Host species is linked to pathogen genotype for the amphibian chytrid fungus (Batrachochytrium dendrobatidis).

Host-pathogen specificity can arise from certain selective environments mediated by both the host and pathogen. Therefore, understanding the degree to which host species identity is correlated with pathogen genotype can help reveal historical host-pathogen dynamics. One animal disease of particular concern is chytridiomycosis, typically caused by the global panzootic lineage of the amphibian chytrid fungus (Batrachochytrium dendrobatidis, Bd), termed Bd-GPL. This pathogen lineage has caused devastating declines in amphibian communities around the world. However, the site of origin for the common ancestor of modern Bd-GPL and the fine-scale transmission dynamics of this lineage have remained a mystery. This is especially the case in North America where Bd-GPL is widespread, but disease outbreaks occur sporadically. Herein, we use Bd genetic data collected throughout the United States from amphibian skin swabs and cultured isolate samples to investigate Bd genetic patterns. We highlight two case studies in Pennsylvania and Nevada where Bd-GPL genotypes are strongly correlated with host species identity. Specifically, in some localities bullfrogs (Rana catesbeiana) are infected with Bd-GPL lineages that are distinct from those infecting other sympatric amphibian species. Overall, we reveal a previously unknown association of Bd genotype with host species and identify the eastern United States as a Bd diversity hotspot and potential site of origin for Bd-GPL.

Population-Level Resistance to Chytridiomycosis is Life-Stage Dependent in an Imperiled Anuran.

Amphibian declines caused by chytridiomycosis have been severe, but some susceptible populations have persisted or even recovered. Resistance to the causal agent Batrachochytrium dendrobatidis (Bd) could result from alleles of the adaptive immune system. During metamorphosis, however, immune systems may not be fully functional, implying that an effective immune response to Bd may be life-stage dependent. We evaluated the susceptibility of the relict leopard frog (Rana onca) sourced from two areas where Bd was present or absent, and where the populations appeared to show differences in pathogen resistance. We evaluated whether population-level resistance manifested across life stages using challenge experiments with late-stage tadpoles (Gosner stage 31-38), metamorphs (stage 45-46), and juvenile frogs. We used three different Bd isolates including one from wild R. onca to challenge juvenile frogs and focused on the isolate from R. onca to challenge tadpoles and resulting metamorphs. We found that juveniles from the Bd exposed population were 5.5 times more likely to survive Bd infection and 10 times more likely to clear infections than those from the area without Bd. In contrast, and regardless of the source area, we observed 98% survivorship of tadpoles, but only 19% survivorship of resulting metamorphs following re-exposure. Given the low survivorship of exposed metamorphs in the laboratory, we speculate on how resistance characteristics, whether adaptive or innate, that do not manifest at each life stage could develop in the wild. We suggest that seasonal high temperatures during times when metamorphosis appears common may modulate the effects of the pathogen during this most susceptible life stage.

Cryptic diversity of a widespread global pathogen reveals expanded threats to amphibian conservation.

Biodiversity loss is one major outcome of human-mediated ecosystem disturbance. One way that humans have triggered wildlife declines is by transporting disease-causing agents to remote areas of the world. Amphibians have been hit particularly hard by disease due in part to a globally distributed pathogenic chytrid fungus (Batrachochytrium dendrobatidis [Bd]). Prior research has revealed important insights into the biology and distribution of Bd; however, there are still many outstanding questions in this system. Although we know that there are multiple divergent lineages of Bd that differ in pathogenicity, we know little about how these lineages are distributed around the world and where lineages may be coming into contact. Here, we implement a custom genotyping method for a global set of Bd samples. This method is optimized to amplify and sequence degraded DNA from noninvasive skin swab samples. We describe a divergent lineage of Bd, which we call BdASIA3, that appears to be widespread in Southeast Asia. This lineage co-occurs with the global panzootic lineage (BdGPL) in multiple localities. Additionally, we shed light on the global distribution of BdGPL and highlight the expanded range of another lineage, BdCAPE. Finally, we argue that more monitoring needs to take place where Bd lineages are coming into contact and where we know little about Bd lineage diversity. Monitoring need not use expensive or difficult field techniques but can use archived swab samples to further explore the history-and predict the future impacts-of this devastating pathogen.

Systematic approach to isolating Batrachochytrium dendrobatidis.

We developed a protocol for isolating the amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) from anurans. We sampled skin tissues from 2 common treefrogs, Pseudacris regilla and P. triseriata, collected from populations with high infection prevalence. We sampled tissues from 3 anatomical ventral regions (thigh, abdomen, and foot) where the pathogen is thought to concentrate. To mitigate potential bacterial contamination, we used a unique combination of 4 antibiotics. We quantified infections on frogs as zoospore equivalents (ZE) using a swabbing approach combined with quantitative real-time polymerase chain reaction. We isolated Bd from 68.9% of frogs sampled from both species. Contamination was low (9.7% of all plates), with most contamination presumed to be fungal. We found positive correlations between successful isolation attempts and infection intensity. Our levels of isolation success were 74% for P. triseriata and 100% for P. regilla once Bd detection intensities reached ≥40 ZE. Of the 3 anatomical regions sampled in both species, we had significantly more success isolating Bd from foot tissue. Our results support published recommendations to focus sampling for Bd infection on feet, particularly webbing.

Batrachochytrium dendrobatidis and the Decline and Survival of the Relict Leopard Frog.

Epizootic disease caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd) is a major driver of amphibian declines, yet many amphibians declined before the pathogen was described. The Relict Leopard Frog, Rana onca (=Lithobates onca), was nearly extinct, with the exception of populations within a few geothermal springs. Growth of Bd, however, is limited by high water temperature, and geothermal springs may have provided refuge during outbreaks of chytridiomycosis. We conducted field surveys and laboratory experiments to assess the susceptibility of R. onca to Bd. In the field, we found Bd at one of the two areas where remnant populations of R. onca still occur, but not in the other. In the laboratory, we infected juvenile frogs from these two areas with two hypervirulent Bd isolates associated with declines in other ranid species. In our experiments, these Bd isolates did not affect survivorship of R. onca and most infections (64%) were cleared by the end of the experiments. We propose that R. onca either has inherent resistance to Bd or has recently evolved such resistance. These results may be important for conservation efforts aimed at establishing new populations of R. onca across a landscape where Bd exists. Resistance, however, varies among life stages, and we also did not assess Bd from the local environment. We caution that the resistance we observed for young frogs under laboratory conditions may not translate to the situation for R. onca in the wild.

Range and niche shifts in response to past climate change in the desert horned lizard (Phrynosoma platyrhinos).

During climate change, species are often assumed to shift their geographic distributions (geographic ranges) in order to track environmental conditions - niches - to which they are adapted. Recent work, however, suggests that the niches do not always remain conserved during climate change but shift instead, allowing populations to persist in place or expand into new areas. We assessed the extent of range and niche shifts in response to the warming climate after the Last Glacial Maximum (LGM) in the desert horned lizard (Phrynosoma platyrhinos), a species occupying the western deserts of North America. We used a phylogeographic approach with mitochondrial DNA sequences to approximate the species range during the LGM by identifying populations that exhibit a genetic signal of population stability versus those that exhibit a signal of a recent (likely post-LGM) geographic expansion. We then compared the climatic niche that the species occupies today with the niche it occupied during the LGM using two models of simulated LGM climate. The genetic analyses indicated that P. platyrhinos persisted within the southern Mojave and Sonoran deserts throughout the latest glacial period and expanded from these deserts northwards, into the western and eastern Great Basin, after the LGM. The climatic niche comparisons revealed that P. platyrhinos expanded its climatic niche after the LGM towards novel, warmer and drier climates that allowed it to persist within the southern deserts. Simultaneously, the species shifted its climatic niche towards greater temperature and precipitation fluctuations after the LGM. We concluded that climatic changes at the end of the LGM promoted both range and niche shifts in this lizard. The mechanism that allowed the species to shift its niche remains unknown, but phenotypic plasticity likely contributes to the species ability to adjust to climate change.

Phylogeography of Beck's Desert Scorpion, Paruroctonus becki, reveals Pliocene diversification in the Eastern California Shear Zone and postglacial expansion in the Great Basin Desert.

The distribution of Beck's Desert Scorpion, Paruroctonus becki (Gertsch and Allred, 1965), spans the 'warm' Mojave Desert and the western portion of the 'cold' Great Basin Desert. We used genetic analyses and species distribution modeling to test whether P. becki persisted in the Great Basin Desert during the Last Glacial Maximum (LGM), or colonized the area as glacial conditions retreated and the climate warmed. Phylogenetic and network analyses of mitochondrial cytochrome c oxidase 1 (cox1), 16S rDNA, and nuclear internal transcribed spacer (ITS-2) DNA sequences uncovered five geographically-structured groups in P. becki with varying degrees of statistical support. Molecular clock estimates and the geographical arrangement of three of the groups suggested that Pliocene geological events in the tectonically dynamic Eastern California Shear Zone may have driven diversification by vicariance. Diversification was estimated to have continued through the Pleistocene, during which a group endemic to the western Great Basin diverged from a related group in the eastern Mojave Desert and western Colorado Plateau. Demographic and network analyses suggested that P. becki underwent a recent expansion in the Great Basin. According to a landscape interpolation of genetic distances, this expansion appears to have occurred from the northwest, implying that P. becki may have persisted in part of the Great Basin during the LGM. This prediction is supported by species distribution models which suggest that climate was unsuitable throughout most of the Great Basin during the LGM, but that small patches of suitable climate may have remained in areas of the Lahontan Trough.

A multilocus perspective on the speciation history of a North American aridland toad (Anaxyrus punctatus).

Interpretations of phylogeographic patterns can change when analyses shift from single gene-tree to multilocus coalescent analyses. Using multilocus coalescent approaches, a species tree and divergence times can be estimated from a set of gene trees while accounting for gene-tree stochasticity. We utilized the conceptual strengths of a multilocus coalescent approach coupled with complete range-wide sampling to examine the speciation history of a broadly distributed, North American warm-desert toad, Anaxyrus punctatus. Phylogenetic analyses provided strong support for three major lineages within A. punctatus. Each lineage broadly corresponded to one of three desert regions. Early speciation in A. punctatus appeared linked to late Miocene-Pliocene development of the Baja California peninsula. This event was likely followed by a Pleistocene divergence associated with the separation of the Chihuahuan and Sonoran Deserts. Our multilocus coalescent-based reconstruction provides an informative contrast to previous single gene-tree estimates of the evolutionary history of A. punctatus.

Wernerius inyoensis, an elusive new scorpion from the Inyo Mountains of California (Scorpiones, Vaejovidae).

A new scorpion species is described from the Inyo Mountains of California (USA). The presence of a strong subaculear spine, along with other characters, places the new species within Wernerius, an incredibly rare genus that until now consisted of only two species. Wernerius inyoensissp. n. can be most easily distinguished from the other members of the genus by smaller adult size, femur and pedipalp dimensions, and differences in hemispermatophore morphology. Previous studies have suggested that the elusive nature of this genus may be attributed to low densities and sporadic surface activity. Herein, we provide another hypothesis, that Wernerius are primarily subterranean. Mitochondrial sequence data are provided for the holotype.

Elucidation of cryptic diversity in a widespread nearctic treefrog reveals episodes of mitochondrial gene capture as frogs diversified across a dynamic landscape.

We investigate the evolutionary history of the wide-ranging Nearctic treefrog Hyla arenicolor through the integration of extensive range-wide sampling, phylogenetic analyses of multilocus genetic data, and divergence dating. Previous phylogeographic studies of this frog documented a potential signature of introgressive hybridization from an ecologically and morphologically divergent sister species. Based on our Bayesian phylogenetic analyses of mitochondrial DNA, we inferred strong phylogeographic structure in H. arenicolor as indicated by seven well-supported clades, five of which correspond to well-defined biogeographic regions. Clades from the Balsas Basin and southwestern Central Mexican Plateau in Mexico, and the Grand Canyon of Arizona, group with the morphologically, behaviorally, and ecologically divergent mountain treefrogs in the H. eximia group, rendering H. arenicolor as paraphyletic. The phylogenetic position of at least two of these three H. arenicolor clades within the H. eximia group, however, is most likely the result of several episodes of introgressive hybridization and subsequent mitochondrial gene capture separated in time and space, as supported by evidence from the nuclear genes. Hyla arenicolor from the Balsas Basin appear to be deeply divergent from other H. arenicolor and represent a distinctly different species. Results suggests that introgressive hybridization events, both ancient and contemporary, coupled with late Neogene vicariance and Pleistocene climate-driven range shifts, have all played a role in the historical diversification of H. arenicolor.

Isolation and characterization of microsatellite markers in the lowland leopard frog (Rana yavapaiensis) and the relict leopard frog (R. onca), two declining frogs of the North American desert southwest.

We characterized 15 microsatellite loci for the lowland leopard frog (Rana yavapaiensis) and the relict leopard frog (R. onca) for future studies of population genetic structure and relatedness. Analysis of 20 individuals from single populations of each species showed that all markers were polymorphic in at least one species. Observed and expected heterozygosities ranged from 0 to 0.94 and from 0.11 to 0.85, respectively, and there were three to 11 alleles per locus. No loci were in linkage disequilibrium, but six loci deviated significantly from Hardy-Weinberg equilibrium, and the presence of a null allele was detected in two of these loci.

Mitochondrial introgression and incomplete lineage sorting through space and time: phylogenetics of crotaphytid lizards.

We investigate the roles of mitochondrial introgression and incomplete lineage sorting during the phylogenetic history of crotaphytid lizards. Our Bayesian phylogenetic estimate for Crotaphytidae is based on analysis of mitochondrial DNA sequence data for 408 individuals representing the 12 extant species of Crotaphytus and Gambelia. The mitochondrial phylogeny disagrees in several respects with a previously published morphological tree, as well as with conventional species designations, and we conclude that some of this disagreement stems from hybridization-mediated mitochondrial introgression, as well as from incomplete lineage sorting. Unidirectional introgression of Crotaphytus collaris (western collared lizard) mitochondria into C. reticulatus (reticulate collared lizard) populations in the Rio Grande Valley of Texas has resulted in the replacement of ancestral C. reticulatus mitochondria over approximately two-thirds of the total range of the species, a linear distance of approximately 270 km. Introgression of C. collaris mitochondria into C. bicinctores (Great Basin collared lizard) populations in southwestern Arizona requires a more complex scenario because at least three temporally separated and superimposed introgression events appear to have occurred in this region. We propose an "introgression conveyor" model to explain this unique pattern of mitochondrial variation in this region. We show with ecological niche modeling that the predicted geographical ranges of C. collaris, C. bicinctores, and C. reticulatus during glacial maxima could have provided enhanced opportunities for past hybridization. Our analyses suggest that incomplete lineage sorting and/or introgression has further confounded the phylogenetic placements of additional species including C. nebrius, C. vestigium, C. insularis, C. grismeri, and perhaps G. copei. Despite many independent instances of interspecific hybridization among crotaphytid lizards, the species continue to maintain morphological and geographic cohesiveness throughout their ranges.

Cryptic Neogene vicariance and Quaternary dispersal of the red-spotted toad (Bufo punctatus): insights on the evolution of North American warm desert biotas.

We define the geographical distributions of mitochondrial DNA (mtDNA) lineages embedded within a broadly distributed, arid-dwelling toad, Bufo punctatus. These patterns were evaluated as they relate to hypothesized vicariant events leading to the formation of desert biotas within western North America. We assessed mtDNA sequence variation among 191 samples from 82 sites located throughout much of the species' range. Parsimony-based haplotype networks of major identified lineages were used in nested clade analysis (NCA) to further elucidate and evaluate shallow phylogeographic patterns potentially associated with Quaternary (Pleistocene-Holocene) vicariance and dispersal. Phylogenetic analyses provided strong support for three monophyletic lineages (clades) within B. punctatus. The geographical distributions of the clades showed little overlap and corresponded to the general boundaries of the Peninsular Desert, and two continental desert regions, Eastern (Chihuahuan Desert-Colorado Plateau) and Western (Mojave-Sonoran deserts), geographically separated along the Rocky Mountains and Sierra Madre Occidental. The observed divergence levels and congruence with postulated events in earth history implicate a late Neogene (latest Miocene-early Pliocene) time frame for separation of the major mtDNA lineages. Evaluation of nucleotide and haplotype diversity and interpretations from NCA reveal that populations on the Colorado Plateau resulted from a recent, likely post-Pleistocene, range expansion from the Chihuahuan Desert. Dispersal across historical barriers separating major continental clades appear to be recent, resulting in secondary contacts in at least two areas. Given the observed contact between major clades, we speculated as to why the observed deep phylogeographic structure has not been eroded during the multiple previous interglacials of the Pleistocene.