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Global wildlife trade spreads emerging infectious diseases that threaten biodiversity. The amphibian chytrid pathogen Batrachochytrium dendrobatidis (Bd) has caused population declines and species extinctions worldwide except in Asia. Fire-bellied toads (Bombina orientalis), exported in large numbers from Asia, are tolerant of Bd and carry hypervirulent ancestral chytrid BdAsia-1 variants. We assayed the virulence of a new isolate of BdAsia-1 on the model Australasian frog host Litoria caerulea. Infected individuals (n = 15) all showed rapid disease progression culminating in death, whereas sham-inoculated individuals (n = 10) presented no clinical signs of disease and all survived (log rank test, χVariantes Patógenas Nuevas de Quitridios y el Mercado Mundial de Anfibios Mascota Resumen El mercado mundial de fauna dispersa enfermedades infecciosas emergentes que amenazan a la biodiversidad. El quitridio patógeno de anfibios Batrachochytrium dendrobatidis (Bd) ha causado declinaciones poblacionales y la extinción de especies en todo el mundo excepto Asia. El sapo Bombina orientalis, exportado en grandes cantidades desde Asia, es tolerante al Bd y carga genéticamente las variantes ancestrales hipervirulentas de quitridio BdAsia-1. Analizamos la virulencia de una nueva cepa de BdAsia-1 con el modelo de la rana australo-asiática hospedera Litoria caerulea. Todos los individuos infectados (n = 15) mostraron una progresión acelerada de la enfermedad que culminaba con la muerte, mientras que los individuos con inoculación simulada (n = 10) no presentaron señales clínicas de la enfermedad y todos sobrevivieron (prueba log de rango, χ全球野生动物贸易引起的新发传染病传播对生物多样性产生了威胁。两栖动物壶菌病原体蛙壶菌 Batrachochytrium dendrobatidis (Bd) 已导致全球除亚洲以外地区的两栖动物种群数量下降及物种灭绝。从亚洲大量出口的火腹蟾蜍 (Bombina orientalis) 对 Bd 有耐受性, 且携带高致病性的祖先型壶菌变体 BdAsia-1。本研究检测了一个新分离的 BdAsia-1菌株对澳大利西亚模式青蛙宿主澳洲绿树蛙 (Litoria caerulea) 的毒力。研究发现, 被感染的个体 (n=15) 均表现出快速的疾病进展且最终死亡, 而假接种的个体 (n=10) 则没有出现疾病临床症状且全部存活(时序检验, χ

Deinococcus is a genus of soil bacteria known for radiation resistance. However, the effects of radiation exposure on its community structure are unknown. We exposed soil to three levels of gamma radiation, 0.1 kGy/h (low), 1 kGy/h (medium), and 3 kGy/h (high), once a week for 6 weeks and then extracted soil DNA for 16S rRNA amplicon sequencing. We found the following: (1) Increasing radiation dose produced a major increase in relative abundance of Deinococcus, reaching ~ 80% of reads at the highest doses. Differing abundances of the various Deinococcus species in relation to exposure levels indicate distinct "radiation niches." At 3 kGy/h, a single OTU identified as D. ficus overwhelmingly dominated the mesocosms. (2) Corresponding published genome data show that the dominant species at 3 kGy/h, D. ficus, has a larger and more complex genome than other Deinococcus species with a greater proportion of genes related to DNA and nucleotide metabolism, cell wall, membrane, and envelope biogenesis as well as more cell cycle control, cell division, and chromosome partitioning-related genes. Deinococcus ficus also has a higher guanine-cytosine ratio than most other Deinococcus. These features may be linked to genome stability and may explain its greater abundance in this apparently competitive system, under high-radiation exposures. (3) Genomic analysis suggests that Deinococcus, including D. ficus, are capable of utilizing diverse carbon sources derived from both microbial cells killed by the radiation (including C5-C12-containing compounds, like arabinose, lactose, N-acetyl-D-glucosamine) and plant-derived organic matter in the soil (e.g., cellulose and hemicellulose). (4) Overall, based on its metagenome, even the most highly irradiated (3 kGy/h) soil possesses a wide range of the activities necessary for a functional soil system. Future studies may consider the resilience and sustainability of such soils in a high-radiation environment.

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.

Animal-associated microbiomes are integral to host health, yet key biotic and abiotic factors that shape host-associated microbial communities at the global scale remain poorly understood. We investigated global patterns in amphibian skin bacterial communities, incorporating samples from 2,349 individuals representing 205 amphibian species across a broad biogeographic range. We analysed how biotic and abiotic factors correlate with skin microbial communities using multiple statistical approaches. Global amphibian skin bacterial richness was consistently correlated with temperature-associated factors. We found more diverse skin microbiomes in environments with colder winters and less stable thermal conditions compared with environments with warm winters and less annual temperature variation. We used bioinformatically predicted bacterial growth rates, dormancy genes and antibiotic synthesis genes, as well as inferred bacterial thermal growth optima to propose mechanistic hypotheses that may explain the observed patterns. We conclude that temporal and spatial characteristics of the host’s macro-environment mediate microbial diversity. National Science Foundation/[DEB-1146284]/NSF/Estados Unidos National Science Foundation/[IOS-1121758]/NSF/Estados Unidos National Science Foundation/[DEB-1310036]/NSF/Estados Unidos John Templeton Foundation/[]/JTF/Estados Unidos Deutsche Forschungsgemeinschaft/[]/DFG/Alemania Deutsche Forschungsgemeinschaft/[VE247/9-1]/DFG/Alemania Coordenação de Aperfeiçoamento de Pessoal de Nível Superior/[]/CAPES/Brasil Fundação de Amparo à Pesquisa do Estado de São Paulo/[#2013/50741-7]/FAPESP/Brasil Conselho Nacional de Desenvolvimento Científico e Tecnológico/[]/CNPq/Brasil Simons Foundation/[429440, WTW]//Estados Unidos Deutscher Akademischer Austauschdienst/[]/DAAD/Alemania Universidad de Costa Rica/[801-B2-029]/UCR/Costa Rica Ministerio de Ciencia, Tecnología y Telecomunicaciones/[849-PINN-2015]/MICITT/Costa Rica National Research Foundation of Korea/[2015R1D1A1A01057282]/NRF/Corea del Sur UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigación en Biología Celular y Molecular (CIBCM) UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigación en Estructuras Microscópicas (CIEMIC)

Proteins encoded by antigen-processing genes (APGs) prepare antigens for presentation by the major histocompatibility complex class I (MHC I) molecules. Coevolution between APGs and MHC I genes has been proposed as the ancestral gnathostome condition. The hypothesis predicts a single highly expressed MHC I gene and tight linkage between APGs and MHC I. In addition, APGs should evolve under positive selection, a consequence of the adaptive evolution in MHC I. The presence of multiple highly expressed MHC I genes in some teleosts, birds, and urodeles appears incompatible with the coevolution hypothesis. Here, we use urodele amphibians to test two key expectations derived from the coevolution hypothesis: 1) the linkage between APGs and MHC I was studied in Lissotriton newts and 2) the evidence for adaptive evolution in APGs was assessed using 42 urodele species comprising 21 genera from seven families. We demonstrated that five APGs (PSMB8, PSMB9, TAP1, TAP2, and TAPBP) are tightly linked (

The increasing number of new fungal species described from all over the world along with the use of genetics to define taxa, has dramatically changed the classification system of early-diverging fungi over the past several decades. The number of phyla established for non-Dikarya fungi has increased from 2 to 17. However, to date, both the classification and phylogeny of the basal fungi are still unresolved. In this article, we review the recent taxonomy of the basal fungi and re-evaluate the relationships among early-diverging lineages of fungal phyla. We also provide information on the ecology and distribution in Mucoromycota and highlight the impact of chytrids on amphibian populations. Species concepts in Chytridiomycota, Aphelidiomycota, Rozellomycota, Neocallimastigomycota are discussed in this paper. To preserve the current application of the genus Nephridiophaga (Chytridiomycota: Nephridiophagales), a new type species, Nephridiophaga blattellae, is proposed.

Proteins encoded by antigen-processing genes (APGs) prepare antigens for presentation by the major histocompatibility complex class I (MHC I) molecules. Coevolution between APGs and MHC I genes has been proposed as the ancestral gnathostome condition. The hypothesis predicts a single highly expressed MHC I gene and tight linkage between APGs and MHC I. In addition, APGs should evolve under positive selection, a consequence of the adaptive evolution in MHC I. The presence of multiple highly expressed MHC I genes in some teleosts, birds, and urodeles appears incompatible with the coevolution hypothesis. Here, we use urodele amphibians to test two key expectations derived from the coevolution hypothesis: 1) the linkage between APGs and MHC I was studied in Lissotriton newts and 2) the evidence for adaptive evolution in APGs was assessed using 42 urodele species comprising 21 genera from seven families. We demonstrated that five APGs (PSMB8, PSMB9, TAP1, TAP2, and TAPBP) are tightly linked (

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., The work was supported by the Polish National Science Centre (Grant No. 2016/23/B/NZ8/00738) to W.B. D.J. was supported by PROTEUS project in Croatia; E.J. was supported by the University of Connecticut Research Foundation; M.Ma. was supported by a KAKENHI Grant-in-Aid for Young Scientists (B) (No. JP16K18613) from the Japan Society for the Promotion of Science; M.Mi. was supported by the Foundation for Polish Science (FNP) START stipend and the Polish National Science Centre Sonatina 3 (Grant No. 2019/32/C/NZ8/00440); J.V. was supported by Project No. KH130360 implemented with the support provided from the National Research, Development and Innovation Fund of Hungary, financed under the KH_18 funding scheme; B.Wa. was funded by the National Research Foundation of Korea (Ministry of Education, 2015R1D1A01057282); Ministry of Science, ICT, and Future Planning (2018R1A2B6006833).

We conduct a phylogeographic and population genetic study of the Asiatic Toad (Bufo gargarizans) to understand its evolutionary history, and the influence of geology and climate of the region. A total of 292 individuals from 94 locations were genotyped for two mitochondrial DNA loci (cytb, ND2 gene) and five nuclear introns (Sox9-2, Rho-3, CCNB2-3, UCH-2, DBI-2). We performed a suite of phylogenetic, population genetic, and divergence dating analyses. The phylogenetic trees constructed using mitochondrial loci inferred B. gargarizans being divided into two major groups: West (China mainland) and Northeast (Northeast China, Russia and Korean Peninsula). As with previous studies of this species, we recover population genetic structure not tied to geographic region. Additionally, we discover a new genetic clade restricted to Northeast Asia that points towards the Korean Peninsula being a glacial refugium during the Pleistocene. The weak phylogeographic pattern of B. gargarizans is likely the result of multiple biological, anthropogenic, and historical—robust dispersal abilities as a consequence of physiological adaptations, human translocation, geologic activity, and glacial cycles of the Pleistocene. We highlight the complex geologic and climatic history of Northeast Asia and encourage further research to understand its impact on the biodiversity in the region.

Globalized infectious diseases are causing species declines worldwide, but their source often remains elusive. We used whole-genome sequencing to solve the spatiotemporal origins of the most devastating panzootic to date, caused by the fungus Batrachochytrium dendrobatidis, a proximate driver of global amphibian declines. We traced the source of B. dendrobatidis to the Korean peninsula, where one lineage, BdASIA-1, exhibits the genetic hallmarks of an ancestral population that seeded the panzootic. We date the emergence of this pathogen to the early 20th century, coinciding with the global expansion of commercial trade in amphibians, and we show that intercontinental transmission is ongoing. Our findings point to East Asia as a geographic hotspot for B. dendrobatidis biodiversity and the original source of these lineages that now parasitize amphibians worldwide., S.J.O., T.W.J.G., L.Br., A.Lo., A.A.C., D.S.S., E.A.C., C.M., J.B., D.M.A., F.C., and M.C.F. were supported through NERC (standard grant NE/K014455/1). S.J.O. acknowledges a Microsoft Azure for Research Sponsorship (subscription ID: ab7cd695-49cf-4a83-910a-ef71603e708b). T.W.J.G., A.Lo., A.A.C., D.S.S., E.A.C., C.M., J.B., D.M.A., F.C., and M.C.F. were also supported by the EU BiodivERsA scheme (RACE, funded through NERC directed grant NE/G002193/1 and ANR08-Biodiversa-002-03) and NERC (standard grant NE/K012509/1). M.C.F., E.A.C., and C.M. acknowledge the Nouragues Travel Grant Program 2014. R.A.F. was supported by an MIT/Wellcome Trust Fellowship. T.W.J.G. was supported by the People’s Trust for Endangered Species and the Morris Animal Foundation (D12ZO002). J.M.G.S. and M.C.F. were supported by the Leverhulme Trust (RPG-2014-273) and the Morris Animal Foundation (D16ZO-022). F.B. was supported by the ERC (grant ERC 260801–Big_Idea). D.M.A. was funded by Wellcome Trust grant 099202. J.V. was supported by the Hungarian Scientific Research Fund (OTKA K77841) and Bolyai János Research Scholarship, Hungarian Academy of Sciences (BO/00579/14/8). D.J.G. was supported by the Conservation Leadership Programme (grant 0134010) with additional assistance from F. Gebresenbet, R. Kassahun, and S. P. Loader. C.S.-A. was supported by Fondecyt Nº11140902 and 1181758. T.M.D.-B. was supported by the Royal Geographical Society and the Royal Zoological Society of Scotland with assistance from M. Hirschfeld and the Budongo Conservation Field Station. B.W. was supported by the National Research Foundation of Korea (2015R1D1A1A01057282). L.F.T. was supported by FAPESP (#2016/25358-3) and CNPq (#300896/2016-6). L.Be., L.F.S., and R.J.W. were supported by the Australian Research Council (FT100100375, DP120100811). A.A.C. was supported by a Royal Society Wolfson Research Merit award. J.H., A.La., and S.M. were funded by the Swedish Research Council Formas (grant no. 2013- 1389-26445-20). C.W. was funded by the National Research Foundation, South Africa. T.Y.J. and T.S.J. acknowledge NSF grant DEB-1601259. W.E.H. was funded by the NSERC Strategic and Discovery grant programs

Bird assemblages are sensitive to changes in landscape composition and the environment, such as those that result from drought. In this study, the relationship between landscape composition and avian functional diversity in traditional agricultural ecosystems in the Civilian Control Zone (CCZ) of Korea was examined. In addition, the resilience of biodiversity to changes in landscape elements resulting from drought conditions was investigated. The traditional agricultural landscape (TAL) of the sites studied was divided into three types: TAL 1 had a high proportion of rice paddies, TAL 2 included large forest areas, and TAL 3 represented areas with drylands. Of these, TAL 1 showed the highest species richness and functional richness, but these measures were most vulnerable to drought. Meanwhile, TAL 2 showed that the bird communities were more tolerant under drought event. This study shows that to conserve and enhance the diversity of birds in traditional agricultural landscapes of Northeast Asia, active management of forest areas is needed to protect bird populations. In addition, commercial pressures to develop this area will require urgent biodiversity conservation plans to protect the unique biodiversity of the Korean CCZ. This study thus provides landscape management guidance for conservation planning., The species richness (SR) and functional diversity (FD) of birds are affected by traditional landscape practices in the vicinity of the Demilitarized Zone (DMZ). SR and FD are greatest around rice paddies when precipitation patterns are normal, but species assemblages lack resilience to drought. Where forests are abundant, SR and FD little change, even as rice paddies dry in drought conditions. As a result, forests play an important role in the traditional agricultural landscape (TAL), and the maintenance of forests in the TAL is key to maintaining species diversity.

To assess the remediation capacity of a leachate channel, we monitored basic environmental parameters such as bathymetry, leachate, and soil characteristics and vegetation coverage. Based on our results, we designed a series of experiments to determine the most suitable remediating plant species for sustainable wastewater treatment. We found that adaptability to water depth may be a prime driver of reduced remediation capacity. Large portions of the leachate channel were deeper than the maximum tolerance range of many candidate emergent macrophytes, resulting in only 16% total vegetation coverage. Among tested species, Typha angustifolia showed the most promising potential for remediation, reaching the highest aboveground biomass (3300 g/m2) and demonstrating maximum concentrations in tissues (34,600 mg/kg of Na, 4013 mg/kg of Mg, 904 mg/kg of P, 639 mg/kg of Mn, 191 mg/kg of Fe and 62 mg/kg of Zn) when grown in leachate filled tank for six months. Typha angustifolia also showed greater tolerance of water depth than Phragmites australis, which previously was planted in leachate channels. Thus, T. angustifolia should be more suitable for the actual water depth of the channel. Additional planting of T. angustifolia will improve the vegetation coverage, the total remediation capacity and sustainability of the leachate channel. Considering water depths of target wetlands when selecting remediation plant will improve remediation ability and sustainability of remediation wetlands.

Amphibians, as the first terrestrial vertebrates, offer a window into early major histocompatibility complex (MHC) evolution. We characterized the MHC class I of two Korean amphibians, the Asiatic toad (Bufo gargarizans) and the Japanese tree frog (Hyla japonica). We found at least four transcribed MHC class I (MHC I) loci, the highest number confirmed in any anuran to date. Furthermore, we identified MHC I transcripts in terrestrial adults, and possibly in aquatic larvae, of both species. We conducted a phylogenetic analysis based on MHC I sequence data and found that B. gargarizans and H. japonica cluster together in the superfamily Nobleobatrachia. We further identified three supertypes shared by the two species. Our results reveal substantial variation in the number of MHC I loci in anurans and suggest that certain supertypes have particular physiochemical properties that may confer pathogen resistance.

Many amphibian species around the world, except in Asia, suffer morbidity and mortality when infected by the emerging infectious pathogenBatrachochytrium 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 orientalisandHyla 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 onL. 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.

Little is known of the effects of ionizing radiation exposure on soil biota. We exposed soil microcosms to weekly bursts of 60Co gamma radiation over six weeks, at three levels of exposure (0.1 kGy/hr/wk [low], 1 kGy/hr/wk [medium] and 3 kGy/hr/wk [high]). Soil DNA was extracted, and shotgun metagenomes were sequenced and characterised using MG-RAST. We hypothesized that with increasing radiation exposure there would be a decrease in both taxonomic and functional diversity. While bacterial diversity decreased, diversity of fungi and algae unexpectedly increased, perhaps because of release from competition. Despite the decrease in diversity of bacteria and of biota overall, functional gene diversity of algae, bacteria, fungi and total biota increased. Cycles of radiation exposure may increase the range of gene functional strategies viable in soil, a novel ecological example of the effects of stressors or disturbance events promoting some aspects of diversity. Moreover, repeated density-independent population crashes followed by population expansion may allow lottery effects, promoting coexistence. Radiation exposure produced large overall changes in community composition. Our study suggests several potential novel radiation-tolerant groups: in addition to Deinococcus-Thermus, which reached up to 20% relative abundance in the metagenome, the phyla Chloroflexi (bacteria), Chytridiomycota (fungi) and Nanoarcheota (archaea) may be considered as radiation-tolerant.

Parasitic chytrid fungi have emerged as a significant threat to amphibian species worldwide, necessitating the development of techniques to isolate these pathogens into sterile culture for research purposes. However, early methods of isolating chytrids from their hosts relied on killing amphibians. We modified a pre-existing protocol for isolating chytrids from infected animals to use toe clips and biopsies from toe webbing rather than euthanizing hosts, and distributed the protocol to interested researchers worldwide as part of the BiodivERsA project RACE – here called the RML protocol. In tandem, we developed a lethal procedure for isolating chytrids from tadpole mouthparts. Reviewing a database of use a decade after their inception, we find that these methods have been widely applied across at least 5 continents, 23 countries and in 62 amphibian species, and have been successfully used to isolate chytrids in remote field locations. Isolation of chytrids by the non-lethal RML protocol occured in 18% of attempts with 207 fungal isolates and three species of chytrid being recovered. Isolation of chytrids from tadpoles occured in 43% of attempts with 334 fungal isolates of one species (Batrachochytrium dendrobatidis) being recovered. Together, these methods have resulted in a significant reduction and refinement of our use of threatened amphibian species and have improved our ability to work with this important group of emerging fungal pathogens.

International audience; In organisms with complex life histories, dramatic changes in microbial community structure may occur with host development and immune system maturation. Amphibian host susceptibility to diseases such as chytridiomycosis may be affected by the reorganization of skin microbial community structure that occurs during metamorphosis. We tracked changes in the bacterial communities inhabiting skin of Korean fire-bellied toads (Bombina orientalis) that we infected as tadpoles with different strains of Batrachochytrium dendrobatidis (Bd), the pathogenic fungus that causes chytridiomycosis. We found that B. orientalis undergoes a major change in skin bacterial community composition between 5 and 15 days following metamorphosis. Richness indices and phylogenetic diversity measures began to diverge earlier, between aquatic and terrestrial stages. Our results further reveal differences in skin bacterial community composition among infection groups, suggesting that the effect of Bd infection on skin microbiome composition may differ by Bd strain. Additional studies are needed to further investigate the structural and temporal dynamics of microbiome shifts during metamorphosis in wild and captive amphibian populations. Analyses of the ontogeny of microbiome shifts may contribute to an understanding of why amphibians vary in their susceptibility to chytridiomycosis.

Some larval amphibians can develop into two distinct morphological types: a small-headed “normal” morph and a rarer broad-headed morph with specialized adaptations to feed on the normal morph. Cannibalism confers nutritional benefits that accelerate development, essential for survival in transient environments, but incurs potential inclusive fitness costs. Selective cannibalism of non-kin thus should be favored. However, subjects may be more vulnerable to contracting disease from non-siblings to which they lack immunological defenses. We investigated kin discrimination and pathogen transmission among Korean salamander (Hynobius leechii) larvae. We placed broad-headed morph larvae into a circular arena together with two normal morph larvae, one their sibling and the other a non-sibling. To test for kin discrimination, we recorded all behavioral interactions among them. To study pathogen transmission, we fed broad-headed larvae either a sibling or non-sibling normal larva that we previously had infected with the bacterium Aeromonas hydrophila. Two days after ingestion, we determined whether the bacterium had systemically infected cannibals by quantitative PCR (qPCR). Broad-headed larvae discriminated between sibling and non-sibling normal larvae, directing aggressive behaviors mostly toward siblings. Infection loads varied more widely among broad-headed larvae that cannibalized non-siblings than those that cannibalized siblings, but the highest infection loads were recorded after ingestion of non-siblings. Cannibalizing non-siblings thus may increase the risk of contracting disease. Broad-headed larvae discriminate most strongly between siblings and non-siblings late in development, when inclusive fitness costs of cannibalizing relatives diminish and vulnerability to novel pathogens transmitted by non-relatives rises.

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.

Suppressant foams used to fight forest fires may leave residual effects on surviving biota that managers need to consider prior to using them. We examined how three fire-suppressant foams (FSFs) (Forexpan S, Phos-Chek-WD881, and Silv-ex) affected seed germination and physiological responses of three plant species. Exposure to FSFs, whether in diluted concentrations or those typical in the field, reduced final germination percentages of seeds grown in petri dishes and within growth chambers. However, the FSFs did not cause total germination failure in any treatment. Inhibition of germination increased with longer exposure times, but only to diluted FSF solutions. Unlike in the laboratory experiments, none of the three FSFs affected seedling emergence when tested in field conditions. Further, we found no evidence of long-term phytotoxic effects on antioxidant enzyme activity nor chlorophyll content of the plant saplings. Therefore, although the three FSFs showed evidence of phytotoxicity to plants in laboratory tests, their actual impact on terrestrial ecosystems may be minimal. We suggest that the benefits of using these FSFs to protect plants in threatened forest ecosystems outweigh their minor risks.

Green roofs recently have garnered much attention as a means to reduce both the absorption of solar energy in summer and heat loss in winter, especially in urban areas with limited space for gardening. Constructed wetland roofs maintain more stable temperature profiles than terrestrial systems because of their slow heat transfer and high heat storage capacity. We found that wetland roofs were particularly efficient at decreasing the temperature of green roof systems on hot days. Wetland plants have high evaporation rates that are associated with their ability to cool buildings. Constructed wetland had excellent water holding ability, requiring less than 400 l water/m2 of irrigation over the entire growing season, which was less than 20% of the expected irrigation requirement for terrestrial systems on green rooftops. Wetland macrophyte species demonstrated high tolerance to flooding and drought and showed great potential for regeneration by rhizomes, suggesting easy maintenance. Plants grown in the constructed wetland accumulated high biomass that can serve as a carbon sink. Wetlands on rooftops would not exceed the weight-bearing capacity of rooftops if water depths are designed and kept under 30 cm. Constructed-wetland roofs offer thermal benefits, a low amount of required irrigation, high tolerance of drought and flood, and flood-control capacities. They also can act as a carbon sink, are easy to manage, and provide other ecological services. Therefore, constructed wetlands are a reasonable choice for green rooftop systems.

Engineered nanoparticles (NPs), increasingly used in industry, enter and migrate through biological ecosystems. NPs may create some acute toxicity, but their overall effects on living organisms remain largely unknown. In particular, the behavior of NPs in natural conditions and their consequent ecological effects are still poorly understood. In this study, we developed methods to test the phytotoxicity of two distinctly different NPs, one aerosol (nano-TiO2), and the other colloidal silver (AgNP), by specifically considering their tendencies to agglomerate and form precipitates. First we examined effects of these NPs on germination and root elongation. While exposure to neither of these NPs resulted in acute toxicity on germination, silver NPs caused significantly decreased root elongation at every concentration we tested. We found that the hydrodynamic diameters of AgNPs were much smaller than those of nano-TiO2, which induced higher uptake and phytotoxicity. Based on the agglomeration behavior of the NPs, greenhouse trials were run using commercial soil, for nano-TiO2, and Hoagland's solution, for AgNP. Phytotoxicity of silver NPs in the mature plants was demonstrated by lower chlorophyll contents, higher superoxide dismutase activity and less fruit productivity, while nano-TiO2 resulted in higher superoxide dismutase activity at the highest concentration (5000mg/kg). Both nano-TiO2 and AgNPs were taken up into plant stems, leaves and fruits. Our results suggest that further studies of the ecological effects of nanoparticles and steps to mitigate appropriate management strategies are required.

Population declines and extinctions of amphibians have been attributed to the chytrid fungus Batrachochytrium dendrobatidis (Bd), especially one globally emerging recombinant lineage ('Bd-GPL'). We used PCR assays that target the ribosomal internal transcribed spacer region (ITS) of Bd to determine the prevalence and genetic diversity of Bd in South Korea, where Bd is widely distributed but is not known to cause morbidity or mortality in wild populations. We isolated Korean Bd strains from native amphibians with low infection loads and compared them to known worldwide Bd strains using 19 polymorphic SNP and microsatellite loci. Bd prevalence ranged between 12.5 and 48.0%, in 11 of 17 native Korean species, and 24.7% in the introduced bullfrog Lithobates catesbeianus. Based on ITS sequence variation, 47 of the 50 identified Korean haplotypes formed a group closely associated with a native Brazilian Bd lineage, separated from the Bd-GPL lineage. However, multilocus genotyping of three Korean Bd isolates revealed strong divergence from both Bd-GPL and the native Brazilian Bd lineages. Thus, the ITS region resolves genotypes that diverge from Bd-GPL but otherwise generates ambiguous phylogenies. Our results point to the presence of highly diversified endemic strains of Bd across Asian amphibian species. The rarity of Bd-GPL-associated haplotypes suggests that either this lineage was introduced into Korea only recently or Bd-GPL has been outcompeted by native Bd strains. Our results highlight the need to consider possible complex interactions among native Bd lineages, Bd-GPL and their associated amphibian hosts when assessing the spread and impact of Bd-GPL on worldwide amphibian populations.

We examined the potential of Azolla japonica as a remediating plant for leachate channels and post-accumulation use as fertilizer for landfill slope. The harvested biomass of Azolla after one month grown in leachate was 254% that of the initial biomass and the predicted annual harvestable biomass of Azolla using a growth model was 32 times that of the initial biomass. Na, Fe, Mn, Mg, and P were accumulated in Azolla at very high concentrations. Such rapid increase of biomass and high accumulation rates suggest that this plant could be an excellent remediating plant. The post-harvest use of Azolla as compost was studied for the management and use of phytoaccumulating Azolla. Metal contents of Azolla compost were below permissible limits for co-composting material. Nitrogen, organic matter, P, and Mg content of the Azolla compost improved the soil condition of the landfill and enhanced ecophysiological responses of the plants. The application of Azolla compost can improve management of sanitary landfills, including the restoration of vegetation. Considering its ease of harvesting, high accumulation rates, harvestable biomass and suitability for composting, Azolla can provide a suitable solution for sustainable management of leachate channels and landfill slopes.

The most archaic lineages of frogs, represented by the New Zealand family Leiopelmatidae, communicate by chemical rather than bioacoustic signals. We analyzed how chemical signatures mediate social recognition in Hamilton’s frog, Leiopelma hamiltoni. In the wild, Hamilton’s frogs live 40 years or more, rarely moving outside small (

Pathogen-driven selection can favour major histocompatibility complex (MHC) alleles that confer immunological resistance to specific diseases. However, strong directional selection should deplete genetic variation necessary for robust immune function in the absence of balancing selection or challenges presented by other pathogens. We examined selection dynamics at one MHC class II (MHC-II) locus across Panamanian populations of the túngara frog, Physalaemus pustulosus , infected by the amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd). We compared MHC-II diversity in highland túngara frog populations, where amphibian communities have experienced declines owing to Bd, with those in the lowland region that have shown no evidence of decline. Highland region frogs had MHC variants that confer resistance to Bd. Variant fixation appeared to occur by directional selection rather than inbreeding, as overall genetic variation persisted in populations. In Bd-infected lowland sites, however, selective advantage may accrue to individuals with only one Bd-resistance allele, which were more frequent. Environmental conditions in lowlands should be less favourable for Bd infection, which may reduce selection for specific Bd resistance in hosts. Our results suggest that MHC selection dynamics fluctuate in túngara frog populations as a function of the favourability of habitat to pathogen spread and the vulnerability of hosts to infection.

Chytridiomycosis, a disease caused by the fungus Batrachochytrium dendrobatidis (Bd), potentially disrupts osmoregulation or respiration across the skin of amphibians it infects, releases toxins into the host, or both. We investigated whether infection with Bd alters water balance or metabolic rate of the hylid frog Litoria raniformis. Frogs were held in laboratory conditions simulating those in which Bd epizootics had been observed in the field. We inoculated six frogs with infective Bd zoospores, held the subjects in individual containers, and compared their course of infection and associated physiological measures with those of six controls. Experimental subjects exhibited clinical signs of chytridiomycosis during the early period of infection, one week after they were inoculated, possibly due to invasion of Bd into the skin. These clinical signs were accompanied by significant inhibition of rehydration through the skin. However, we detected no changes in metabolic rate attributable to chytridiomycosis after one week. Five months after inoculation, all but one of the infected subjects had survived. Molecular testing confirmed that surviving frogs, although aclinical, still were infected. Control and infected subjects showed no difference in water balance or metabolism. These results provide evidence of inhibited rehydration in individuals exhibiting clinical signs of chytridiomycosis. However, aclinical chytridiomycosis does not severely affect amphibian skin function. Frogs that survive infection by Bd, even if they remain infected, may suffer no significant impairment in their physiological responses. The disease progression, with initial clinical signs of chytridiomycosis followed by apparent full recovery, is consistent with an adaptive immune response to Bd infection. Further research is needed to determine how Bd causes clinical chytridiomycosis and the immunological mechanisms by which hosts respond to Bd.

Japanese quail (Coturnix coturnix japonica) have finely-tuned kin-recognition abilities that may serve a role in mate choice. To investigate whether these abilities are expressed in other life stages, we examined patterns of association among quail chicks in the laboratory four days after hatching. Hatchlings were reared either in mixed-family groups, consisting of siblings and nonsiblings, or in pure-family groups, consisting just of siblings. Chicks were highly social in testing sessions, as reflected in their clumped spatial distributions, and interacted more with their siblings than with nonsiblings. Siblings in each of four mixed-family groups preferentially associated with one another over nonsiblings. No discrimination was evident among differently marked siblings in four purefamily groups. Thus, at an early age, quail discriminate between siblings and nonsiblings despite social experience of both. Recognition abilities that develop prior to or soon after hatching may facilitate cooperation among chicks, may enable siblings to track each others' traits as they mature, or both.

To clarify the mechanism by which tadpoles of the American toad (Bufo americanus) recognize siblings, tadpoles were reared from eggs either (1) with siblings only, (2) in isolation from conspecifics, or (3) exposed to both siblings and non-siblings. Tadpoles reared with siblings and those reared in isolation preferentially associated with siblings in a laboratory test pool. However, tadpoles that were continuously exposed to siblings and non-siblings showed no significant sibling association unless they had been reared in sibling groups during early development. Tadpoles reared in isolation discriminated paternal — but not maternal — half-siblings from full-siblings. Although sibling preferences can develop in the absence of post-embryonic interactions with conspecifics, early experience appears to be important in the normal ontogeny of sibling recognition. Possibly the mother contributes some factor to her offspring that enables them to recognize each other as siblings. Zusammenfassung Kaulquappen der amerikanischen Krote (Bufo americanus) wurden unter verschiedenen Bedingungen gehalten und dann im Labor auf ihre Fahigkeit getestet, Vollgeschwister von Halbgeschwistern und Fremden zu unterscheiden. Ausschlieslich mit Geschwistern aufgezogene Kaulquappen schlossen sich Geschwistern an, nicht aber fremden Nicht-Geschwistern. Das liegt aber nicht nur am Bekanntsein, denn sie machten keinen Unterschied zwischen bekannten und unbekannten Geschwistern. Auch Kaulquappen, die von fruhen Embryonal-stadien an isoliert aufwuchsen, verhielten sich so; Geschwisterbevorzugung bedarf also keiner nach-embryonalen Erfahrung. Isoliert aufgewachsene Kaulquappen bevorzugten Vollgeschwister signifikant gegenuber Halbgeschwistern vaterlicherseits, nicht aber gegenuber solchen mutterlicherseits. Es scheint, das ein nicht-genetischer, von der Mutter stammender Faktor fur die Geschwistererkennung masgeblich ist. Kaulquappen, die standig Kontakt mit Geschwistern und Fremden hatten, zogen Geschwister nicht den bekannten Fremden vor. Diesen Unterschied machten sie aber, wenn sie zunachst nur Kontakt mit Geschwistern hatten und erst spater auch Kontakt mit Fremden bekamen. Wuchsen sie in Kontakt mit Geschwistern und Nicht-Geschwistern auf, so schlossen sie sich nicht an Geschwister an, wenn sie spater in reinen Geschwistergruppen gehalten wurden. Es scheint, das Kaulquappen fruh in der Ontogenese im Kontakt mit Art-genossen Merkmale von diesen erlernen und sich spater an solche Individuen halten, die diese Merkmale aufweisen. (Oder sie nehmen im Kontakt dieses Merkmal selber an und weisen dann alle dasselbe Kennzeichen auf.) Kaulquappen, die ausschlieslich mit Geschwistern aufwuchsen, Ziehen diese alien Fremden vor, gleichgultig ob die Fremden aus dem gleichen oder einem anderen Laichgewasser stammen. Im Wahlversuch ziehen sie aber Fremde aus dem gleichen Gewasser vor. Das deutet darauf hin, das Kaulquappen aus dem gleichen Gewasser naher miteinander verwandt sind als solche aus verschiedenen Gewassern.

and Summary Previous studies demonstrated that American toad (Bufo americanus) larvae reared with their siblings or in social isolation subsequently show a tendency to preferentially associate with siblings over non-siblings; if, however, tadpoles are exposed both to siblings and to non-siblings during early development, they do not show a sibling preference. One possible explanation for these results is that individuals, as a consequence of their early interactions, develop convergent recognition traits. To test this hypothesis, eggs of sibling cohorts were divided among two or three mixed group tanks, each tank comprising a different set of 8 sibships. Also, portions of some sib groups were reared in tanks in which they were exposed only to siblings. After 24–51 days, tadpoles were tested for tendencies to associate with familiar siblings over unfamiliar siblings or unfamiliar non-siblings. Analyses of spatial distributions of tadpole groups in a test pool suggest that individuals did not discriminate between familiar siblings and siblings reared in another tank. As in previous tests, tadpoles preferentially associated with familiar siblings over unfamiliar non-siblings, but this effect was most pronounced between sibships that had no prior shared exposure. The results provide no evidence to indicate either that recognition characters of siblings reared in different social environments diverged, or that recognition characters of non-siblings reared in similar social environments converged. Thus, social environment may influence the sibling preferences of toad larvae by generalizing the template with which recognition is assessed, rather than by altering the recognition labels. Data from other species suggest that recognition labels may vary under some conditions, however. Zusammenfassung Fruhere Untersuchungen haben gezeigt, das Kaulquappen der amerikanisdien Krote (Bufo americanus), die mit ihren Geschwistern oder in Isolation aufgezogen wurden, spater ihre Geschwister Nicht-Geschwistern vorzogen. Dies ist nicht der Fall, wenn sie in ihrer fruhen Entwicklung Geschwistem und Nicht-Geschwistern ausgesetzt sind. Eine mogliche Erklarung fur diese Ergebnisse ist, das die Individuen infolge der fruhen Interaktionen sich angleichende Erkennungsmerkmale entwickeln. Um diese Hypothese zu prufen, wurden Eier von Geschwistergelegen in zwei oder drei Aquarien gebracht, von denen jedes einen unterschiedlichen Satz von 8 Gelegen enthielt. Ebenso wurden Larven aus einigen der Geschwistergelege unter Bedingungen aufgezogen, in denen sie nur mit Geschwistem Kontakt hatten. Nach 24–51 Tagen wurde untersucht, ob die Kaulquappen bekannte Geschwister den unbekannten Geschwistem oder unbekannten Nichtgeschwistern vorziehen. Die raumliche Verteilung der Kaulquappengruppen wurde in einem Testbecken analysiert. Die Ergebnisse besagten, das die Individuen nicht zwischen bekannten und unbekannten Geschwistem unterschieden. Wie auch in fruheren Experimenten schlossen sich die Kaulquappen bekannten Geschwistem an, nicht aber unbekannten Nicht-Geschwistern. Dieser Effekt war am deutlichsten in Gruppen, die vorher keinen Kontakt miteinander hatten. Die Ergebnisse weisen weder darauf hin, das Erkennungsmerkmale von Geschwistem, die in unterschiedlichen sozialen Bedingungen aufgezogen wurden, sich auseinanderentwickeln, noch das Erkennungsmerkmale von Nicht-Geschwistern, die unter denselben sozialen Bedingungen aufgezogen wurden, sich angleichen. Daher beeinflust das soziale Umfeld die Kaulquappen in ihrer Wahl der Geschwister vielleicht eher durch eine Verallgemeinerung der Erkennungsschablone als durch die Veranderung der Erkennungszeichen. Ergebnisse von anderen Arten haben jedoch gezeigt, das Erkennungszeichen sich unter gewissen Bedingungen verandern konnen.

Leiopelma hochstetteri belongs to a singular anuran lineage endemic to New Zealand that diverged from other frogs about 200 million years ago. The species now is reduced to a series of isolated populations in the northern half of the North Island and Great Barrier Island. We have used mitochondrial and nuclear sequence data to examine the genetic affinities of extant populations of L. hochstetteri. Phylogenetic reconstructions reveal that populations are highly structured. Each of the geographically isolated populations harbours independent mtDNA lineages as well as different degrees of nuDNA differentiation. Moreover, molecular dating reveals that this structure originated in the early Pleistocene. This pattern of genetic structure likely results from unfavourable climatic conditions during the Pleistocene combined with the low dispersal ability of the species. Isolated populations in forested refugia existed even in the southern part of the distribution of the species during glacial cycles. Previously published variation in chromosome numbers and isozyme data are consistent with the new evidence. We identified 13 evolutionary significant units (ESUs) that should serve as the focus for future management and conservation of this species.

The amphibian fauna of New Zealand consists of three native species (Leiopelma spp.), and three Litoria species introduced from Australia in the last 140 years. We conducted a molecular phylogeographical study that aimed to identify the Australian origins of two species, Litoria aurea and Litoria raniformis. We used partial sequences of the mitochondrial cytochrome oxidase I (cox1) gene from 59 specimens sampled from across the range of both species to identify the probable source populations for the New Zealand introductions, and to describe the current genetic diversity among New Zealand Litoria populations. Our genetic data suggest that L. aurea was introduced into the North Island of New Zealand from two regions in Australia, once from the northern part of coastal New South Wales and once from the southern part of coastal New South Wales. Our data indicate that L. raniformis introductions originated from the Melbourne region of southern Victoria and once established in the South Island of New Zealand, the species subsequently spread throughout both islands. In addition, we found a distinct haplotype in L. raniformis from Tasmania that strongly suggests, contrary to earlier reports, that this species was not introduced into New Zealand from Tasmania. Finally, we identified two very distinctive mitochondrial lineages of L. raniformis within the mainland Australia distribution, which may be previously unrecognized species.

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.

The Oriental fire-bellied toad (Bombina orientalis) is a commonly used study organism, but knowledge of its evolutionary history is incomplete. We analyze sequence data from four genetic markers (mtDNA genes encoding cytochrome c oxidase subunit I, cytochrome b, and 12S–16S rRNA; nuDNA gene encoding recombination activating gene 2) from 188 individuals across its range in Northeast Asia to elucidate phylogeographic patterns and to identify the historic events that shaped its evolutionary history. Although morphologically similar across its range, B. orientalis exhibits phylogeographic structure, which we infer was shaped by geologic, climatic, and anthropogenic events. Phylogenetic and divergence-dating analyses recover four genetically distinct groups of B. orientalis: Lineage 1—Shandong Province and Beijing (China); Lineage 2—Bukhan Mountain (Korea); Lineage 3—Russia, Northeast China, and northern South Korea; and Lineage 4—South Korea. Lineage 2 was previously unknown. Additionally, we discover an area of secondary contact on the Korean Peninsula, and infer a single dispersal event as the origin of the insular Jeju population. Skyline plots estimate different population histories for the four lineages: Lineages 1 and 2 experienced population decreases, Lineage 3 remained stable, while Lineage 4 experienced a sharp increase during the Holocene. The timing of the population expansion of Lineage 4 coincides with the advent of rice cultivation, which may have facilitated the increase in population size by providing additional breeding habitat.

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.

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.

The pathogenic chytrid fungusBatrachochytrium dendrobatidis(Bd) can cause precipitous population declines in its amphibian hosts. Responses of individuals to infection vary greatly with the capacity of their immune system to respond to the pathogen. We used a combination of comparative and experimental approaches to identify major histocompatibility complex class II (MHC-II) alleles encoding molecules that foster the survival of Bd-infected amphibians. We found that Bd-resistant amphibians across four continents share common amino acids in three binding pockets of the MHC-II antigen-binding groove. Moreover, strong signals of selection acting on these specific sites were evident among all species co-existing with the pathogen. In the laboratory, we experimentally inoculated Australian tree frogs with Bd to test how each binding pocket conformation influences disease resistance. Only the conformation of MHC-II pocket 9 of surviving subjects matched those of Bd-resistant species. This MHC-II conformation thus may determine amphibian resistance to Bd, although other MHC-II binding pockets also may contribute to resistance. Rescuing amphibian biodiversity will depend on our understanding of amphibian immune defence mechanisms against Bd. The identification of adaptive genetic markers for Bd resistance represents an important step forward towards that goal.

Bioacoustic signals appear to be so essential to the social communication of anuran amphibians that other sensory modalities have been largely ignored. We studied the abilities of Leiopelma hamiltoni, a species evolutionarily basal to most living anurans, to communicate by means of chemosignals. We collected frogs in the field, held them in captivity for 72 h, and then tested their preferences for substrates that they had marked themselves to those marked by conspecifics. Individuals preferred paper towels that they had marked themselves to those marked by frogs collected from other home ranges. Preferences were greater, on average, when the conspecific had been collected farther away. Frogs did not discriminate between their own odor and those of other individuals with which they had shared a home range. Individuals preferred their own odor to a blank unmarked towel but also avoided odors of unfamiliar conspecifics when paired with a blank. The discovery of chemical communication in an archaic anuran offers a window into how frogs may have communicated before mechanisms of bioacoustic signaling evolved. We suggest, however, that chemical signaling may be widespread in anuran amphibians. Copyright 2004.

Communication by acoustic signals has been extensively studied in anuran amphibians, but other sensory modalities have been largely ignored. We show here that the frog Leiopelma hamiltoni communicates through fecal chemosignals. When given a choice between their own and other individuals' feces, subjects spent more time near their own feces. Further, this effect was greatest when the conspecific was larger in body size, suggesting that information about size as well as individuality is communicated. Time spent near conspecific feces correlated negatively with the distance between the collection sites of the frogs. This correlation may reflect differential responses to the feces of frogs of varied levels of kinship and social familiarity: frogs may avoid nonrelatives and unfamiliar conspecifics. To test the hypothesis that frogs alter fecal production upon exposure to conspecific feces, we presented subjects with either one smear of their own and one smear of a conspecific's feces or two smears of their own feces. Frogs did not defecate more when exposed to conspecific feces. However, when the frogs did defecate, they placed their feces closer to the conspecific's feces than to their own. This supports our hypothesis that feces serve as signals to conspecifics. Visual and tactile cues were eliminated in our experiments. Our results show that the frog L. hamiltoni communicates with conspecifics through chemical signals. We suggest that chemical signaling may be widespread in anuran amphibians.

Patterns of habitat-use are used widely as management guides in the conservation of wildlife. However, even for relatively well-studied species, such the Green and Golden Bell Frog Litoria aurea, our knowledge of specific habitat requirements is lacking.This study sought to compare the patterns of breeding habitat shown by L. aurea in New South Wales (NSW), Victoria and New Zealand (where it is a feral species), with those described in an earlier study of Pyke and White (1996), to review the conservation status of this species in Victoria,and to relate its observed patterns of distribution and habitat-use to the distribution of the introduced Plague Minnow (Gambusia holbrooki in Australia; G. affinis in New Zealand).We found that L. aurea used similar breeding habitats in NSW,Victoria and New Zealand.Across these areas its breeding is almost completely restricted to water bodies that are still, relatively unshaded, and low in salinity (i.e.

The pathogenic chytrid fungus, Batrachochytrium dendrobatidis (denoted Bd), causes large-scale epizootics in naive 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.

New Zealand's native frogs (genus Leiopelma) are considered to be archaic amphibians of exceptional scientific interest that appear to have remained virtually unchanged for 160-200 million years. They are among the rarest extant amphibians and are highly restricted in distribution, confined to isolated, highly disjunct, populations on the North Island and a few small offshore islands in Cook Strait. Previous investigations have suggested, based on patterns of allozyme variation, that the Stephens Island frog (Leiopelma hamiltoni) and Archey's frog (L. archeyi) are sister taxa to the exclusion of the Maud Island frog, a species in close geographical proximity to the Stephens Island frog and previously viewed as a population of this species. As a consequence of these data, a new species, L. pakeka, the Maud Island Frog, has been described. This new species definition has dramatically enhanced the conservation status of L. hamiltoni, of which there are probably fewer than 150 individuals. In this study we re-examine the systematics of the Leiopelmatidae using mtDNA sequence analyses. Partial 12 S ribosomal RNA and cytochrome b (Cyt b) gene sequences were obtained for 57 frogs from six populations representing all four extant Leiopelma species. Contrary to previous reports we find L. pakeka and L. hamiltoni to be monophyletic. The amount of variation evident between these present species (