Amphibian Genus Research Articles

A combined palaeomodelling approach reveals the role as selective refugia of the Mediterranean peninsulas

The northern Mediterranean reptiles and amphibians show contrasting biogeographic histories during the Plio-Pleistocene. The influence of European climate changes on the evolution of the biogeographic ranges of taxa with a rich fossil record is determined herein combining different proxies to obtain well-supported palaeoclimatic scenarios. Ecological Niche Modelling through the ensemble modelling approach was used to reconstruct the climatic niche of selected reptile and amphibian genera, combining modern occurrences with seven independent bioclimatic variables. The obtained model was then projected on past scenarios using fossil data to crosscheck the validity of the climatic explanation for the selective extirpations. Palaeobotanical proxies and the hypsodonty of mammals were used to reconstruct the humidity pattern of the last million years in the studied area. The climate of the Italian Peninsula during the Last Glacial Maximum was proved herein to be unsuitable for many reptile taxa, which persisted in this area until the Early or Late Pleistocene, whereas they survived till the present day in the Iberian and/or Balkans peninsulas. On the contrary, all the proxies agree that Italy was comparably more humid than the other peninsulas during the Pliocene and Pleistocene, therefore allowing the prolonged survival of amphibian taxa (including the still living Salamandrina and Speleomantes), which became extinct or extirpated elsewhere in Europe. An intensification of the conservation efforts concerning amphibians, particularly sensitive to the global climate change, is recommended in the Italian Peninsula, which acted as an amphibian refugium for Europe due to its peculiar climatic history.

Effects of agricultural lands on the distribution pattern of genus diversity for neotropical terrestrial vertebrates

The expansion and intensification of agricultural lands (i.e., croplands and pasture areas) have become increasing threats to species diversity around the world. However, genus diversity should be considered for biodiversity conservation due to the important role that phylogenetic diversity and evolutionary history play shaping biodiversity patterns, particularly in the Neotropics, the most species-rich realm. Here, we explored agricultural land effects on distribution patterns of terrestrial vertebrate diversity at the genus level using cases from the Neotropics. We compiled distribution data on genera of amphibians, birds, mammals, and reptiles and explored the effects of agricultural lands on genus diversity distribution patterns for terrestrial vertebrates in the region using distribution modelling. Geography Weighting Regression (GWR) was used to explore the spatially variable relationship between agricultural lands and genus diversity. We found that there are significant, non-stationary relationships between agricultural lands and genus diversity in the Neotropics. The non-stationary relationship between croplands and genus diversity was stronger than that between pasture areas and genus diversity. Furthermore, these effects may depend on changes in genus groups (i.e., amphibians, birds, mammals, and reptiles) and biomes. The relationship was the strongest for the croplands and reptile diversity pair at the genus levels. The relationship between both croplands and pasture areas and reptile diversity was the largest in temperate broadleaf and mixed forests. Given the influence of crop and pasturelands on vertebrate distribution patterns, we argue that agriculture development should be considered in the spatial prioritization of conservation for vertebrates in the Neotropics. Agricultural lands should be used as environmental variables for distribution modelling, and understanding the impacts of agricultural lands on biodiversity is critical for effective conservation management in the Neotropics.

Chromosome Mapping of U2 snDNA in Species of Leptodactylus (Anura, Leptodactylidae)

Small nuclear RNA (snRNA) is a class of molecules involved in the processing of pre-mRNA and in regulatory cell processes. snRNAs are always associated with a set of specific proteins. The complexes are referred to as small nuclear ribonucleoproteins, and spliceosome U RNAs are their most common snRNA components. The repetitive sequences of U snDNAs have been cytogenetically mapped in several species of Arthropoda, fishes, and mammals; however, their distribution remains unknown in amphibians. Here, we show results of FISH mapping of U2 snDNA repetitive sequences in species of the amphibian genus Leptodactylus to reveal the distribution patterns of this sequence in their karyotypes. The probe hybridized in the metacentric chromosome pair 6 in Leptodactylus fuscus, L. gracilis, L. latrans, L. chaquensis, L. petersii, L. podicipinus, and L. brevipes. A different pattern was observed in L. labyrinthicus with hybridization signals in 4 chromosome pairs. The same localization of U2 gene sequences in most of the species analyzed suggests a relatively conserved pattern and a similarity of the chromosome 6 among these species of Leptodactylus.

A review of mono- and bispecific genera of Amphibians worldwide

Monospecific and bispecific genera are of particular interest in studies of taxonomic diversity and speciation evolution. Here, the distribution patterns of mono- and bispecific amphibians worldwide are investigated, with some discussion of on the conservation implications also presented. Based on an online database search (available from the American Museum of Natural History, New York), we found that the mean number of mono- and bispecific genera was similar among zoogeographic regions, with a total of 120 mono- (95 Anura, 17 Caudata, 8 Gymnophiona) and 65 bispecific (48 Anura, 10 Caudata, 7 Gymnophiona) genera. Out of 73 known amphibian families worldwide, only 35.6 % of them do not contain any mono- or bispecific genera. The frequency of mono- or bispecific genera by family was not significantly different among Anura, Caudata and Gymnophiona. There was a general tendency for the number of mono- and bispecific genera of amphibians to be positively correlated with the total number of genera in that family. In Anura, there was a preponderance of mono-specific genera in Afrotropical and Neotropical regions. Concerning bispecific genera, there was a clear preponderance in the Neotropical region for anurans. There was a positive correlation between the number of threatened genera (according to the IUCN Red List) in both the mono- and bispecific groups and the relative number of species in each taxon, thus showing that taxonomical speciosity clearly influences the frequency of occurrence of mono- and bispecific taxa in each family and order. In this regard, Anura dominated in both the number of worldwide described mono- and bispecific taxa as well as in that of the threatened ones according to IUCN Red List .

Identification and characterization of phospholipases A2 from the skin secretion of Pithecopus azureus anuran

The present work reports the isolation, characterization and the complete sequence of a phospholipase A2 (PLA2) present in the skin secretion of Pithecopus azureus. Among several peptides and small proteins previously described by our group from some species belonging to this amphibian genus (formerly named Phyllomedusa), a 15 kDa N-glycosylated protein showing PLA2 activity was purified, assayed, sequenced and named Pa-PLA2. The Pithecopus azureus skin phospholipase A2 polypeptide chain is composed by 125 amino acid residues linked by seven disulfide bonds and two N-glycosylated sites (N67 and N108). The Pa-PLA2 enzymatic activity was qualitatively evaluated and compared to classical viperid PLA2 showing that both, native and deglycosylated Pa-PLA2 forms, are catalytically functional. The tridimensional molecular model of Pa-PLA2 indicates that the observed glycan moieties are suggestively placed far from the active site of that enzyme and therefore having little or no significant role on the direct interaction of the Pa-PLA2 catalytic pocket and its substrates.

Occupancy modelling reveals a highly restricted and fragmented distribution in a threatened montane frog (Philoria kundagungan) in subtropical Australian rainforests

In the last several decades, habitat loss, overexploitation, invasive organisms, disease, pollution and, more recently, climate change have led to catastrophic declines in amphibian biodiversity. Montane amphibian species, particularly those with narrow distributions and specific habitat requirements are likely to be at particular risk under future climate change scenarios. Despite this, fundamental ecological data are lacking for most of these species. Philoria kundagungan is a poorly known representative of a range-restricted genus of montane amphibians from eastern Australia. Using an occupancy framework, we conducted repeated call playback surveys at 32 sites across the mountainous regions of south-east Queensland and north-eastern New South Wales, Australia, to investigate: (1) the current extent of this species’ geographic range, and (2) environmental predictors of this species’ presence. We found that P. kundagungan has a highly restricted and fragmented distribution, being limited to ~11 km2 of potentially suitable habitat, and that its presence is strongly associated with high elevation (>800 m). Our survey protocol resulted in a high probability of detection (>70%) at occupied sites from relatively few visits. From these baseline data, future studies will have the ability to determine changes in this species’ geographic range and occupancy rate in response to the ever-increasing threats faced by P. kundagungan, thereby supporting more effective conservation strategies and policy decisions.

An interaction between climate change and infectious disease drove widespread amphibian declines.

Climate change might drive species declines by altering species interactions, such as host-parasite interactions. However, few studies have combined experiments, field data, and historical climate records to provide evidence that an interaction between climate change and disease caused any host declines. A recently proposed hypothesis, the thermal mismatch hypothesis, could identify host species that are vulnerable to disease under climate change because it predicts that cool- and warm-adapted hosts should be vulnerable to disease at unusually warm and cool temperatures, respectively. Here, we conduct experiments on Atelopus zeteki, a critically endangered, captively bred frog that prefers relatively cool temperatures, and show that frogs have high pathogen loads and high mortality rates only when exposed to a combination of the pathogenic chytrid fungus (Batrachochytrium dendrobatidis) and high temperatures, as predicted by the thermal mismatch hypothesis. Further, we tested various hypotheses to explain recent declines experienced by species in the amphibian genus Atelopus that are thought to be associated with B.dendrobatidis and reveal that these declines are best explained by the thermal mismatch hypothesis. As in our experiments, only the combination of rapid increases in temperature and infectious disease could account for the patterns of declines, especially in species adapted to relatively cool environments. After combining experiments on declining hosts with spatiotemporal patterns in the field, our findings are consistent with the hypothesis that widespread species declines, including possible extinctions, have been driven by an interaction between increasing temperatures and infectious disease. Moreover, our findings suggest that hosts adapted to relatively cool conditions will be most vulnerable to the combination of increases in mean temperature and emerging infectious diseases.

Evidences for a shared history for spectacled salamanders, haplotypes and climate

The so-called glacial refugia, formed during the Pleistocene climatic oscillations, played a major role in shaping the distribution of European species, triggering migrations or isolating populations. Many of these events were recently investigated by genetic data, mainly for the European Last Glacial stage, in the Iberic, Italian and Greek-Balkan peninsulas. The amphibian genus Salamandrina, the most ancient living salamandrid lineage, was widespread in Europe until the climatic oscillations of Miocene probably forced it to shelter in the only suitable territory at that time, the Apennines. Nowadays this genus is endemic of peninsular Italy with two parapatric species, S. perspicillata and S. terdigitata, sharing an area of secondary contact formed after the Last Glacial Maximum. Climate is generally identified as the key factor for the interpretation of genetic data. In this research, we directly measure climate influences on the two Salamandrina known species through Ensemble Modelling techniques and post-modelling GIS analyses, integrating updated genetic data in this process. Our results confirm the hypotheses of southwards (and subsequent northwards) shifts, identify glacial refugia and corridors used for the post-glacial re-colonization. Finally, we map a contact zone deserving more sampling effort to disentangle the introgression and hybridization observed.

Cryptic diversity in Amazonian frogs: Integrative taxonomy of the genus Anomaloglossus (Amphibia: Anura: Aromobatidae) reveals a unique case of diversification within the Guiana Shield

Lack of resolution on species boundaries and distribution can hamper inferences in many fields of biology, notably biogeography and conservation biology. This is particularly true in megadiverse and under-surveyed regions such as Amazonia, where species richness remains vastly underestimated. Integrative approaches using a combination of phenotypic and molecular evidence have proved extremely successful in reducing knowledge gaps in species boundaries, especially in animal groups displaying high levels of cryptic diversity like amphibians. Here we combine molecular data (mitochondrial 16S rRNA and nuclear TYR, POMC, and RAG1) from 522 specimens of Anomaloglossus, a frog genus endemic to the Guiana Shield, including 16 of the 26 nominal species, with morphometrics, bioacoustics, tadpole development mode, and habitat use to evaluate species delineation in two lowlands species groups. Molecular data reveal the existence of 18 major mtDNA lineages among which only six correspond to described species. Combined with other lines of evidence, we confirm the existence of at least 12 Anomaloglossus species in the Guiana Shield lowlands. Anomaloglossus appears to be the only amphibian genus to have largely diversified within the eastern part of the Guiana Shield. Our results also reveal strikingly different phenotypic evolution among lineages. Within the A. degranvillei group, one subclade displays acoustic and morphological conservatism, while the second subclade displays less molecular divergence but clear phenotypic divergence. In the A. stepheni species group, a complex evolutionary diversification in tadpole development is observed, notably with two closely related lineages each displaying exotrophic and endotrophic tadpoles.

A few problems in the generic nomenclature of insects and amphibians, with recommendations for the publication of new generic nomina in zootaxonomy and comments on taxonomic and nomenclatural databases and websites.

Dahanukar et al. (2016a) proposed the nomen Walkerana for a new genus of amphibians, but shortly after (2016b) they replaced it by the new nomen Sallywalkerana, believing that their nomen Walkerana was preoccupied by a generic nomen of orthopterans. This was unjustified because the orthopteran nomen 'Walkerella' Otte & Perez-Gelabert, 2009a and its new replacement nomen 'Walkerana' Otte & Perez-Gelabert, 2009b were both nomina nuda. These recent examples of nomenclatural errors in generic nomenclature are just a few among many in recent zootaxonomic publications. This opportunity is taken to make some general methodological recommendations, in several domains (availability, homonymy, synonymy, neonymy, length and palatability of nomina), for the publication of new generic nomina in zootaxonomy. However, the absence of a comprehensive database and website providing all the relevant information necessary to establish the nomenclatural status of all zoological generic and subgeneric nomina is a brake on the efforts that can be made to avoid nomenclatural errors in zoological generic nomenclature. The international community of taxonomists should seek at establishing such a database and website.

Biology of tiny animals: three new species of minute salamanders (Plethodontidae: Thorius) from Oaxaca, Mexico.

We describe three new species of minute salamanders, genus Thorius, from the Sierra Madre del Sur of Oaxaca, Mexico. Until now only a single species, T. minutissimus, has been reported from this region, although molecular data have long shown extensive genetic differentiation among geographically disjunct populations. Adult Thorius pinicola sp. nov., T. longicaudus sp. nov., and T. tlaxiacus sp. nov. are larger than T. minutissimus and possess elliptical rather than oval nostrils; T. pinicola and T. longicaudus also have longer tails. All three new species occur west of the range of T. minutissimus, which has the easternmost distribution of any member of the genus. The new species are distinguished from each other and from other named Thorius in Oaxaca by a combination of adult body size, external morphology and osteology, and by protein characters (allozymes) and differences in DNA sequences. In addition, we redescribe T. minutissimus and a related species, T. narisovalis, to further clarify the taxonomic status of Oaxacan populations and to facilitate future studies of the remaining genetically differentiated Thorius that cannot be satisfactorily assigned to any named species. Populations of all five species considered here appear to have declined dramatically over the last one or two decades and live specimens are difficult to find in nature. Thorius may be the most endangered genus of amphibians in the world. All species may go extinct before the end of this century.

Genetic analysis reveals candidate species in the Scinax catharinae clade (Amphibia: Anura) from Central Brazil.

Scinax (Anura: Hylidae) is a species-rich genus of amphibians (113 spp.), divided into five species groups by morphological features. Cladistic analyses however revealed only two monophyletic clades in these groups: Scinax catharinae and Scinax ruber. Most species from the S. catharinae clade are found in Atlantic rainforest, except for Scinax canastrensis,S. centralis, S. luizotavioi, S. machadoi,S. pombali and S. skaios. In the present work, specimens of Scinax collected in Chapada dos Guimarães, central Brazil, were morphologically compatible with species from theS. catharinae group. On the other hand, genetic analysis based on mitochondrial (16S and 12S) and nuclear (rhodopsin) sequences revealed a nucleotide divergence of 6 to 20% between Scinax sp. and other congeners from the Brazilian savannah (Cerrado). Accordingly, Bayesian inference placed Scinax sp. in the S. catharinae clade with high support values. Hence, these findings strongly indicate the presence of a new species in the S. catharinae clade from the southwestern portion of the Brazilian savannah. To be properly validated as a novel species, detailed comparative morphological and bioacustic studies with other taxa from Brazil such asS. canastrensis, S. centralis, S. luizotavioi, S. machadoi, S. pombali and S. skaios are required.

Cytogenetic analysis of Scinaxauratus and Scinaxeurydice (Anura, Hylidae) with emphasis on cytotaxonomy.

Scinax Wagler, 1830 is a species-rich genus of amphibians with relatively few detailed chromosomal reports. In this work, cytogenetic analyses of Scinax auratus (Wied-Neuwied, 1821) and Scinax eurydice (Bokermann, 1968) were carried out based on conventional (Giemsa staining, Ag-NOR and C-banding) and cytomolecular (base-specific fluorochrome staining and fluorescence in situ hybridization – FISH of ribosomal probes) techniques. Both species shared the same karyotype, location of active nucleolar organizer regions on pair 11 and GC-rich heterochromatin, as reported for most species in Scinax ruber clade. Interpopulation chromosomal variation was observed in Scinax eurydice, indicating the occurrence of cryptic species. The mapping of 18S ribosomal genes by FISH is reported for the first time in both species.

Amphibian and reptilian distribution patterns in the transitional zone between the Euro-Siberian and Central Asia Subrealms

There exist some controversies over the larger zoogeographic divisions of the arid areas of Central Asia, whose characteristics include complex ecological environments, complex fauna origins and unique patterns of animal distribution. The aim of this study was to determine, using quantitative analysis, the distribution patterns of amphibians and reptiles in the arid areas of Central Asia, whose various physiographical regions were divided into 17 Operative Geographical Units (OGUs). Based on the presence or absence of 52 amphibian and reptile genera in the 17 OGUs, and by making use of the Czekanowski Similarity Index, the Baroni-Urbani and Buser’s Similarity Index, and the strong and weak boundary test, we studied the biotic boundaries within these contested regions. In accordance with our results, the classification dendrogram was divided into two main branches. One branch is composed of the northern OGUs of the Altai Mountains which are a part of the Euro-Siberian Subrealm. The other branch includes all of the OGUs south of the Altai Mountains, which belong to the Central Asia Subrealm. There is a significantly weak biotic boundary (DW>0, GW>GS, P<0.01) between the Euro-Siberian Subrealm and the Central Asia Subrealm that corresponded to the transitional zones. The boundary between the two subrealms runs along the Altai Mountains, the Sayan Mountains, the Hangai Mountains and the Mongolian Dagurr Mountains. The boundaries between the main branches in the Central Asia Subrealm are weak, reflecting the widespread existence of transitional zones in the arid areas of Central Asia. The Tianshan Mountains should be elevated to form its own separate region, “the Middle Asian Mountain Region”, which, due to its special fauna and environment, would be classified at the same level as the Mongolia-Xinjiang Region. With the approach of creating a cluster analysis dendogram based upon the genera of amphibians and reptiles, the relationship of these higher level zoogeographical divisions was successfully resolved and the error of long-branch attraction was also avoided. With our clustering dendrogram as the foundation, the independence test was applied to strong and weak boundaries, and this resolved the problem of where to attribute the transition areas and revealed as well the barrier effect that physical, geographic boundaries have upon amphibians and reptiles. The approach of combining genera clustering analysis with a statistical boundary test should be applicable not only to the distribution patterns of other animal groups, but also to delineating large-scale zoogeographical divisions.

A checklist of the amphibians and reptiles of Honduras, with additions,comments on taxonomy, some recent taxonomic decisions, and areas of further studies needed.

An updated checklist of the amphibians and reptiles of Honduras is provided. The list includes three amphibian species (Ptychohyla euthysanota, Bolitoglossa odonnelli, Oedipina chortiorum) and two reptile species (Laemanctus waltersi [elevated from subspecies status], Epictia phenops) not included in the most recent checklist of the amphibians and reptiles of the country. Also, one amphibian genus and species (Triprion petasatus) is removed from the country list and one Honduran lizard (Ctenosaura praeocularis) is synonymized with an older name. Comments where more study is needed are also included where pertinent. Authors, dates, and original spellings of the higher-level taxonomy of all taxa covered herein are also given. A total of 401 species (137 amphibians and 264 reptiles) are now known from the country with 111 species (27.7%) being Honduran endemics (52 amphibians and 59 reptiles).

A new endemic lineage of the Andean frog genusTelmatobius(Anura, Telmatobiidae) from the western slopes of the central Andes

The amphibian genus Telmatobius is a diverse group of species that inhabits the Andes. This study analysed the phylogenetic relationships of 19 species described from the central Andes of Chile and Bolivia, and 12 undescribed populations of Chile. A molecular phylogeny based on mitochondrial DNA 16S and cytochrome b shows that the Chilean species belong to three groups: (1) the Telmatobius marmoratus group, widespread in the Chilean and Bolivian Altiplano; (2) the Telmatobius hintoni group, including the species Telmatobius philippii, Telmatobius fronteriensis, and Telmatobius huayra, occurring in the south-western Altiplano of Chile and Bolivia, and (3) the Telmatobius zapahuirensis group, a new clade which also includes Telmatobius chusmisensis, Telmatobius dankoi, and Telmatobius vilamensis, restricted to western slopes of the Andes, and which was recovered as more closely related to the T. hintoni group than the T. marmoratus group. The divergence times between clades were traced to the late Pleistocene. The molecular phylogeny also confirmed that the groups of the Altiplano and western Andes slopes form a clade separated from the species that inhabit the eastern Andes (Telmatobius verrucosus and Telmatobius bolivianus groups), supporting the forest origin of the Altiplano groups proposed by several previous authors. © 2014 The Linnean Society of London

Trimerorhachis (Amphibia: Temnospondyli) from the Lower Permian of Texas and New Mexico: cranial osteology, taxonomy and biostratigraphy

The morphology and taxonomy of the Early Permian temnospondyl amphibian genus Trimerorhachis are revised. Using previously undescribed material from Texas, numerous details of the cranial osteology of T. insignis Cope, 1878 are redescribed. Emphasis is placed on the structure of the palate, the sutures of the skull roof, and on the variation of some phylogenetically important character-states. The taxonomic revision, which makes particular use of morphometric characters, supports the four species previously considered to be valid, and adds a fifth species. T. sandovalensis Berman & reisz, 1980 from the Cutler Formation of New Mexico is probably the most primitive species and does not share any unambiguous derived character-states with the other species. T. insignis – the type species – is the only one known from the Nocona and Petrolia formations of Texas. In the later formations up to the Middle Clear Fork Group, there are two lineages diverging from the T. insignis morphology in opposite directions. One, here designated T. greggi sp. nov. , has larger orbits and a shorter snout and occurs in the Lower, and probably also the Middle, Clear Fork. The other is T. mesops Cope, 1896, which has smaller orbits and a longer snout and occurs in the Lower and Middle Clear Fork and possibly the earlier Waggoner Ranch Formation of Texas. T. rogersi olson, 1955 is a possible late population of T. mesops type that occurs in the late Middle Clear Fork. The simplest model is that there was one short-snouted Trimerorhachis lineage from the base of the Permian to the top of the Petrolia Formation (contemporaneous with the long-snouted Neldasaurus in Archer City Formation beds), followed by two diverging lineages showing some character displacement from the Waggoner Ranch Formation upwards, but possibly incipiently present in the Nocona/Petrolia populations.

Helminth Infracommunity Structure ofLeptodactylus melanonotus(Anura) in Tres Palos, Guerrero, and Other Records for This Host Species in Mexico

The amphibian genus Leptodactylus includes around 50 species, of which only 2 are distributed in Mexico; the helminth fauna of these 2 species is poorly known. As part of a research program on amphibian parasites in Mexico from 1997 to 2005, 281 sabinal frogs Leptodactylus melanonotus from 42 localities in 11 Mexican states were examined from a helminthological perspective. A total of 20 taxa of helminths-7 digeneans (5 adults, 2 larvae) and 13 nematodes (8 adults, 5 larvae)-was found to infect this amphibian host species. These data represent 105 new locality records, and 11 taxa are recorded in L. melanonotus for the first time. Infracommunity analyses of the sabinal frogs from Tres Palos indicated that these hosts are depauperate. The helminth community is dominated by specialist species, with Cosmocerca podicipinus the most common in almost 50% of the infracommunities. Percutaneous infection and predator-prey interactions were the 2 most common infection routes by helminths in frogs from Tres Palos, with 79% of the parasites recruited via skin penetration. Finally, our results show that the helminth fauna parasitizing L. melanonotus throughout Mexico has low similarity with the helminth fauna of leptodactylids studied comprehensively in South America, with only 2 digeneans and 3 nematodes being shared by hosts from both regions. As a result of our survey, the number of helminth species parasitizing L. melanonotus increased to 34. Considering its native distribution range, this number is now 36 with the inclusion of the nematodes Oswaldocruzia costaricensis and Cruzia empera in Costa Rica.

Five novel antimicrobial peptides from the Kuhl’s wart frog skin secretions, Limnonectes kuhlii

Five novel antimicrobial peptides (temporin-LK1, rugosin-LK1, rugosin-LK2, gaegurin-LK1, and gaegurin-LK2) are purified and characterized from Kuhl's wart frog skin secretions, Limnonectes kuhlii. They share obvious similarity to temporin, rugosin, and gaegurin antimicrobial peptide family, respectively. Their amino acid sequences were determined by Edman degradation and mass spectrometry, and further confirmed by cDNA cloning. Nine cDNA sequences encoding precursors of these five purified antimicrobial peptides and other four hypothetical antimicrobial peptides were cloned from the skin cDNA library of L. kuhlii. The deduced precursors are composed of a predicted signal peptide, an acidic spacer peptide, and a mature antimicrobial peptide. Most of them showed strong antimicrobial activities against Gram-positive and Gram-negative bacteria and fungi. The current work identified and characterized three families of antimicrobial peptides from L. kuhlii skins and confirmed that the genus of Limnonectes amphibians share similar antimicrobial peptide families with the genus of Rana amphibians. In addition, a unique antimicrobial peptide (temporin-LK1) with 17 residues including four phenylalanines, which is significantly different from other temporins (16 residues, one or two phenylalanines), was identified in this work. Such unique structure might provide novel template or leading structure to design antimicrobial agents.

Empirical and simulation evaluations of an abundance estimator using unmarked individuals of cryptic forest-dwelling taxa

Conservation and management programs use populations of sentinel taxa, such as stream-associated amphibians, as indicator species due to their perceived sensitivity to environmental change. Estimating population size with traditional tools such as mark–recapture estimators may be impractical for forest-dwelling stream-associated amphibians, many of which are cryptic and have low detection probabilities. In addition, sampling techniques can alter habitat conditions, particularly with repeat sampling, and compromise inferences about management impacts. We used simulated and empirical data and N-mixture models to estimate detection probabilities and abundances for two amphibian genera, giant Dicamptodon and torrent Rhyacotriton salamanders, sampled with a less-invasive approach than other methods. We surveyed forested headwater streams located in western Washington, USA, for salamanders 7 July–27 August 2008. We assessed model sensitivity to changes in animal abundance (5 and 15) and detection probability (0.05–0.5), as well as study design alternatives including number of sample plots (50–150) and number of sampling visits (2–4). We also evaluated the effects of stream temperature and stream order on detection probability using data collected from forested streams in Washington, USA. Precision of detection probability estimates improved as the number of plots and sampling occasions increased. Variability of estimated population sizes decreased with higher detection probability, although species abundance had little effect on precision of detection probability estimates. Detection probability estimated from empirical data ranged from 0.07 to 0.65 for giant salamanders and 0.06–0.67 for torrent salamanders. Giant salamander detection probability was positively associated with stream temperature regardless of stream order, and was higher in second- and third-order streams than first-order streams. Detection probability for torrent salamanders varied with stream temperature, order, and the interaction of those covariates, with detection increasing with temperature for second- and third-order streams but showing a flat or decreasing trend for first-order streams. N-mixture models, paired with careful consideration of study design alternatives, can produce robust estimates of abundance and obviate the traditional reliance on indices of relative abundance for many rare and sensitive taxa. Use of a less invasive technique for sampling stream-associated amphibians resulted in sufficient animal captures for suitable model performance. Our simulation results can inform study design and direct efficient allocation of field effort.