C. pucherani , we describe the new genus Drymotoxeres for this species.

The Auk

Unraveling the effects of sex and dispersal: Ozark big-eared bat (Corynorhinus townsendii ingens) conservation genetics

The Ozark big-eared bat (Corynorhinus townsendii ingens) is federally listed as endangered and is found in only a small number of caves in eastern Oklahoma and northwestern Arkansas. Previous studies suggested site fidelity of females to maternity caves; however, males are solitary most of the year, and thus specific information on their behavior and roosting patterns is lacking. Population genetic variation often provides the necessary data to make inferences about gene flow or mating behavior within that population. We used 2 types of molecular data: DNA sequences from the mitochondrial D loop and alleles at 5 microsatellite loci. Approximately 5% of the population, 24 males and 39 females (63 individuals), were sampled. No significant differentiation between 5 sites was present in nuclear microsatellite variation, but distribution of variation in maternally inherited markers differed among sites. This suggests limited dispersal of female Ozark big-eared bats and natal philopatry. Areas that experience local extinctions are unlikely to be recolonized by species that show strong site fidelity. These results provide a greater understanding of the population dynamics of Ozark big-eared bats and highlight the importance of cave protection relative to maintaining genetic integrity during recovery activities for this listed species. ?? 2005 American Society of Mammalogists.

Journal of Mammalogy

Conservation genetics of North American freshwater mussels Amblema and Megalonaias

Freshwater bivalves are among the most endangered groups of organisms in North America. Efforts to protect the declining mussel fauna are confounded by ambiguities associated with recognition of distinct evolutionary entities or species. This, in part, is due to the paucity of reliable morphological characters for differentiating taxa. We have employed allozymes and DNA sequence data to search for diagnosably distinct evolutionary entities within two problematic genera of unionid mussels, Amblema and Megalonaias. Within the genus Amblema three species are recognized based on our DNA sequence data for the mitochondrial 16S rRNA and allozyme data (Amblema neislerii, A. plicata, and A. elliotti). Only one taxonomically distinct entity is recognized within the genus Megalonaias—M. nervosa. Megalonaias boykiniana of the Apalachicolan Region is not diagnosable and does not warrant specific taxonomic status. Interestingly, Megalonaias from west of the Mississippi River, including the Mississippi, exhibited an allozyme and mtDNA haplotype frequency shift suggestive of an east-west dichotomy. The results of this study eliminate one subspecies of Amblema and increase the range of A. plicata. This should not affect the conservation status of “currently stable” assigned to A. plicata by Williams et al. (1993). The conservation status of A. elliotti needs to be reexamined because its distribution appears to be limited to the Coosa River System in Alabama and Georgia.

Conservation Biology

Genomic insights into isolation of the threatened Florida crested caracara (Caracara plancus)

We conducted a population genomic study of the crested caracara ( Caracara plancus ) using samples ( n = 290) collected from individuals in Florida, Texas, and Arizona, United States. Crested caracaras are non-migratory raptors ranging from the southern tip of South America to the southern United States, including a federally protected relict population in Florida long thought to have been isolated since the last ice age. Our objectives were to evaluate genetic diversity and population structure of Florida’s apparently isolated population and to evaluate taxonomic relationships of crested caracaras at the northern edge of their range. Using DNA purified from blood samples, we conducted double-digest restriction site associated DNA sequencing and sequenced the mitochondrial ND2 gene. Analyses of population structure using over 9,000 SNPs suggest that two major clusters are best supported, one cluster including only Florida individuals and the other cluster including Arizona and Texas individuals. Both SNPs and mitochondrial haplotypes reveal the Florida population to be highly differentiated genetically from Arizona and Texas populations, whereas, Arizona and Texas populations are moderately differentiated from each other. The Florida population’s mitochondrial haplotypes form a separate monophyletic group, while Arizona and Texas populations share mitochondrial haplotypes. Results of this study provide substantial genetic evidence that Florida’s crested caracaras have experienced long-term isolation from caracaras in Arizona and Texas and thus, represent a distinct evolutionary lineage possibly warranting distinction as an Evolutionarily Significant Unit (ESU) or subspecies. This study will inform conservation strategies focused on long-term survival of Florida’s distinct, panmictic population.

Journal of Heredity

Biogeographical history and coalescent species delimitation of Pacific island skinks (Squamata: Scincidae: Emoia cyanura species group)

Aim A prevailing hypothesis for how Pacific islands organisms have obtained their extant distributions is that of a stepping-stone model, in which populations originate from Papua New Guinea in the western Pacific and gradually disperse eastward. Here, we test this model using a spatiotemporal framework for Emoia cyanura and E. impar , two species within the Emoia cyanura species group (ECSG; Family: Scincidae). We further assess species limits within the group, utilizing novel coalescent methods. Location Pacific Islands. Methods We obtained DNA sequence data from one mitochondrial and three nuclear markers for 117 individuals, representing seven of the nine species within the ECSG. These data were analysed for concordance with the stepping-stone model using estimation of population structure, divergence dates, and historical biogeographical range. To assess hypotheses of independent lineages within each widespread species, we also employed the Bayesian Phylogenetics & Phylogeography (BPP) program to define operational taxonomic units in *BEAST. Results Population structure analyses consistently found individuals from western island groups representing divergent populations, with central and eastern populations demonstrating minimal genetic variation. Phylogenetic hypotheses support a western origin for E. cyanura and E. impar , while biogeographical and divergence time estimations predict a recent and rapid expansion out of the western Pacific. The BPP and *BEAST analyses found evidence for five independent lineages within E. impar and five independent lineages within E. cyanura / E. pseudocyanura . Main conclusions In contrast to the expectations of a stepping-stone model, E. cyanura and E. impar each exhibit the genetic signature of a rapid radiation during the mid to late Pleistocene, with evidence for newly identified lineages, mainly on western islands. Of these recovered lineages, we propose three to be elevated to species status. These findings expand our understanding of endemic Pacific biota, which are subject to conservation threats from human impacts and climate change.

Journal of Biogeography

Sperm quality biomarkers complement reproductive and endocrine parameters in investigating environmental contaminants in common carp (Cyprinus carpio) from the Lake Mead National Recreation Area

Lake Mead National Recreational Area (LMNRA) serves as critical habitat for several federally listed species and supplies water for municipal, domestic, and agricultural use in the Southwestern U.S. Contaminant sources and concentrations vary among the sub-basins within LMNRA. To investigate whether exposure to environmental contaminants is associated with alterations in male common carp ( Cyprinus carpio ) gamete quality and endocrine- and reproductive parameters, data were collected among sub-basins over 7 years (1999–2006). Endpoints included sperm quality parameters of motility , viability, mitochondrial membrane potential, count, morphology, and DNA fragmentation; plasma components were vitellogenin (VTG), 17ß-estradiol, 11-keto-testosterone, triiodothyronine, and thyroxine. Fish condition factor, gonadosomatic index, and gonadal histology parameters were also measured. Diminished biomarker effects were noted in 2006, and sub-basin differences were indicated by the irregular occurrences of contaminants and by several associations between chemicals (e.g., polychlorinated biphenyls, hexachlorobenzene , galaxolide, and methyl triclosan) and biomarkers (e.g., plasma thyroxine, sperm motility and DNA fragmentation). By 2006, sex steroid hormone and VTG levels decreased with subsequent reduced endocrine disrupting effects. The sperm quality bioassays developed and applied with carp complemented endocrine and reproductive data, and can be adapted for use with other species.

Lake Mead National Recreation Area