Integration of Indigenous Research Methodologies, Traditional Ecological Knowledge and molecular scatology in an assessment of mesocarnivore presence, diet and habitat use on Yurok Ancestral Lands.

Partnerships between Tribes and researchers in wildlife monitoring and application of Traditional Ecological Knowledge (TEK) have taken a variety of forms, and some scholars have noted a need for culturally sensitive approaches. Guided by Indigenous Research Methodologies, this research is coupled with Yurok TEK, or hlkelonah 'ue-megetohl ('to take care of the earth'), enabling an applied, culturally sensitive approach in partnership with the Yurok Tribe. We present results from a molecular scatology study of wildlife within the ancestral territory of the Yurok Tribe. Scats were collected opportunistically on road transects. All samples (N = 132) were analysed via DNA barcoding and results matched to documented 'Oohl 'we-toh (Yurok language) names to determine the depositor species (N = 8). Though there were four focal mesocarnivore species in our study, only bobcat (Chmuuek; Lynx rufus) and gray fox (Wergers; Urocyon cinereoargenteus) were detected as depositor species. Post hoc analyses were conducted to explore distribution, habitat use and selection in a use-availability context, and food habits of these two species. We found almost complete separation of bobcat and gray fox use of transects, as well as indication of partitioning of vegetation cover types and food. We demonstrate an integrated framework of Western and Indigenous sciences that allows the Indigenous researcher to transcend structured academic disciplinary boundaries. Our approach can be modified for partnerships between Tribes, agencies, academics and students for wildlife monitoring in broader geographic regions in various research applications.

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.

Effects of hunting on mating, relatedness, and genetic diversity in a puma population.

Hunting mortality can affect population abundance, demography, patterns of dispersal and philopatry, breeding, and genetic diversity. We investigated the effects of hunting on the reproduction and genetic diversity in a puma population in western Colorado, USA. We genotyped over 11,000 single nucleotide polymorphisms (SNPs), using double-digest, restriction site-associated DNA sequencing (ddRADseq) in 291 tissue samples collected as part of a study on the effects of hunting on puma population abundance and demography in Colorado from 2004 to 2014. The study was designed with a reference period (years 1-5), during which hunting was suspended, followed by a treatment period (years 6-10), in which hunting was reinstated. Our objectives were to examine the effects of hunting on: (1) paternity and male reproductive success; (2) the relatedness between pumas within the population, and (3) genetic diversity. We found that hunting reduced the average age of male breeders. The number of unique fathers siring litters increased each year without hunting and decreased each year during the hunting period. Mated pairs were generally unrelated during both time periods, and females were more closely related than males. Hunting was also associated with increased relatedness among males and decreased relatedness among females in the population. Finally, genetic diversity increased during the period without hunting and decreased each year when hunting was present. This study demonstrates the utility of merging demographic data with large-scale genomic datasets in order to better understand the consequences of management actions. Specifically, we believe that this study highlights the need for long-term experimental research in which hunting mortality is manipulated, including at least one non-harvested control population, as part of a broader adaptive, zone management scheme.

Identification of a novel polyomavirus in wild Sonoran Desert rodents of the family Heteromyidae.

Rodents are the largest and most diverse group of mammals. Covering a wide range of structural and functional adaptations, rodents successfully occupy virtually every terrestrial habitat, and they are often found in close association with humans, domestic animals, and wildlife. Although a significant amount of research has focused on rodents' prominence as known reservoirs of zoonotic viruses, there has been less emphasis on the viral ecology of rodents in general. Here, we utilized a viral metagenomics approach to investigate polyomaviruses in wild rodents from the Baja California peninsula, Mexico, using fecal samples. We identified a novel polyomavirus in fecal samples from two rodent species, a spiny pocket mouse (Chaetodipus spinatus) and a Dulzura kangaroo rat (Dipodomys simulans). These two polyomaviruses represent a new species in the genus Betapolyomavirus. Sequences of this polyomavirus cluster phylogenetically with those of other rodent polyomaviruses and two other non-rodent polyomaviruses (WU and KI) that have been identified in the human respiratory tract. Through our continued work on seven species of rodents, we endeavor to explore the viral diversity associated with wild rodents on the Baja California peninsula and expand on current knowledge of rodent viral ecology and evolution.

Scat as a source of DNA for population monitoring.

Sampling fecal droppings (scat) to genetically identify individual animals is an established method for monitoring mammal populations and could be highly useful for monitoring reptile populations. Whereas existing protocols for obtaining DNA from reptile scat focus on analyses of whole, fresh scat deposited during animal handling, the collection of scat naturally deposited by reptiles in situ, as required for non-invasive population monitoring, requires protocols to extract highly degraded DNA. Using surface swabs from such scats can reduce PCR inhibition and increase genotyping success. We report on three related but independently designed studies of DNA analyses from scat swabs of herbivorous reptiles under natural desert conditions: two free-ranging desert tortoise species (Agassiz's desert tortoise, Gopherus agassizii, California, US, and Morafka's desert tortoise, G. morafkai, Arizona, US) and the common chuckwalla (Sauromalus atar) (Arizona, US, and Sonora, MX). We analyzed samples from both tortoise species with the same set of 16 microsatellites and chuckwalla samples with four mtDNA markers; studies also varied in swab preservation medium and DNA extraction method. Microsatellite amplification success per sample, defined as ≥9 loci with amplification, was 15% for the study of Agassiz's desert tortoise and for the study of 42% Morafka's desert tortoise. For chuckwallas, we successfully amplified and sequenced 50% of samples. We recovered fragments up to 400 bp for tortoises and 980 bp for chuckwallas from scat swab samples. This study indicates that genotypes can successfully be obtained from swabs of scat from herbivorous reptiles collected in the field under natural environmental conditions and emphasizes that repeat amplifications are necessary for the genetic identification of individuals from non-invasive samples.

Give and take: Effects of genetic admixture on mutation load in endangered Florida panthers.

Genetic admixture is a biological event inherent to genetic rescue programs aimed at the long-term conservation of endangered wildlife. Although the success of such programs can be measured by the increase in genetic diversity and fitness of subsequent admixed individuals, predictions supporting admixture costs to fitness due to the introduction of novel deleterious alleles are necessary. Here, we analyzed nonsynonymous variation from conserved genes to quantify and compare levels of mutation load (i.e. proportion of deleterious alleles and genotypes carrying these alleles) among endangered Florida panthers and non-endangered Texas pumas. Specifically, we used canonical (i.e. non-admixed) Florida panthers, Texas pumas, and F1 (canonical Florida × Texas) panthers dating from a genetic rescue program and Everglades National Park panthers with Central American ancestry resulting from an earlier admixture event. We found neither genetic drift nor selection significantly reduced overall proportions of deleterious alleles in the severely bottlenecked canonical Florida panthers. Nevertheless, the deleterious alleles identified were distributed into a disproportionately high number of homozygous genotypes due to close inbreeding in this group. Conversely, admixed Florida panthers (either with Texas or Central American ancestry) presented reduced levels of homozygous genotypes carrying deleterious alleles but increased levels of heterozygous genotypes carrying these variants relative to canonical Florida panthers. Although admixture is likely to alleviate the load of standing deleterious variation present in homozygous genotypes, our results suggest that introduced novel deleterious alleles (temporarily present in heterozygous state) in genetically rescued populations could potentially be expressed in subsequent generations if their effective sizes remain small.

Sex-specific markers undetected in green sunfish Lepomis cyanellus using restriction-site associated DNA sequencing.

We used restriction-site associated DNA sequencing for SNP discovery and genotyping of known-sex green sunfish Lepomis cyanellus DNA samples to search for sex-diagnostic single nucleotide polymorphisms (SNPs) and restriction-site associated sequences present in one sex and absent in the other. The bioinformatic analyses discovered candidate SNPs and sex-specific restriction-site associated sequences that fit patterns of male or female heterogametic sex determination systems. However, when primers were developed and tested, no candidates reliably identified phenotypic sex. The top performing SNP candidate (ZW_218) correlated with phenotypic sex 63.0% of the time and the presence-absence loci universally amplified in both sexes. We recommend further investigations that interrogate a larger fraction of the L. cyanellus genome. Additionally, studies on the effect of temperature and rearing density on sex determination, as well as breeding of sex-reversed individuals, could provide more insights into the sex determination system of L. cyanellus.

Complex evolutionary history of felid anelloviruses.

Anellovirus infections are highly prevalent in mammals, however, prior to this study only a handful of anellovirus genomes had been identified in members of the Felidae family. Here we characterise anelloviruses in pumas (Puma concolor), bobcats (Lynx rufus), Canada lynx (Lynx canadensis), caracals (Caracal caracal) and domestic cats (Felis catus). The complete anellovirus genomes (n = 220) recovered from 149 individuals were diverse. ORF1 protein sequence similarity network analysis coupled with phylogenetic analysis, revealed two distinct clusters that are populated by felid-derived anellovirus sequences, a pattern mirroring that observed for the porcine anelloviruses. Of the two-felid dominant anellovirus groups, one includes sequences from bobcats, pumas, domestic cats and an ocelot, and the other includes sequences from caracals, Canada lynx, domestic cats and pumas. Coinfections of diverse anelloviruses appear to be common among the felids. Evidence of recombination, both within and between felid-specific anellovirus groups, supports a long coevolution history between host and virus.

Agriculture creates subtle genetic structure among migratory and nonmigratory populations of burrowing owls throughout North America.

Population structure across a species distribution primarily reflects historical, ecological, and evolutionary processes. However, large-scale contemporaneous changes in land use have the potential to create changes in habitat quality and thereby cause changes in gene flow, population structure, and distributions. As such, land-use changes in one portion of a species range may explain declines in other portions of their range. For example, many burrowing owl populations have declined or become extirpated near the northern edge of the species' breeding distribution during the second half of the 20th century. In the same period, large extensions of thornscrub were converted to irrigated agriculture in northwestern Mexico. These irrigated areas may now support the highest densities of burrowing owls in North America. We tested the hypothesis that burrowing owls that colonized this recently created owl habitat in northwestern Mexico originated from declining migratory populations from the northern portion of the species' range (migration-driven breeding dispersal whereby long-distance migrants from Canada and the United States became year-round residents in the newly created irrigated agriculture areas in Mexico). We used 10 novel microsatellite markers to genotype 1,560 owls from 36 study locations in Canada, Mexico, and the United States. We found that burrowing owl populations are practically panmictic throughout the entire North American breeding range. However, an analysis of molecular variance provided some evidence that burrowing owl populations in northwestern Mexico and Canada together are more genetically differentiated from the rest of the populations in the breeding range, lending some support to our migration-driven breeding dispersal hypothesis. We found evidence of subtle genetic differentiation associated with irrigated agricultural areas in southern Sonora and Sinaloa in northwestern Mexico. Our results suggest that land use can produce location-specific population dynamics leading to subtle genetic structure even in the absence of dispersal barriers.

Novel Circoviruses Detected in Feces of Sonoran Felids.

Sonoran felids are threatened by drought and habitat fragmentation. Vector range expansion and anthropogenic factors such as habitat encroachment and climate change are altering viral evolutionary dynamics and exposure. However, little is known about the diversity of viruses present in these populations. Small felid populations with lower genetic diversity are likely to be most threatened with extinction by emerging diseases, as with other selective pressures, due to having less adaptive potential. We used a metagenomic approach to identify novel circoviruses, which may have a negative impact on the population viability, from confirmed bobcat (Lynx rufus) and puma (Puma concolor) scats collected in Sonora, Mexico. Given some circoviruses are known to cause disease in their hosts, such as porcine and avian circoviruses, we took a non-invasive approach using scat to identify circoviruses in free-roaming bobcats and puma. Three circovirus genomes were determined, and, based on the current species demarcation, they represent two novel species. Phylogenetic analyses reveal that one circovirus species is more closely related to rodent associated circoviruses and the other to bat associated circoviruses, sharing highest genome-wide pairwise identity of approximately 70% and 63%, respectively. At this time, it is unknown whether these scat-derived circoviruses infect felids, their prey, or another organism that might have had contact with the scat in the environment. Further studies should be conducted to elucidate the host of these viruses and assess health impacts in felids.

De Novo Assembly and Annotation from Parental and F1 Puma Genomes of the Florida Panther Genetic Restoration Program.

In the mid-1990s, the population size of Florida panthers became so small that many individuals manifested traits associated with inbreeding depression (e.g., heart defects, cryptorchidism, high pathogen-parasite load). To mitigate these effects, pumas from Texas were introduced into South Florida to augment genetic variation in Florida panthers. In this study, we report a de novo puma genome assembly and annotation after resequencing 10 individual genomes from partial Florida-Texas-F1 trios. The final genome assembly consisted of ∼2.6 Gb and 20,561 functionally annotated protein-coding genes. Foremost, expanded gene families were associated with neuronal and embryological development, whereas contracted gene families were associated with olfactory receptors. Despite the latter, we characterized 17 positively selected genes related to the refinement of multiple sensory perceptions, most notably to visual capabilities. Furthermore, genes under positive selection were enriched for the targeting of proteins to the endoplasmic reticulum, degradation of mRNAs, and transcription of viral genomes. Nearly half (48.5%) of ∼6.2 million SNPs analyzed in the total sample set contained putative unique Texas alleles. Most of these alleles were likely inherited to subsequent F1 Florida panthers, as these individuals manifested a threefold increase in observed heterozygosity with respect to their immediate, canonical Florida panther predecessors. Demographic simulations were consistent with a recent colonization event in North America by a small number of founders from South America during the last glacial period. In conclusion, we provide an extensive set of genomic resources for pumas and elucidate the genomic effects of genetic rescue on this iconic conservation success story.

Genetic assessment of a bighorn sheep population expansion in the Silver Bell Mountains, Arizona.

BACKGROUND: The isolated population of desert bighorn sheep in the Silver Bell Mountains of southern Arizona underwent an unprecedented expansion in merely four years. We hypothesized that immigration from neighboring bighorn sheep populations could have caused the increase in numbers as detected by Arizona Game and Fish Department annual aerial counts. METHODS: We applied a multilocus genetic approach using mitochondrial DNA and nuclear microsatellite markers for genetic analyses to find evidence of immigration. We sampled the Silver Bell Mountains bighorn sheep before (2003) and during (2015) the population expansion, and a small number of available samples from the Gila Mountains (southwestern Arizona) and the Morenci Mine (Rocky Mountain bighorn) in an attempt to identify the source of putative immigrants and, more importantly, to serve as comparisons for genetic diversity metrics. RESULTS: We did not find evidence of substantial gene flow into the Silver Bell Mountains population. We did not detect any new mitochondrial haplotypes in the 2015 bighorn sheep samples. The microsatellite analyses detected only one new allele, in one individual from the 2015 population that was not detected in the 2003 samples. Overall, the genetic diversity of the Silver Bell Mountains population was lower than that seen in either the Gila population or the Morenci Mine population. DISCUSSION: Even though the results of this study did not help elucidate the precise reason for the recent population expansion, continued monitoring and genetic sampling could provide more clarity on the genetic demographics of this population.

Genome-Wide Analysis of SNPs Is Consistent with No Domestic Dog Ancestry in the Endangered Mexican Wolf (Canis lupus baileyi).

The Mexican gray wolf (Canis lupus baileyi) was historically distributed throughout the southwestern United States and northern Mexico. Extensive predator removal campaigns during the early 20th century, however, resulted in its eventual extirpation by the mid 1980s. At this time, the Mexican wolf existed only in 3 separate captive lineages (McBride, Ghost Ranch, and Aragón) descended from 3, 2, and 2 founders, respectively. These lineages were merged in 1995 to increase the available genetic variation, and Mexican wolves were reintroduced into Arizona and New Mexico in 1998. Despite the ongoing management of the Mexican wolf population, it has been suggested that a proportion of the Mexican wolf ancestry may be recently derived from hybridization with domestic dogs. In this study, we genotyped 87 Mexican wolves, including individuals from all 3 captive lineages and cross-lineage wolves, for more than 172000 single nucleotide polymorphisms. We identified levels of genetic variation consistent with the pedigree record and effects of genetic rescue. To identify the potential to detect hybridization with domestic dogs, we compared our Mexican wolf genotypes with those from studies of domestic dogs and other gray wolves. The proportion of Mexican wolf ancestry assigned to domestic dogs was only between 0.06% (SD 0.23%) and 7.8% (SD 1.0%) for global and local ancestry estimates, respectively; and was consistent with simulated levels of incomplete lineage sorting. Overall, our results suggested that Mexican wolves lack biologically significant ancestry with dogs and have useful implications for the conservation and management of this endangered wolf subspecies.

Life histories predict genetic diversity and population structure within three species of octopus targeted by small-scale fisheries in Northwest Mexico.

The fishery for octopus in Northwest Mexico has increased to over 2,000 tons annually, but to date the specific composition of the catch has been ignored. With at least three main species targeted by artisanal fisheries in the region with distinct life histories, the lack of basic biological information about the distribution, metapopulation size and structure of each species could impede effective fisheries management to avoid overexploitation. We tested if different life histories of three species of octopus could help predict observed patterns of genetic diversity, population dynamics, structure and connectivity and how this information could be relevant to the sustainable management of the fishery. We sequenced two mitochondrial genes and genotyped seven nuclear microsatellite loci to identify the distribution of each species in 20 locations from the Gulf of California and the west coast of the Baja California peninsula. We tested five hypotheses derived from population genetic theory based on differences in the fecundity and dispersal potential for each species. We discovered that Octopus bimaculoides with low fecundity and direct development (without a planktonic phase) had lower average effective population size and genetic diversity, but higher levels of kinship, population structure, and richness of private alleles, than the other two species. These features indicated limited dispersal and high local recruitment. In contrast, O. bimaculatus and O. hubbsorum with higher fecundity and planktonic phase as paralarvae had higher effective population size and genetic diversity, and overall lower kinship and population structure than O. bimaculoides. These observations supported higher levels of gene flow over a larger geographical scale. O. bimaculatus with the longest planktonic paralarval duration and therefore larger dispersal potential had differences in the calculated parameters possibly associated with increased connectivity. We propose O. bimaculoides is more susceptible to over exploitation of small, isolated populations and could have longer recovery times than the other two species. This species may benefit from distinct fishery management within each local population. O. bimaculatus and O. hubbsorum may benefit from fishery management that takes into account metapopulation structure over larger geographic scales and the directionality and magnitude of larval dispersal driven by ocean currents and population connectivity among individuals of each locality. The distribution of each species and variations in their reproductive phenology is also important to consider when establishing marine reserves or seasonal fishing closures.

Evolutionary and Functional Mitogenomics Associated With the Genetic Restoration of the Florida Panther.

Florida panthers are endangered pumas that currently persist in reduced patches of habitat in South Florida, USA. We performed mitogenome reference-based assemblies for most parental lines of the admixed Florida panthers that resulted from the introduction of female Texas pumas into South Florida in 1995. With the addition of 2 puma mitogenomes, we characterized 174 single nucleotide polymorphisms (SNPs) across 12 individuals. We defined 5 haplotypes (Pco1-Pco5), one of which (Pco1) had a geographic origin exclusive to Costa Rica and Panama and was possibly introduced into the Everglades National Park, Florida, prior to 1995. Haplotype Pco2 was native to Florida. Haplotypes Pco3 and Pco4 were exclusive to Texas, whereas haplotype Pco5 had an undetermined geographic origin. Phylogenetic inference suggests that haplotypes Pco1-Pco4 diverged ~202000 (95% HPDI = 83000-345000) years ago and that haplotypes Pco2-Pco4 diverged ~61000 (95% HPDI = 9000-127000) years ago. These results are congruent with a south-to-north continental expansion and with a recent North American colonization by pumas. Furthermore, pumas may have migrated from Texas to Florida no earlier than ~44000 (95% HPDI = 2000-98000) years ago. Synonymous mutations presented a greater mean substitution rate than other mitochondrial functional regions: nonsynonymous mutations, tRNAs, rRNAs, and control region. Similarly, all protein-coding genes were under predominant negative selection constraints. We directly and indirectly assessed the presence of potential deleterious SNPs in the ND2 and ND5 genes in Florida panthers prior to and as a consequence of the introduction of Texas pumas. Screenings for such variants are recommended in extant Florida panthers.

Molecular detection of bacteria in the families Rickettsiaceae and Anaplasmataceae in northern crested caracaras (Caracara cheriway).

Bacterial pathogens of the families Anaplasmataceae and Rickettsiaceae are often spread to humans or other animals from bites from infected arthropod hosts. Recently, an increasing number of studies have implicated migratory birds in the circulation of these pathogens through the spread of arthropod vectors. However, few studies have examined the potential for resident bird populations to serve as reservoirs for these zoonoses. In this study, we used nested PCRs of the GroESL and 17 kDa genes to screen for Anaplasmataceae and Rickettsiaceae, respectively, in a resident population of the northern crested caracara (Caracara cheriway) from Florida (n=55). Additionally, a small number (n=6) of captive individuals from Texas were included. We identified one individual (1.64%) positive for Rickettsia felis and one (1.64%) positive for Ehrlichia chaffeensis; both these individuals were from Florida. Presence of these pathogens demonstrates that these birds are potential hosts; however, the low prevalence of infections suggests that these populations likely do not function as an ecological reservoir.

Assessing models of speciation under different biogeographic scenarios; an empirical study using multi-locus and RNA-seq analyses.

Evolutionary biology often seeks to decipher the drivers of speciation, and much debate persists over the relative importance of isolation and gene flow in the formation of new species. Genetic studies of closely related species can assess if gene flow was present during speciation, because signatures of past introgression often persist in the genome. We test hypotheses on which mechanisms of speciation drove diversity among three distinct lineages of desert tortoise in the genus Gopherus. These lineages offer a powerful system to study speciation, because different biogeographic patterns (physical vs. ecological segregation) are observed at opposing ends of their distributions. We use 82 samples collected from 38 sites, representing the entire species' distribution and generate sequence data for mtDNA and four nuclear loci. A multilocus phylogenetic analysis in *BEAST estimates the species tree. RNA-seq data yield 20,126 synonymous variants from 7665 contigs from two individuals of each of the three lineages. Analyses of these data using the demographic inference package ∂a∂i serve to test the null hypothesis of no gene flow during divergence. The best-fit demographic model for the three taxa is concordant with the *BEAST species tree, and the ∂a∂i analysis does not indicate gene flow among any of the three lineages during their divergence. These analyses suggest that divergence among the lineages occurred in the absence of gene flow and in this scenario the genetic signature of ecological isolation (parapatric model) cannot be differentiated from geographic isolation (allopatric model).

Testing Taxon Tenacity of Tortoises: evidence for a geographical selection gradient at a secondary contact zone.

We examined a secondary contact zone between two species of desert tortoise, Gopherus agassizii and G. morafkai. The taxa were isolated from a common ancestor during the formation of the Colorado River (4-8 mya) and are a classic example of allopatric speciation. However, an anomalous population of G. agassizii comes into secondary contact with G. morafkai east of the Colorado River in the Black Mountains of Arizona and provides an opportunity to examine reinforcement of species' boundaries under natural conditions. We sampled 234 tortoises representing G. agassizii in California (n - 103), G. morafkai in Arizona (n - 78), and 53 individuals of undetermined assignment in the contact zone including and surrounding the Black Mountains. We genotyped individuals for 25 STR loci and determined maternal lineage using mtDNA sequence data. We performed multilocus genetic clustering analyses and used multiple statistical methods to detect levels of hybridization. We tested hypotheses about habitat use between G. agassizii and G. morafkai in the region where they co-occur using habitat suitability models. Gopherus agassizii and G. morafkai maintain independent taxonomic identities likely due to ecological niche partitioning, and the maintenance of the hybrid zone is best described by a geographical selection gradient model.

Concurrent speciation in the eastern woodland salamanders (Genus Plethodon): DNA sequences of the complete albumin nuclear and partial mitochondrial 12s genes.

Salamanders of the North American plethodontid genus Plethodon are important model organisms in a variety of studies that depend on a phylogenetic framework (e.g., chemical communication, ecological competition, life histories, hybridization, and speciation), and consequently their systematics has been intensively investigated over several decades. Nevertheless, we lack a synthesis of relationships among the species. In the analyses reported here we use new DNA sequence data from the complete nuclear albumin gene (1818 bp) and the 12s mitochondrial gene (355 bp), as well as published data for four other genes (Wiens et al., 2006), up to a total of 6989 bp, to infer relationships. We relate these results to past systematic work based on morphology, allozymes, and DNA sequences. Although basal relationships show a strong consensus across studies, many terminal relationships remain in flux despite substantial sequencing and other molecular and morphological studies. This systematic instability appears to be a consequence of contemporaneous bursts of speciation in the late Miocene and Pliocene, yielding many closely related extant species in each of the four eastern species groups. Therefore we conclude that many relationships are likely to remain poorly resolved in the face of additional sequencing efforts. On the other hand, the current classification of the 45 eastern species into four species groups is supported. The Plethodon cinereus group (10 species) is the sister group to the clade comprising the other three groups, but these latter groups (Plethodon glutinosus [28 species], Plethodon welleri [5 species], and Plethodon wehrlei [2 species]) probably diverged from each other at approximately the same time.

Novel primers for complete mitochondrial cytochrome b gene sequencing in mammals.

Sequence-based species identification relies on the extent and integrity of sequence data available in online databases such as GenBank. When identifying species from a sample of unknown origin, partial DNA sequences obtained from the sample are aligned against existing sequences in databases. When the sequence from the matching species is not present in the database, high-scoring alignments with closely related sequences might produce unreliable results on species identity. For species identification in mammals, the cytochrome b (cyt b) gene has been identified to be highly informative; thus, large amounts of reference sequence data from the cyt b gene are much needed. To enhance availability of cyt b gene sequence data on a large number of mammalian species in GenBank and other such publicly accessible online databases, we identified a primer pair for complete cyt b gene sequencing in mammals. Using this primer pair, we successfully PCR amplified and sequenced the complete cyt b gene from 40 of 44 mammalian species representing 10 orders of mammals. We submitted 40 complete, correctly annotated, cyt b protein coding sequences to GenBank. To our knowledge, this is the first single primer pair to amplify the complete cyt b gene in a broad range of mammalian species. This primer pair can be used for the addition of new cyt b gene sequences and to enhance data available on species represented in GenBank. The availability of novel and complete gene sequences as high-quality reference data can improve the reliability of sequence-based species identification.