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Conservation genetics of evolutionary lineages of the endangered mountain yellow-legged frog, Rana muscosa (Amphibia: Ranidae), in southern California

Severe population declines led to the listing of southern California Rana muscosa (Ranidae) as endangered in 2002. Nine small populations inhabit watersheds in three isolated mountain ranges, the San Gabriel, San Bernardino and San Jacinto. One population from the Dark Canyon tributary in the San Jacinto Mountains has been used to establish a captive breeding population at the San Diego Zoo Institute for Conservation Research. Because these populations may still be declining, it is critical to gather information on how genetic variation is structured in these populations and what historical inter-population connectivity existed between populations. Additionally, it is not clear whether these populations are rapidly losing genetic diversity due to population bottlenecks. Using mitochondrial and microsatellite data, we examine patterns of genetic variation in southern California and one of the last remaining populations of R. muscosa in the southern Sierra Nevada. We find low levels of genetic variation within each population and evidence of genetic bottlenecks. Additionally, substantial population structure is evident, suggesting a high degree of historical isolation within and between mountain ranges. Based on estimates from a multi-population isolation with migration analysis, these populations diversified during glacial episodes of the Pleistocene, with little gene flow during population divergence. Our data demonstrate that unique evolutionary lineages of R. muscosa occupy each mountain range in southern California and should be managed separately. The captive breeding program at Dark Canyon is promising, although mitigating the loss of neutral genetic diversity relative to the natural population might require additional breeding frogs.

California

The interface between rare plant management and genetics: Conserving Pitcher’s thistle (Cirsium pitcheri) in the Great Lakes region, USA

Background and Aims Tools showing best conservation practices are becoming critical for rare plant conservation as populations become isolated through habitat fragmentation and changing ecosystem processes. We illustrate the importance of reintroduction and assisted gene flow, using reintroduction of the USA federally threatened Pitcher’s thistle ( Cirsium pitcheri ), a monocarpic perennial endemic to the western Great Lakes sand dunes. We evaluate the success of experimental reintroductions of this species into its dynamic coastal environment and address fundamental issues for rare plant reintroduction, including questions of maintenance of genetic diversity, founder size, introduction methods and assisted gene flow effectiveness. Methods In 1994 we initiated experimental reintroductions at three new locations along a habitat successional gradient using 4200 seeds collected from 54 maternal lines. At each site, seed sources were distributed among replicate blocks that were split by sowing method (sowing vs broadcasting) to examine establishment success. We monitored population demography for 30 years, assessing genetic variation in 2009 and regionally from 1997 to 2014. Key Results Two of three populations persisted for 30 years from a single-seeding founder event. Reintroduction populations had greater expected heterozygosity than regional native populations. Despite persistence, moderate inbreeding coefficients showed that reintroduction populations have not achieved an effective size to reduce the likelihood of inbreeding depression. However, the mid-successional reintroduction had the lowest kinship of all populations sampled, indicating a healthy restoration. Seed sowing produced three times as many seedlings as broadcasting, but seed source did not affect germination success. Persistence has been facilitated by local migration to suitable habitat patches, an important metapopulation process. Conclusions Seeds can be an effective method for reintroducing monocarpic plants in high-quality habitats. However, low numbers of reproductive adults and moderate inbreeding illustrate the need for repeated additions of plants or seed to reduce inbreeding to improve rare plant genetic evolutionary potential.

Indiana

Conservation genetics of the endangered Isle Royale gray wolf

The small group of wolves on Isle Royale has been studied for over three decades as a model of the relationship between large carnivores and their prey. During the last ten years the population declined from 50 individuals to as few as 12 individuals. The causes of this decline may be food shortages, disease, or reduced genetic variability. We address the issues of genetic variability and relationships of Isle Royale wolves using allozyme electrophoresis, mtDNA restriction-site analysis, and multilocus hypervariable minisatellite DNA analysis (genetic fingerprinting). Our results indicate that approximately 50% of the allozyme heterozygosity has been lost in the island population, a decline similar to that expected if no immigration had occurred from the mainland. The genetic fingerprinting data indicate that the seven sampled Isle Royale wolves are as similar as captive populations of siblings. Surprisingly, the Isle Royale wolves have an mDNA genotype that is very rare on the mainland, being found in only one of 144 mainland wolves ThFF suggests that the remaining Isle Royale wolves areprobably derived from a single female founder.

Michigan

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

Conservation genetics of Lake Superior brook trout: Issues, questions, and directions

Parallel efforts by several genetic research groups have tackled common themes relating to management concerns about and recent rehabilitation opportunities for coaster brook trout Salvelinus fontinalis in Lake Superior. The questions that have been addressed include the evolutionary and genetic status of coaster brook trout, the degree of relatedness among coaster populations and their relationship to riverine tributary brook trout populations, and the role and effectiveness of stocking in maintaining and restoring coasters to Lake Superior. Congruent genetic results indicate that coasters are an ecotype (life history variant) rather than an evolutionarily significant unit or genetically distinct strain. Regional structure exists among brook trout stocks, coasters being produced from local populations. Introgression of hatchery genes into wild populations appears to vary regionally and may relate to local population size, habitat integrity, and anthropogenic pressures. Tracking the genetic diversity and integrity associated with captive breeding programs is helping to ensure that the fish used for stocking are representative of their source populations and appropriate for rehabilitation efforts. Comparative analysis of shared samples among collaborating laboratories is enabling standardization of genotype scoring and interpretation as well as the development of a common toolkit for assessing genetic structure and diversity. Incorporation of genetic data into rehabilitation projects will facilitate monitoring efforts and subsequent adaptive management. Together, these multifaceted efforts provide comprehensive insights into the biology of coaster brook trout and enhance restoration options. ?? Copyright by the American Fisheries Society 2008.

Conference Paper

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 managed ungulate populations

Natural populations of many species are increasingly impacted by human activities. Perturbations are particularly pronunced for large ungulates due in part to sport and commercial harvest, to reductions and fragmentation of native habitat, and as the result of reintroductions. These perturbations affect population size, sex and age composition, and population breeding structure, and as a consequence affect the levels and partitioning of genetic variation. Three case histories highlighting long-term ecological genetic research on mule deer Odocoileus hemionus (Rafinesque, 1817), white-tailed deer O . virginianus (Zimmermann, 1780), and Alpine ibex Capra i. ibex Linnaeus, 1758 are presented. Joint examinations of population ecological and genetic data from several populations of each species reveal: (1) that populations are not in genetic equilibrium, but that allele frequencies and heterozygosity change dramatically over time and among cohorts produced in successive years, (2) populations are genetically structured over short and large geographic distances reflecting local breeding structure and patterns of gene flow, respectively; however, this structure is quite dynamic over time, due in part to population exploitation, and (3) restocking programs are often undertaken with small numbers of founding individuals resulting in dramatic declines in levels of genetic variability and increasing levels of genetic differentiation among populations due to genetic drift. Genetic characteristics have and will continue to provide valuable indirect sources of information relating enviromental and human perturbations to changes in population processes.

Acta Theriologica

Cgsim: An R package for simulation of population genetics for conservation and management applications

Wildlife conservation and management increasingly considers genetic information to plan, understand and evaluate implemented population interventions. These actions commonly include conservation translocation and population reductions through removals. Change in genetic variation in response to management actions can be unintuitive due to the influence of multiple interacting drivers (e.g. genetic drift, life history traits, environmental stochasticity). Simulation is an excellent tool to understand the predicted consequences of different proposed or implemented actions. However, the genetic simulators that are robust to a wide variety of life history traits also have a steep learning curve to appropriately parameterize common management actions. To fill this gap, we have developed cgsim, an R package for simulating the genetic consequences of common management interventions for populations of wildlife species. We developed a set of functions to specifically understand the effects of four main aspects of managing small, declining or isolated populations: loss of genetic diversity to drift, augmenting existing populations (e.g. translocation), population reduction through targeted removals and population catastrophes driven by stochastic extrinsic forces. Our single population simulation model is individual-based, and flexible to a range of life history traits. Here we validate cgsim through comparison of simulations to theoretical expectations of genetic diversity loss and illustrate its applied utility by focusing on a recently published empirical example for the Greater Sage-Grouse. Cgsim is available as an R package at: https://doi.org/10.5066/P1BXBEXJ .

Molecular Ecology Resources

A metapopulation strategy to support long term conservation of genetic diversity in Department of the Interior bison

Once numbering in the tens of millions, plains bison ( Bison bison bison ) were nearly driven to extinction with only a few hundred individuals remaining by the late 19th century. Plains bison have since recovered to approximately 20,000 animals managed in conservation herds throughout North America, yet substantial challenges to their recovery remain. The Department of the Interior (DOI) is working with diverse partners to steward approximately 11,000 bison in 18 conservation herds across 12 states. Most herds exist in areas without native predators, and removals are required to keep herd sizes at or below carrying capacity. The loss of genetic diversity within bison, and the fact that most DOI herds are relatively small and isolated from each other with no opportunity for natural gene flow, raises concerns about maintaining genetic diversity over the long term. Connecting populations through gene flow (i.e., creating a metapopulation) can minimize loss of genetic diversity, both within and across populations. Management of DOI bison conservation herds has historically varied across bureaus and conservation units. Adopting a national perspective on bison conservation was identified as a priority in the 2008 Department of the Interior Bison Conservation Initiative (BCI). The concept of metapopulation management as a potential tool to maximize the conservation of genetic diversity among DOI herds was first described in this 2008 Initiative and was specifically encouraged in the 2010 DOI Bison Conservation Genetics Workshop report (Dratch and Gogan 2010). In the 2020 BCI, the DOI re-affirmed its commitment to conserving bison as native, North American wildlife. This document establishes a framework for a nationally coordinated strategy for bison managed by the DOI to support the genetic conservation goals outlined in the 2020 BCI. This is a decisionmaking framework that guides managers through the process of determining when and how to consider translocations. Decisions and actions within the framework are informed by analysis and interpretation of data housed in an integrated, relational database that will be initially populated with the most current data and updated annually thereafter. It provides science-based guidance on how to conserve DOI bison genetic diversity through strategic translocations, while also considering cattle introgression and bison health. We illustrate how this Strategy can be used to guide the establishment of new conservation herds and discuss what it means to be a DOI partner. Finally, this is intended to be used as a living document that will evolve as needs and technologies change.

Science Report

The ties that bind the sagebrush biome: Integrating genetic connectivity into range-wide conservation of greater sage-grouse

Conserving genetic connectivity is fundamental to species persistence, yet rarely is made actionable into spatial planning for imperilled species. Climate change and habitat degradation have added urgency to embrace connectivity into networks of protected areas. Our two-step process integrates a network model with a functional connectivity model, to identify population centres important to maintaining genetic connectivity then to delineate those pathways most likely to facilitate connectivity thereamong for the greater sage-grouse ( Centrocercus urophasianus ), a species of conservation concern ranging across eleven western US states and into two Canadian provinces. This replicable process yielded spatial action maps, able to be prioritized by importance to maintaining range-wide genetic connectivity. We used these maps to investigate the efficacy of 3.2 million ha designated as priority areas for conservation (PACs) to encompass functional connectivity. We discovered that PACs encompassed 41.1% of cumulative functional connectivity—twice the amount of connectivity as random—and disproportionately encompassed the highest-connectivity landscapes. Comparing spatial action maps to impedances to connectivity such as cultivation and woodland expansion allows both planning for future management and tracking outcomes from past efforts.

western United States

Genetic diversity goals and targets have improved, but remain insufficient for clear implementation of the post-2020 global biodiversity framework

Genetic diversity among and within populations of all species is necessary for people and nature to survive and thrive in a changing world. Over the past three years, commitments for conserving genetic diversity have become more ambitious and specific under the Convention on Biological Diversity’s (CBD) draft post-2020 global biodiversity framework (GBF). This Perspective article comments on how goals and targets of the GBF have evolved, the improvements that are still needed, lessons learned from this process, and connections between goals and targets and the actions and reporting that will be needed to maintain, protect, manage and monitor genetic diversity. It is possible and necessary that the GBF strives to maintain genetic diversity within and among populations of all species, to restore genetic connectivity, and to develop national genetic conservation strategies, and to report on these using proposed, feasible indicators.

Conservation Genetics

A new era of genetic diversity conservation through novel tools and accessible data

As the foundation of biodiversity, genetic diversity is necessary for species to adapt to ecological changes, such as impacts from disease, invasive species, and climate change. Genetic diversity also supports ecosystem resilience and societal innovations. Unfortunately, declines in genetic diversity have been frequently observed in populations of wild and domestic species. Yet the field of population genetics is often neglected in national and global environmental policy and has been underutilized in biodiversity monitoring and conservation action.

Frontiers in Ecology and the Environment

Validation of eDNA markers for New Zealand mudsnail surveillance and initial eDNA monitoring at Mississippi River Basin sites

The performance of newly developed New Zealand mudsnail ( Potamopyrgus antipodarum ; NZMS) genetic markers for environmental (eDNA) analysis of water were compared across two laboratories. The genetic markers were tested in four quantitative polymerase chain reaction assays targeting two regions of the NZMS mitochondrial genome, specifically the cytochrome c oxidase subunit 1 (coi) and cytochrome b (cytb) genes. In a blind study, analysts tested each sample eight times with each assay. There were 10 expected-negative samples from the Black River in La Crosse, Wisconsin, 10 expected-positive samples from the Black Earth Creek in Black Earth, Wisconsin, and 10 known-positive samples from the Black River spiked with NZMS DNA. Previously extracted samples, kept at the Upper Midwest Environmental Sciences Center, were pooled by sample location and then equal quantities were distributed between the Upper Midwest Environmental Sciences Center and the Molecular Conservation Genetics Laboratory at the University of Wisconsin-Stevens Point for analysis. The assays tested were (1) the assay targeting cytb with a minor groove binder probe described by Goldberg and others (2013), (2) the cytb assay with a modified double-quenched probe, (3) an assay targeting coi with a double-quenched probe, and (4) a duplex reaction combining the modified cytb assay and the coi assay. Samples were considered positive for the presence of NZMS DNA when quantitative polymerase chain reaction amplification and probe signal was higher than the normalized threshold value above baseline fluorescence. For the duplex assay, samples were considered positive only when both probe signals were higher than the normalized threshold value above baseline fluorescence. Positive results were then confirmed by sequencing the products. All four assays detected the DNA of NZMS in all expected-positive and known-positive samples in both labs. The modified cytb assay, the coi assay, and the duplex assay all failed to detect the DNA of NZMS in all expected-negative samples in both labs. The cytb assay, as described by Goldberg and others (2013), failed to detect the DNA of NZMS in all expected-negative samples for the Molecular Conservation Genetics Laboratory, but some reactions resulted in positive detection in late cycles for 9 of the 10 expected-negative samples at the Upper Midwest Environmental Sciences Center. Amplicons for expected-negative samples with positive reactions were sent for sequencing, and none were confirmed as NZMS. Six amplicons failed to give readable sequences, and three gave sequences without similarity to any known sequence in GenBank. Amplicons from each assay for one representative positive sample were sequenced and identified as NZMS with greater than 99 percent identity. The duplex assay was chosen as the most efficient assay and was used at the Upper Midwest Environmental Sciences Center to analyze triplicate samples from 29 streams in Wisconsin, 8 streams in Illinois, and 8 streams in Iowa. In order to verify results, additional triplicate samples were collected from two of the streams in Iowa and two of the streams in Wisconsin for analysis at the Molecular Conservation Genetics Laboratory. All samples at all sites were negative for NZMS DNA.

Illinois, Iowa, Wisconsin

Distribution and population genetics of walleye and sauger

Conserving genetic diversity and local adaptations are management priorities for wild populations of exploited species, which increasingly are subject to climate change, habitat loss, and pollution. These constitute growing concerns for the walleye Sander vitreus, an ecologically and economically valuable North American temperate fish with large Laurentian Great Lakes' fisheries. This study compares genetic diversity and divergence patterns across its widespread native range using mitochondrial (mt) DNA control region sequences and nine nuclear DNA microsatellite (μsat) loci, examining historic and contemporary influences. We analyze the genetic and morphological characters of a putative endemic variant– “blue pike” S. v. “glaucus” –described from Lakes Erie and Ontario, which became extinct. Walleye with turquoise-colored mucus also are evaluated, since some have questioned whether these are related to the “blue pike”.

BMC Evolutionary Biology

Population genetics of museum specimens indicate decreasing genetic resiliency: The case of two bumble bees of conservation concern

Genetic resiliency is the likelihood that populations retain sufficient genetic diversity to respond to environmental change. It is rarely examined through time in conservation genetic studies due to challenges of acquiring and sequencing historical specimens. Focusing on populations of two sibling bumble bee species of conservation concern with different recent patterns of decline, we used museum specimens collected between 1960 and 2020 and 15 microsatellite markers to assess genetic resiliency (allelic richness, expected heterozygosity, and inbreeding) through time and across geographic space. We find evidence of decreasing allelic richness through time, starting at least 30 years before observed abundance declines in one species and at least 20 years before present in a species with apparently stable abundance. We also found increasing expected heterozygosity through time, indicating increased inbreeding, in the putatively stable species. We demonstrate that genetic measurements taken from specimens collected through time can be used to detect population decline in imperiled species before decreases in abundance are detected. We also demonstrate the importance of interpreting population genetic metrics within the context of historical patterns to assess species' conservation statuses. Finally, we discuss the limitations of currently available population genetic methods, including the influence of isolation by distance and sampling density on measurements of genetic structure, and the influence of demographic characteristics and choice of genetic markers on estimates of genetic diversity and structure. We call for further development of individual-based modeling methods to measure genetic structure, as opposed to commonly applied population-based metrics, to overcome these limitations.

Biological Conservation