Lineages that have invaded subterranean environments have repeatedly evolved remarkable adaptations to life in darkness. However, observational and experimental studies in additional natural systems are needed to further our understanding of repeated evolution and convergence. In Texas, a radiation of groundwater salamanders (genus Eurycea), with independent invasions of subterranean karstic environments, offers an opportunity to investigate phenotypic convergence, parallel evolution, and the enhancement and regression of sensory systems. Adaptations to a troglobitic life in this clade include morphological, behavioral, and physiological changes within and among species. Intraspecific and interspecific variation in morphology in response to the selective pressures of life underground allows for detailed examination of physical, behavioral, and physiological changes associated with subterranean adaptation within a comparative phylogenetic framework. We find a correlated change between two sensory systems repeated across multiple subterranean Eurycea lineages: the degeneration of the eye and the expansion of the mechanosensory lateral line. The increase in anterior neuromast organs in subterranean lineages was positively correlated with the expression of pax6 (Paired-box 6), a conserved transcription factor important for vertebrate neurogenesis. Our results show a decreasing trend of PAX6 labeling in the neuromasts of adult surface salamanders (Eurycea nana) relative to the maintained labeling in subterranean species (Eurycea rathbuni). These lateral line enhancements are correlated with reductions in the development of optic systems in subterranean salamander lineages. Altogether, our findings provide a starting point for future evolutionary developmental investigations examining the genetic underpinnings of adaptive, repeated evolution in a novel system.

PAX6 is well known as a transcription factor that drives eye development in animals as widely divergent as flies and mammals. In addition to its localization in eyes, PAX6 expression has been reported in the central nervous system, the pancreas, testes, Merkel cells, nasal epithelium, developing cells of the inner ear, and embryonic submandibular salivary gland. Here we show that PAX6 also appears to be present in the mechanosensory neuromasts of the lateral line system in paedomorphic salamanders of the genus Eurycea. Using immunohistochemistry and confocal microscopy to examine a limited number of larvae of two species, listed by the United States of America’s federal government as threatened (E. nana) or endangered (E. rathbuni), we found that anti-PAX6 antibody labeled structures that were extranuclear, and labeling was most intense in the apical appendages of the hair cells of the neuromast. This extranuclear localization raises the possibility of an as yet undescribed function for PAX6 as a cytoskeleton-associated protein.

Background The subgenus Syrrhophus (genus Eleutherodactylus) contains >40 species of small, direct-developing frogs that occur at low to moderate elevations from Texas through Mexico and into Guatemala and Belize, with two species in western Cuba. Morphological conservatism and phenotypic convergence have made species delimitation challenging and resulted in a complicated taxonomic history. Since 2015, molecular systematic work has uncovered eleven new species from western Mexico and one from eastern Mexico, but current taxonomy still underestimates species level diversity and there is confusion surrounding the validity and boundary of several species. Methods We used phylogenetic analysis of 16S ribosomal RNA (rRNA) sequences, multivariate statistical analysis of morphological data, and bioacoustic analysis of male advertisement calls to discover two additional unnamed species of Eleutherodactylus from Central and Western Mexico. We describe those species here. Results Eleutherodactylus (Syrrhophus) humboldti sp. nov. is described from the Quaternary Valle de Bravo volcanic field of the Eje Neovolcánico in Central Mexico. This species is sister to E. maurus and is 3% divergent in 16S. Eleutherodactylus (Syrrhophus) jamesdixoni sp. nov. is described from the Sierra Madre Occidental of western Mexico. This species is sister to E. nitidus and is 3% divergent. We provide color photographs, advertisement call recordings, and molecular diagnoses of these new species and their sister species to aid future workers.

Background.The subgenus Syrrhophus (genus Eleutherodactylus) contains >40 species of small, directdeveloping frogs that occur at low to moderate elevations from Texas through Mexico and into Guatemala and Belize, with two species in western Cuba.Morphological conservatism and phenotypic convergence have made species delimitation challenging and resulted in a complicated taxonomic history.Since 2015, molecular systematic work has uncovered eleven new species from western Mexico and one from eastern Mexico, but current taxonomy still underestimates species level diversity and there is confusion surrounding the validity and boundary of several species. Methods.We used phylogenetic analysis of 16S ribosomal RNA (rRNA) sequences, multivariate statistical analysis of morphological data, and bioacoustic analysis of male advertisement calls to discover two additional unnamed species of Eleutherodactylus from Central and Western Mexico.We describe those species here.Results.Eleutherodactylus (Syrrhophus) humboldti sp.nov. is described from the Quaternary Valle de Bravo volcanic field of the Eje Neovolcánico in Central Mexico.This species is sister to E. maurus and is 3% divergent in 16S.Eleutherodactylus (Syrrhophus) jamesdixoni sp.nov. is described from the Sierra Madre Occidental of western Mexico.This species is sister to E. nitidus and is 3% divergent.We provide color photographs, advertisement call recordings, and molecular diagnoses of these new species and their sister species to aid future workers.

Delineating genetically distinct population segments of threatened species and quantifying population connectivity are important steps in developing effective conservation and management strategies aimed at preventing extinction. The gopher frog (Rana capito) is a xeric-adapted, pond-breeding species endemic to the Gulf and Atlantic coastal plains of the southeastern United States. This species has experienced extensive habitat loss and fragmentation in the formerly widespread longleaf pine-wiregrass savanna where it lives, resulting in individual abundance declines and population extinctions throughout its range. We used individual-based clustering methods along with Bayesian inference of historical migration based on almost 1500 multilocus microsatellite genotypes to examine genetic structure in this taxon. Clustering analyses identified panhandle and peninsular populations in Florida as distinct genetic clusters separated by the Aucilla River, consistent with the division between the Coastal Plain and peninsular mitochondrial lineages, respectively. Analysis of historical migration indicated an east–west population divergence event followed by immigration to the east. Together, our results indicate that the genetically distinct Coastal Plain and peninsular Florida lineages should be considered separately for conservation and management purposes.

Over the last two decades, mitochondrial DNA (mtDNA) sequence data, as well as analyses of nuclear DNA based on the multispecies coalescent model, have increasingly been used to delimit species, sometimes based on limited sampling and without other supporting evidence. We have argued elsewhere that the uncritical use of these approaches has resulted in unnecessary and unwarranted taxonomic changes that have real and long-lasting consequences for science and society. Unfortunately, these arguments have been misrepresented by Burbrink and Ruane's Point of View on "contemporary'' species delimitation in this issue. Here, we discuss the role of models in species delimitation research, and again argue that careful consideration of model assumptions (and their potential violation) is necessary when inferring population history and delimiting species. We echo recent calls for targeted, thorough geographic sampling across contact zones and into parapatric ranges to test for reproductive isolation and draw inferences about the evolutionary independence (or lack thereof) of populations that exchange genes. Finally, despite our very different views on how best to identify species and when to make taxonomic changes, we end by highlighting areas where we agree with Burbrink and Ruane's Point of View and offer suggestions for future research.

Senticolis triaspis is a widespread polytypic colubrid snake that ranges from the southwestern United States southward along the Pacific Coast and part of the Atlantic Coast of Mexico to Costa Rica. Three subspecies have been described based on differences in color pattern and scutellation: S. t. intermedia, S. t. mutabilis, and S. t. triaspis. The last taxonomic revision was completed in 1960. Here, we present an integrative taxonomic analysis of geographic variation using multiple sources of evidence: mitochondrial DNA (817 bp ND4 from 62 samples), traditional morphology (meristic and mensural characters), geometric morphometrics of head shape, and bioclimatic estimates of environmental niche similarity. Molecular data revealed two deeply divergent mitochondrial lineages corresponding to northern and southern clades and eight well-supported clades within these two main groups. Head shape and niche models also showed north–south differences, while traditional morphological analyses did not discriminate among mtDNA lineages. Thus, our study suggests there are at least two candidate species within Senticolis triaspis based on the mtDNA divergence, differences in head shape, and non-overlapping ecological niches. However, we refrain from making taxonomic recommendations pending the addition of nuclear DNA sequence data, finer sampling at contact zones, and additional morphological data; these data are warranted to provide a more robust, stable species delimitation.

Movement behavior is an important aspect of animal ecology but is challenging to study in species that are unobservable for some portion of their lives, such as those inhabiting subterranean environments. Using four years of robust-design capture-recapture data, we examined the probability of movement into subterranean habitat by a population of endangered Barton Springs salamanders (Eurycea sosorum), a species that inhabits both surface and subterranean groundwater habitats. We tested the effects of environmental variables and body size on survival and temporary emigration, using the latter as a measure of subterranean habitat use. Based on 2,046 observations of 1,578 individuals, we found that temporary emigration was higher for larger salamanders, 79% of which temporarily emigrated into subterranean habitat between primary sampling intervals, on average. Body size was a better predictor of temporary emigration and survival compared to environmental covariates, although coefficients from lower ranked models suggested turbidity and dissolved oxygen may influence salamander movement between the surface and subsurface. Surface population dynamics are partly driven by movement below ground and therefore surface abundance estimates represent a fraction of the superpopulation. As such, while surface habitat management remains an important conservation strategy for this species, periodic declines in apparent surface abundance do not necessarily indicate declines of the superpopulation associated with the spring habitat.

The exceptional groundwater community inhabiting the karstic Edwards--Trinity Aquifer system in central Texas has inspired generations of biologists seeking to understand diversification in an extreme environment. Since the late 1990s, molecular genetic tools have increasingly been used to uncover hidden diversity and infer the evolutionary history of groundwater species inhabiting the Edwards-Trinity system. The field of phylogeography-the study of the spatial distribution of genealogical lineages within and among intraspecific populations and closely related species-has provided unparalleled insight into patterns of Edwards-Trinity groundwater biodiversity. Similar to other global groundwater biodiversity hotspots, phylogeographic studies in the Edwards-Trinity Aquifer system have documented exceptionally high levels of endemism and strong population structure due to isolation across naturally fragmented habitat. Cryptic species (two or more morphologically similar but genetically distinct species) have been discovered in a number of phylogeographic investigations, including Eurycea salamanders, Dionda minnows, and Stygobromus amphipods. A number of these species are threatened or endangered with extinction due to habitat loss and degradation resulting from urbanization. Accurately delimiting species boundaries has had significant implications for biodiversity and groundwater conservation in the Edwards-Trinity region because the Endangered Species Act has been used to regulate unrestricted groundwater withdrawal in the eastern Edwards Aquifer where listed species are found. New developments in deoxyribonucleic acid (DNA) sequencing technology coupled with advancements in model-based inference will provide powerful tools for furthering our understanding of Edwards-Trinity biodiversity and predicting its response to a rapidly changing environment.

Groundwater-dependent species are among the least-known components of global biodiversity, as well as some of the most vulnerable because of rapid groundwater depletion at regional and global scales. The karstic Edwards-Trinity aquifer system of west-central Texas is one of the most species-rich groundwater systems in the world, represented by dozens of endemic groundwater-obligate species with narrow, naturally fragmented distributions. Here, we examine how geomorphological and hydrogeological processes have driven population divergence and speciation in a radiation of salamanders (Eurycea) endemic to the Edwards-Trinity system using phylogenetic and population genetic analysis of genome-wide DNA sequence data. Results revealed complex patterns of isolation and reconnection driven by surface and subsurface hydrology, resulting in both adaptive and nonadaptive population divergence and speciation. Our results uncover cryptic species diversity and refine the borders of several threatened and endangered species. The US Endangered Species Act has been used to bring state regulation to unrestricted groundwater withdrawals in the Edwards (Balcones Fault Zone) Aquifer, where listed species are found. However, the Trinity and Edwards-Trinity (Plateau) aquifers harbor additional species with similarly small ranges that currently receive no protection from regulatory programs designed to prevent groundwater depletion. Based on regional climate models that predict increased air temperature, together with hydrologic models that project decreased spring-flow, we conclude that Edwards-Trinity salamanders and other codistributed groundwater-dependent organisms are highly vulnerable to extinction within the next century.

We report new occurrence records for stygobiontic invertebrates from the Edwards and Trinity aquifers in Blanco, Hays, and Travis counties of central Texas, USA. Our collection includes seven species from four families: Caecidoteareddelli (Steeves, 1968), Asellidae; Crangonyxnr.pseudogracilis Bousfield, 1958, Stygobromusbalconis (Hubricht, 1943), Stygobromusbifurcatus (Holsinger, 1967), and Stygobromusrusselli (Holsinger, 1967), Crangonyctidae; Sphalloplanamohri Hyman, 1938, Kenkiidae; and Cirolanides sp., Cirolanidae. Specimens of Caecidoteareddelli and Crangonyxnr.pseudogracilis are new records for Hays County and Travis county, respectively. Specimens of an undescribed species of Cirolanides were collected from a well in Hays County and from two localities in Travis County.

It has come to our attention that in Table 2, four records of Cirolanides sp. were mistakenly labeled as having been catalogued in the University of Texas Insect Collections (UTIC), when in fact they are catalogued in the Aquifer Biology Collection at the Edwards Aquifer Research and Data Center at Texas State University, San Marcos, Texas. All other information about the specimens is correct.The CORRECT Table is as follows:

We present 7 new occurrence records for the Barton Springs Salamander (Eurycea sosorum Chippindale, Price & Hillis, 1993) from Hays and Travis counties, Texas, USA, including the first for this species from the Trinity Aquifer. Eurycea sosorum is listed as endangered under the Endangered Species Act of 1973 due to ongoing threats from urbanization and aquifer overdraft throughout its narrow range. Although this species is more widely distributed than when it was first described in 1993, its range is still exceptionally small, restricted to portions of only two watersheds (Onion and Barton creeks) in one of the fastest-growing metropolitan areas in the United States (Austin, Texas). Under any ecologically-relevant criterion that is based on the best available scientific evidence, this species remains in danger of extinction throughout its range.

The Sierra Madre del Sur (SMS) of southern Mexico is known for its exceptionally high levels of amphibian endemism, but knowledge of species’ ranges in the region is incomplete. Here, we report new locality records for Eleutherodactylus syristes (Hoyt, 1965) from the states of Oaxaca and Guerrero. Previously, this species was known only from the Pacific slopes of the Sierra de Miahuatlán and Mixteca Alta in Oaxaca. These new occurrence records extend the known range of this species by approximately 335 km from the type locality and increase the elevational range, indicating it is more widespread in the SMS. Eleutherodactylus syristes is currently listed as endangered by the IUCN based on its limited distribution and ongoing threats to its persistence.

We conducted a first survey of the amphibians and reptiles of the Nectandra Cloud Forest Reserve in June 2010, during the rainy season in Costa Rica. We found a total of 30 species of amphibians and reptiles in 15 families during our weeklong survey, including 15 frogs, 2 salamanders, 7 lizards, and 6 snakes. We compare our results to those of a previous survey of the nearby San Ramón Reserve.

We describe two new species of Eleutherodactylus, subgenus Syrrhophus, from two separate mountain ranges in western Mexico. Eleutherodactylus grunwaldi sp. nov. inhabits the Sierra de Manantlán in Colima and Jalisco from 1300 to 2200 m, whereas E. wixarika sp. nov. is known from a single locality in the Sierra Huichola of northern Jalisco at 2400 m, but is probably more widespread. Eleutherodactylus grunwaldi is readily distinguishable from most members of mainland Syrrhophus by a combination of its large size, broad, truncate digital pads more than three times the narrowest part of the digit, and a black and green marbled color pattern. This species is saxicolous, inhabiting limestone outcrops, and has been found in caves during the dry season. Eleutherodactylus wixarika is a moderate sized species, most similar to E. teretistes, E. pallidus and E. modestus. It is distinguished from all other members of the subgenus by the combination a tuberculate, reddish dorsum, lack of compact lumbar glands, and expanded digital pads less than twice the width of the narrowest part of the digit. This species inhabits areas with secondary vegetation in pine forest. Males of both species call at night during the rainy season. The advertisement call of both species consists of a short, narrow band, pure-tone note organized into a discrete train at a rate of about six times per minute. Spectral and temporal acoustic properties differ between species. The subgenus Syrrhophus of the genus Eleutherodactylus is one of the most poorly studied groups of frogs in Mexico but probably one of the most diverse.

Young species complexes that are widespread across ecologically disparate regions offer important insights into the process of speciation because of their relevance to how local adaptation and gene flow influence diversification. We used mitochondrial DNA and up to 28 152 genomewide single nucleotide polymorphisms from polytypic barking frogs ( C raugastor augusti complex) to infer phylogenetic relationships and test for the signature of introgressive hybridization among diverging lineages. Our phylogenetic reconstructions suggest (i) a rapid P liocene– P leistocene radiation that produced at least nine distinct lineages and (ii) that geographic features of the arid C entral M exican P lateau contributed to two independent northward expansions. Despite clear lineage differentiation (many private alleles and high between‐lineage F ST scores), D ‐statistic tests, which differentiate introgression from ancestral polymorphism, allowed us to identify two putative instances of reticulate gene flow. Partitioned D ‐statistics provided evidence that these events occurred in the same direction between clades but at different points in time. After correcting for geographic distance, we found that lineages involved in hybrid gene flow interactions had higher levels of genetic variation than independently evolving lineages. These findings suggest that the nature of hybrid compatibility can be conserved overlong periods of evolutionary time and that hybridization between diverging lineages may contribute to standing levels of genetic variation.