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Plethodontid salamanders possess numerous courtship glands. Previous studies have shown that the glands are more prominent in male individuals than females, and often experience periods of atrophy and hypertrophy throughout the year that correlate to the nonmating and mating seasons, respectively. We sampled male and female Eurycea bislineata throughout the year to test the hypothesis that external nasal glands are courtship glands. External nasal glands are paired, branched tubular glands that extend from excretory ducts dorsal to the nares to terminal secretory units posterior to the eyes. We found that the glands hypertrophy and stain/react more intensely with histochemical procedures during the mating season. Hypertrophy of the glands is more pronounced in males, and seasonal variation in epithelial height of external nasal glands of males is significantly correlated to that of seasonal variation in mental gland epithelial height, a known courtship gland found in males, when compared throughout the year. This correlation was not as strong in females, confirming sexual dimorphism of external nasal glands in terms of seasonal variation. We found no ultrastructural differences between male and female external nasal glands. In all specimens, the glandular tubules were lined by a simple, columnar epithelium that was packed with secretory granules that often obscured other cytoplasmic contents.

We review the use of reproduction technologies (RTs) to support the sustainable management of threatened Caudata (salamanders) and Gymnophiona (caecilian) biodiversity in conservation breeding programs (CBPs) or through biobanking alone. The Caudata include ∼760 species with ∼55% threatened, the Gymnophiona include ∼215 species with an undetermined but substantial number threatened, with 80% of Caudata and 65% of Gymnophiona habitat unprotected. Reproduction technologies include: (1) the exogenous hormonal induction of spermatozoa, eggs, or mating, (2) in vitro fertilisation, (3) intracytoplasmic sperm injection (ICSI), (4) the refrigerated storage of spermatozoa, (5) the cryopreservation of sperm, cell or tissues, (6) cloning, and (7) gonadal tissue or cell transplantation into living amphibians to eventually produce gametes and then individuals. Exogenous hormone regimens have been applied to 11 Caudata species to stimulate mating and to 14 species to enable the collection of spermatozoa or eggs. In vitro fertilisation has been successful in eight species, spermatozoa have been cryopreserved in seven species, and in two species in vitro fertilisation with cryopreserved spermatozoa has resulted in mature reproductive adults. However, the application of RTs to Caudata needs research and development over a broader range of species. Reproduction technologies are only now being developed for Gymnophiona, with many discoveries and pioneering achievement to be made. Species with the potential for repopulation are the focus of the few currently available amphibian CBPs. As Caudata and Gymnophiona eggs or larvae cannot be cryopreserved, and the capacity of CBPs is limited, the perpetuation of the biodiversity of an increasing number of species depends on the development of RTs to recover female individuals from cryopreserved and biobanked cells or tissues.

IntroductionAtrial fibrillation (AF) is a prevalent disease considerably contributing to the worldwide cardiovascular burden. For patients at high thromboembolic risk (CHA2DS2-VASc ≥3) and not suitable for chronic oral anticoagulation, owing to history of major bleeding or other contraindications, left atrial appendage occlusion (LAAO) is indicated for stroke prevention, as it lowers patient’s ischaemic burden without augmentation in their anticoagulation profile.Methods and analysisStand-Alone Left Atrial appendage occlusion for throMboembolism prevention in nonvalvular Atrial fibrillatioN DiseasE Registry (SALAMANDER) will be conducted in 10 heart surgery and cardiology centres across Poland to assess the outcomes of LAAO performed by fully thoracoscopic-epicardial, percutaneous-endocardial or hybrid endo-epicardial approach. The registry will include patients with nonvalvular AF at a high risk of thromboembolic and bleeding complications (CHA2DS2-VASc Score ≥2 for males, ≥3 for females, HASBLED score ≥2) referred for LAAO. The first primary outcome is composite procedure-related complications, all-cause death or major bleeding at 12 months. The second primary outcome is a composite of ischaemic stroke or systemic embolism at 12 months. The third primary outcome is the device-specific success assessed by an independent core laboratory at 3–6 weeks. The quality of life (QoL) will be assessed as well based on the QoL EQ-5D-5L questionnaire. Medication and drug adherence will be assessed as well.Ethics and disseminationBefore enrolment, a detailed explanation is provided by the investigator and patients are given time to make an informed decision. The patient’s data will be protected according to the requirements of Polish law, General Data Protection Regulation (GDPR) and hospital Standard Operating Procedures. The study will be conducted in accordance with the Declaration of Helsinki. Ethical approval was granted by the local Bioethics Committee of the Upper-Silesian Medical Centre of the Silesian Medical University in Katowice (decision number KNW/0022/KB/284/19). The results will be published in peer-reviewed journals and presented during national and international conferences.Trial registration numberNCT05144958.

Alongside exotic reptiles, amphibians, such as toads, frogs, salamanders, and newts, are nowadays considered popular pets worldwide. As reported for other exotic pet animals, amphibians are known to harbor numerous gastrointestinal parasites. Nonetheless, very little data are available on captive amphibian parasitic diseases. In this study, we applied direct saline fecal smears (DSFS) to examine in total 161 stool samples from 41 different amphibian species belonging to the orders Anura and Caudata. In addition, carbolfuchsin-smear (CFS) staining (n = 74 samples) was used to detect amphibian Cryptosporidium oocysts. Also, complete dissections of deceased amphibians (n = 107) were performed to specify parasite infections and to address parasite-associated pathogenicity. Overall, examined amphibian fecal samples contained 12 different parasite taxa. The order Rhabditida with the species Rhabdias spp. and Strongyloides spp. were the most prevalent nematode species (19.3%), followed by flagellated protozoans (8.7%), Amphibiocapillaria spp./Neocapillaria spp. (7.5%), Oswaldocruzia spp. (4.3%), Blastocystis spp. (3.1%), Cosmocerca spp. (3.1%), oxyurids (Pharyngonoidae) (3.1%), spirurids (1.2%), un-sporulated coccidian oocysts (0.6%), Tritrichomonas spp. (0.6%), Karotomorpha spp. (0.6%), and Cryptosporidium spp. (0.6%). One CFS-stained fecal sample (1.4%) was positive for Cryptosporidium oocysts. Within dissected amphibians, 31 (48.4%) of the anurans and 11 (26.2%) of the salamanders were infected with gastrointestinal parasites. One cutaneous Pseudocapillaroides xenopi infection was diagnosed in an adult African clawed frog (Xenopus laevis). Etiologically, 17 (15.9%) of them died due to severe parasitic and/or bacterial infections (e.g., Chryseobacterium indologenes, Citrobacter freudii, Sphingobacterium multivorum, Klebsiella pneumoniae). High prevalence and pathological findings of several clinical amphibian parasitoses call for more detailed investigation on gastrointestinal parasite-derived molecular mechanisms associated with detrimental lesions or even death.

A new species of the genus Tylototriton sensu lato from Tongzi County, Guizhou Province, China was described. Molecular phylogenetic analyses based on mitochondrial 16S and ND2 gene sequences indicated the new species as the most closely related species of T. dabienicus in Henan. The new species could be identified from its congeners by a combination of the following morphological characters: (1) body size medium (TOL 120.5–135.1 mm and SVL 61.1–65.9 mm in males, and TOL 123.5–127.6 mm and SVL 66.7–69.2 mm in females); (2) gular fold present; (3) the tail length shorter than the snout-vent length; (4) the distal ends and ventral surfaces of digits, peripheral area of cloaca, and the lower margin of tail orange; (5) the distal tips of the limbs greatly overlapping when the fore and hind limbs being pressed along the trunk; (6) fingertips reaching to the level beyond the snout when the forelimbs being stretched forward; (7) nodule-like warts on body sides continuous and no obvious. The new species is known only from the montane forests of Huanglian Nature Reserve, Tongzi County, Guizhou Province, China. We recommend the new species to be listed as Critically Endangered.

Complex parenting has been proposed to contribute to the evolutionary success of vertebrates. However, the evolutionary routes to complex parenting and the role of parenting in vertebrate diversity are still contentious. Although basal vertebrates provide clues to complex reproduction, these are often understudied. Using 181 species that represent all major lineages of an early vertebrate group, the salamanders and newts (Caudata, salamanders henceforth) here we show that fertilisation mode is tied to parental care: male-only care occurs in external fertilisers, whereas female-only care exclusively occurs in internal fertilisers. Importantly, internal fertilisation opens the way to terrestrial reproduction, because fertilised females are able to deposit their eggs on land, and with maternal care provision, the eggs could potentially develop outside the aquatic environment. Taken together, our results of a semi-aquatic early vertebrate group propose that the diversity and follow-up radiation of terrestrial vertebrates are inherently associated with a complex social behaviour, parenting.

The new nematode species, Falcaustra hanzaki n. sp. and Urodelnema takanoensis n. sp. (Cosmocercoidea: Kathlaniidae), were found from the intestine of giant salamanders in Kyoto Prefecture, Japan. The first species is featured by the number and arrangement of caudal papillae (3 pairs of precloacal papillae, 8 pairs of postcloacal papillae and a single ventral precloacal papilla), the presence of a single pseudosucker, spicules equal in size (520-638 μm long), and V-shaped gubernaculum in males; vulva situated about 3/5 of a body in females. The second species is characterized by the number and arrangement of caudal papillae (5 pairs of precloacal papillae, 6 pairs of postcloacal papillae and a single ventral precloacal papilla), spicules equal in size (403-593 μm long), V-shaped gubernaculum in males; vulva situated about 3/5 of a body in females. The molecular phylogenetic analysis was performed using the partial 18S and 28S ribosomal DNA and the internal transcribed spacers 1 region in the nuclear DNA. This phylogenetic study raised a question about the validity of Family Kathlaniidae and related families of Cosmocercoidea.

In 2019, two wild-caught adult female eastern hellbenders (

Females of some Asian salamanders of the genus Hynobius deposit in streams their eggs embedded in a translucent envelope called an 'egg sac'. The edges of the envelope exhibit a spectacular blue-to-yellow iridescent glow, which instantaneously disappears when the sac is removed from water. First, our scanning electron microscopy analyses reveal that the inner surface of the 100 μm-thick envelope displays striations (length scale of about 3 μm), which are themselves covered by much smaller (190 ± 30 nm) and quasi-periodic corrugations. The latter could constitute a surface diffraction grating generating iridescence by light interference. Second, our transmission electron microscopy and focused-ion-beam scanning electron microscopy analyses show that the bulk of the egg sac wall is composed of meandering fibres with a quasi-periodic modulation of 190 ± 60 nm along the thickness of the envelope, generating a photonic crystal. Third, Fourier power analyses of 450 electron microscopy images with varying incident angles indicate that changing the surrounding medium from water to air shifts most of the backscattered power spectrum to the ultraviolet range, hence, explaining that the egg sac loses visible iridescence when removed out of the water. Fourth, the results of our photography and optical spectroscopy experiments of submerged and emerged egg sacs rule out the possibility that the iridescence is due to a thin film or a multilayer, whereas the observed non-specular response is compatible with the backscattering expected from surface diffraction gratings and volumetric photonic crystals with spatial 1D modulation. Finally, although we mention several potential biological functions of the egg sac structural colours and iridescence, we emphasise that these optical properties might be the by-products of the envelope material internal structure selected during evolution for its mechanical properties.

A new stream salamander species, Batrachuperus daochengensis sp. nov., from southwestern China, is described herein based on morphological and molecular evidence. Molecular phylogeny derived from the mitochondrial gene together with previous nuclear data revealed that B. daochengensis sp. nov. is sister to B. yenyuanensis. The new species differs from all other species of the genus by the following combination of characters: brown horny epidermis on tips of fingers and toes absent; tubercles on palms and soles absent; costal grooves 12; dorsal brown, mottled with blackish spots; fingers 2-3-4-1 in order of decreasing length; tips of longest digits of fore- and hindlimbs largely separated by one to two costal spaces when adpressed towards each other along sides of body. The new species is currently known in the central and southern Shaluli Mountains in southwestern China.

Systems of genetic sex determination and the homology of sex chromosomes in different taxa vary greatly across vertebrates. Much progress remains to be made in understanding systems of genetic sex determination in non-model organisms, especially those with homomorphic sex chromosomes and/or large genomes. We used reduced representation genome sequencing to investigate genetic sex determination systems in the salamander family Cryptobranchidae (genera Cryptobranchus and Andrias), which typifies both of these inherent difficulties. We tested hypotheses of male- or female-heterogamety by sequencing hundreds of thousands of anonymous genomic regions in a panel of known-sex cryptobranchids and characterized patterns of presence/absence, inferred zygosity, and depth of coverage to identify sex-linked regions of these 56 gigabase genomes. Our results strongly support the hypothesis that all cryptobranchid species possess homologous systems of female heterogamety, despite maintenance of homomorphic sex chromosomes over nearly 60 million years. Additionally, we report a robust, non-invasive genetic assay for sex diagnosis in Cryptobranchus and Andrias which may have great utility for conservation efforts with these endangered salamanders. Co-amplification of these W-linked markers in both cryptobranchid genera provides evidence for long-term sex chromosome stasis in one of the most divergent salamander lineages. These findings inform hypotheses about the ancestral mode of sex determination in salamanders, but suggest that comparative data from other salamander families are needed. Our results further demonstrate that massive genomes are not necessarily a barrier to effective genome-wide sequencing and that the resulting data can be highly informative about sex determination systems in taxa with homomorphic sex chromosomes.

Background Chinese giant salamander Andrias davidianus is an endangered species. The success of artificial breeding provides a useful way to protect this species. However, the method to identify the sex and mechanism of sex determination were unclear which hinder the improvement of the artificial breeding. Detection of a sex specific marker provides an effective approach to identify genetic sex and investigate the sex determination mechanism. Results We used restriction-site-associated DNA (RAD) sequencing to isolate a sex-specific genetic marker in A. davidianus to expand knowledge of the sex determination mechanism. Four male and four female specimens were subjected to RAD sequencing, which generated 934,072,989 reads containing approximately 134.4 Gb of sequences. The first round of comparison of the assembled sequence against the opposite sex raw reads revealed 19,097 female and 17,994 male unmatched sequences. Subsequently, 19,097 female sequences were subjected to a BLAST search against male genomic data, which revealed 308 sequences unmapped to the male genome. One hundred of these were randomly selected and validated by PCR in five male and five female specimens, and four putative sex-specific sequences were produced. Further validation was performed by PCR in another 24 females and 24 males, and all female individuals exhibited the expected specific bands, while the males did not. To apply the sex-specific marker, three specimens reversed from genetic female to physiological male were found in a group exposed to elevated temperature, and 13 individuals reversed from genetic male to physiological female were obtained in a 17β-estradiol exposed group. Conclusion This is the first report of a sex-specific marker in A. davidianus and may have potential for elucidation of its sex determination mechanism and, hence, its conservation. Electronic supplementary material The online version of this article (10.1186/s12864-019-5771-5) contains supplementary material, which is available to authorized users.

Understanding polymorphism is a central problem in evolution and ecology, and alternative reproductive tactics (ARTs) provide compelling examples for studying the origin and maintenance of behavioral and morphological variation. Much attention has been given to examples where "parasitic" individuals exploit the reproductive investment of "bourgeois" individuals, but some ARTs are instead maintained by environmental heterogeneity, with alternative tactics exhibiting differential fitness in discontinuous reproductive niches. We use genomic, behavioral, karyological, and field observational data to demonstrate one such example in plethodontid salamanders. These ARTs ("searching" and "guarding" males) are associated with different reproductive niches and, unlike most other examples in amphibians, demonstrate substantial morphological differences and inflexibility within a reproductive season. Evidence suggests the existence of these ARTs within three putative species in the two-lined salamander (Eurycea bislineata) species complex, with other members of this clade fixed for one of the two tactics. We highlight directions for future research in this system, including the relationship between these ARTs and parental care.

The Chinese giant salamander, Andrias davidianus, is the largest amphibian species in the world; it is thus an economically and ecologically important species. The skin of A. davidianus exhibits complex adaptive structural and functional adaptations to facilitate survival in aquatic and terrestrial ecosystems. Here, we report the first full-length amphibian transcriptome from the dorsal skin of A. davidianus, which was assembled using hybrid sequencing and the PacBio and Illumina platforms. A total of 153,038 transcripts were hybrid assembled (mean length of 2039 bp and N50 of 2172 bp), and 133,794 were annotated in at least one database (nr, Swiss-Prot, KEGG, KOGs, GO, and nt). A total of 58,732, 68,742, and 115,876 transcripts were classified into 24 KOG categories, 1903 GO term categories, and 46 KEGG pathways (level 2), respectively. A total of 207,627 protein-coding regions, 785 transcription factors, 27,237 potential long non-coding RNAs, and 8299 simple sequence repeats were also identified. The hybrid-assembled transcriptome recovered more full-length transcripts, had a higher N50 contig length, and a higher annotation rate of unique genes compared with that assembled in previous studies using next-generation sequencing. The high-quality full-length reference gene set generated in this study will help elucidate the genetic characteristics of A. davidianus skin and aid the identification of functional skin proteins.

Caudata is an order of amphibians with great variation in genome size, which can reach enormous dimensions in salamanders. In this work, we analysed the activity of transposable elements (TEs) in the transcriptomes obtained from female and male gonads of the Chinese fire-bellied newt, Cynops orientalis, a species with a genome about 12-fold larger than the human genome. We also compared these data with genomes of two basal sarcopterygians, coelacanth and lungfish. In the newt our findings highlighted a major impact of non-LTR retroelements and a greater total TE activity compared to the lungfish Protopterus annectens, an organism also characterized by a giant genome. This difference in TE activity might be due to the presence of young copies in newt in agreement also with the increase in the genome size, an event that occurred independently and later than lungfish. Moreover, the activity of 33 target genes encoding proteins involved in the TE host silencing mechanisms, such as Ago/Piwi and NuRD complex, was evaluated and compared between the three species analysed. These data revealed high transcriptional levels of the target genes in both newt and lungfish and confirmed the activity of NuRD complex genes in adults. Finally, phylogenetic analyses performed on PRDM9 and TRIM28 allowed increasing knowledge about the evolution of these two key genes of the NuRD complex silencing mechanism in vertebrates. Our results confirmed that the gigantism of the newt genomes may be attributed to the activity and accumulation of TEs.

In vertebrates, the main tissue devoted to energy storage is the adipose tissue. In salamanders, energy reserves can also be stored in the adipose tissues of the tail. Therefore, we evaluated if energy storage in salamanders' tails is related to individual body condition, life cycle and environmental constraints. We calculated a scaled measure of tail width for 345 salamanders belonging to six Mediterranean taxa exhibiting wide phylogenetic, behavioural and ecological variation. We related this measure to the Scaled Mass Index (SMI), a body condition index which reliably predicts body fat. We found significant relationships between the SMI and scaled tail width in the terrestrial Spectacled salamander and Alpine salamanders, independently of sex. At the same time, we found that energy storage in the tail is maximum in Alpine Salamanders, which experience reduced activity periods and restricted access to resources. Conversely, we found a significant effect of sex in Imperial cave salamanders, where females store reserves in the tail to counterbalance resource investment in parental care, and in Corsican Brook Newts, where the reproductive function of males' tails may imply a greater tail width. Finally, in the biphasic Great Crested Newt, tail width was not related to SMI in both sexes.

Water quality under tourism disturbance was simulated through controlling the water intake of the ecological breeding ponds of Chinese giant salamander (通过控制大鲵仿生态繁育池进水量模拟旅游干扰下的水质溶解氧特征,采用红外数字监控系统研究大鲵繁殖行为(产卵与护卵)及繁殖力(相对产卵量、受精率与孵化率)特点,分析它们与水质的关系,探讨旅游干扰导致的水质变化对大鲵繁殖行为及繁殖力的影响。结果表明: 与对照组相比,旅游干扰下大鲵的产卵行为与繁殖力未受到显著影响,但雄鲵护卵行为中的扇尾与搅动时间显著延长,以提高水中溶解氧浓度,满足大鲵胚胎发育需求;此外,旅游干扰下受精卵的孵化时间显著延长,但孵化率未受到显著影响。雄鲵护卵行为变化与受精卵孵化时间延长可能是大鲵对旅游干扰导致的水质变化的一种主动响应。.

Semen of high to moderate quality was collected following the hormonal induction of North American giant salamanders Cryptobranchus alleganiensis. Oocytes from one female yielded the first C. alleganiensis produced while maintained in aquaria under human care and the first externally fertilising salamander produced with cryopreserved spermatozoa and IVF. Further research is needed with North American giant salamanders to establish reliable techniques to produce large numbers of viable offspring, along with the application of cryopreserved spermatozoa.

Salamanders of the tribe Bolitoglossini Hallowell are a highly diversified group of amphibians, and their helminth parasite fauna has been scarcely studied. Some species of plethodontid salamanders distributed along the Trans-Mexican Volcanic Belt, in central Mexico, were sampled, and their helminth parasites were recovered for taxonomic identification. Specimens of a pharyngodonid nematode from 2 species of bolitoglossines of the genus Pseudoeurycea Taylor were morphologically identified as Batracholandros salamandrae (Schad, 1960) Petter and Quentin, 1976. These specimens were studied in further detail through light and scanning electron microscopy and were sequenced for 2 ribosomal genes and 1 mitochondrial gene to test the hypothesis of whether B. salamandrae is a species widely distributed in salamanders across the Nearctic biogeographic region, or if it represents a cryptic species complex. Our molecular results revealed that these specimens consisted of 2 genetic lineages in concordance with host species, although with slight morphological differences among specimens in each of them. A thorough study, including the generation of molecular data from individuals from other areas of North America, and the examination of type specimens, is required to test the reliability of these morphological differences and to corroborate the species identity of the 2 genetic lineages.

Polyphenisms, where two or more alternative, environmentally-cued phenotypes are produced from the same genotype, arise through variability in the developmental rate and timing of phenotypic traits. Many of these developmental processes are controlled or influenced by endogenous hormones, such as glucocorticoids, which are known to regulate a wide array of vertebrate ontogenetic transitions. Using the mole salamander, Ambystoma talpoideum, as a model, we investigated the role of glucocorticoids in regulating facultative paedomorphosis, an ontogenetic polyphenism where individuals may delay metamorphosis into terrestrial adults. Instead, individuals reproduce as aquatic paedomorphic adults. Paedomorphosis often occurs when aquatic conditions remain favorable, while metamorphosis typically occurs in response to deteriorating or "stressful" aquatic conditions. Since glucocorticoids are central to the vertebrate stress response and are known to play a central role in regulating obligate metamorphosis in amphibians, we hypothesized that they are key regulators of paedomorphic life history strategies. To test this hypothesis, we compared development of larvae in outdoor mesocosms exposed to Low, Medium, and High exogenous doses of corticosterone (CORT). Results revealed that body size and the proportion of paedomorphs were both inversely proportional to exogenous CORT doses and whole-body CORT content. Consistent with known effects of CORT on obligate metamorphosis in amphibians, our results link glucocorticoids to ontogenetic transitions in facultatively paedomorphic salamanders. We discuss our results in the context of theoretical models and the suite of environmental cues known to influence facultative paedomorphosis.

Background Regeneration of complex patterned structures is well described amongst, although limited to a small sampling of, amphibians. This limitation impedes our understanding of the full range of regenerative competencies within this class of vertebrates, according to phylogeny, developmental life stage and age. To broaden the phylogenetic breath of this research, we characterized the regenerative capacity of the Texas blind salamander (Eurycea rathbuni), a protected salamander native to the Edwards Aquifer of San Marcos, Texas and colonized by the San Marcos Aquatic Resource Center. As field observations suggested regenerative abilities in this population, the forelimb stump of a live captured female was amputated in the hopes of restoring the structure, and thus locomotion in the animal. Tails were clipped from two males to additionally document tail regeneration. Results We show that the Texas blind salamander exhibits robust limb and tail regeneration, like all other studied Plethodontidae. Regeneration in this species is associated with wound epithelium formation, blastema formation and subsequent patterning and differentiation of the regenerate. Conclusions The study has shown that the Texas blind salamander is a valuable model to study regenerative processes, and that therapeutic surgeries offer a valuable means to help maintain and conserve this vulnerable species. This article is protected by copyright. All rights reserved.

Synopsis Courtship behavior in salamanders is often complex and involves well-documented communication from males to females in multiple sensory modalities. Historically, behaviors exhibited during the major stages of courtship have been predominately framed as a male acting and signaling to “persuade” a passive female to participate in courtship and remain with him until sperm release is completed. In this review, we use courtship descriptions for lungless salamanders (Plethodontidae) as a case study to illustrate this historical bias of a male-centered perspective. We then re-examine the literature and summarize the many ways females are active participants during plethodontid courtships. We also relate female behaviors to the types of female-to-male communication that may occur. For example, females have been documented to approach a male and initiate courtship, participate in mutual head rubbing, and step astride the male’s tail to begin the tail-straddling walk (a key courtship behavior observed in all plethodontids). Additionally, females have glands that may produce chemical signals that males respond to during courtship. We conclude that communication during courtship is more accurately described as a two-way interaction where each partner’s behavior is coordinated with the other’s via multi-modal signaling. Shifting the lens through which we view courtship and behavior provides insight into which female behaviors and anatomical features are most likely to be used for communication with males.

Sialic acids (Sia) are terminal components of glycoconjugates that are involved in molecular and cellular interactions in the olfactory system. Diverse glycoconjugates are expressed in the salamander olfactory projection; however, their sialylation and the linkage of Sia to underlying sugars remain largely unknown. The present study aimed to determine the expression of Sia linked to galactose (Gal)-N-acetylglucosamine and N-acetylgalactosamine (GalNAc) in the olfactory bulbs of three species of salamanders using lectin binding. Abundant distribution of sialoglycoconjugates was observed in the salamander olfactory bulb by lectins, Sambucus sieboldiana (SSA) and Maackia amurensis (MAM). Moreover, SSA and MAM showed heterogeneous bindings in the primary olfactory projection of Cynops pyrrhogaster and C. orientalis. Lectin reactivities obviously decreased in all layers of the olfactory bulb after sialidase digestion, indicating selective binding to sialoglycoconjugates. Next, we examined the expression of the subterminal sugar residues, Gal and GalNAc, after terminal Sia removal. Desialylation in the olfactory bulb enhanced the reactivity of Jacalin and Vicia villosa (VVA) lectins that recognize Gal and GalNAc respectively. Together with the binding of SSA and MAM, Sia linked to Gal and GalNAc might be a major component of sialoglycoconjugates in the salamander olfactory projection.

Salamanders (Urodela) have among the largest vertebrate genomes, ranging in size from 10 to 120 pg. Although changes in genome size often occur randomly and in the absence of selection pressure, non-random patterns of genome size variation are evident among specific vertebrate lineages. Here we report i) an unexpected relationship between genome size (C-value) and species richness at the family taxonomic level in salamander clades; ii) C-value and species richness both correlate with clade crownage but not with diversification rates; iii) C-value is strongly associated with geographical area. Consistent with this observation we found that iv) genome size in salamanders is also strongly related to climatic niche rate. Finally, we report a relationship between C-value diversity and family-level species diversity as measured by the coefficient of variation of genome size within clades. Based on these results we propose that variation in C-value and genomic organization (heterochromatin content and gene synteny) might underlie variation in species richness in agreement with the Geographical Area Hypothesis. These observations suggest that changes in C-value, and hence changes in the amount and/or organization of non-coding DNA in the vertebrate genome, are associated with the allelic incompatibility believed to drive reproductive isolation and speciation in salamanders.

The Chinese giant salamander Andrias davidianus is a protected amphibian with high nutritional and economic value. Understanding its sex determination mechanism is important for improving culture techniques and sex control in breeding. However, little information on the characterization of critical genes involved in sex is available. Herein, sequencing of ovary and test produced 40,783,222 and 46,128,902 raw reads, respectively, which were jointly assembled into 80,497 unigenes. Of these, 36,609 unigenes were annotated, of which 8907 were female-biased and 10,385 were male-biased. Several sex-related pathways were observed, including the Wnt signaling pathway. After elevated temperature and estrogen exposure, neomale and neofemale specimens were identified by a female-specific marker for the first time. RT-qPCR analysis showed the expression profile of ten selected sex-biased genes to be exhibited consistently in male and neomale and in female and neofemale, with the exception of the Amh and TfIIIa genes. Results suggested that these genes may play important roles in A. davidianus sex determination and gonad development. This provides a basis for further investigation of the molecular mechanisms of sex determination in amphibians.

Variation in environmental conditions can result in disparate associations between hosts and microbial symbionts. As such, it is imperative to evaluate how environmental variables (e.g., habitat quality) can influence host-associated microbiome composition. Within wildlife conservation programs, captive conditions can negatively influence the establishment and maintenance of "wild-type" microbiotas within a host. Alternative microbial communities can result in the proliferation of disease among captive stock or upon reintroduction. Hellbenders (Cryptobranchus alleganiensis) are a threatened salamander for which extensive captive management is currently employed. Using metabarcoding, we characterized the skin microbiota of wild and captive hellbenders from two subspecies in the state of Missouri, the eastern (C. a. alleganiensis) and the Ozark hellbender (C. a. bishopi). Both subspecies in our study included wild adults and captive juveniles that were collected from the wild as eggs. Our objectives were to investigate differences in the skin microbial communities' richness/diversity, composition, and functional profiles of microbes between wild and captive individuals. Captive eastern hellbenders possessed richer communities than wild cohorts, whereas the opposite pattern was observed within the Ozark subspecies. We found significant microbial community structure between wild and captive populations of both subspecies. Microbiota structure translated into differences in the predicted metagenome of wild and captive individuals as well. As such, we can expect captive hellbenders to experience alternative microbial structure and function upon reintroduction into the wild. Our study provides a baseline for the effect of captivity on the skin microbial communities of hellbenders, and highlights the need to incorporate microbiota management in current captive-rearing programs.

Males and females face different selection pressures due to a sexually biased investment into reproduction. This often results in different morphologies. Sexual size dimorphisms (SSD) can give us important hints on the evolution and biology of a species. Salamanders are a perfectly suited system for investigating SSD, including a diversity of reproductive modes and behaviors, and patterns of SSD combined with life history traits in a phylogenetic context help us to understand the evolution of these processes. Because spectacled salamanders (genus Salamandrina) are the phylogenetically most basal taxon of the Salamandridae, they play a key role in reconstructing the evolutionary pattern of SSD. Combining extensive external and skeletal measurements of the cranium, limbs, and the pelvic girdle using high-resolution micro Computer Tomography (μCT) yielded an integrative analysis of expressed SSD of morphology and osteology of Salamandrina perspicillata. Multivariate analysis of external characters showed that males generally had larger cloacae, heads, and limbs relative to body size, while females had larger trunks. Analysis of osteology confirmed this pattern but also revealed new dimorphic characters in the cranium and the pelvic girdle. Dimorphic characters in external morphology and osteology are likely linked to the different reproductive roles of the sexes and support sexual rather than ecological selection as the primary force acting on the phenotype of the phylogenetically basal salamandrids.

Abnormal embryos of Chinese giant salamander (Andrias davidianus, CGS) were observed in an imitating ecologic breeding system at 20°C. The aim of the present study was to investigate the relationship of bacterial infection on the early embryonic development of CGS. The ratio of abnormal embryos at 10 days after embryos incubated was 35.0% ± 2.1%, 35.6% ± 2.3% and 34.7% ± 3.4% in six breeding system farms of Hanzhong city in the years 2014, 2015, and 2016, respectively. However, in the standard imitating ecology breeding mesocosm, the proportion of abnormal embryos was about 5%. Six bacteria species in the egg water of the early-dead embryos and eight bacteria species in the gut of healthy CGSs were detected and identified by PCR and 16S rRNA gene sequence homology analysis. All bacteria species in the egg water were also found in the cloaca contents. Cetobacterium somerae and Hafnia alvei, which individually can cause embryo death, were first isolated from egg water and gut of CGSs. Further, the egg jelly membrane and the egg water of embryos did not inhibit bacteria survival and the bacteria could individually lead to CGS larva death. These results suggest that bacteria in the eggs of CGS may derive from the gut and that high-velocity flow of water through nest may decrease bacterial infection of egg in the imitating ecologic culture system.

Accurate measures of species abundance are essential to identify conservation strategies. N-mixture models are increasingly used to estimate abundance on the basis of species counts. In this study we tested whether abundance estimates obtained using N-mixture models provide consistent results with more traditional approaches requiring capture (capture-mark recapture and removal sampling). We focused on endemic, threatened species of amphibians and reptiles in Italy, for which accurate abundance data are needed for conservation assessments: the Lanza’s Alpine salamander Salamandra lanzai, the Ambrosi’s cave salamander Hydromantes ambrosii and the Aeolian wall lizard Podarcis raffonei. In visual counts, detection probability was variable among species, ranging between 0.14 (Alpine salamanders) and 0.60 (cave salamanders). For all the species, abundance estimates obtained using N-mixture models showed limited differences with the ones obtained through capture-mark-recapture or removal sampling. The match was particularly accurate for cave salamanders in sites with limited abundance and for lizards, nevertheless non-incorporating heterogeneity of detection probability increased bias. N-mixture models provide reliable abundance estimates that are comparable with the ones of more traditional approaches, and offer additional advantages such as a smaller sampling effort and no need of manipulating individuals, which in turn reduces the risk of harming animals and spreading diseases.

Seasonally breeding species exhibit cyclical changes in circulating steroid hormone profiles that correspond with changes to their reproductive behavior and ecology. Such information is critical to the conservation of imperiled and data-deficient species, such as the eastern hellbender salamander (Cryptobranchus alleganiensis alleganiensis). We determined changes in plasma testosterone (T), dihydrotestosterone (DHT), 11-ketotestosterone (11-KT), 11-ketoandrostenedione (11-KA), dehydroepiandrosterone (DHEA), cortisol, corticosterone, and progesterone (P4) during a four-month period preceding breeding in adult male and female eastern hellbenders. This pre-breeding period is characterized by increased diel movement and aggression by both sexes, follicular development and yolk production in females, and sperm production, territoriality, and nest site establishment in males. In both males and females, we observed a progressive increase in circulating T and DHT during the pre-reproductive season, both peaking in August (17 days before breeding), but concentrations of both hormones were higher in males. Conversely, 11-KT was higher in females, but did not vary significantly by date. These results suggest that T and DHT are the predominant androgens in eastern hellbenders and are likely important regulators of reproductive processes in both males and females. The detection of significant quantities of DHT and 11-KT in females is particularly interesting, considering that unlike T, neither of these androgens can be converted to estrogens. Therefore, it seems possible that aggression or some aspect of reproduction in the female eastern hellbender may be directly mediated by androgen signaling. Baseline cortisol did not vary throughout the pre-breeding period but was higher in females than males, and also became highly variable in females leading up to breeding. Progesterone, 11-KA, DHEA, and corticosterone were rarely or never detected, and thus, do not appear to be important during the pre-reproductive season. This study provides a physiological framework for future studies of hellbender reproductive biology, which could ultimately be important for their conservation.

Examination of the holotype of Hynobius stejnegeri Dunn, 1923 revealed that the species is identical with H. yatsui Oyama, 1947 , recently resurrected from H. naevius (Temminck and Schlegel, 1838). The name H. yatsui is therefore relegated to a subjective junior synonym of H. stejnegeri. The latter name, however, has been widely applied to an amber-colored or tortoise-shell salamander from Kyushu, southwestern Japan, which now requires a new name. We, thus, describe the ambercolored salamander as a new species Hynobius ikioi. The new species is phylogenetically very close to H. amakusaensis Nishikawa and Matsui, 2014 and H. osumiensis Nishikawa and Matsui, 2014 , both also from Kyushu, but is easily distinguished from them by its uniquely bi-colored dorsum.

Intermediate filament proteins are structural components of the cellular cytoskeleton with cell-type specific expression and function. Glial fibrillary acidic protein (GFAP) is a type III intermediate filament protein and is up-regulated in glia of the nervous system in response to injury and during neurodegenerative diseases. In the retina, GFAP levels are dramatically increased in Müller glia and are thought to play a role in the extensive structural changes resulting in Müller cell hypertrophy and glial scar formation. In spite of similar changes to the morphology of Xenopus Müller cells following injury, we found that Xenopus lack a gfap gene. Other type III intermediate filament proteins were, however, significantly induced following rod photoreceptor ablation and retinal ganglion cell axotomy. The recently available X. tropicalis and X. laevis genomes indicate a small deletion most likely resulted in the loss of the gfap gene during anuran evolution. Lastly, a survey of representative species from all three extant amphibian orders including the Anura (frogs, toads), Caudata (salamanders, newts), and Gymnophiona (caecilians) suggests that deletion of the gfap locus occurred in the ancestor of all Anura after its divergence from the Caudata ancestor around 290 million years ago. Our results demonstrate that extensive changes in Müller cell morphology following retinal injury do not require GFAP in Xenopus, and other type III intermediate filament proteins may be involved in the gliotic response.

The reduction in fecundity associated with the evolution of viviparity may have far-reaching implications for the ecology, demography, and evolution of populations. The evolution of a polygamous behaviour (e.g. polyandry) may counteract some of the effects underlying a lower fecundity, such as the reduction in genetic diversity. Comparing patterns of multiple paternity between reproductive modes allows us to understand how viviparity accounts for the trade-off between offspring quality and quantity. We analysed genetic patterns of paternity and offspring genetic diversity across 42 families from two modes of viviparity in a reproductive polymorphic species, Salamandra salamandra. This species shows an ancestral (larviparity: large clutches of free aquatic larvae), and a derived reproductive mode (pueriparity: smaller clutches of larger terrestrial juveniles). Our results confirm the existence of multiple paternity in pueriparous salamanders. Furthermore, we show the evolution of pueriparity maintains, and even increases, the occurrence of multiple paternity and the number of sires compared to larviparity, though we did not find a clear effect on genetic diversity. High incidence of multiple paternity in pueriparous populations might arise as a mechanism to avoid fertilization failures and to ensure reproductive success, and thus has important implications in highly isolated populations with small broods.

The hynobiid salamander genus

It is generally accepted that most non-mammal tetrapods have a hinge-like jaw operation restricted to vertical opening and closing movements. Many mammal jaw joints, by contrast, operate in more complex, three-dimensional ways, involving not only vertical, but also propalinal (rostro-caudal) and transverse (lateral) movements. Data on intraoral food processing in lissamphibians and sauropsids has prompted a generally accepted view that these groups mostly swallow food unreduced, and that in those cases where lissamphibians and sauropsids chew, they mostly use simple vertical jaw movements for food processing. The exception to that generally accepted view being some propalinal chewing in sauropsids. We combined 3D kinematics and morphological analyses from biplanar high-speed video fluoroscopy and micro-CT to determine how the paedomorphic salamander Siren intermedia treats captured food. We discovered that S. intermedia not only uses intraoral food processing, but that the elaborated morphology of its jaw joint facilitates mandibular motions in all three planes, resulting in complex three-dimensional chewing. Thus, our data challenge the commonly held view that complex three-dimensional chewing movements are exclusive to mammals, by suggesting that complex chewing mechanisms might evolved early in tetrapod evolution.

There is considerable variation among studies that evaluate how amphibian populations respond to global climate change. We used 23 years of annual survey data to test whether changes in climate have caused predictable shifts in the phenology and population characteristics of adult spotted salamanders (Ambystoma maculatum) during spring breeding migrations. Although we observed year-to-year correlation between seasonal climate variables and salamander population characteristics, there have not been long-term, directional shifts in phenological or population characteristics. Warm winters consistently resulted in early migration dates, but across the 23-year study, there was no overall shift towards warmer winters and thus no advanced migration timing. Warm summers and low variability in summer temperatures were correlated with large salamander body sizes, yet an overall shift towards increasing body sizes was not observed despite rising summer temperatures during the study. This was likely due to the absence of long-term changes of within-year variation in summer temperatures, which was a stronger determinant of body size than summer temperature alone. Climate-induced shifts in population characteristics were thus not observed for this species as long-term changes in important seasonal climate variables were not observed during the 23-years of the study. Different amphibian populations will likely be more resilient to climate change impacts than others, and the probability of amphibians exhibiting long-term population changes will depend on how seasonal climate change interacts with a species' life history, phenology, and geographic location. Linking a wide range of seasonal climatic conditions to species or population characteristics should thus improve our ability for explaining idiosyncratic responses of species to climate change.

Unisexual

The Hyrcanian Forests present a unique Tertiary relict ecosystem, covering the northern Elburz and Talysh Ranges (Iran, Azerbaijan), a poorly investigated, unique biodiversity hotspot with many cryptic species. Since the 1970s, two nominal species of Urodela, Hynobiidae, Batrachuperus (later: Paradactylodon) have been described: Paradactylodon persicus from northwestern and P. gorganensis from northeastern Iran. Although P. gorganensis has been involved in studies on phylogeny and development, there is little data on the phylogeography, systematics, and development of the genus throughout the Hyrcanian Forests; genome-wide resources have been entirely missing. Given the huge genome size of hynobiids, making whole genome sequencing hardly affordable, we aimed to publish the first transcriptomic resources for Paradactylodon from an embryo and a larva (9.17 Gb RNA sequences; assembled to 78,918 unigenes). We also listed 32 genes involved in vertebrate sexual development and sex determination. Photographic documentation of the development from egg sacs across several embryonal and larval stages until metamorphosis enabled, for the first time, comparison of the ontogeny with that of other hynobiids and new histological and transcriptomic insights into early gonads and timing of their differentiation. Transcriptomes from central Elburz, next-generation sequencing (NGS) libraries of archival DNA of topotypic P. persicus, and GenBank-sequences of eastern P. gorganensis allowed phylogenetic analysis with three mitochondrial genomes, supplemented by PCR-amplified mtDNA-fragments from 17 museum specimens, documenting

Ultrasound imaging has more frequently been used in veterinary medicine of amphibians and reptiles. In this study, we have verified the usefulness of ultrasound imaging in pregnancy determination of the fire salamander Salamandra salamandra. We have also undertaken to estimate the number of larvae and their developmental stage directly in the oviducts. Three gravid females from Lower Silesia (southern Poland) were examined. Due to the small size of the scanned animals, and the particular arrangement of embryos in the oviducts and ultrasound beams dispersal, the method proved to be inaccurate. Therefore, the minimum number of well-visualized larvae was determined. The maximum number of larvae was established on the basis of the visible fragments of embryos. After birth, we found that the number of larvae born was included in the „min-max” range in only one case. In the remaining two salamanders the number of larvae was higher than estimated in 3 to 7 individuals. The results showed that ultrasound imaging allows the minimum number of larvae in salamander; oviducts to be specified. However, total length measurements were possible only for single and clearly visible embryos.

Lhx9 is an LIM (named for the first three proteins in which the domain was found, Lin-11, Isl1 and Mec-3) homeodomain protein involved in development and differentiation of the gonad. In this study, we isolated the full-length Lhx9 and Lhx9 α from Andrias davidianus, detected the tissue distribution and analysed the methylation of the promoters. We identified Lhx9 of 1411 bp and Lhx9 α of 1153-bp length, differing in the 3 $^{\prime }$ -flanking region, encoding 399 and 330 amino acids, respectively. The Lhx9 gene was detected primarily in liver, ovary and heart with moderate expression in brain, pituitary, intestine and spleen, and low expression in the remaining examined tissues, while Lhx9 α expression was high in heart, pituitary and liver, and low in spleen and stomach. Significantly higher Lhx9 expression was observed in ovary than in testis, with no differences in Lhx9 α expression between testis and ovary observed. Bisulphite sequencing revealed significantly higher methylation in testis compared to ovary. The methylation level of CpG sites –733, –673, –615 and –594 exhibited significantly higher levels in testis than in ovary, which was negatively correlated with Lhx9 expression. The methylation and expression patterns suggested that promoter methylation suppressed expression of Lhx9 in A. davidianus.

Phylogeographic studies frequently result in the elevation of subspecific taxa to species given monophyly, or the synonymy of subspecies that are not monophyletic. However, given limited or incongruent datasets, retention of subspecies can be useful to describe hypothesized incipient species or to illustrate interesting biological phenomena driving morphological diversity. Four subspecific taxa have been used to describe largely allopatric geographic variation within the species Pseudotriton ruber, a plethodontid salamander occupying stream and spring habitats across eastern North America: P. r. vioscai occurs in lowland Coastal Plain habitats, while P. r. ruber, P. r. nitidus, and P. r. schencki occupy upland regions in and around the Appalachian Mountains. Pseudotriton ruber co-occurs through its distribution with the aposematic newt Notophthalmus viridescens, and both species are hypothesized to be part of a Müllerian mimicry complex. In this study, we sequenced regions of two mitochondrial (cytochrome b, NADH dehydrogenase subunit 2) and one single copy nuclear protein-coding gene (pro-opiomelanocortin) from individuals sampled across much of the distribution of P. ruber and then used maximum-likelihood and Bayesian phylogenetic inference to test the monophyly of subspecies, reconstruct biogeographic history, and make inferences about morphological evolution. Phylogeographic hypotheses from mitochondrial and nuclear datasets described structure among populations of P. ruber which separated Coastal Plain and upland Appalachian populations, but subspecies were not monophyletic. Biogeographic reconstruction estimated the ancestor of all populations to have occupied and initially diverged in the Coastal Plain during the Pliocene (∼3.6mya), before one lineage subsequently invaded upland areas of Appalachia. Bold bright coloration of high elevation subspecies P. r. nitidus and P. r. schencki appears to have evolved twice. We hypothesize that the Müllerian mimicry complex with N. viridescens and P. ruber may provide a selective mechanism driving the co-evolution of striking bright and dull morphological variation among populations of both species. While P. ruber subspecies were not consistent with our criteria for diagnosing species (monophyly) and therefore could not be elevated to species, we advocate for the retention of subspecies because they describe hypotheses about an incipient species (P. r. vioscai) and how Müllerian mimicry may shape morphological diversity of species.

Gene co-option is a major force in the evolution of novel biological functions. In plethodontid salamanders, males deliver proteinaceous courtship pheromones to the female olfactory system or transdermally to the bloodstream. Molecular studies identified three families of highly duplicated, rapidly evolving pheromones (PRF, PMF, and SPF). Analyses for Plethodon salamanders revealed pheromone mixtures of primarily PRF and PMF. The current study demonstrates that in Desmognathus ocoee--a plesiomorphic species with transdermal delivery--SPF is the major pheromone component representing >30% of total protein. Chromatographic profiles of D. ocoee pheromones were consistent from May through October. LC/MS-MS analysis suggested uniform SPF isoform expression between individual male D. ocoee. A gene ancestry for SPF with the Three-Finger Protein superfamily was supported by intron-exon boundaries, but not by the disulfide bonding pattern. Further analysis of the pheromone mixture revealed paralogs to peptide hormones that contained mutations in receptor binding regions, such that these novel molecules may alter female physiology by acting as hormone agonists/antagonists. Cumulatively, gene co-option, duplication, and neofunctionalization have permitted recruitment of additional gene families for pheromone activity. Such independent co-option events may be playing a key role in salamander speciation by altering male traits that influence reproductive success.

International audience; Temperature affects the physiological functions of ectotherms. To maintain optimal body temperature and ensure physiological performance, these organisms can use behavioral adjustments to keep the body temperature in their specific temperature range, so-called preferred temperature (Tpref). It is therefore crucial to describe and understand how Tpref vary within and amongst populations to predict the effects of climate change of altitudinal range shifts in organisms. We aimed at determining the altitudinal variations in Tpref in three ectothermic species (the Pyrenean brook salamander – a semi-aquatic and thigmothermic amphibian – the European common lizard and the wall lizard – both heliothermic species). Using an experimental approach where Tpref were measured along a temperature gradient in laboratory conditions, we used a cross-sectional approach to compare the variation of Tpref measured in populations sampled along the altitudinal gradient in the Pyrenees. We hypothesized a complex and highly variable intra-specific response of Tpref along geographical clines, with a positive relationship between Tpref and altitude (as predicted by the countergradient variation), the reverse pattern (referring to the adaptation of local optima hypothesis), or no relationship at all. Our results corroborated partially the countergradient hypothesis in the salamander (middle to high elevation part). At high altitude level, individuals may compensate for lower opportunities of favorable conditions by choosing a high temperature which maximizes their activities. However, populations from low elevation level hence better supported the adaptation of local optima hypothesis, such as both lizard species, for which Tpref tended instead to decrease with altitude. Lizards from cold climates may be physiologically adapted to low temperature, with the ability to reach optimal functioning at lower temperature than individuals from low altitude. Our findings suggest that predicting future niche models should therefore take into account the variability of Tpref, where species might be differently affected by global warming.

We characterized the Andrias davidianus T-box 1 (Tbx1) gene. Tbx1 expression was high in testis and low in other examined tissues. Immunohistochemistry detected tbx 1 expression in somatic and germ cells 62 days post-hatching (dph), prior to gonad differentiation. At 210 dph, after gonad differentiation, tbx1 was expressed in spermatogonia and testis somatic cells and in granulosa cells in ovary. Tbx1 expression was up-regulated in ovary after high temperature treatment. In the neomale, tbx 1 expression showed a similar profile to normal males, and vice-versa for genetic male. Over-expression of tbx1 in females after injection of TBX1 protein down-regulated the female-biased genes cyp19a and foxl2 and up-regulated the male-biased amh gene. When tbx 1 was knocked down by tbx 1/siRNA, cyp19a and foxl2 expression was up-regulated, and expression of amh , cyp26a , dmrt1 , and wt1 was down-regulated. Results suggest that tbx1 influenced sex-related gene expression and participates in regulation of A. davidianus testis development.

Reconstructing the locomotion of extinct vertebrates offers insights into their palaeobiology and helps to conceptualize major transitions in vertebrate evolution

Evolutionary theory predicts the evolution of metamorphosis over paedomorphosis (the retention of larval traits at the adult stage) in response to life in unfavourable habitats and to the benefits of dispersal. Although many organisms are canalised into obligatory complex or simple life cycles, some species of newts and salamanders can express both processes (facultative paedomorphosis). Previous research highlighted the detrimental effect of fish on both metamorphic and paedomorphic phenotypes, but it remains unknown whether predation risk could induce shifts from paedomorphosis to metamorphosis, whether behavioural avoidance could be an alternative strategy to metamorphosis and whether these responses could be sex-biased. Testing these hypotheses is important because metamorphosed paedomorphs are dispersal individuals which could favour the long-term persistence of the process by breeding subsequently in more favourable waters. Therefore, we quantified the spatial behaviour and timing of the metamorphosis of facultative paedomorphic palmate newts Lissotriton helveticus in response to predation risk. We found that fish induced both male and female paedomorphs to hide more often, but behavioural avoidance was not predictive of metamorphosis. Paedomorphs did not metamorphose more in the presence of fish, yet there was an interaction between sex and predation risk in metamorphosis timing. These results improve our understanding of the lower prevalence of paedomorphs in fish environments and of the female-biased sex ratios in natural populations of paedomorphic newts. Integrating sex-dependent payoffs of polyphenisms and dispersal across habitats is therefore essential to understand the evolution of these processes in response to environmental change.

Animal sex pheromone systems often exist as multicomponent signals [1–11] to which chemical cues have been added over evolutionary time. Little is known on why and how additional molecules become recruited and conserved in an already functional pheromone system. Here, we investigated the evolutionary trajectory of a series of 15 kDa proteins—termed persuasins—that were co-opted more recently alongside the ancient sodefrin precursor-like factor (SPF) courtship pheromone system in salamanders [9, 12]. Expression, genomic, and molecular phylogenetic analyses show that persuasins originated from a gene that is expressed as a multi-domain protein in internal organs where it has no pheromone function but underwent gene duplication and neofunctionalization. The subsequent evolution combined domain loss and the introduction of a proteolytic cleavage site in the duplicated gene to give rise to two-domain cysteine rich proteins with structural properties similar to SPF pheromones [12]. An expression shift to the pheromone-producing glands, where expression of persuasins was immediately spatiotemporally synchronized with the already available pheromone system, completed the birth of a new pheromone. Electrostatic forces between members of both protein families likely enhance co-localization and simultaneous activation of different female olfactory neurons, explaining why persuasins immediately had a selective advantage. In line with this, behavioral assays show that persuasins increase female receptivity on their own but also exert a cumulative or synergistic effect in combination with SPF, clearly reinforcing the pheromone system as a whole. Our study reveals molecular remodeling of an existing protein architecture as an evolutionary mechanism for functional reinforcement of animal pheromone systems. Little is known about how additional molecules are recruited to functional pheromone systems. Maex et al. show how a new salamander courtship pheromone, persuasin, was co-opted alongside an ancient pheromone after a gene duplication and shift in expression. Subsequent evolution led the molecule along a pathway that reinforced the existing pheromone.

Amphibian skin is unique among vertebrate classes, containing a large number of multicellular exocrine glands that vary among species and have diverse functions. The secretions of skin glands contain a rich array of bioactive compounds including antimicrobial peptides (AMPs). Such compounds are important for amphibian innate immune responses and may protect some species from chytridiomycosis, a lethal skin disease caused by the fungal pathogens, Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal). While the bioactivity of skin secretions against Bd has been assessed for many amphibian taxa, similar studies are lacking for Bsal, a chytrid fungus that is especially pathogenic for salamanders. We studied the skin glands and their potential functions in the aquatic salamander, Three-toed Amphiuma (Amphiuma tridactylum). Skin secretions of captive adult salamanders were analyzed by RP-HPLC and tested against the growth of Bd and Bsal using in-vitro assays. We found that compounds within collected skin secretions were similar between male and female salamanders and inhibited the growth of Bd and Bsal. Thus, skin secretions that protect against Bd may also provide protection against Bsal. Histological examination of the skin glands of preserved salamanders revealed the presence of enlarged granular glands concentrated within caudal body regions. A site of potential gland specialization was identified at the tail base and may indicate specialized granular glands related to courtship and communication.

Telocytes (TCs) and their telopodes (Tps) have been found in various organs of many mammals, including in lower animals. However, knowledge of TCs in lower animals is still very limited. This study identified TCs and their Tps in the ileum of the Chinese giant salamander, Andrias davidianus (Amphibia: Caudata), by transmission electron microscopy. The TCs/Tps were found near epithelial cells, glandular cells and unmyelinated nerve fibres. Moreover, exosomes were also found to be present in between TCs/Tps and these cells.