Are fecal samples an appropriate proxy for amphibian intestinal microbiota?

The intestinal microbiota, an invisible organ supporting a host's survival, has essential roles in metabolism, immunity, growth, and development. Since intestinal microbiota influences a host's biology, application of such data to wildlife conservation has gained interest. There are standard protocols for studying the human intestinal microbiota, but no equivalent for wildlife. A major challenge is sampling the intestinal microbiota in an effective, unbiased way. Fecal samples are a popular proxy for intestinal microbiota because collection is non-invasive and allows for longitudinal sampling. Yet it is unclear whether the fecal microbiota is representative of the intestinal microbiota. In wildlife studies, research on the sampling methodology is limited. In this study focusing on amphibians, we characterize and compare the microbiota (small intestine, large intestine, and feces) of two Hong Kong stream-dwelling frog species: Lesser Spiny Frog (Quasipaa exilispinosa) and Hong Kong Cascade Frog (Amolops hongkongensis). We found that the microbiota of both species are similar at the level of phylum and family, but diverge at the level of genus. When we assessed the performance of fecal microbiota in representing the intestinal microbiota in these two species, we found that (1) the microbiota of the small and large intestine differs significantly, (2) feces are not an appropriate proxy of either intestinal sections, and (3) a set of microbial taxa significantly differs between sample types. Our findings raise caution equating fecal and intestinal microbiota in stream-dwelling frogs. Sampling feces can avoid sacrifice of an animal, but researchers should avoid over-extrapolation and interpret results carefully.

Fine-scale geographic difference of the endangered Big-headed Turtle (Platysternon megacephalum) fecal microbiota, and comparison with the syntopic Beale's Eyed Turtle (Sacalia bealei).

BACKGROUND: Studies have elucidated the importance of gut microbiota for an organism, but we are still learning about the important influencing factors. Several factors have been identified in helping shape the microbiome of a host, and in this study we focus on two factors-geography and host. We characterize the fecal microbiota of the Big-headed Turtle (Platysternon megacephalum) and compare across a relatively fine geographic scale (three populations within an 8-km radius) and between two syntopic hosts (P. megacephalum and Sacalia bealei). Both species are endangered, which limits the number of samples we include in the study. Despite this limitation, these data serve as baseline data for healthy, wild fecal microbiotas of two endangered turtle species to aid in conservation management. RESULTS: For geography, the beta diversity of fecal microbiota differed between the most distant sites. The genus Citrobacter significantly differs between sites, which may indicate a difference in food availability, environmental microbiota, or both. Also, we identify the common core microbiome for Platysternon across Hong Kong as the shared taxa across the three sites. Additionally, beta diversity differs between host species. Since the two species are from the same site and encounter the same environmental microbiota, we infer that there is a host effect on the fecal microbiota, such as diet or the recruitment of host-adapted bacteria. Lastly, functional analyses found metabolism pathways (KEGG level 1) to be the most common, and pathways (KEGG level 3) to be statistically significant between sites, but statistically indistinguishable between species at the same site. CONCLUSIONS: We find that fecal microbiota can significantly differ at a fine geographic scale and between syntopic hosts. Also, the function of fecal microbiota seems to be strongly affected by geographic site, rather than species. This study characterizes the identity and function of the fecal microbiota of two endangered turtle species, from what is likely their last remaining wild populations. These data of healthy, wild fecal microbiota will serve as a baseline for comparison and contribute to the conservation of these two endangered species.

Revisiting the genetic diversity and population structure of the endangered Green Sea Turtle (Chelonia mydas) breeding populations in the Xisha (Paracel) Islands, South China Sea.

The Green Sea Turtle (Chelonia mydas) is an umbrella species in the South China Sea, a Chinese national first-level protected wild animal, and the only sea turtle that nests in waters around China. The largest C. mydas nesting ground is distributed in the Xisha (Paracel) Islands, which plays a vital role in the survival of sea turtle populations in the region. This study reveals the genetic diversity and population structure of the breeding population of C. mydas in the Xisha (Paracel) Islands using three mitochondrial markers. A total of 15 D-loop, five Cytochrome b (Cyt b), and seven Cytochrome C Oxidase subunit I (COI) haplotypes were identified in the breeding population of C. mydas in the Xisha (Paracel) Islands. D-loop haplotypes are distributed in clades III, IV, and VIII of the C. mydas mitochondrial control region. It is the first time that one haplotype from Clade IV was found in this C. mydas population, and five new D-loop haplotypes were also identified. The haplotype and nucleotide diversity were calculated for each marker: D-loop (0.415 haplotype diversity, 0.00204 nucleotide diversity), Cyt b (0.140, 0.00038) and COI (0.308, 0.00083). The average genetic distance (p) of each molecular marker was less than 0.01. Neutral detection and nucleotide mismatch analysis suggested that the breeding population of C. mydas in the Xisha (Paracel) Islands did not experience a population expansion event in recent history. It is recommended that a sea turtle protection area be established in the Xisha (Paracel) Islands area to strengthen protection and effectively protect the uniqueness and sustainability of the breeding population of C. mydas in the South China Sea.

Re-description of the genus Heterobiantes and its type species H. geniculatus (Opiliones: Laniatores: Epedanidae) with additional genital and external morphology data.

The genus Heterobiantes Roewer, 1912 and its type species Epedanus geniculatus Pocock, 1903 are redescribed based on the type specimen currently housed within the British Museum of Natural History (BMNH), London, along with new specimens captured in Hong Kong, providing updated morphological data and male genital description. Closer inspections reveal how the species is different from other similar genera within Epedaninae and rationalizes its placement as a monotypic genus within the sub-family. Genetic data are also included.

Using eDNA techniques to find the endangered big-headed turtle (Platysternon megacephalum).

We evaluate the efficacy of environmental DNA (eDNA) techniques to locate wild populations and estimate the population size of the endangered big-headed turtle (Platysternon megacephalum) in Hong Kong. The results from this study are important for identifying priority sites for protection and further research. Additionally, we assess the impact of two environmental variables (temperature and pH) on eDNA quantity. We surveyed 34 streams for three years, sampling four times each year. Four new populations were first identified with eDNA analysis, and then verified by field surveys. Our multi-year survey highlights that eDNA detection can be inconsistent over time, even in streams with known populations. There was no significant relationship between eDNA quantity and the environmental variables tested. Lastly, our results suggest that eDNA methods remain promising to estimate population size, since number of positive detections were positively correlated with population size in streams with known populations. We conclude that eDNA methods are powerful, but care must be taken when interpreting field results as they are affected by species ecology and environmental conditions.

Taxonomic Revision of the Genus Lactifluus (Russulales, Basidiomycota) of South Korea.

Lactifluus (Pers.) Roussel is an ectomycorrhizal genus that was recently recognized to be distinct from the genus Lactarius. To date, 226 Lactifluus species have been reported worldwide. Misidentification of Lactifluus species is common because of intraspecific morphological variation, cryptic diversity, and the limited number of taxonomic keys available. Molecular data are indispensable for species delimitation; a multilocus phylogenetic analysis showed that most Asian Lactifluus species are not conspecific with morphologically similar species present on other continents. In particular, Korea has misused European and North American Lactifluus names. In this study, we evaluated the taxonomy of Lactifluus in Korea using both morphological and multilocus molecular (ITS, nrLSU, rpb1, and rpb2) data. We examined 199 Lactifluus specimens collected between 1980 and 2016, and a total of 24 species across the four Lactifluus subgenera were identified. All Korean species are distinct and clearly separated from European and North American species. Five taxa corresponded to previously described species from Asia and the remaining 19 taxa are confirmed as new species. Herein, we provide keys to the Korean Lactifluus species within their subgenera, molecular phylogenies, a summary of diversity, and detailed description of the new species.

Phylogeographic study of the Bufo gargarizans species complex, with emphasis on Northeast Asia.

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. A total of 292 individuals from 94 locations were genotyped for two mitochondrial loci (cytb, ND2) and five nuclear introns (Sox9-2, Rho-3, CCNB2-3, UCH-2, and DBI-2), and 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: China mainland and Northeast Asia (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 factors - 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.

Comparative Analysis of the Fecal Microbiota of Wild and Captive Beal's Eyed Turtle (Sacalia bealei) by 16S rRNA Gene Sequencing.

The Beal's eyed turtle (Sacalia bealei) is threatened with extinction due to hunting for large-scale trade. In Hong Kong, there are some of the world's remaining wild populations of S. bealei, as well as a breeding colony. This breeding colony is at the core of conservation efforts (captive breeding, reintroduction programs). Therefore, we would like to know how captivity, in particular diet, affects the gut microbiota. Using high-throughput 16S rRNA gene sequencing, we comparatively analyzed the fecal microbiota of wild and captive S. bealei. We found that wild S. bealei have higher alpha diversity than captive S. bealei, but the difference was not significant. Significant differences were found in ß-diversity; at the phylum level, wild S. bealei have higher relative abundances of Proteobacteria and captive S. bealei have higher relative abundances of Firmicutes. At the genus level, Cetobacterium and Citrobacter are more abundant in wild S. bealei, while Clostridium spp. are significantly more abundant in captive S. bealei. These results suggest conditions in captivity, with diet being a major factor, influence the gut microbiota of S. bealei. The connection between diet and health has always been considered for captive animals, and in this study we use the gut microbiota as an another tool to assess health.

Influence of Season and Soil Properties on Fungal Communities of Neighboring Climax Forests (Carpinus cordata and Fraxinus rhynchophylla).

Trees in forest ecosystems constantly interact with the soil fungal community, and this interaction plays a key role in nutrient cycling. The diversity of soil fungal communities is affected by both environmental factors and host tree species. We investigated the influence of both of these factors by examining the total fungal communities in the rhizospheric soil of climax tree species that have similar ecological roles (Carpinus cordata, an ectomycorrhizal [ECM] tree, and Fraxinus rhynchophylla, an arbuscular mycorrhizal [AM] tree) in temperate forests with continental climates of Mt. Jeombong, South Korea. Fungal communities were assessed by Illumina-MiSeq sequencing the internal transcribed spacer (ITS) region of environmental DNA, and comparing their environmental factors (season and soil properties). We found that soil fungi of the two forest types differed in terms of community structure and ecological guild composition. The total fungal community composition changed significantly with seasons and soil properties in the F. rhynchophylla forest, but not in the C. cordata forest. However, potassium and carbon were significantly correlated with fungal diversity in both forests, and a positive correlation was found only between symbiotrophs of C. cordata and the carbon to nitrogen (C/N) ratio. Thus, the effects of environmental factors on soil fungal communities depended on the host trees, but some factors were common in both forests. Our results indicate that individual tree species should be considered when anticipating how the fungal communities will respond to environmental change.

Successional Change of the Fungal Microbiome Pine Seedling Roots Inoculated With Tricholoma matsutake.

The pine mushroom (Tricholoma matsutake; Agaricales, Tricholomataceae) is an ectomycorrhizal fungus that produces a commercially valuable, edible mushrooms. Attempts to artificially cultivate T. matsutake has so far been unsuccessful. One method used to induce T. matsutake to produce fruiting bodies of in the wild is shiro (mycelial aggregations of T. matsutake) transplantation. In vitro ectomycorrhization of T. matsutake with seedlings of Pinus densiflora has been successful, but field trials showed limited production of fruiting bodies. Few studies have been done to test what happens after transplantation in the wild, whether T. matsutake persists on the pine seedling roots or gets replaced by other fungi. Here, we investigated the composition and the interaction of the root fungal microbiome of P. densiflora seedlings inoculated with T. matsutake over a 3 year period after field transplantation, using high-throughput sequencing. We found a decline of T. matsutake colonization on pine roots and succession of mycorrhizal fungi as P. densiflora seedlings grew. Early on, roots were colonized by fast-growing, saprotrophic Ascomycota, then later replaced by early stage ectomycorrhiza such as Wilcoxina. At the end, more competitive Suillus species dominated the host roots. Most of the major OTUs had negative or neutral correlation with T. matsutake, but several saprotrophic/plant pathogenic/mycoparasitic species in genera Fusarium, Oidiodendron, and Trichoderma had positive correlation with T. matsutake. Four keystone species were identified during succession; two species (Fusarium oxysporum, and F. trincintum) had a positive correlation with T. matsutake, while the other two had a negative correlation (Suillus granulatus, Cylindrocarpon pauciseptatum). These findings have important implications for further studies on the artificial cultivation of T. matsutake.

Integrating and updating wildlife conservation in China.

China has about 11% of the world's total wildlife species, so strengthening China's wildlife conservation is of great significance to global biodiversity. Despite some successful cases and conservation efforts, 21.4% of China's vertebrate species are threatened by human activities. The booming wildlife trade in China has posed serious threat to wildlife in China and throughout the world, while leading to a high risk of transmission of infectious zoonotic diseases. China's wildlife conservation has faced a series of challenges, two of which are an impractical, separated management of wildlife and outdated protected species lists. Although the Wildlife Protection Law of China was revised in 2016, the issues of separated management remain, and the protected species lists are still not adequately revised. These issues have led to inefficient and overlapping management, waste of administrative resources, and serious obstacles to wildlife protection. In this article, we analyze the negative effects of current separated management of wildlife species and outdated protected species lists, and provide some suggestions for amendment of the laws and reform of wildlife management system.

Large-Scale Hybridisation as an Extinction Threat to the Suweon Treefrog (Hylidae: Dryophytes suweonensis).

Amphibians are in the midst of a sixth mass extinction, and human activities play a major role in pushing species towards extinction. Landscape anthropisation has impacts that indirectly threaten species, in addition to the obvious destruction of natural habitats. For instance, land modification may bring human-commensal species in contact with sister-clades from which they were previously isolated. The species in these new contact zones are then able to hybridise to the point of reaching lineage fusion, through which the gene pool of the two species merges and one of the parental lineages becomes extirpated. Here, we documented the patterns of hybridisation between the spatially restricted D. suweonensis and the widespread D. japonicus. On the basis of the analysis of Cytochrome c oxidase subunit I mitochondrial DNA sequences (404 individuals from 35 sites) and six polymorphic microsatellites (381 individuals from 34 sites), we revealed a generalised, bi-directional, and geographically widespread hybridisation between the two species. Evidence of fertile back-crosses is provided by relatively high numbers of individuals in cyto-nuclear disequilibrium, as well as the presence of hybrid individuals further south than the species distribution limit, determined on the basis of call properties. Hybridisation is an additional threat to the endangered D. suweonensis.

Genetic diversity and inferred dispersal history of the Schlegel's Japanese Gecko (Gekko japonicus) in Northeast Asia based on population genetic analyses and paleo-species distribution modelling.

To understand the genetic diversity and dispersal history of Schlegel's Japanese gecko (Gekko japonicus), we performed genetic analyses and paleo-species distributional modelling. For the genetic analysis, we analyzed mitochondrial DNA (mtDNA) (cytochrome b [Cytb] and NADH dehydrogenase 2 [ND2]) and seven microsatellite loci of 353 individuals from 11 populations (2 east coast China, 4 west and central coast Japan and 5 Korea). For the paleo-species distribution modelling, we used 432 occurrence data points (125 China, 291 Japan and 16 Korea) over the Pleistocene and Holocene. China is inferred to be the source population, which had higher genetic diversity (mtDNA) and more private alleles (mtDNA) compared to Japanese and Korean populations. Differences between the three counties were very small in the mtDNA haplotype network despite some genetic structure among the three countries. Microsatellite analysis inferred that genetic exchange has actively occurred among the Chinese, Japanese and Korean populations. Suitable habitats in Japan should have been plentiful by the mid-Holocene, but have only recently become available in Korea. These results suggest that dispersal of G. japonicus occurred after the Holocene warming from the east coast of China to the west and central coasts of Japan and Korea, and gene flow is actively occurring among the three countries.

The diversity and ecological roles of Penicillium in intertidal zones.

Members of the genus Penicillium are commonly isolated from various terrestrial and marine environments, and play an important ecological role as a decomposer. To gain insight into the ecological role of Penicillium in intertidal zones, we investigated the Penicillium diversity and community structure using a culture-dependent technique and a culture independent metagenomic approach using ITS (ITS-NGS) and partial ß-tubulin (BenA-NGS) as targets. The obtained isolates were tested for halotolerance, enzyme activity, and polycyclic aromatic hydrocarbons (PAHs) degradation. A total of 96 Penicillium species were identified from the investigated intertidal zones. Although the BenA-NGS method was efficient for detecting Penicillium, some species were only detected using conventional isolation and/or the ITS-NGS method. The Penicillium community displayed a significant degree of variation relative to season (summer and winter) and seaside (western and southern coast). Many Penicillium species isolated in this study exhibited cellulase and protease activity, and/or degradation of PAHs. These findings support the important role of Penicillium in the intertidal zone for nutrient recycling and pollutant degradation.

Macrolepiota in Korea: New Records and a New Species.

The genus Macrolepiota (Agaricales, Basidiomycota) is easy to recognize at the genus level because of big, fleshy basidiocarps with squamules covering the pileus; a single or double annulus; and big, thick-walled basidiospores with a germ pore. However, morphological identification is often unreliable in Macrolepiota due to similar morphological features among species. Due to the uncertainty of previous morphological identification in the genus Macrolepiota, it is necessary to re-examine Korean Macrolepiota using molecular data. We re-examined 34 Macrolepiota specimens collected from 2012 to 2018 in Korea using a reverse taxonomic approach, whereby species identification was first done based on the internal transcribed spacer (ITS) region analysis, followed by morphological confirmation. We identified the presence of four species: M. detersa, M. mastoidea, M. procera, and M. umbonata sp. nov. Two species (M. detersa and M. mastoidea) were previously unrecorded from Korea and M. umbonata is a new species. Detailed descriptions of all four species and taxonomic key are provided in this study. Macrolepiota procera and M. umbonata are distributed through the country, but M. detersa and M. mastoidea are distributed only in limited areas. According to our results, the combination of ITS locus and morphology proved to be a robust approach to evaluate the taxonomic status of Macrolepiota species in Korea. Additional surveys are needed to verify the species diversity and clarify their geographic distribution.

Reproductive ecology of the endangered Beal's-eyed turtle, Sacalia bealei.

The Beal's-eyed turtle (Sacalia bealei) is endemic to southeastern China and endangered due to poaching and habitat loss. Knowledge of S. bealei ecology is lacking and this study provides baseline information of its reproduction in a natural environment. We studied the reproductive ecology of S. bealei using X-ray, spool-and-line tracking, and direct observation. Six nesting females were successfully tracked and their nesting behaviors are documented in detail. Females produced a mean clutch size of 2.2 eggs (range 1-3). The hard-shelled eggs were ellipsoidal with a mean length of 45.50 mm, a mean width of 23.20 mm, and mean weight of 14.8 g. The relative clutch mass was 9.47%, while the relative egg mass was 4.60%. The mean incubation period was 94.7 days with a mean nest temperature of 25.08 °C. Hatchlings had a mean weight of 9.7 g, carapace length of 40.1 mm, carapace width of 33.3 mm, carapace height of 17.4 mm, plastron length of 31.6 mm, and plastron width of 25.4 mm. The results of this study provide important information to inform conservation plans and ex-situ breeding for this endangered species.

Northward dispersal of sea kraits (Laticauda semifasciata) beyond their typical range.

Marine reptiles are declining globally, and recent climate change may be a contributing factor. The study of sea snakes collected beyond their typical distribution range provides valuable insight on how climate change affects marine reptile populations. Recently, we collected 12 Laticauda semifasciata (11 females, 1 male) from the waters around southern South Korea-an area located outside its typical distribution range (Japan, China including Taiwan, Philippines and Indonesia). We investigated the genetic origin of Korean specimens by analyzing mitochondrial cytochrome b gene (Cytb) sequences. Six individuals shared haplotypes with a group found in Taiwan-southern Ryukyu Islands, while the remaining six individuals shared haplotypes with a group encompassing the entire Ryukyu Archipelago. These results suggest L. semifasciata moved into Korean waters from the Taiwan-Ryukyu region via the Taiwan Warm Current and/or the Kuroshio Current, with extended survival facilitated by ocean warming. We highlight several contributing factors that increase the chances that L. semifasciata establishes new northern populations beyond the original distribution range.

Diversity and abundance of human-pathogenic fungi associated with pigeon faeces in urban environments.

Pathogenic fungi are a growing health concern worldwide, particularly in large, densely populated cities. The dramatic upsurge of pigeon populations in cities has been implicated in the increased incidence of invasive fungal infections. In this study, we used a culture-independent, high-throughput sequencing approach to describe the diversity of clinically relevant fungi (CRF) associated with pigeon faeces and map the relative abundance of CRF across Seoul, Korea. In addition, we tested whether certain geographical, sociological and meteorological factors were significantly associated with the diversity and relative abundance of CRF. Finally, we compared the CRF diversity of fresh and old pigeon faeces to identify the source of the fungi and the role of pigeons in dispersal. Our results demonstrated that both the composition and relative abundance of CRF are unevenly distributed across Seoul. The green area ratio and the number of multiplex houses were positively correlated with species diversity, whereas wind speed and number of households were negatively correlated. The number of workers and green area ratio were positively correlated with the relative abundance of CRF, whereas wind speed was negatively correlated. Because many CRF were absent in fresh faeces, we inferred that most species cannot survive the gastrointestinal tract of pigeons and instead are likely transmitted through soil or air and use pigeon faeces as a substrate for proliferation.

Diversity and phylogeography of Northeast Asian brown frogs allied to Rana dybowskii (Anura, Ranidae).

We investigated the species diversity and phylogeography of the Northeast Asian brown frogs allied to Rana dybowskii (the R. dybowskii species complex: R. dybowskii, R. pirica, and R. uenoi) using four mitochondrial and three nuclear loci. Phylogenetic analyses confirmed the existence of three distinct species in this complex; using extensive molecular data, we confirm the validity of Rana uenoi recognized as a distinct species, and infer R. dybowskii and R. pirica to be sister species. Also, we included populations from previously unsampled regions in Northeast China, and identified them to be R. dybowskii. While many species in Northeast Asia diverged due to Pleistocene glaciation, divergence-dating analyses inferred older, Miocene speciation in the R. dybowskii species complex. Ancestral area reconstruction identified the orogenic movement of the Changbai Mountain Range and the opening of the Sea of Japan/East Sea being major events influencing allopatric speciation.

Disappearance of endangered turtles within China's nature reserves.

China ranks first among Northern hemisphere countries for species richness, but approximately 43% of its species are threatened [1], with harvesting being the major threat to vertebrates [2]. To protect its biodiversity, China has established about 2,700 nature reserves covering 1.46 million km2 ( about 15% of China's territory, a percentage higher than the world average [3]). With increasing habitat destruction and harvesting, nature reserves are the final refugia for threatened species. However, many Chinese nature reserves are poorly managed, leaving them vulnerable to poaching and other human encroachment [4]. In this study, we conducted a 12-year (2002-2013) case study on turtles to illustrate the damaging impacts China's nature reserves have on wildlife conservation. We discovered that poaching occurred in all of the 56 reserves surveyed, resulting in dramatically reduced turtle populations. In a majority of the reserves, the reserve staff themselves were involved in poaching. Although nature reserves were created to protect plants and animals, they have become part of the problem due to weak enforcement of rules.