Jonathan J. Fong

Genetic evidence for a high diversity and wide distribution of endemic strains of the pathogenic chytrid fungus Batrachochytrium dendrobatidis in wild Asian amphibians.

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.

The chelonian trade in the largest pet market in China: scale, scope and impact on turtle conservation

China is the largest consumer of turtles in the world and international trade has been cited as the greatest threat to Asian turtles. Two main types of trade in live turtles occur in China: for food and traditional Chinese medicine, and for pets, including those for release by Buddhists. The food trade involves the largest quantities of turtles. In recent years, however, the international pet turtle trade has increased dramatically. Yuehe Pet Market in Guangzhou is the largest pet market in China. selling live chelonians and other animals. To understand the potential impacts of the pet trade on chelonians we conducted seven surveys in Yuehe Pet Market from August 2006 to March 2008. Over 39,000 individual chelonians of 61 species were recorded (19.1% of the global total of 319 species). Fifteen (24.6%) of these species are native to China and 46 (75.4%) are native to other countries. Two are designated as grade II key state-protected species in China. Thirty-eight (62.3%) are CITES listed species (four in CITES Appendix I, 26 in CITES II and eight in CITES III). Four are categorized on the IUCN Red List as Critically Endangered, 16 as Endangered and 19 as Vulnerable. Our surveys indicate that increasing demand and the illegal international pet trade could be having a severe impact on chelonian conservation, and we make recommendations for law enforcement and conservation.

A phylogenomic approach to vertebrate phylogeny supports a turtle-archosaur affinity and a possible paraphyletic lissamphibia.

In resolving the vertebrate tree of life, two fundamental questions remain: 1) what is the phylogenetic position of turtles within amniotes, and 2) what are the relationships between the three major lissamphibian (extant amphibian) groups? These relationships have historically been difficult to resolve, with five different hypotheses proposed for turtle placement, and four proposed branching patterns within Lissamphibia. We compiled a large cDNA/EST dataset for vertebrates (75 genes for 129 taxa) to address these outstanding questions. Gene-specific phylogenetic analyses revealed a great deal of variation in preferred topology, resulting in topologically ambiguous conclusions from the combined dataset. Due to consistent preferences for the same divergent topologies across genes, we suspected systematic phylogenetic error as a cause of some variation. Accordingly, we developed and tested a novel statistical method that identifies sites that have a high probability of containing biased signal for a specific phylogenetic relationship. After removing putatively biased sites, support emerged for a sister relationship between turtles and either crocodilians or archosaurs, as well as for a caecilian-salamander sister relationship within Lissamphibia, with Lissamphibia potentially paraphyletic.

Evaluating phylogenetic informativeness and data-type usage for new protein-coding genes across Vertebrata

As a resource for vertebrate phylogenetics, we developed 75 new protein-coding genes using a combination of expressed sequence tags (ESTs) available in Genbank, and targeted amplification of complementary DNA (cDNA). In addition, we performed three additional analyses in order to assess the utility of our approach. First, we profiled the phylogenetic informativeness of these new markers using the online program PhyDesign. Next, we compared the utility of four different data-types used in phylogenetics: nucleotides (NUCL), amino acids (AA), 1st and 2nd codon positions only (N12), and modified sequences to account for codon degeneracy (DEGEN1; Regier et al., 2010). Lastly, we use these new markers to construct a vertebrate phylogeny and address the uncertain relationship between higher-level mammal groups: monotremes, marsupials, and placentals. Our results show that phylogenetic informativeness of the 75 new markers varies, both in the amount of phylogenetic signal and optimal timescale. When comparing the four data-types, we find that the NUCL data-type, due to the high level of phylogenetic signal, performs the best across all divergence times. The remaining three data-types (AA, N12, DEGEN1) are less subject to homoplasy, but have greatly reduced levels of phylogenetic signal relative to NUCL. Our phylogenetic inference supports the Theria hypothesis of mammalian relationships, with marsupials and placentals being sister groups.

Delimitation of Russula Subgenus Amoenula in Korea Using Three Molecular Markers

Abstract Distinguishing individual Russula species has been difficult due to extensive phenotypic plasticity and obscure morphological and anatomical discontinuities. Due to highly similar macroscopic features, such as the presence of a red-cap, species identification within the Russula subgenus Amoenula is particularly difficult. Three species of the subgenus Amoneula have been reported in Korea. We used a combination of morphology and three molecular markers, the internal transcribed spacer (ITS), 28S nuclear ribosomal large subunit (LSU), and RNA polymerase II gene (RPB2), for identification and study of the genetic diversity of Russula subgenus Amoenula in Korea. We identified only two species in Korea (R. mariae and R. violeipes); these two species were indistinguishable according to morphology and LSU, but were found to be reciprocally monophyletic species using ITS and RPB2. The markers, ITS, LSU, and RPB2, have been tested in the past for use as DNA barcoding markers, and findings of our study suggest that ITS and RPB2 had the best performance for the Russula subgenus Amoneula.

Early 1900s Detection of Batrachochytrium dendrobatidis in Korean Amphibians

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.

Identifying airborne fungi in Seoul, Korea using metagenomics

Fungal spores are widespread and common in the atmosphere. In this study, we use a metagenomic approach to study the fungal diversity in six total air samples collected from April to May 2012 in Seoul, Korea. This springtime period is important in Korea because of the peak in fungal spore concentration and Asian dust storms, although the year of this study (2012) was unique in that were no major Asian dust events. Clustering sequences for operational taxonomic unit (OTU) identification recovered 1,266 unique OTUs in the combined dataset, with between 223᾿96 OTUs present in individual samples. OTUs from three fungal phyla were identified. For Ascomycota, Davidiella (anamorph: Cladosporium) was the most common genus in all samples, often accounting for more than 50% of all sequences in a sample. Other common Ascomycota genera identified were Alternaria, Didymella, Khuskia, Geosmitha, Penicillium, and Aspergillus. While several Basidiomycota genera were observed, Chytridiomycota OTUs were only present in one sample. Consistency was observed within sampling days, but there was a large shift in species composition from Ascomycota dominant to Basidiomycota dominant in the middle of the sampling period. This marked change may have been caused by meteorological events. A potential set of 40 allergyinducing genera were identified, accounting for a large proportion of the diversity present (22.5᾿7.2%). Our study identifies high fungal diversity and potentially high levels of fungal allergens in springtime air of Korea, and provides a good baseline for future comparisons with Asian dust storms.

Mitochondrial variation of the “eyed” turtles (Sacalia) based on known-locality and trade specimens

Southern China Normal University ; Natural Science Foundation of China [30260019, 30660026]; Science and Technology and National Key L; National Science Foundation EAPSI program ; University of California (Berkeley) Museum of Vertebrate Zoology ; Sloan Foundation This is UCMP Contribution [1961]

Ventral coloration differentiation and mitochondrial sequences of the Chinese Cobra (Naja atra) in Taiwan

Differences in coloration between eastern and western populations of the Chinese cobra (Naja atra) in Taiwan have been noted by snake collectors, snake keepers, and users of Chinese traditional medicine, but have never been verified by scientific research. In this study, we quantified the amount of black pigment on ventral scales, and found prominent differences in ventral coloration of populations across Taiwan; populations in eastern Taiwan have black ventral scales while populations in the west are predominantly white. Previous studies have shown a similar east-west population differentiation in regards to venom components. We supplement these data with mitochondrial control region sequences, which show extremely low nucleotide diversity. Black-ventral and white-ventral snakes share major haplotypes and show no genetic differentiation. Nevertheless, moderate Fst and low Nm values between populations indicate low levels of gene flow. With a morphological fixation earlier than mitochondrial sequences on a relatively short evolutionary time scale, ventral coloration is potentially a result of local adaptation. Based upon the results of this study, along with traditional observations, we strongly recommend treating each of the four populations of the Chinese cobra in Taiwan as distinct ESUs. Reintroducing confiscated snakes from the illegal trade back into the wild needs to be halted to prevent artifical gene flow.

Penicillium jejuense sp. nov., isolated from the marine environments of Jeju Island, Korea.

Three strains of an unidentified Penicillium species were isolated during a fungal diversity survey of marine environments in Korea. These strains are described here as a new species following a multigene phylogenetic analyses of nuc rDNA internal transcribed spacer barcodes (ITS1–5.8S–ITS2), genes for β-tubulin, calmodulin and RNA polymerase II second largest subunit, and observation of macro-and micromorphological characters. Phylogenetic analyses revealed that the three strains formed a strongly supported monophyletic group distinct from previously reported species of section Aspergilloides. Morphologically this species can be distinguished from its sister species, P. crocicola, by the reverse color on Czapek yeast autolysate agar, abundant production of sclerotia on malt extract agar and colony characters on yeast extract sucrose agar. We name this new species P. jejuense, after the locality where it was discovered. At 25 C for 7 d, P. jejuense colonies grew to 55–60 mm on CYA, 45–48 mm on MEA, 48–52 mm on YES and 23–26 mm on CREA. Conidia (2.2–3.4 × 2.0–2.6 μm) and sclerotia (160–340 × 125–210 μm) were globose to ellipsoidal.