Chytridiomycosis, a disease that has caused amphibian population declines globally and elevated many species of anurans to endangered or threatened status, has recently been declared an internationally notifiable disease. Batrachochytrium dendrobatidis (Bd), the amphibian chytrid fungus causing this disease, has not been previously reported in Korea or on mainland Asia. Thirty-six frog specimens representing 7 species were collected from the wild in South Korea and examined for Bd using standard PCR. Bd was detected in 14 (38.8%) samples from 3 species (Bufo gargarizans, Hyla japonica, and Rana catesbiana). Skin sections from all 14 PCR-positive frogs were examined using 2 staining techniques: haematoxylin and eosin (H&E) and Bd immunoperoxidase (IPX). In histological sections, zoosporangia were found in 6 frogs, with lower sensitivity for H&E (21%) than for IPX (46%). Intensity of infection, based on histopathology, was low in all frogs. These results confirm that Bd is present in South Korea and, hence, on the Asian mainland. Studies are urgently required to determine the impact of chytridiomycosis on Korean amphibians, and to map the distribution of Bd in Korea and other Asian mainland countries. KEY WORDS: Chytridiomycosis · Batrachochytrium dendrobatidis · Amphibian decline · Korea · Fungus Resale or republication not permitted without written consent of the publisherDis Aquat Org 86: [9][10][11][12][13] 2009 nerable (Matsui 2004), and Hynobius yangi is classified as endangered (Stuart 2008) MATERIALS AND METHODSFrogs were collected opportunistically from the northern part of South Korea between June 11 and December 13, 2007 (see Table 1). During nocturnal and occasional diurnal surveys, frogs were captured individually by hand, with a new pair of disposable latex gloves being used for each frog in order to avoid cross-contamination between individuals . The animals were sacrificed by soaking them in a bath of 0.05% aqueous tricaine methane sulfonate (MS-222). We aseptically cut small pieces of skin from the abdomen, inguinal region, and web between toes; we then divided this into 2, and placed 1 in 10% formalin for histological analysis and the other in a 1.5 ml microtube for DNA extraction. Instruments were flamed after dissection of each specimen to prevent cross-contamination. DNA was immediately extracted with Gene Releaser (Bio Ventures). The PCR assay used species-specific primers (Bd1a and Bd2a) located within internal transcribed spacer ITS1 and ITS2 to amplify the 5.8S region of nuclear rDNA (Annis et al. 2004). The PCR products were examined using 1.0% agarose gel and some of the positive bands were cut and sequenced to confirm the Bd sequence. Each sample was tested in triplicate, and was only recorded as positive if all 3 replicates indicated the presence of Bd. For PCR positive amphibians, histological sections of formalin fixed skin were stained using 2 techniques: haematoxylin and eosin (H&E) and Bd immunoperoxidase (IPX), the latter technique using polyclonal antibodies speci...

Korean salamanders of the genus Hynobius are currently classified into 3 species, H. leechii, H. quelpaertensis, and H. yangi. To investigate the phylogenetic relationship of these species, we analyzed the partial sequence of mitochondrial cytochrome b gene (907 bp) of 197 specimens from 43 regions in South Korea. Of these specimens, 93 were additionally examined with 12S rRNA (799 bp). Based on the partial sequence of the mitochondrial cytochrome b gene and 12S rRNA, 89 and 36 haplotypes were defined, respectively, consisting of six subclades (H. leechii, H. quelpaertensis, H. yangi, HC1, HC2, and HC3). Among these subclades, the three subclades (HC1, HC2, and HC3) were clearly separated from the 3 previously reported species in the genus Hynobius. Pairwise sequence divergence between the six subclades ranged from 6.3 to 11.2% in cytochrome b gene and 2.0 to 4.3% in 12S rRNA. These results indicate there may be more divergent populations than the three currently described. Moreover, the estimation of divergence time revealed that the Hynobius species in South Korea diverged during the Miocene epoch, approximately 9 -5 MYA. In addition, we confirmed the distribution of the three known species (H. leechii, H. quelpaertensis, and H. yangi) and determined the distributions of new, distinct groups (or subclades; HC1, HC1, and HC3). To more accurately establish the taxonomic status and population structure, further genetic, morphological, and ecological studies will be needed.

We describe a new species of lentic-breeding Hynobius salamander from the Naro Islands, near the village of Bongrae-myeon, Goheung-gun, Jeollanam-do, South Korea, on the basis of results of morphological, ecological and genetic analyses. Hynobius unisacculus sp. nov. is distinguished from its congeners by a combination of the following morphological attributes: (1) comparatively small size (adult SVL up to 61 mm; range 38.3-60.3 mm in males and 37.5-59.9 mm in females); (2) relatively slender short limbs; tips of fore- and hindlimbs adpressed on body never meeting, but separated by a large gap (gap of -3.0 to -1.5 costal folds in males and -3.5 to -1.5 in females); (3) comparatively short tail (TL/SVL ratio in adult males varying from 0.54-0.98, in adult females from 0.55 to 0.89), tail flattened and with a low dorsal fin extending to the posterior one-third of tail length; (4) usually 11 (occasionally 12) costal grooves; (5) in adults, dark brown dorsum with indistinct bronze or dark copper spots, lighter greyish-white or pinkish belly; (6) well developed fifth toe; (7) comparatively shallow vomerine tooth series with 13-23 vomerine teeth; (8) small, pigmented ova, located in one, occasionally two, strings in a small, curved egg sac with folded envelope, lacking distinct mucous stalks or whiptail-like structures on both ends. The molecular differentiation among Korean Hynobius is high; Hynobius unisacculus sp. nov. is genetically highly divergent from the morphologically similar H. leechii, H. yangi and H. quelpaertensis: pairwise distances are 9.7%, 9.1% and 8.0% of sequence divergence at the COI mtDNA gene respectively, and 10.9%, 10.9% and 9.4% of sequence divergence at the cyt b mtDNA gene, respectively. At present, the new species is known from coastal areas and offshore islands in southeastern part of Jeollanam-do in South Korea. We suggest the species should be considered as Vulnerable (Vu2a) in accordance with IUCN's Red List categories. Our study supports the presence of undiagnosed taxonomic diversity among Korean Hynobius.

We studied and compared the age structure, body size, and growth rates of field populations of two Korean salamander species (Hynobius yangi and Hynobius quelpaertensis) to elucidate important aspects of basic population dynamics of these two endemic Hynobius species. In both populations, females were sexually mature at three years of age, while H. yangi and H. quelpaertensis males matured at two and three years of age, respectively. Both males and females of H. yangi and H. quelpaertensis attained a maximum age of 11 years and 10 years, respectively. In both species, the snout-vent length (SVL) and body weight (BW) of the females were greater than those of the males. The SVL, BW, and asymptotic SVL of both male and female H. yangi were smaller than those of H. quelpaertensis. The adult growth rates after sexual maturation of male and female H. yangi were lower than those of H. quelpaertensis, possibly resulting in the smaller body size of the former, although overall growth coefficients were not significantly different between the two species. We also compared the age structure and growth rates of three Korean and three Japanese species of Hynobius.

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