Black Salamander Research Articles

A Latitudinal Variation in Growth Rate Caused by the Differences in the Length of the Period Suitable for Larval Growth in the Japanese Black Salamander, Hynobius nigrescens.

In ectotherms, body size differences between latitudes resulting from shorter activity periods at higher latitudes may disappear due to higher growth rates. Although such latitudinal variations have been examined for various taxa, only a few studies have examined such variations in Japanese vertebrates. Hynobius nigrescens is widely distributed in the northern part of Honshu, Japan, and although their larval period in the wild is shorter at high latitudes, there is no latitudinal variation in the body size of juveniles in the wild. Therefore, larvae may grow faster at high latitudes, and they can grow into juveniles with body sizes similar to those at low latitudes. To examine whether the growth rate of larvae is faster at high latitudes, we reared groups at 15°C and 20°C. We found that the larval period was shorter and the growth rate was greater at high latitudes. Next, we examined whether the body sizes of reared individuals differed between latitudes. We found that the body sizes of metamorphosed juveniles were smaller at high latitudes. Some ectotherms are known to feed more at high latitudes, leading to the disappearance of latitudinal differences in body size between latitudes. In this study, we provided the same amount of food regardless of latitude, which might have caused the differences in body size between latitudes. Our study suggested that the high growth rate at relatively high latitudes can compensate for the short activity period, leading to similar body sizes of juveniles among latitudes.

Temporal Changes in Geographic Range and Population Size of the Santa Cruz Black Salamander

Abstract The Santa Cruz black salamander Aneides niger is a priority 3 California species of special concern with a restricted geographic range confined to the Santa Cruz Mountains. Anecdotal observations suggest that the species was relatively abundant in the early 1900s, but it has become more difficult to find in the past few decades. To better understand if the species has undergone population size fluctuations, we analyzed mitochondrial and nuclear sequence data to examine levels of genetic variation and phylogeographic structure, and test for signatures of population size change. We then reconstructed the climatic suitability for the species to 1) determine if past climate fluctuations could have influenced range size and genetic diversity, and 2) estimate the effects of future climate change on geographic range suitability as a proxy for possible future population size change. Genetic analyses detected low levels of genetic variation and a general lack of genetic structuring, suggesting a recent genetic bottleneck. While neutrality tests of individual loci were nonsignificant, skyline plot and isolation-with-migration analyses detected a relatively recent reduction in population size. Interpretation of these genetic results should consider the limited number of localities and individuals sampled for this species. Climatic suitability for Santa Cruz black salamanders was much lower during the last glacial maximum, which could be the cause of the detected historical change in population size. Future projections of climatic suitability under a high-emission scenario suggest a dramatic geographic range restriction to coastal areas. These projections highlight the need for the protection of coastal habitat patches to preserve existing coastal populations, and to maintain connectivity between coastal and inland habitats to allow the westward movement of populations and genes in response to climate change.

Dark future for a black salamander: effects of climate change and conservation implications for an endemic alpine amphibian

Climate change is threatening several montane species across the world, including a large number of endemics, needing the development of forward-looking conservation strategies to foster their future survival. In this context, Species Distribution Models (SDMs) represent a useful method to forecast changes in species’ habitat suitability under different scenarios of global warming, often advising conservation frameworks with credible, defensible and repeatable information. In this paper, we estimate the environmental and bioclimatic suitability for an endemic mountain amphibian (Salamandra lanzai) in the western European Alps through an SDM approach, considering both current and future scenarios, to address short- and long-term management and conservation actions, and to update the current IUCN extinction risk assessment. The ensemble model forecasts predict a dramatic decline of the climatically suitable area for the Lanza’s alpine salamander in the next 20–40 years, even considering an optimistic CO2 emissions scenario, leading to a theoretical extinction of this species in 2100 in case the worst global warming prediction will be actualised. This underlines the urgent need for up-to-date conservation and management strategies to ensure the successful mitigation of climate change effects on S. lanzai, especially by adapting and improving the network of protected areas, immediately removing additional threats and identifying possible management actions able to increase fine-scale habitat suitability and connectivity among populations. In addition, a significant range contraction in the future has to be considered when assessing the extinction risk for this species, possibly exacerbating the effect of other threatening factors, such as the spread of lethal pathogens. Keywords: Salamandra lanzai, ensemble models, environmental suitability, bioclimatic suitability, future projections

Non-tuberculous mycobacterial disease associated with Mycobacterium montefiorense in salamanders.

Mycobacterium montefiorense is one of the causes of non-tuberculous mycobacterial infections in moray eels and salamanders. Although M. montefiorense infection could be a threat to salamanders, little information is available regarding this pathogen and associated infection. This study aimed to provide fundamental information regarding M. montefiorense and its infection in salamanders. Nine M. montefiorense strains isolated from three species of salamanders, namely, Japanese black salamander (Hynobius nigrescens), Hakuba salamander (H. hidamontanus), and Tohoku hynobiid salamander (H. lichenatus), between 2010 and 2018, were characterized based on phenotypic and genetic examination. We also pathologically observed salamanders infected with the M. montefiorense strains, including Hakuba salamanders and Tohoku hynobiid salamanders. The microbiological and chemical characteristics of the M. montefiorense salamander and an eel strain (reference strain) matched. Susceptibility testing for antimicrobials suggested that clarithromycin may be effective. Regarding disinfectants, phtharal, peracetic acid, glutaral, sodium hypochlorite, and benzalkonium chloride may be effective. Phylogenetic analyses revealed that the strains isolated from salamanders in 2014 and 2018 were genetically closely related, which could indicate an outbreak. The main gross findings in infected salamanders include skin ulcerative lesions or nodules in the enlarged liver. Microscopically, multifocal to coalescent granulomatous lesions composed of massive macrophages containing numerous acid-fast bacilli were prominently observed in the liver. This study contributes to our understanding of the genetic diversity and phenotypic characteristics of M. montefiorense, as well as the pathology of the infection.

Spatial Variation in Egg Sac Transparency of the Japanese Black Salamander, Hynobius nigrescens, Related to Environmental Gradients in the Ryohaku Mountains, Ishikawa Prefecture

Spatial Variation in Egg Sac Transparency of the Japanese Black Salamander, Hynobius nigrescens, Related to Environmental Gradients in the Ryohaku Mountains, Ishikawa Prefecture

An assessment of aquatic amphibian biodiversity on the California Lost Coast

One of the most difficult aspects of studying intact amphibian communities is that they tend to occupy isolated areas within inaccessible terrain—factors that both protect watersheds from development and disturbance while also making them difficult to study. We conducted an extensive survey of the freshwater herpetofauna of the remote King Range National Conservation Area in Northern California using a combination of visual encounter surveys and environmental DNA (eDNA) sampling. We found twelve species of native aquatic amphibians and the western pond turtle (Actinemys marmorata), and no introduced amphibians. Detection probabilities for the four most commonly encountered species, giant salamanders (Dicamptodon sp), foothill yellow-legged frogs (Rana boylii), western toads (Anaxyrus boreas), and black salamanders (Aneides flavipunctatus), were affected by substrate and canopy cover, but the effects of these habitat characteristics on detection probability were species specific. Neither survey method, visual encounter surveys nor eDNA sampling, was more effective than the other, and our study suggests that the use of visual encounter surveys in conjunction with eDNA sampling may counteract the shortcomings of either when done individually. Five species were found using both methods, seven were only encountered during visual encounter surveys, and one recorded only from eDNA sampling. DNA samples from two taxa, toads and giant salamanders, could not be resolved to species. Toad species identity was assigned to the only member of the candidate species with a species range known to overlap the study area; the other three candidate species occupy restricted ranges far from the study area. Neither of the two giant salamander candidate species have known species ranges overlapping the study area. One, the California giant salamander (D. ensata), is known to occur within 100 km. However, there is a paucity of genetic material in GenBank DNA library for both the California giant salamander and the coastal giant salamander (D. tenebrosus), a widely distributed species with a range overlapping the study area, which could lead to inaccurate assignment of eDNA fragments.

Taxonomic revision of black salamanders of the Aneides flavipunctatus complex (Caudata: Plethodontidae).

We present a taxonomic revision of the black salamander (Aneides flavipunctatus) complex of northwestern California and extreme southeastern Oregon. The revision is based on a number of published works as well as new molecular and morphological data presented herein. The subspecies Aneides flavipunctatus niger Myers & Maslin 1948 is raised in rank to a full species. It is isolated far to the south of the main range on the San Francisco Peninsula, south and west of San Francisco Bay. Another geographically isolated set of populations occurs well inland in Shasta County, northern CA, mainly in the vicinity of Shasta Lake. It is raised from synonymy and recognized as Aneides iecanus (Cope 1883). The remaining taxa occur mainly along and inland from the coast from the vicinity of the Russian River and Lake Berryessa/Putah Creek, north to the vicinity of the Smith River near the Oregon border and more inland along the Klamath and Trinity Rivers and tributaries into Oregon. The northern segment of this nearly continuous range is named Aneides klamathensis Reilly and Wake 2019. We use molecular data to provide a detailed examination of a narrow contact zone between the northern A. klamathensis and the more southern A. flavipunctatus in southern Humboldt County in the vicinity of the Van Duzen and main fork of the Eel rivers. To the south is the remnant of the former species and it takes the name Aneides flavipunctatus (Strauch 1870). It is highly diversified morphologically and genetically and requires additional study.

Using iNaturalist in a Coverboard Protocol to Measure Data Quality: Suggestions for Project Design

We evaluated the accuracy of data records generated by citizen scientist participants using iNaturalist in a coverboard sampling scheme, a common method to detect herpetofauna, involving 17 species of amphibians and reptiles. We trained and observed 10 volunteers working over an eight-month period at a study site in Sonoma County, California, USA. A total number of 1,169 observations were successfully uploaded to the iNaturalist database by volunteers including a new locality for Black Salamander. Volunteers were generally successful in collecting and uploading data for verification but had more difficulties detecting smaller, camouflaged salamanders and photographing quick-moving lizards. Errors associated with uploading smartphone data were reduced with volunteer experience. We evaluated all observations and determined that 82% could be verified to the species level based on the photograph. Forty-five percent of the observations made it to the iNaturalist “research grade” quality using their crowdsourcing tools. Independently we evaluated the crowdsourcing accuracy of the research grade observations and found them to be 100% accurate to the species level. A variety of factors (herpetofauna group, species, photograph quality) played a role in determining whether observations were elevated to research grade. Volunteer screening and training protocols emphasizing smartphones that have appropriate battery and data storage capacity eliminated some issues. Our results suggest that a variety of factors can help scientists and resource managers improve the experiences and quality of the data in citizen science biodiversity programs.

Draft Genome Sequence of Mycobacterium montefiorense Isolated from Japanese Black Salamander (Hynobius nigrescens).

ABSTRACTMycobacterium montefiorense is a member of the Mycobacterium simiae complex, the largest group of nontuberculous mycobacteria. Here, we report the genome sequence of M. montefiorense isolate BS, isolated from diseased Japanese black salamander (Hynobius nigrescens) reared in an aquarium in Japan. This is the first reported case of an M. montefiorense infection in an amphibian.

Age, Sexual Dimorphism, and Growth Rates in the Black Salamander,Aneides flavipunctatus(Plethodontidae)

To understand how rates of growth interact to result in sexual dimorphism in the Black salamander, Aneides flavipunctatus, I conducted a mark–recapture study in Mendocino County, California. Five hundred captures (420 unique animals plus 80 recaptures over four occasions) were measured (body length, head width, head length) and released. Adult males and females are not sexually dimorphic in body length, but are dimorphic in head width; males have wider heads compared to females. From 80 recaptures, growth rates were determined for body length and head size. As expected, juveniles grow faster relative to adults; growth rates decrease as body size increases. Adult males and females have similar growth rates of body length. Males and juveniles have greater head-width growth rates compared to adult females. Head size dimorphism in A. flavipunctatus is a result of a higher head growth rate in males at sexual maturity relative to females. Because body growth rates are not significantly different between adult m...

Ontogenetic changes of trunk muscle structure in the Japanese black salamander (Hynobius nigrescens).

We investigated ontogenetic changes in the trunk muscles of the Japanese black salamander (Hynobius nigrescens) before, during and after metamorphosis. Given that amphibians change their locomotive patterns with metamorphosis, we hypothesized that they may also change the structure of their trunk muscles. The trunk muscles were macroscopically observed, and the weight ratios of each trunk muscle group were quantified at six different developmental stages. Immediately after hatching, we found that the lateral hypaxial muscle was composed of one thick M. ventralis, from ventral edge of which M. transversus abdominis arose later, followed by M. obliquus externus and M. rectus abdominis. The weight ratios of the dorsal and abdominal muscles to the trunk muscles increased with growth. We suggest that a single thick and large lateral hypaxial muscle facilitates swimming during early developmental stages. The increase in the weight ratios of the dorsal and abdominal muscles with growth possibly assists with gravity resistance necessary for terrestrial life.

Cryptic diversity and biogeographical patterns within the black salamander (Aneides flavipunctatus) complex

AbstractAimPhylogeographical structure in the black salamander (Aneides flavipunctatus) was inferred using two independent genetic datasets. Concordance between the datasets was sought in order to evaluate earlier suggestions of species‐level breaks and evidence of vicariance and long‐term isolation within the complex. We hypothesized that major phylogeographical breaks would either correspond to current tectonic plate boundaries or to historical geological processes.LocationNorth‐western California and southern Oregon (USA).MethodsThree mitochondrial DNA (mtDNA) genes were sequenced for 240 black salamanders from 136 localities, and up to 13 nuclear DNA loci were sequenced for 145 black salamanders representing 93 localities. Phylogenetic analysis of our mitochondrial dataset was performed to recover major lineages, while spatial clustering analysis of our nuclear dataset was utilized to identify points of concordance with our mtDNA phylogeny. Levels of gene flow were estimated for all contact zones.ResultsOur mitochondrial phylogeny recovered four major lineages. Cluster analysis of our nuclear dataset is consistent with a four‐population scheme, with the boundaries matching those of the mtDNA lineages. Gene flow across a contact zone in southern Humboldt between two of the populational units is extremely limited (2Nm < 1). In what is identified as the Central Core population, two distinctive subpopulations were delineated based on nuclear data, but mitochondrial data are discordant.Main conclusionsThe Aneides flavipunctatus complex comprises at least four species‐level units. Two of the boundaries between these units are associated with current tectonic plate boundaries. The contact zone between our Northwest and Central Core populations lies adjacent to the Mendocino Triple Junction (MTJ), where the Humboldt and North American plates meet, while the area separating the Santa Cruz and Central Core populations corresponds to the boundary between the Pacific and Humboldt tectonic plates. The phylogeographical break within the Central Core population lies in a region in which uplift occurred that is associated with the historical position of the migrating MTJ.

Genetic Diversity of Black Salamanders (Aneides flavipunctatus) across Watersheds in the Klamath Mountains

Here we characterize the genetic structure of Black Salamanders (Aneides flavipunctatus) in the Klamath Mountains of northwestern California and southwestern Oregon using mitochondrial and nuclear DNA sequences. We hypothesized that the Sacramento, Smith, Klamath, and Rogue River watersheds would represent distinct genetic populations based on prior ecological results, which suggest that Black Salamanders avoid high elevations such as the ridges that separate watersheds. Our mitochondrial results revealed two major lineages, one in the Sacramento River watershed, and another containing the Klamath, Smith, and Rogue River watersheds. Clustering analyses of our thirteen nuclear loci show the Sacramento watershed population to be genetically distinctive. Populations in the Klamath, Smith, and Rogue watersheds are also distinctive but not as differentiated and their boundaries do not correspond to watersheds. Our historical demographic analyses suggest that the Sacramento population has been isolated from the Klamath populations since the mid-Pleistocene, with negligible subsequent gene flow (2 Nm ≤ 0.1). The Smith and Rogue River watershed populations show genetic signals of recent population expansion. These results suggest that the Sacramento River and Klamath River watersheds served as Pleistocene refugia, and that the Rogue and Smith River watersheds were colonized more recently by northward range expansion from the Klamath.

Defining evolutionary boundaries across parapatric ecomorphs of Black Salamanders (Aneides flavipunctatus) with conservation implications

The accurate delimitation of evolutionary population units represents an important component in phylogeographic and conservation genetic studies. Here, we used a combined population assignment and historical demographic approach to study a complex of ecomorphologically distinctive populations of Black Salamanders (Aneides flavipunctatus) that are parapatrically distributed and meet at a three-way contact zone in north-western California. We used mitochondrial tree-based and multilocus clustering methods to evaluate a priori two- (Northern and Southern) and three (Northern, Coast and Inland) population hypotheses derived from previous studies. Mitochondrial results were consistent with the two- and three-population hypotheses, while the nDNA clustering results supported only the two-population hypothesis. Historical demographic analyses and mtDNA gene divergence estimates revealed that the Northern and Southern populations split during the Pliocene (2-5 Ma). Subdivision of the Southern population into Coast and Inland populations was estimated to be late Pleistocene (0.24 Ma), although our mtDNA results suggested a Pliocene divergence. Effective gene flow estimates (2N(e)m) suggest that either the two- or three-population hypotheses remain valid. However, our results unexpectedly revealed that the Northern population might instead represent two parapatric populations that separated nearly 4 Ma. These results are surprising because the Pliocene divergence between these ecomorphologically conservative forms is similar or older than for the ecomorphologically divergent Coast and Inland sister populations. We conclude that Black Salamanders in north-western California belong to at least three or four populations or species, and these all meet criteria for being Evolutionary Significant Units or 'ESUs' and therefore warrant conservation consideration.

CXCL14-like immunoreactivity in growth hormone-containing cells of urodele pituitaries

Immunohistochemical techniques were employed to investigate the distribution of a chemokine, namely, CXCL14-like immunoreactivity in the axolotl (Ambystoma mexicanum) and Japanese black salamander (Hynobius nigrescens) pituitaries. CXCL14-immunoreactive cells concentrated at an area of the pars distalis adjacent to the pars intermedia. We found that these cells correspond to the cells immunoreactive to an antibody against rat growth hormone (GH). Immunoelectron microscopy indicated that the CXCL14-like substance and GH coexisted on the secretory granules in the axolotl pituitary. Western blot analysis of axolotl pituitary extracts revealed the anti-human CXCL14 antibody labeled an approximately 16.6-kDa band that was not labeled by the anti-GH antibody. The CXCL14-like substance in the pars distalis may participate in GH functions in these species.

Ontogeny of ENaC expression in the gills and the kidneys of the Japanese black salamander (Hynobius nigrescens Stejneger)

A full-length cDNA cloning and tissue distribution of epithelial sodium channel (ENaC) protein were studied during ontogeny by immunohistochemistry in the external gills, and the kidney, pronephros and mesonephros, of the Japanese black salamander, Hynobius nigrescens (Family Hynobiidae; a primitive caudate species). The amino acid sequence of Hynobius ENaCα is 64 and 63% identical to Bufo ENaCα and Rat ENaCα, respectively. In aquatic larva salamander at the digit differentiation stage, Hynobius ENaCα mRNA was expressed in the external gills and pronephros. In the adult, the mRNA was expressed in the skin and the mesonephros. In the larvae, juvenile, and adult specimens, Hynobius ENaCα immunoreactivity was observed at the apical cell membrane of the external gills, late parts of the distal tubules, and mesonephric duct in the kidney. Colocalization of the apical Hynobius ENaCα and the basolateral Na(+) ,K(+) -ATPase was observed in the tubular cells of pronephros and mesonephros. These results suggest that Hynobius ENaCα plays an important role in the regulation of sodium transport in the external gills and pronephros of aquatic larvae, and in the skin and mesonephros of terrestrial adult. This is the first study to indicate ENaC expression during ontogeny in amphibians. Since no orthologs or paralogs for ENaC have been found, so far, in databases of the genomes of teleosts, it is assumed that ENaC might have played a role in terrestriality during the evolution of early tetrapods, the origin of lissamphibians.

Cellular localization of a putative Na+/H+ exchanger 3 during ontogeny in the pronephros and mesonephros of the Japanese black salamander (Hynobius nigrescens Stejneger)

The cloning of cDNA and an examination of the tissue distribution of Na(+)/H(+) exchanger 3 (NHE3) were carried out in the Japanese black salamander, Hynobius nigrescens. The cellular localization of Hynobius NHE3 was examined by in situ hybridization and immunohistochemistry during ontogeny in the nephron of the pronephros and mesonephros of the salamander. The partial amino acid sequence of Hynobius NHE3 was 81% and 72% identical to rat NHE3 and stingray NHE3, respectively. Hynobius NHE3 mRNA and protein were exclusively expressed along the late portion of the distal tubule to the anterior part of the pronephric duct of premetamorphic larvae (IY stages 43-50). NHE3 mRNA was expressed in the pronephros but not in the external gills in the larvae at the digit differentiation stage (IY stage 50). In the adult, mRNA was strongly expressed in the mesonephros but not in the ventral and dorsal skin. In juvenile and adult specimens, NHE3 immunoreactivity was observed at the apical membrane of the initial parts of the distal tubules of the mesonephric kidney. Immunohistochemical and in situ hybridization studies suggested that Na(+) absorption coupled with H(+) secretion via NHE3 occurred in the distal nephron of the pronephros and mesonephros. This is the first study to indicate NHE3 expression during ontogeny in amphibians.

Adding More Ecology into Species Delimitation: Ecological Niche Models and Phylogeography Help Define Cryptic Species in the Black Salamander (Aneides flavipunctatus)

Being able to efficiently and accurately delimit species is one of the most basic and important aspects of systematics because species are the fundamental unit of analysis in biogeography, ecology, and conservation. We present a rationale and approach for combining ecological niche modeling, spatially explicit analyses of environmental data, and phylogenetics in species delimitation, and we use our methodology in an empirical example focusing on Aneides flavipunctatus, the black salamander (Caudata: Plethodontidae), in California. We assess the relationships between genetic, environmental, and geographic distance among populations. We use 11 climatic variables and point locality data from public databases to create ecological niche models. The suitability of potential contact zones between parapatric lineages is also assessed using the data from ecological niche modeling. Phylogenetic analyses of portions of the mitochondrial genome reveal morphologically cryptic mitochondrial lineages in this species. In addition, we find that patterns of genetic divergence are strongly associated with divergence in the ecological niche. Our work demonstrates the ease and utility of using spatial analyses of environmental data and phylogenetics in species delimitation, especially for groups displaying fine-scaled endemism and cryptic species.

A Solution to the Large Black Salamander Problem (Genus Bolitoglossa) in Costa Rica and Panamá

Several populations of large (adult standard length, 43–134 mm) black salamanders of the widespread neotropical genus Bolitoglossa (Plethodontidae) are known from the cordilleras of western Panamá and Costa Rica. These populations constitute at least seven species, including two recently described (B. anthracina, B. copia), one described long ago that remains poorly known (B. nigrescens), and three described herein as new. The long-recognized, wide-ranging B. robusta, which is distinguished by a pale, pigmented ring around the tail base and a unique combination of maxillary and vomerine tooth counts, may occur sympatrically with four of the other taxa. Differences in head and body form, adult size, cranial osteology, and maxillary and vomerine tooth counts separate all recognized taxa from one another. These results confirm and indeed increase the exceedingly high diversity of salamander species known from the Cordillera Talamanca-Barú of Costa Rica and Panamá, diversity that now rivals that found anywhere else in the tropics.

Lack of Genetic Diversity in Salamandra lanzai Revealed by CytochromebGene Sequences

The black salamander, Salamandra lanzai, exists in isolated and fragmented populations in the Cottian Alps. To examine the degree of intra- and interpopulation divergence, mitochondrial cytochrome b sequences were obtained from samples representing all populations of S. lanzai. No genetic variation was observed either within or among populations. The presence of a single lineage suggests historical explanations closely related to Pleistocene events.