Variation in the Slimy Salamander (Plethodon spp.) Skin and Gut-Microbial Assemblages Is Explained by Geographic Distance and Host Affinity.

A multicellular host and its microbial communities are recognized as a metaorganism-a composite unit of evolution. Microbial communities have a variety of positive and negative effects on the host life history, ecology, and evolution. This study used high-throughput amplicon sequencing to characterize the complete skin and gut microbial communities, including both bacteria and fungi, of a terrestrial salamander, Plethodon glutinosus (Family Plethodontidae). We assessed salamander populations, representing nine mitochondrial haplotypes ('clades'), for differences in microbial assemblages across 13 geographic locations in the Southeastern United States. We hypothesized that microbial assemblages were structured by both host factors and geographic distance. We found a strong correlation between all microbial assemblages at close geographic distances, whereas, as spatial distance increases, the patterns became increasingly discriminate. Network analyses revealed that gut-bacterial communities have the highest degree of connectedness across geographic space. Host salamander clade was explanatory of skin-bacterial and gut-fungal assemblages but not gut-bacterial assemblages, unless the latter were analyzed within a phylogenetic context. We also inferred the function of gut-fungal assemblages to understand how an understudied component of the gut microbiome may influence salamander life history. We concluded that dispersal limitation may in part describe patterns in microbial assemblages across space and also that the salamander host may select for skin and gut communities that are maintained over time in closely related salamander populations.

Re-evaluation of the Wehrle's salamander (Plethodon wehrlei Fowler and Dunn) species group (Caudata: Plethodontidae) using genomic data, with the description of a new species.

Woodland salamanders of the genus Plethodon are characterized by strong ecological and morphological conservatism. One assemblage, the Wehrle's salamander (Plethodon wehrlei Fowler Dunn) species group, is distributed from New York to Tennessee, USA, and includes several morphological variants, four of which are sufficiently distinct to have been recognized as species in the past. For many years after two of these species were placed in synonymy, only P. wehrlei and P. punctatus Highton were recognized. A recent phylogeographic study using mitochondrial DNA and nuclear DNA uncovered considerable genetic diversity within the group and conservatively resurrected one of the previously synonymized forms (P. dixi Pope Fowler). However, their analysis could not resolve all relationships among remaining populations of P. wehrlei, leaving the taxon paraphyletic. We re-evaluated the evolutionary history of this group using genomic data, recovered strong support for at least five distinct clades, and corroborated previously reported relationships. We also collected morphological data and demonstrated morphological distinctiveness for four of the five clades that we herein recognize as species. We resurrect the synonymized name P. jacksoni Newman to represent the southern clades of P. wehrlei in southwestern Virginia and North Carolina exclusive of P. dixi. In addition, we describe a yellow-spotted form of P. wehrlei endemic to the Cumberland Plateau as a new species. Although our proposed changes rectify the paraphyly of P. wehrlei, our sampling was not sufficient to resolve potential taxonomic issues remaining within the species herein recognized as P. jacksoni.

Methodological considerations for detection of terrestrial small-body salamander eDNA and implications for biodiversity conservation.

Environmental DNA (eDNA) can be used as an assessment tool to detect populations of threatened species and provide fine-scale data required to make management decisions. The objectives of this project were to use quantitative PCR (qPCR) to: (i) detect spiked salamander DNA in soil, (ii) quantify eDNA degradation over time, (iii) determine detectability of salamander eDNA in a terrestrial environment using soil, faeces, and skin swabs, (iv) detect salamander eDNA in a mesocosm experiment. Salamander eDNA was positively detected in 100% of skin swabs and 66% of faecal samples and concentrations did not differ between the two sources. However, eDNA was not detected in soil samples collected from directly underneath wild-caught living salamanders. Salamander genomic DNA (gDNA) was detected in all qPCR reactions when spiked into soil at 10.0, 5.0, and 1.0 ng/g soil and spike concentration had a significant effect on detected concentrations. Only 33% of samples showed recoverable eDNA when spiked with 0.25 ng/g soil, which was the low end of eDNA detection. To determine the rate of eDNA degradation, gDNA (1 ng/g soil) was spiked into soil and quantified over seven days. Salamander eDNA concentrations decreased across days, but eDNA was still amplifiable at day 7. Salamander eDNA was detected in two of 182 mesocosm soil samples over 12 weeks (n = 52 control samples; n = 65 presence samples; n = 65 eviction samples). The discrepancy in detection success between experiments indicates the potential challenges for this method to be used as a monitoring technique for small-bodied wild terrestrial salamander populations.

Draft Genome Sequence of Lactobacillus crispatus JCM5810, Which Can Reduce Campylobacter jejuni Colonization in Chicken Intestine.

We present the 2.05-Mb draft genome sequence ofLactobacillus crispatusJCM5810, a chicken intestinal isolate with the ability to reduceCampylobacter jejunicolonization in chickens. The genome sequence will provide insights on the probiotic mechanisms ofL. crispatusJCM5810.

A metagenomics-based approach to the top-down effect on the detritivore food web: a salamanders influence on fungal communities within a deciduous forest.

The flow of energy within an ecosystem can be considered either top-down, where predators influence consumers, or bottom-up, where producers influence consumers. Plethodon cinereus (Red-backed Salamander) is a terrestrial keystone predator who feeds on invertebrates within the ecosystem. We investigated the impact of the removal of P. cinereus on the detritivore food web in an upland deciduous forest in northwest Ohio, U.S.A. A total of eight aluminum enclosures, each containing a single P. cinereus under a small log, were constructed in the deciduous forest. On Day 1 of the experiment, four salamanders were evicted from four of the eight enclosures. Organic matter and soil were collected from the center of each enclosure at Day 1 and Day 21. From each sample, DNA was extracted, fungal-specific amplification performed, and 454 pyrosequencing was used to sequence the nuclear ribosomal internal transcribed spacer (ITS2) region and partial ribosomal large subunit (LSU). Changes in overall fungal community composition or species diversity were not statistically significant between treatments. Statistically significant shifts in the most abundant taxonomic groups of fungi were documented in presence but not absence enclosures. We concluded that P. cinereus does not affect the overall composition or diversity of fungal communities, but does have an impact on specific groups of fungi. This study used a metagenomics-based approach to investigate a missing link among a keystone predator, P. cinereus, invertebrates, and fungal communities, all of which are critical in the detritivore food web.

Validation of phylogenetic signals in amplified fragment length data: testing the utility and reliability in closely related taxa.

BACKGROUND: Discriminating taxa with the nuclear marker, amplified fragment length polymorphism (AFLP) has been accomplished for various organisms in economic, ecological, and evolutionary studies. The protocol available for AFLP generation does not require prior knowledge of the genome; however, it is often extensively modified to fit the needs of the researcher. Modification of this protocol for new labs is intimidating and time-consuming, particularly for taxa in which AFLP have not been previously developed. Furthermore, determining what constitutes quality output during different stages of fragment generation is not well defined and this may further hinder the use AFLP by new researchers. FINDINGS: We present a step-by-step AFLP protocol, using flourophore-labeled primers for use with automated sequencers, including examples of both successful and unsuccessful results. We sufficiently normalized peak intensity and standardized allele calling across all samples for each primer combination. Repeatability was assessed with a phylogenetic tree in which replicate samples clustered together using the minimum evolution procedure. We found differences greater than 10% in allele position among replicated samples would cause replicates to no longer cluster. To minimize offset allele positions, we suggest that researchers analyze different primer combinations at the same time using multiple dyes with the automated sequencer to minimize mismatched alleles across replicates. CONCLUSION: For researchers wanting to use AFLP, this molecular technique is difficult and time-consuming to develop. Clarifying what constitutes quality output for each step in AFLP generation will help to reduce redundant trials in protocol development and, in turn, advance the discipline of population genetics.

The importance of comparative phylogeography in diagnosing introduced species: a lesson from the seal salamander, Desmognathus monticola.

BACKGROUND: In most regions of the world human influences on the distribution of flora and fauna predate complete biotic surveys. In some cases this challenges our ability to discriminate native from introduced species. This distinction is particularly critical for isolated populations, because relicts of native species may need to be conserved, whereas introduced species may require immediate eradication. Recently an isolated population of seal salamanders, Desmognathus monticola, was discovered on the Ozark Plateau, approximately 700 km west of its broad continuous distribution in the Appalachian Mountains of eastern North America. Using Nested Clade Analysis (NCA) we test whether the Ozark isolate results from population fragmentation (a natural relict) or long distance dispersal (a human-mediated introduction). RESULTS: Despite its broad distribution in the Appalachian Mountains, the primary haplotype diversity of D. monticola is restricted to less than 2.5% of the distribution in the extreme southern Appalachians, where genetic diversity is high for other co-distributed species. By intensively sampling this genetically diverse region we located haplotypes identical to the Ozark isolate. Nested Clade Analysis supports the hypothesis that the Ozark population was introduced, but it was necessary to include haplotypes that are less than or equal to 0.733% divergent from the Ozark population in order to arrive at this conclusion. These critical haplotypes only occur in < 1.2% of the native distribution and NCA excluding them suggest that the Ozark population is a natural relict. CONCLUSION: Our analyses suggest that the isolated population of D. monticola from the Ozarks is not native to the region and may need to be extirpated rather than conserved, particularly because of its potential negative impacts on endemic Ozark stream salamander communities. Diagnosing a species as introduced may require locating nearly identical haplotypes in the known native distribution, which may be a major undertaking. Our study demonstrates the importance of considering comparative phylogeographic information for locating critical haplotypes when distinguishing native from introduced species.

Effects of meal size, meal type, and body temperature on the specific dynamic action of anurans.

Specific dynamic action (SDA), the increase in metabolism stemming from meal digestion and assimilation, varies as a function of meal size, meal type, and body temperature. To test predictions of these three determinants of SDA, we quantified and compared the SDA responses of nine species of anurans, Bombina orientalis, Bufo cognatus, Ceratophrys ornata, Dyscophus antongilli, Hyla cinerea, Kassina maculata, Kassina senegalensis, Pyxicephalus adspersus, and Rana catesbeiana subjected to meal size, meal type, and body temperature treatments. Over a three to seven-fold increase in meal size, anurans experienced predicted increases in postprandial rates of oxygen consumption (VO(2)) the duration of elevated VO(2) and SDA. Meal type had a significant influence on the SDA response, as the digestion and assimilation of hard-bodied, chitinous crickets, mealworms, and superworms required 76% more energy than the digestion and assimilation of soft-bodied earthworms, waxworms, and neonate rodents. Body temperature largely effected the shape of the postprandial metabolic profile; peak VO(2) increased and the duration of the response decreased with an increase in body temperature. Variation in body temperature did not significantly alter SDA for four species, whereas both H. cinerea and R. catesbeiana experienced significant increases in SDA with body temperature. For 13 or 15 species of anurans ranging in mass from 2.4 to 270 g, SMR, postprandial peak VO(2) and SDA scaled with body mass (log-log) with mass exponents of 0.79, 0.93, and 1.05, respectively.