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Cutaneous bacteria may play an important role in the resistance of amphibians to the pathogenic fungus Batrachochytrium dendrobatidis (Bd). Microbial communities resident on hosts’ skin show topographical diversity mapping to skin features, as demonstrated by studies of the human microbiome. We examined skin microbiomes of wild and captive fire-bellied toads (Bombina orientalis) for differences across their body surface. We found that bacterial communities differed between ventral and dorsal skin. Wild toads showed slightly higher bacterial richness and diversity in the dorsal compared to the ventral region. On the other hand, captive toads hosted a higher richness and diversity of bacteria on their ventral than their dorsal skin. Microbial community composition and relative abundance of major bacterial taxonomic groups also differed between ventral and dorsal skin in all populations. Furthermore, microbiome diversity patterns varied as a function of their Bd infection status in wild toads. Bacterial richness and diversity was greater, and microbial community structure more complex, in wild than captive toads. The results suggest that bacterial community structure is influenced by microhabitats associated with skin regions. These local communities may be differentially modified when interacting with environmental bacteria and Bd. A better understanding of microbiome variation across skin regions will be needed to assess how the skin microbiota affects the abilities of amphibian hosts to resist Bd infection, especially in captive breeding programs.

Little is known of how fungal distribution ranges vary with elevation. We studied fungal diversity and community composition from 740 to 2940 m above sea level on Mt. Norikura, Japan, sequencing the ITS2 region. There was a clear trend, repeated across each of the fungal phyla (Basidiomycota, Ascomycota, Zygomycota, Chytridomycota and Glomeromycota), and across the whole fungal community combined, towards an increased elevational range of higher elevation OTUs, conforming to the elevational Rapoport pattern. It appears that fungi from higher elevation environments are more generalized ecologically, at least in terms of climate-related gradients. These findings add to the picture from latitudinal studies of fungal ranges, which also suggest that the classic Rapoport Rule (broader ranges at higher latitudes) applies on a geographical scale. However, there was no mid-elevation maximum in diversity in any of the phyla studied, and different diversity trends for the different phyla, when different diversity indices were used. In terms of functional guilds, on Norikura there were trends towards increased saprotrophism (Zygomycota), symbiotrophism (Basidiomycota), symbiotrophism and saprotrophism (Ascomycota) and pathotrophism (Chytridiomycota) with elevation. The causes of each of these trends require further investigation from an ecological and evolutionary viewpoint.

The pathogenic chytrid fungus, Batrachochytrium dendrobatidis (denoted Bd), causes large-scale epizootics in naïve amphibian populations. Intervention strategies to rapidly respond to Bd incursions require sensitive and accurate diagnostic methods. Chytridiomycosis usually is assessed by quantitative polymerase chain reaction (qPCR) amplification of amphibian skin swabs. Results based on this method, however, sometimes yield inconsistent results on infection status and inaccurate scores of infection intensity. In Asia and other regions where amphibians typically bear low Bd loads, swab results are least reliable. We developed a Bd-sampling method that collects zoospores released by infected subjects into an aquatic medium. Bd DNA is extracted by filters and amplified by nested PCR. Using laboratory colonies and field populations of Bombina orientalis, we compare results with those obtained on the same subjects by qPCR of DNA extracted from swabs. Many subjects, despite being diagnosed as Bd-negative by conventional methods, released Bd zoospores into collection containers and thus must be considered infected. Infection loads determined from filtered water were at least 1000 times higher than those estimated from swabs. Subjects significantly varied in infection load, as they intermittently released zoospores, over a 5-day period. Thus, the method might be used to compare the infectivity of individuals and study the periodicity of zoospore release. Sampling methods based on water filtration can dramatically increase the capacity to accurately diagnose chytridiomycosis and contribute to a better understanding of the interactions between Bd and its hosts.

Genes of the major histocompatibility complex (MHC) that underlie the adaptive immune system may allow vertebrates to recognize their kin. True kin-recognition genes should produce signalling products to which organisms can respond. Allelic variation in the peptide-binding region (PBR) of MHC molecules determines the pool of peptides that can be presented to trigger an immune response. To examine whether these MHC peptides also might underlie assessments of genetic similarity, we tested whether Xenopus laevis tadpoles socially discriminate between pairs of siblings with which they differed in PBR amino acid sequences. We found that tadpoles (four sibships, n = 854) associated preferentially with siblings with which they were more similar in PBR amino acid sequence. Moreover, the strength of their preference for a conspecific was directly proportional to the sequence similarity between them. Discrimination was graded, and correlated more closely with functional sequence differences encoded by MHC class I and class II alleles than with numbers of shared haplotypes. Our results thus suggest that haplotype analyses may fail to reveal fine-scale behavioural responses to divergence in functionally expressed sequences. We conclude that MHC–PBR gene products mediate quantitative social assessment of immunogenetic similarity that may facilitate kin recognition in vertebrates.

Abstract Proteins encoded by antigen-processing genes (APGs) provide major histocompatibility complex (MHC) class I (MHC-I) with antigenic peptides. In mammals, polymorphic multigenic MHC-I family is served by monomorphic APGs, whereas in certain nonmammalian species both MHC-I and APGs are polymorphic and coevolve within stable haplotypes. Coevolution was suggested as an ancestral gnathostome feature, presumably enabling only a single highly expressed classical MHC-I gene. In this view coevolution, while optimizing some aspects of adaptive immunity, would also limit its flexibility by preventing the expansion of classical MHC-I into a multigene family. However, some nonmammalian taxa, such as salamanders, have multiple highly expressed MHC-I genes, suggesting either that coevolution is relaxed or that it does not prevent the establishment of multigene MHC-I. To distinguish between these two alternatives, we use salamanders (30 species from 16 genera representing six families) to test, within a comparative framework, a major prediction of the coevolution hypothesis: the positive correlation between MHC-I and APG diversity. We found that MHC-I diversity explained both within-individual and species-wide diversity of two APGs, TAP1 and TAP2, supporting their coevolution with MHC-I, whereas no consistent effect was detected for the other three APGs (PSMB8, PSMB9, and TAPBP). Our results imply that although coevolution occurs in salamanders, it does not preclude the expansion of the MHC-I gene family. Contrary to the previous suggestions, nonmammalian vertebrates thus may be able to accommodate diverse selection pressures with flexibility granted by rapid expansion or contraction of the MHC-I family, while retaining the benefits of coevolution between MHC-I and TAPs.

Contrasting with birds and mammals, poikilothermic vertebrates often have homomorphic sex chromosomes, possibly resulting from high rates of sex-chromosome turnovers and/or occasional X–Y recombination. Strong support for the latter mechanism was provided by four species of European tree frogs, which inherited from a common ancestor (∼5 Ma) the same pair of homomorphic sex chromosomes (linkage group 1, LG1), harboring the candidate sex-determining gene Dmrt1. Here, we test sex linkage of LG1 across six additional species of the Eurasian Hyla radiation with divergence times ranging from 6 to 40 Ma. LG1 turns out to be sex linked in six of nine resolved cases. Mapping the patterns of sex linkage to the Hyla phylogeny reveals several transitions in sex-determination systems within the last 10 My, including one switch in heterogamety. Phylogenetic trees of DNA sequences along LG1 are consistent with occasional X–Y recombination in all species where LG1 is sex linked. These patterns argue against one of the main potential causes for turnovers, namely the accumulation of deleterious mutations on nonrecombining chromosomes. Sibship analyses show that LG1 recombination is strongly reduced in males from most species investigated, including some in which it is autosomal. Intrinsically low male recombination might facilitate the evolution of male heterogamety, and the presence of important genes from the sex-determination cascade might predispose LG1 to become a sex chromosome.

Many amphibian species around the world, except in Asia, suffer morbidity and mortality when infected by the emerging infectious pathogen Batrachochytrium dendrobatidis (Bd). A lineage of the amphibian chytrid fungus isolated from South Korean amphibians (BdAsia-1) is evolutionarily basal to recombinant global pandemic lineages (BdGPL) associated with worldwide amphibian population declines. In Asia, the Bd pathogen and its amphibian hosts have coevolved over 100 years or more. Thus, resilience of Asian amphibian populations to infection might result from attenuated virulence of endemic Bd lineages, evolved immunity to the pathogen or both. We compared susceptibilities of an Australasian amphibian, Litoria caerulea , known to lack resistance to BdGPL, with those of three Korean species, Bufo gargarizans , Bombina orientalis and Hyla japonica , after inoculation with BdAsia-1, BdGPL or a blank solution. Subjects became infected in all experimental treatments but Korean species rapidly cleared themselves of infection, regardless of Bd lineage. They survived with no apparent secondary effects. By contrast, L. caerulea , after infection by either BdAsia-1 or BdGPL, suffered deteriorating body condition and carried progressively higher Bd loads over time. Subsequently, most subjects died. Comparing their effects on L. caerulea , BdAsia-1 induced more rapid disease progression than BdGPL. The results suggest that genomic recombination with other lineages was not necessary for the ancestral Bd lineage to evolve hypervirulence over its long period of coevolution with amphibian hosts. The pathogen's virulence may have driven strong selection for immune responses in endemic Asian amphibian host species.

The pathogenic fungus Batrachochytrium dendrobatidis (Bd) is a major conservation concern because of its role in decimating amphibian populations worldwide. We used quantitative PCR to screen 244 museum specimens from the Korean Peninsula, collected between 1911 and 2004, for the presence of Bd to gain insight into its history in Asia. Three specimens of Rugosa emeljanovi (previously Rana or Glandirana rugosa), collected in 1911 from Wonsan, North Korea, tested positive for Bd. Histology of these positive specimens revealed mild hyperkeratosis -- a non-specific host response commonly found in Bd-infected frogs -- but no Bd zoospores or zoosporangia. Our results indicate that Bd was present in Korea more than 100 years ago, consistent with hypotheses suggesting that Korean amphibians may be infected by endemic Asian Bd strains.

Self/non-self recognition mechanisms underlie the development, immunology and social behaviour of virtually all living organisms, from bacteria to humans. Indeed, recognition processes lie at the core of how social cooperation evolved. Much evidence suggests that the major histocompatibility complex (MHC) both facilitates nepotistic interactions and promotes inbreeding avoidance. Social discrimination based on MHC differences has been demonstrated in many vertebrates but whether the labels used in discrimination are directly associated with the MHC, rather than with other genes with which it covaries, has remained problematic. Furthermore, effects of familiarity on natural preferences have not been controlled in most previous studies. Here we show that African clawed frog (Xenopus laevis) tadpoles discriminate among familiar full siblings based on MHC haplotype differences. Subjects (N=261) from four parental crosses preferred siblings with which they shared MHC haplotypes to those with no MHC haplotypes in common. Using only full siblings in experimental tests, we controlled for genetic variation elsewhere in the genome that might influence schooling preferences. As test subjects were equally familiar with stimulus groups, we conclude that tadpole discrimination involves a self-referent genetic recognition mechanism whereby individuals compare their own MHC type with those of conspecifics.

Chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), has been implicated in population declines and species extinctions of amphibians around the world. Susceptibility to the disease varies both within and among species, most likely attributable to heritable immunogenetic variation. Analyses of transcriptional expression in hosts following their infection by Bd reveal complex responses. Species resistant to Bd generally show evidence of stronger innate and adaptive immune system responses. Major histocompatibility complex (MHC) class I and class II genes of some susceptible species are up-regulated following host infection by Bd, but resistant species show no comparable changes in transcriptional expression. Bd-resistant species share similar pocket conformations within the MHC-II antigen-binding groove. Among susceptible species, survivors of epizootics bear alleles encoding these conformations. Individuals with homozygous resistance alleles appear to benefit by enhanced resistance, especially in environmental conditions that promote pathogen virulence. Subjects that are repeatedly infected and subsequently cleared of Bd can develop an acquired immune response to the pathogen. Strong directional selection for MHC alleles that encode resistance to Bd may deplete genetic variation necessary to respond to other pathogens. Resistance to chytridiomycosis incurs life-history costs that require further study.