Genomic Data Reveal Conserved Female Heterogamety in Giant Salamanders with Gigantic Nuclear Genomes

A grant from the One-University Open Access Fund at the University of Kansas was used to defray the author's publication fees in this Open Access journal. The Open Access Fund, administered by librarians from the KU, KU Law, and KUMC libraries, is made possible by contributions from the offices of KU Provost, KU Vice Chancellor for Research & Graduate Studies, and KUMC Vice Chancellor for Research. For more information about the Open Access Fund, please see http://library.kumc.edu/authors-fund.xml.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.Cryptobranchid Interest GroupFresno Chaffee ZooSaint Louis Zoo WildCare InstituteUniversity of Kentucky G.F. Ribble FundNational Science Foundation (DEB-0949532 to D.W.W. and DGE-3048109801 to P.M.H.

Quantitative Behavioral Analysis of First Successful Captive Breeding of Endangered Ozark Hellbenders

Understanding behaviors associated with reproductive events is vital to management of captive breeding programs for threatened and endangered species. The Ozark hellbender (Cryptobranchus alleganiensis bishopi) is a federally endangered aquatic salamander with only one successful captive breeding program (the Saint Louis Zoo's Ron Goellner Center for Hellbender Conservation). Although anecdotal observations have been reported for hellbender reproductive behavior from field observations, no quantitative assessments have been made. We quantified hellbender behavior from video-recordings of three successful breeding events at the Saint Louis Zoo that occurred in 2012, including aggressive, sexual, social, and locomotory behaviors. We used transition matrices to organize these data into kinematic diagrams that illustrated behavioral sequences for five time periods: pre-oviposition (2 nights), first oviposition night, inter-oviposition night, second oviposition night, and post-oviposition. General activity and agonistic behaviors increased moderately through the first oviposition night, peaked during inter-oviposition, and declined abruptly following the second oviposition night. Agonistic behavior included bites, charges, chases, and flight. Female-female aggression was common. Surfacing (presumably for accessory air breathing) followed intense activity. Presumed courtship behaviors (tail swishing and circling) occurred at low rates. During oviposition, females remained in the nest box for 1–2+ h. We encourage managers of captive breeding programs to use quantitative behavioral analyses to pin-point critical time periods and conditions for successful reproduction

Evaluation of Microorganisms Cultured from Injured and Repressed Tissue Regeneration Sites in Endangered Giant Aquatic Ozark Hellbender Salamanders

Investigation into the causes underlying the rapid, global amphibian decline provides critical insight into the effects of changing ecosystems. Hypothesized and confirmed links between amphibian declines, disease, and environmental changes are increasingly represented in published literature. However, there are few long-term amphibian studies that include data on population size, abnormality/injury rates, disease, and habitat variables to adequately assess changes through time. We cultured and identified microorganisms isolated from abnormal/injured and repressed tissue regeneration sites of the endangered Ozark Hellbender, Cryptobranchus alleganiensis bishopi, to discover potential causative agents responsible for their significant decline in health and population. This organism and our study site were chosen because the population and habitat of C. a. bishopi have been intensively studied from 1969–2009, and the abnormality/injury rate and apparent lack of regeneration were established. Although many bacterial and fungal isolates recovered were common environmental organisms, several opportunistic pathogens were identified in association with only the injured tissues of C.a. bishopi. Bacterial isolates included Aeromonas hydrophila, a known amphibian pathogen, Granulicetella adiacens, Gordonai terrae, Stenotrophomonas maltophilia, Aerococcus viridans, Streptococcus pneumoniae and a variety of Pseudomonads, including Pseudomonas aeruginosa, P. stutzeri, and P. alcaligenes. Fungal isolates included species in the genera Penicillium, Acremonium, Cladosporium, Curvularia, Fusarium, Streptomycetes, and the Class Hyphomycetes. Many of the opportunistic pathogens identified are known to form biofilms. Lack of isolation of the same organism from all wounds suggests that the etiological agent responsible for the damage to C. a. bishopi may not be a single organism. To our knowledge, this is the first study to profile the external microbial consortia cultured from a Cryptobranchid salamander. The incidence of abnormalities/injury and retarded regeneration in C. a. bishopi may have many contributing factors including disease and habitat degradation. Results from this study may provide insight into other amphibian population declines

Influence of immunogenetics, sex and body condition on the cutaneous microbial communities of two giant salamanders.

The complex association between hosts and microbial symbionts requires the implementation of multiple approaches to evaluate variation in host physiology. Within amphibians, heterogeneity in immunogenetic traits and cutaneous microbiota is associated with variation in disease resistance. Ozark (Cryptobranchus alleganiensis bishopi) and eastern hellbenders (C. a. alleganiensis) provide a model system to assess variation in host traits and microbial communities. Ozark hellbenders have experienced declines throughout their range, are federally endangered and experience wound retardation that is absent in the eastern subspecies. Previous microbial investigations indicate differentiation in the composition of the skin microbiota of both hellbender subspecies, but it is not clear whether these patterns are concurrent with diversity in the major histocompatibility complex (MHC) genes. We characterized the MHC IIB and the skin microbiota of hellbenders in Missouri, where both subspecies co-occur though not sympatric. We compared the microbiota composition and MHC diversity between both subspecies and investigated whether individual-level MHC diversity, sex and body condition were associated with microbiota composition. Overall, MHC IIB diversity was lower in Ozark hellbenders compared to the eastern subspecies. Multivariate statistical comparisons identified microbiota differentiation between Ozark and eastern hellbenders. MHC IIB allele presence/absence, allele divergence, body composition and sex defined grouping of hellbender microbiotas within populations. Differentiation of the cutaneous microbiotas and MHC IIB genes between eastern and Ozark hellbenders suggests that differences exist in immunity between the two subspecies. This study demonstrates how simultaneous assessments of host genetic traits and microbiotas can inform patterns of microbial community structure in natural systems

Abundance and harvest proportion of river turtles in Missouri

Freshwater turtle populations are declining worldwide, yet managers have little information about the effects of commercial turtle harvests. In Missouri, the snapping turtle (Chelydra serpentina), smooth softshell (Apalone mutica), and spiny softshell (Apalone spinifera) are harvested commercially in the Missouri River. In 2011 and 2012, we conducted mark–recapture of these species to estimate abundance on the Missouri River and two unharvested tributaries, the Osage and Gasconade Rivers. We conducted mock harvests, applying capture methods of the state's primary commercial harvester, to estimate plausible expected harvest proportions. Snapping turtle abundance per 2 km was lower at harvested units ( = 15; SE = 7.1; unharvested: = 90; SE = 40.3). Smooth softshell abundance was greater at harvested units ( = 59; SE = 7.9; unharvested: = 14; SE = 28.1), although the difference was not significant. Mean unique spiny softshell captures were similar at harvested ( = 18; SE = 4.3) and unharvested ( = 17; SE = 9.7) units. Expected harvest proportions averaged 23% across species (SE = 5%; range = 6%–79%), exceeding sustainable rates reported for turtles. Our results suggest that on a small scale, using these methods, harvesters can remove a substantial portion of river turtle populations

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Characterization of the Cutaneous Bacterial Communities of Two Giant Salamander Subspecies

Pathogens currently threaten the existence of many amphibian species. In efforts to combat global declines, researchers have characterized the amphibian cutaneous microbiome as a resource for disease management. Characterization of microbial communities has become useful in studying the links between organismal health and the host microbiome. Hellbender salamanders (Cryptobranchus alleganiensis) provide an ideal system to explore the cutaneous microbiome as this species requires extensive conservation management across its range. In addition, the Ozark hellbender subspecies (Cryptobranchus alleganiensis bishopi) exhibits chronic wounds hypothesized to be caused by bacterial infections, whereas the eastern hellbender (Cryptobranchus alleganiensis alleganiensis) does not. We assessed the cutaneous bacterial microbiome of both subspecies at two locations in the state of Missouri, USA. Through 16S rRNA gene-based amplicon sequencing, we detected more than 1000 distinct operational taxonomic units (OTUs) in the cutaneous and environmental bacterial microbiome. Phylogenetic and abundance-based dissimilarity matrices identified differences in the bacterial communities between the two subspecies, but only the abundance-based dissimilarity matrix identified differences between wounds and healthy skin on Ozark hellbenders. The higher abundance of OTUs on Ozark wounds suggests that commensal bacteria present on the skin and environment may be opportunistically colonizing the wounds. This brief exploration of the hellbender cutaneous bacterial microbiome provides foundational support for future studies seeking to understand the hellbender cutaneous bacterial microbiome and the role of the bacterial microbiota on chronic wounds of Ozark hellbenders

Genetic relationships of hellbenders in the Ozark highlands of Missouri and conservation implications for the Ozark subspecies (Cryptobranchus alleganiensis bishopi)

Abstract The hellbender (Cryptobranchus alleganiensis) is an obligately aquatic salamander that is in decline due to habitat loss and disease. Two subspecies of hellbender have been described based on morphological characteristics: C. a. alleganiensis (eastern subspecies) and C. a. bishopi (Ozark hellbender). Current conservation strategies include captive propagation for restorative releases even though information regarding the current levels of genetic variability and structure within populations is not sufficient to effectively plan for conservation of the genetic diversity of the species. To investigate patterns of population structure in the hellbender, we genotyped 276 hellbenders from eight Missouri River drainages, representing both subspecies. Our results showed low levels of within-drainage diversity but strong population structure among rivers, and three distinct genetic clusters. F ST values ranged from 0.00 to 0.61 and averaged 0.40. Our results confirmed previous reports that C. a. bishopi and C. a. alleganiensis are genetically distinct, but also revealed an equidistant relationship between two groups within C. a. bishopi and all populations of C. a. alleganiensis. Current subspecies delineations do not accurately incorporate genetic structure, and for conservation purposes, these three groups should be considered evolutionarily significant units

Heavy Metals, Hematology, Plasma Chemistry, and Parasites in Adult Hellbenders (Cryptobranchus Alleganiensis)

Ozark (Cryptobranchus alleganiensis bishopi) and eastern hellbenders (C. a. alleganiensis) from seven rivers in Missouri, USA, were collected to investigate essential information on hematology, parasites, and plasma chemistry and levels of select heavy metals (Hg, Pb, Cd, Cr, and Co) in the animals\u27 blood. The body masses of Ozark hellbenders were much smaller than those of eastern hellbenders. Blood parasites were detected in Ozark hellbenders, but not eastern hellbenders. The higher frequency in occurrence of eosinophils in Ozark hellbenders (8.8-16.8%) than in eastern hellbenders (highest at 6.6%) might result from the infection of parasites. Seven of the 18 hematology and plasma parameters (hematocrit, basophils, eosinophils, K, P, Ca, and chloride) showed significant differences between subspecies. The blood levels of heavy metals Co, Hg, and Pb differed significantly between subspecies. Ozark hellbenders had higher blood levels of Co (p \u3c 0.001), while blood levels of Hg and Pb were higher in eastern hellbenders. The levels of chromium (Cr) and cadmium (Cd) were not different between subspecies and among rivers. The eastern hellbenders at Niangua River and the Ozark hellbenders at the North Fork of the White River had lower Hg levels compared to eastern and Ozark hellbenders at other sites. All together, our findings provide important baseline information for managing this endangered species