Thomas D. Mullins

Population identification of western hemisphere shorebirds throughout the annual cycle

Identification of relationships among geographically distinct populations of migratory species can provide an understanding of breeding and natal philopatry, migration pathways, and population mixing during winter. We used random amplified polymorphic DNA (RAPD) analyses to search for markers specific to difficult‐to‐differentiate shorebird species (e.g. long‐billed dowitcher Limnodromus scolopaceus and short‐billed dowitcher L. griseus) as well as geographically distinct breeding populations of Hudsonian godwits Limosa haemastica, red‐necked phalaropes Phalaropus lobatus, semipalmated plovers Charadrius semipalmatus, dunlin Calidris alpina, pectoral sandpipers C. melanotos, semipalmated sandpipers C. pusilla and western sandpipers C. mauri. Markers clearly differentiated all shorebird species. Estimates of population differentiation varied greatly among species (FST= 0.095–0.685) and correlated with interspecific variation in philopatry and geographical separation of breeding populations. We assigned individuals to putative breeding locales with greater certainty in well‐differentiated species than in poorly differentiated species. Our findings indicate specific phylogeographical structure varies among species, which has strong implications for conservation of habitats within migratory corridors. We suggest that RAPDs are useful in identifying geographical populations of migratory species and that molecular markers should be considered for tracking migratory birds throughout the annual cycle.

Multiplexed microsatellite recovery using massively parallel sequencing

Conservation and management of natural populations requires accurate and inexpensive genotyping methods. Traditional microsatellite, or simple sequence repeat (SSR), marker analysis remains a popular genotyping method because of the comparatively low cost of marker development, ease of analysis and high power of genotype discrimination. With the availability of massively parallel sequencing (MPS), it is now possible to sequence microsatellite‐enriched genomic libraries in multiplex pools. To test this approach, we prepared seven microsatellite‐enriched, barcoded genomic libraries from diverse taxa (two conifer trees, five birds) and sequenced these on one lane of the Illumina Genome Analyzer using paired‐end 80‐bp reads. In this experiment, we screened 6.1 million sequences and identified 356 958 unique microreads that contained di‐ or trinucleotide microsatellites. Examination of four species shows that our conversion rate from raw sequences to polymorphic markers compares favourably to Sanger‐ and 454‐based methods. The advantage of multiplexed MPS is that the staggering capacity of modern microread sequencing is spread across many libraries; this reduces sample preparation and sequencing costs to less than

Geographic variation and genetic structure in Spotted Owls

400 (USD) per species. This price is sufficiently low that microsatellite libraries could be prepared and sequenced for all 1373 organisms listed as ‘threatened’ and ‘endangered’ in the United States for under

Subspecific relationships and genetic structure in the spotted owl

0.5 M (USD).

Range-wide phylogeographic analysis of the spotted frog complex (Rana luteiventris and Rana pretiosa) in northwestern North America

We examined genetic variation, populationstructure, and definition of conservation unitsin Spotted Owls (Strix occidentalis). Spotted Owls are mostly non-migratory,long-lived, socially monogamous birds that havedecreased population viability due to theiroccupation of highly-fragmented latesuccessional forests in western North America. To investigate potential effects of habitatfragmentation on population structure, we usedrandom amplified polymorphic DNA (RAPD) toexamine genetic variation hierarchicallyamong local breeding areas, subregionalgroups, regional groups, and subspeciesvia sampling of 21 breeding areas (276individuals) among the three subspecies ofSpotted Owls. Data from 11 variable bandssuggest a significant relationship betweengeographic distance among local breeding groupsand genetic distance (Mantel r = 0.53, P< 0.02) although multi-dimensional scaling ofthree significant axes did not identifysignificant grouping at any hierarchical level. Similarly, neighbor-joining clustering ofManhattan distances indicated geographicstructure at all levels and identified MexicanSpotted Owls as a distinct clade. RAPDanalyses did not clearly differentiate NorthernSpotted Owls from California Spotted Owls. Among Northern Spotted Owls, estimates ofpopulation differentiation (FST) rangedfrom 0.27 among breeding areas to 0.11 amongregions. Concordantly, within-group agreementvalues estimated via multi-response permutationprocedures of Jaccards distances ranged from0.22 among local sites to 0.11 among regions. Pairwise comparisons of FST andgeographic distance within regions suggestedonly the Klamath region was in equilibrium withrespect to gene flow and genetic drift. Merging nuclear data with recent mitochondrialdata provides support for designation of anEvolutionary Significant Unit for MexicanSpotted Owls and two overlapping ManagementUnits for Northern and California Spotted Owls.

Evidence for recent population bottlenecks in northern spotted owls (Strix occidentalis caurina).

Hierarchical genetic structure was examined in the three geographically-defined subspecies of spotted owl (Strix occidentalis) to define relationships among subspecies and quantify variation within and among regional and local populations. Sequences (522 bp) from domains I and II of the mitochondrial control region were analyzed for 213 individuals from 30 local breeding areas. Results confirmed significant differences between northern spotted owls and the other traditional geographically defined subspecies but did not provide support for subspecific level differences between California and Mexican spotted owls. Divergence times among subspecies estimated with a 936 bp portion of the cytochrome b gene dated Northern and California/Mexican spotted owl divergence time to 115,000–125,000 years ago, whereas California/Mexican spotted owl divergence was estimated at 15,000 years ago. Nested clade analyses indicated an association between California spotted owl and Mexican spotted owl haplotypes, implying historical contact between the two groups. Results also identified a number of individuals geographically classified as northern spotted owls (S. o. caurina) that contained haplotypes identified as California spotted owls (S. o. caurina). Among all northern spotted owls sampled (n=131), 12.9% contained California spotted owl haplotypes. In the Klamath region, which is the contact zone between the two subspecies, 20.3% (n=59) of owls were classified as California spotted owls. The Klamath region is a zone of hybridization and speciation for many other taxa as well. Analyses of population structure indicated gene flow among regions within geographically defined subspecies although there was significant differentiation among northern and southern regions of Mexican spotted owls. Among all areas examined, genetic diversity was not significantly reduced except in California spotted owls where the southern region consists of one haplotype. Our results indicate a stable contact zone between northern and California spotted owls, maintaining distinct subspecific haplotypes within their traditional ranges. This supports recovery efforts based on the traditional subspecies designation for the northern spotted owl. Further, although little variation was found between California and Mexican spotted owls, we suggest they should be managed separately because of current isolation between groups.

SSR_pipeline: A Bioinformatic Infrastructure for Identifying Microsatellites From Paired-End Illumina High-Throughput DNA Sequencing Data

The dynamic geological and climatic history of northwestern North America has made it a focal region for phylogeography. We conducted a range-wide phylogeographic analysis of the spotted frog complex (Rana luteiventris and Rana pretiosa) across its range in northwestern North America to understand its evolutionary history and the distribution of clades to inform conservation of R. pretiosa and Great Basin R. luteiventris, candidates for listing under the US Endangered Species Act. Mitochondrial DNA sequence data from a segment of the cytochrome b gene were obtained from 308 R. luteiventris and R. pretiosa from 96 sites. Phylogenetic analysis revealed one main R. pretiosa clade and three main R. luteiventris clades, two of which overlapped in southeastern Oregon. The three R. luteiventris clades were separated from each other by high levels of sequence divergence (average of 4.75-4.97%). Two divergent clades were also uncovered within the Great Basin. Low genetic variation in R. pretiosa and the southeastern Oregon clade of R. luteiventris suggests concern about their vulnerability to extinction.

Introgression and dispersal among spotted owl (Strix occidentalis) subspecies

The northern spotted owl (Strix occidentalis caurina) is one of the most controversial threatened subspecies ever listed under the US Endangered Species Act. Despite protection of its remaining forest habitat, recent field studies show continued declines of northern spotted owls. One potential threat to northern spotted owls which has not yet been shown is loss of genetic variation from population bottlenecks. Bottlenecks can increase the probability of mating among related individuals, potentially causing inbreeding depression, and can decrease adaptive potential. Here we report evidence for recent bottlenecks in northern spotted owls using a large genetic dataset (352 individuals and 10 microsatellite loci). The signature of bottlenecks was strongest in the Washington Cascade Mountains, in agreement with field data. Our results provide independent evidence that northern spotted owls have recently declined, and suggest that loss of genetic variation is an emerging threat to the subspecies’ persistence. Reduced effective population size (Ne) shown here in addition to field evidence for demographic decline highlights the increasing vulnerability of this bird to extinction.

EVIDENCE FOR POPULATION BOTTLENECKS AND SUBTLE GENETIC STRUCTURE IN THE YELLOW RAIL

SSR_pipeline is a flexible set of programs designed to efficiently identify simple sequence repeats (e.g., microsatellites) from paired-end high-throughput Illumina DNA sequencing data. The program suite contains 3 analysis modules along with a fourth control module that can automate analyses of large volumes of data. The modules are used to 1) identify the subset of paired-end sequences that pass Illumina quality standards, 2) align paired-end reads into a single composite DNA sequence, and 3) identify sequences that possess microsatellites (both simple and compound) conforming to user-specified parameters. The microsatellite search algorithm is extremely efficient, and we have used it to identify repeats with motifs from 2 to 25 bp in length. Each of the 3 analysis modules can also be used independently to provide greater flexibility or to work with FASTQ or FASTA files generated from other sequencing platforms (Roche 454, Ion Torrent, etc.). We demonstrate use of the program with data from the brine fly Ephydra packardi (Diptera: Ephydridae) and provide empirical timing benchmarks to illustrate program performance on a common desktop computer environment. We further show that the Illumina platform is capable of identifying large numbers of microsatellites, even when using unenriched sample libraries and a very small percentage of the sequencing capacity from a single DNA sequencing run. All modules from SSR_pipeline are implemented in the Python programming language and can therefore be used from nearly any computer operating system (Linux, Macintosh, and Windows).

Subspecies Status and Population Genetic Structure in Piping Plover (Charadrius melodus)

Population genetics plays an increasingly important role in the conservation and management of declining species, particularly for defining taxonomic units. Subspecies are recognized by several conservation organizations and countries and receive legal protection under the US Endangered Species Act (ESA). Two subspecies of spotted owls, northern (Strix occidentalis caurina) and Mexican (S. o. lucida) spotted owls, are ESA‐listed as threatened, but the California (S. o. occidentalis) spotted owl is not listed. Thus, determining the boundaries of these subspecies is critical for effective enforcement of the ESA. We tested the validity of previously recognized spotted owl subspecies by analysing 394 spotted owls at 10 microsatellite loci. We also tested whether northern and California spotted owls hybridize as suggested by previous mitochondrial DNA studies. Our results supported current recognition of three subspecies. We also found bi‐directional hybridization and dispersal between northern and California spotted owls centered in southern Oregon and northern California. Surprisingly, we also detected introgression of Mexican spotted owls into the range of northern spotted owls, primarily in the northern part of the subspecies’ range in Washington, indicating long‐distance dispersal of Mexican spotted owls. We conclude with a discussion of the conservation implications of our study.