Molecular phylogenetic status of the Korean goral and Japanese serow based on partial sequences of the mitochondrial cytochrome b gene

To investigate the molecular phylogenetic status of the Korean goral, Nemorhaedus caudatus raddeanus, and Japanese serow, Capricornis crispus, we determined partial sequences of the mitochondrial cytochrome b gene of twelve Korean gorals and sixteen Japanese serows, and compared them with those of the major lineages of Rupicaprini species including two other Nemorhaedus species and two other Capricornis species. The Korean gorals examined possessed two haplotypes with only one nucleotide difference between them, while the Japanese serows showed slightly higher sequence diversity with five haplotypes. Genetic distances and molecular phylogenetic trees indicated that there is considerable genetic divergence between the Korean goral and N. caudatus (the Chinese goral) [Groves and Shields (1996)], but virtually none between Korean and Russian gorals. The Korean and Russian gorals may therefore be distinct from the Chinese goral. The data highlight the importance of conservation of the goral populations of these regions, and the need to reconsider the taxonomic status of Korean and Russian gorals. Our study also clearly demonstrated sufficient genetic distance between serows and gorals to justify their assignment to separate genera. Of the three species of Capricornis, the Formosan serow, C. swinhoei is more closely related to C. sumatraensis than to the Japanese serow, suggesting that the Formosan serow is a distinct species. Preliminary data on intraspecific genetic variation in the Japanese serow are also presented.     

Cross-species amplification of Bovidae microsatellites and low diversity of the endangered Korean goral

The Korean goral (Nemorhaedus caudatus) is an endangered species of wild goat. The conservation and management of this species could benefit from a better understanding of its genetic diversity and structure. Cross-species amplification of 34 Bovidae microsatellite loci was tested on a panel of 6 Korean gorals and 10 domestic goats. After polymerase chain reaction (PCR) optimization, 29 (85.3%) microsatellite loci amplified successfully for the Korean gorals and 27 (79.4%) for the domestic goats. Of the amplified products, 16 (55.2%) were polymorphic in the Korean goral and 22 (81.5%) in domestic goats. Nei's unbiased mean heterozygosity and mean allele number per locus were, respectively, 0.356 and 2.6 in the Korean goral and 0.636 and 4.8 in domestic goats. Low genetic diversity in the Korean gorals observed in this preliminary microsatellite survey suggests an urgent need for further detailed study of genetic diversity in Korean goral populations and a population management strategy based on these studies. Current results of cross-species amplification of domestic Bovidae microsatellites could be employed for the necessary population genetic studies on the Korean goral and other endangered Caprinae species.     

Considering threats to population viability of the endangered Korean long-tailed goral (Naemorhedus caudatus) using VORTEX

Population viability analysis (PVA) has frequently been used in conservation biology to predict extinction rates for threatened or endangered species. In this study, we used VORTEX to model Korean long-tailed goral (Naemorhedus caudatus) using previously collected ecological data. We focused on modelling population extinction, mean population size and heterozygosity. The minimum viable population size was found to be at least 50 gorals for 100 years, regardless of carrying capacity. However, populations with fewer than 50 gorals could not remain successful in the model. Inbreeding depression, catastrophes and supplementation also affected patterns of population extinction, mean population size and heterozygosity. Supplementation with new individuals had the strongest effect on extinction, mean population size and heterozygosity, followed by initial population size, inbreeding, catastrophes and carrying capacity. These results suggest that a supplementation by extra goral individuals from goral proliferation facilities would be the most helpful means for the restoration programme. More Korean goral-specific information regarding demographic and habitat parameters is needed for further PVA of the species.     

秦岭地区中段死亡斑羚空间分布规律初探

In recent years, Chinese goral (Nemorhaedus griseus) carcasses were frequently found in the mid-Qinling Mountains. In order to assess the spatial distribution pattern of goral carcasses, data of the individuals from Shaanxi's Fourth Giant Panda Survey was used in this study to analyse how the gorals/goral carcasses sites were related to altitude, slope, aspect, river, and road through quantitative analyses. The results showed:(1) the correlation between the sites of goral carcasses and topographic factors was relatively significant. Goral carcasses tend to be found at relatively low altitude (1 200-2 400 m) and mild slope (6°-25°). The chi-square test also showed that the aspect of the goral carcasses sites was significantly different from the goral sites. Most of the goral carcasses were found at south-facing/half southfacing slopes. (2) The correlation between the sites of goral carcasses and the distance to river was significant. The kernel density analysis and Z-test further showed that there were significant differences between the gorals/goral carcasses sites and the distance to river. Most of the goral carcasses sites were close to river (< 100 m). (3) The correlation between the sites of goral carcasses and the distance to road was not significant. Kernel density analysis and Z-test showed that there was no significant difference between the sites of gorals/goral carcasses and the distance to road. This study proposed several suggestions about how to strengthen the protection of gorals in the future.     

Species and sex identification of the Korean goral (Nemorhaedus caudatus) by molecular analysis of non-invasive samples

Korean long-tailed goral (Nemorhaedus caudatus) is one of the most endangered species in South Korea. However, detailed species distribution and sex ratio data on the elusive goral are still lacking due to difficulty of identification of the species and sex in the field. The primary aim of this study was to develop an economical PCR-RFLP method to identify species using invasive or non-invasive samples from five Korean ungulates: goral (N. caudatus), roe deer (Capreolus pygargus), feral goat (Capra hircus), water deer (Hydropotes inermis) and musk deer (Moschus moschiferus). The secondary aim was to find more efficient molecular sexing techniques that may be applied to invasive or non-invasive samples of ungulate species. We successfully utilized PCR-RFLP of partial mitochondrial cytochrome b gene (376 bp) for species identification, and sex-specific amplification of ZFX/Y and AMELX/Y genes for sexing. Three species (goral, goat and water deer) showed distinctive band patterns by using three restriction enzymes (XbaI, StuI or SspI). Three different sexing primer sets (LGL331/335 for ZFX/Y gene; SE47/48 or SE47/53 for AMELX/Y gene) produced sex-specific band patterns in goral, goat and roe deer. Our results suggest that the molecular analyses of non-invasive samples might provide us with potential tools for the further genetic and ecological study of Korean goral and related species.     

小兴安岭通河林区斑羚冬季食性分析

2004年1～3月在小兴安岭通河林区,使用粪便显微组织学分析方法并结合野外食痕调查,对斑羚（Naemorhedus goral）的冬季食性进行了分析研究。结果表明,斑羚冬季主要以枯草、落叶和当年生枝条为食,共取食20属,22种植物。通过频率转换分析发现,莎草科的羊胡子苔草（Carex callitrichos）和杜鹃花科的兴安杜鹃（Rhododendron dauricum）是其冬季的大宗食物,占食物的百分比分别为31．72％和18．14％。此外,斑羚冬季对苔藓、杨柳科和木犀科植物的取食频次也较高,其百分比分别为7．30％、6．87％和5．25％．     

Isolation and characterization of 15 microsatellite loci in the Korean goral (Nemorhaedus caudatus)

Microsatellites have been applied in a variety of fields, including conservation genetics. Species‐specific microsatellites are considered as more powerful genetic markers to generate an accurate genetic composition of a species itself. Accordingly, we characterized 15 polymorphic microsatellite loci from Korean goral, Nemorhaedus caudatus, one of the most endangered species in South Korea. The new markers should benefit future studies of the endangered species of other Asian gorals and their relatives for the study of genetic diversity and potential conservation management.     

Estimating mountain goat abundance using DNA from fecal pellets

Non-invasive collection of tissue samples to obtain DNA for microsatellite genotyping required to estimate population size has been used for many wildlife species but rarely for ungulates. We estimated mountain goat (Oreamnos americanus) population size on a mountain complex in southwestern British Columbia by identification of individuals using DNA obtained from fecal pellet and hair samples collected during 3 sampling sessions. We identified 55 individuals from 170 samples that were successfully genotyped, and estimated a population of 77 mountain goats (SE = 7.4). Mean capture probability was 0.38 (SE = 0.037) per session. Our technique provides one of the first statistically rigorous estimates of abundance of an ungulate species using DNA derived primarily from fecal pellets. Our technique enables managers to obtain minimum counts or population estimates of ungulates in areas of low sightability that can be used for conservation and management. © 2011 The Wildlife Society.     

Genetic integrity and individual identification-based population size estimate of the endangered long-tailed goral,Naemorhedus caudatus from Seoraksan National Park in South Korea,based on a non-invasive genetic approach

ABSTRACT

The long-tailed goral (also called the Amur goral) Naemorhedus caudatus (subfamily Caprinae), a vulnerable and protected species designated by IUCN and CITES, has sharply been declining in the population size and is now becoming critically endangered in South Korea. This species has been conserved as a natural monument by the Korean Cultural Heritage Administration since 1968. In this study, using 78 fecal DNA samples with a non-invasive genetic approach, we assessed the genetic integrity and individual identification-based population size for the goral population from Seoraksan National Park representing the largest wild population in Korea. Using the successfully isolated 38 fecal DNA, phylogeographic and population genetic analyses were performed with mitochondrial DNA control region (CR) sequences and nine microsatellite loci. We found seven CR haplotypes, of which five were unique to the Seoraksan population, considering previously determined haplotypes in Korean populations. The Seoraksan population showed higher haplotype diversity (0.777?±?0.062) and mean number of alleles (4.67?±?1.563) relative to southern populations in Korea reported from previous studies, with no signal of a population bottleneck. Microsatellite-based individual identification estimate based on probability of identity (PID) indicated a population size of ≥30 in this population. Altogether, we suggest that for future management efforts of this species in the Seoraksan National Park, conserving its genetic integrity as an ‘endemic’ lineage, and curbing a decrease in its number through mitigating habitat destruction might be key to secure the population for the long term.     

韩国青羊分布的变化

Amur goral(Nemorhaedus caudatus)is a rare species among Asian mammals. It is listed as Vulnerable in the 1996 IUCN Red List of the Threatened Animals[1], and designated as the natural monument (No. 217) in Republic of Korea[2]. This species is distributed in Northeast and Southwest of China, south of the Russian Far East and the Korean peninsula[3-5].But subspecies are different with inhabitants in China, which occur 5 species of gorals[4].     

赤斑羚和斑羚核型比较研究

本文对赤斑羚（ＮａｅｍｏｒｈｅｄｕｓＣｒａｎｂｒｏｏｋｉ）和斑羚（Ｎ．ｇｏｒａｌｇｒｉｓｅｕｓ）的染色体Ｇ带、Ｃ带和Ａｇ－ＮＯＲｓ的数目、分布等作了较详细的比较研究。赤斑羚２ｎ＝５６全部为近端着丝粒染色体,Ｎ．Ｆ＝５４;斑羚２ｎ＝５４,除Ｎｏ．３是亚中着丝粒染色体外,具有丰富的异染色质;二者Ｇ带带纹相似程度高,其Ｎｏ．３长臂Ｇ带带纹相似。斑羚的Ｎｏ．３短臂与赤斑羚Ｎｏ．２７近端着丝粒染色体的大小、     

Effects of time and rainfall on PCR success using DNA extracted from deer fecal pellets

Non-invasive wildlife research using DNA from feces has become increasingly popular. Recent studies have attempted to solve problems associated with recovering DNA from feces by investigating the influence of factors such as season, diet, collection method, preservation method, extraction protocol, and time. To our knowledge, studies of this nature have not addressed DNA degradation over time in wet environments, and have not been performed on fecal pellets of ungulates. Therefore, our objective was to determine the length of time a fecal pellet from a Sitka black-tailed deer (Odocoileus hemionus sitkensis) could remain in the field in a temperate rainforest environment before the DNA became too degraded for individual identification. Pellets were extracted from the rectum of recently killed deer and placed in an environment protected from rainfall and in an environment exposed to rainfall. Pellets from each treatment group were sampled at intervals of 2, 7, 14, 21, and 28 days after deer harvest. DNA was extracted from sampled pellets and individual samples were genotyped using microsatellite markers. Amplification failure and errors (dropout and false alleles) were recorded to determine extent of DNA degradation. Eighty percent of samples in the protected environment and 22% of samples in the exposed environment were successfully genotyped during the 28-day experiment. With no samples being successfully genotyped in the exposed environment after 7 days, our study showed that rainfall significantly increases degradation rates of DNA from ungulate pellets.     

圈养赤斑羚夏季日食量及表观消化率的探讨

<正>野生动物营养学是野生动物生态学和管理学的重要组成部分,是了解野生和圈养野生动物种群生存和繁殖的核心内容之一(Robbins,1983)。动物采食是一个复杂的、动态的、生物和非生物因素相互作用和相互影响的过程(Van Soest,1994),同时受动物自身、环境和饲料等多种因素的影响,有时还存在互作(NRC,1996)。采食量决定营养物质的摄入量,决定着动物能够从环境中获得营养物     

Effects of time and environmental conditions on the quality of DNA extracted from fecal samples for genotyping of wild deer in a warm temperate broad-leaved forest

Extraction of DNA from non-invasive samples (feces) has been used increasingly in genetic research on wildlife. For effective and reliable genetic analyses, knowledge about which samples should be selected in the field is essential. For this reason, we examined the process of DNA degradation in feces of deer. We collected fresh fecal pellets from three wild deer living in a warm temperate forest. We then assessed the effects of time (3, 5, and 10 days) under three environmental conditions (on the forest floor, on exposed ground, and inside the laboratory) on the rates of correct genotyping (CG), amplification failure (NA), genotyping error among positive amplification (ER), false alleles (FA), and allelic dropout (AD) of 15 microsatellite loci. The rate of CG significantly decreased, and those of NA and FA increased with increasing lapse of time. Rates of CG tended to be highest and those of NA, ER, FA, and AD to be lowest in feces kept inside, followed by those on the forest floor. Suitability of samples for DNA extraction was lowest in fecal pellets left on exposed ground, and we suspect that rain may hasten DNA degradation. NA rate could serve as a reliable indicator of the quality of fecal pellets because it was significantly positively correlated with ER rate. For efficient genetic analyses using deer feces in warm temperate zones, we recommend collecting fecal pellets within 3 days of defecation, during periods without rainfall and from under the cover of trees.     

Reclassification of the serows and gorals: the end of a neverending story?

相似文献   

Species- and sex-specific multiple pcr amplifications of partial cytochromeb gene andZfx/Zfy introns from invasive and non-invasive samples of Korean ungulates

There are five representative ungulates (e.g., goral,Nemorhaedus caudatus; goat,Capra hircus; roe deer,Capreolus pygargus; water deer,Hydropotes inermis; musk deer,Moschus moschiferus) in the wild of South Korea. Their fecal morphologies are similar to each other between species or sexes, and therefore it is very difficult to identify the species or sex. To distinguish the species and sex of the elusive animals, we developed species- and sex-specific multiple PCR amplifications using pairs of primer sets. Partial cytochromeb gene andZfx/Zfy introns were targeted for the PCR amplifications. For species identification using a multiple primer set (BJGL-F1/GT-F1/RD-F1/WD-F1/MD-F1 and BJGL-R/GT-R/RD-R/WD-R /MD-R), all (n=21/21) of the invasive samples were correctly identified. About 71% (n=15/21) of the non-invasive samples were successfully identified. The multiple primer set could determine each species which showed a unique sized PCR fragment. For sex identification using a multiple primer set (BJSexX-F/Y-F and BJSex-R1), about 90% (n=19/21) of invasive samples were correctly determined. About 62% (n=13/21) of the non-invasive samples were successfully identified. Using the multiple primer set, both X-and Y-chromosome linked bands for males (n=21) and only X-chromosome linked band for females (n=11) could be detected. The results would be applicable to the species and sex identification of the Korean and the Far-East Asian wild ungulates.     

Diet analysis in piscivorous birds: What can the addition of molecular tools offer?

In trophic studies on piscivorous birds, it is vital to know which kind of dietary sample provides the information of interest and how the prey can be identified reliably and efficiently. Often, noninvasively obtained dietary samples such as regurgitated pellets, feces, and regurgitated fish samples are the preferred source of information. Fish prey has usually been identified via morphological analysis of undigested hard parts, but molecular approaches are being increasingly used for this purpose. What remains unknown, however, is which dietary sample type is best suited for molecular diet analysis and how the molecular results compare to those obtained by morphological analysis. Pellets, feces, and regurgitated fish samples of Great Cormorants (Phalacrocorax carbo sinensis) were examined for prey using both morphological hard part analysis and molecular prey identification. The sample types and methods were compared regarding number of species detected (overall and per sample) as well as the prey species composition and its variability among individual samples. Via molecular analysis, significantly higher numbers of prey species were detected in pellets, feces, and fish samples. Of the three sample types, pellets contained the most comprehensive trophic information and could be obtained with the lowest sampling effort. Contrastingly, dietary information obtained from feces was least informative and most variable. For all sample types, the molecular approach outperformed morphological hard part identification regarding the detectable prey spectrum and prey species composition. We recommend the use of pellets in combination with molecular prey identification to study the diet of piscivorous birds.     

The potential use of identification of skeletal remains for the early detection of Ammotragus: an exotic ungulate species in Southern Spain

The main aim of our work was to evaluate osteometry as a complementary tool for the early detection of Ammotragus lervia, an exotic ungulate, which currently shows an expanding trend in southeastern Spain. For this purpose, 142 metacarpi and 123 metatarsi from seven Iberian ungulate species were determined by means of a classification function. In a general way, this function works, but regarding related species (those very similar from a morphometrical view), correct determination was reached in only 44.4–90% of the cases. However, in these cases, we can use auxiliary criteria like sexually dimorphic traits, and reach 100% correct identification of bones.     

Development of Bacteroides 16S rRNA Gene TaqMan-Based Real-Time PCR Assays for Estimation of Total, Human, and Bovine Fecal Pollution in Water

Bacteroides species are promising indicators for differentiating livestock and human fecal contamination in water because of their high concentration in feces and potential host specificity. In this study, a real-time PCR assay was designed to target Bacteroides species (AllBac) present in human, cattle, and equine feces. Direct PCR amplification (without DNA extraction) using the AllBac assay was tested on feces diluted in water. Fecal concentrations and threshold cycle were linearly correlated, indicating that the AllBac assay can be used to estimate the total amount of fecal contamination in water. Real-time PCR assays were also designed for bovine-associated (BoBac) and human-associated (HuBac) Bacteroides 16S rRNA genes. Assay specificities were tested using human, bovine, swine, canine, and equine fecal samples. The BoBac assay was specific for bovine fecal samples (100% true-positive identification; 0% false-positive identification). The HuBac assay had a 100% true-positive identification, but it also had a 32% false-positive rate with potential for cross-amplification with swine feces. The assays were tested using creek water samples from three different watersheds. Creek water did not inhibit PCR, and results from the AllBac assay were correlated with those from Escherichia coli concentrations (r² = 0.85). The percentage of feces attributable to bovine and human sources was determined for each sample by comparing the values obtained from the BoBac and HuBac assays with that from the AllBac assay. These results suggest that real-time PCR assays without DNA extraction can be used to quantify fecal concentrations and provide preliminary fecal source identification in watersheds.     

Development of Bacteroides 16S rRNA gene TaqMan-based real-time PCR assays for estimation of total, human, and bovine fecal pollution in water

Bacteroides species are promising indicators for differentiating livestock and human fecal contamination in water because of their high concentration in feces and potential host specificity. In this study, a real-time PCR assay was designed to target Bacteroides species (AllBac) present in human, cattle, and equine feces. Direct PCR amplification (without DNA extraction) using the AllBac assay was tested on feces diluted in water. Fecal concentrations and threshold cycle were linearly correlated, indicating that the AllBac assay can be used to estimate the total amount of fecal contamination in water. Real-time PCR assays were also designed for bovine-associated (BoBac) and human-associated (HuBac) Bacteroides 16S rRNA genes. Assay specificities were tested using human, bovine, swine, canine, and equine fecal samples. The BoBac assay was specific for bovine fecal samples (100% true-positive identification; 0% false-positive identification). The HuBac assay had a 100% true-positive identification, but it also had a 32% false-positive rate with potential for cross-amplification with swine feces. The assays were tested using creek water samples from three different watersheds. Creek water did not inhibit PCR, and results from the AllBac assay were correlated with those from Escherichia coli concentrations (r2= 0.85). The percentage of feces attributable to bovine and human sources was determined for each sample by comparing the values obtained from the BoBac and HuBac assays with that from the AllBac assay. These results suggest that real-time PCR assays without DNA extraction can be used to quantify fecal concentrations and provide preliminary fecal source identification in watersheds.