Bottleneck effects on the sika deer Cervus nippon population in Hokkaido, revealed by ancient DNA analysis

The population size of the sika deer Cervus nippon on Hokkaido Island of Japan had been remarkably reduced because of heavy hunting pressure since the beginning of Meiji Period and effects of heavy snow in 1879 and 1881. After that, the number of sika deer in Hokkaido has increased gradually due to the protection by the Hokkaido government. In the present study, in order to investigate the bottleneck effects, we analyzed ancient mitochondrial DNA (mtDNA) on sika deer bones excavated from archaeological sites just before Meiji Period. On 86 of 113 bones from 13 archaeological sites of Ainu Culture Period (17-19th centuries), 602 base-pair fragments of the mtDNA control region were successfully sequenced. Consequently, we found three new haplotypes (g-, h- and i-types) which had not been identified in modern sika deer. In addition, four haplotypes (a-, b-, c- and d-types) identified from modern sika deer were also found in the archaeological deer. The new haplotypes and previously reported hapoltypes from sika deer of Hokkaido were phylogenetically much closer to each other, compared with those of modern sika deer from Honshu, Kyushu and the Chinese continent. Geographical distribution patterns of haplotypes of the ancient population were different from those of the modern population in Hokkaido. Our findings indicated that their genetic diversity was reduced through the bottleneck and that population structures of sika deer were changed widely in Hokkaido due to genetic drift.     

中国大陆梅花鹿mtDNA控制区序列变异及种群遗传结构分析

测定了37只中国大陆梅花鹿(Cervus nippon)不同种群mtDNA控制区5′端351 bp的序列，共发现23个变异位点，定义了5种单元型。分子变异分析表明，中国大陆梅花鹿出现了显著的种群分化(Φm＝0．45，Fst＝0．60，P＜0．001)，支持把分布于东北、华南和四川的梅花鹿种群归入各自独立的管理单元。中国大陆、日本南部和日本北部之间无共享单元型，且有25个鉴别位点。最小跨度网络图(Minimum spannlng network，MSN)和基于最大似然法和邻接法的系统发生分析均把单元型聚类为对应于中国大陆、日本南部和日本北部的三个单系，其中中国大陆和日本南部梅花鹿有相对较近的亲缘关系，支持日本梅花鹿的祖先通过至少两个大陆桥从亚洲迁移到日本的观点。     

Genetic variation and population structure of the Japanese sika deer (Cervus nippon) in the Tohoku District based on mitochondrial D-loop sequences

The sika deer (Cervus nippon) once inhabited the entire Tohoku District, the northeastern part of the main island of Japan. Currently, they are isolated as three discontinuous populations on Mt. Goyo, the Oshika Peninsula, and Kinkazan Island. To assess the genetic diversity and relationships among the sika deer populations in the Tohoku District, we analyzed the mitochondrial DNA D-loop sequences from 177 individuals. We detected a total of five haplotypes. Three haplotypes were present in the population from Mt. Goyo at a haplotype diversity of 0.235 ± 0.061, two haplotypes in the population from the Oshika Peninsula at 0.171 ± 0.064, and only one haplotype was detected in the population from the Kinkazan Island. A significant genetic differentiation was observed among all population pairs. Collectively, our data supports the observed population bottlenecks in the past. Four of the five haplotypes were specific to one of the three populations, whereas only one haplotype was shared between the Mt. Goyo and the Oshika Peninsula populations. This common haplotype may indicate a common ancestral population in the Tohoku District. Conversely, the D-loop haplotypes were completely different among the Kinkazan Island and Oshika Peninsula populations. The lack of a shared haplotype indicates that female gene flow between the two populations is very limited and that the 0.6 km strait acts as a strong barrier.     

Two Genetically Distinct Lineages of the Sika Deer, Cervus nippon, in Japanese Islands: Comparison of Mitochondrial D-Loop Region Sequences

To investigate genetic diversity among populations of the sika deer, Cervus nippon, nucleotide sequences (705–824 bases) of the mitochondrial D-loop regions were determined in animals from 13 localities in the Japanese islands. Phylogenetic trees constructed by the sequences indicated that the Japanese sika deer is separated into two distinct lineages: the northern Japan group (the Hokkaido island and most of the Honshu mainland) and the southern Japan group (a part of the southern Honshu mainland, the Kyushu island, and small islands around the Kyushu island). All sika deer examined in this study shared four to seven units of repetitive sequences (37 to 40 bases each) within the D-loop sequences. The number of tandem repeats was different among the populations, and it was specific to each population. Six or seven repeats occurred in populations of the northern Japan group, while four or five repeats occurred in populations of the southern Japan group. Each repeat unit included several nucleotide substitutions, compared with others, and 26 types were identified from 31 animals. Sequences of the first, second, and third units in arrays were clearly different between the northern and the southern groups. Based on these D-loop data, colonization and separation of the sika deer populations in the Japanese islands were estimated to have occurred less than 0.5 million years before present. Our results provide an invaluable insight into better understanding the evolutionary history, phylogeny, taxonomy, and population genetics of the sika deer.     

Seroprevalence of fasciolosis in Hokkaido sika deer (Cervus nippon yesoensis) from Hokkaido Prefecture,Japan revealed by ELISA using recombinant cathepsin L1

Fasciolosis, a zoonotic disease caused by liver flukes of the genus Fasciola, has been reported in Hokkaido (Yezo) sika deer (Cervus nippon yesoensis) in Hokkaido Prefecture, Japan; however, the actual seroprevalence in the animal has not been adequately evaluated. The objective of the present study was to analyze the seroprevalence of the disease among Hokkaido sika deer. Recombinant cathepsin L1 (rCatL1) was used as an antigen for an indirect enzyme-linked immunosorbent assay (ELISA) to detect antibodies against Fasciola flukes. The sensitivity and specificity of the ELISA were 84.6% and 100%, respectively. The average seroprevalence in 1109 Hokkaido sika deer from 20 locations in Hokkaido Prefecture was 43.9%. Mature deer showed higher seroprevalence than younger individuals; however, even younger animals may act as a reservoir for the disease. Monitoring infection levels in the Hokkaido sika deer population is important not only for the livestock industry, but also for preventing human fasciolosis.     

Two genetically distinct lineages of the sika deer, Cervus nippon, in Japanese islands: comparison of mitochondrial D-loop region sequences

To investigate genetic diversity among populations of the sika deer, Cervus nippon, nucleotide sequences (705-824 bases) of the mitochondrial D-loop regions were determined in animals from 13 localities in the Japanese islands. Phylogenetic trees constructed by the sequences indicated that the Japanese sika deer is separated into two distinct lineages: the northern Japan group (the Hokkaido island and most of the Honshu mainland) and the southern Japan group (a part of the southern Honshu mainland, the Kyushu island, and small islands around the Kyushu island). All sika deer examined in this study shared four to seven units of repetitive sequences (37 to 40 bases each) within the D-loop sequences. The number of tandem repeats was different among the populations, and it was specific to each population. Six or seven repeats occurred in populations of the northern Japan group, while four or five repeats occurred in populations of the southern Japan group. Each repeat unit included several nucleotide substitutions, compared with others, and 26 types were identified from 31 animals. Sequences of the first, second, and third units in arrays were clearly different between the northern and the southern groups. Based on these D-loop data, colonization and separation of the sika deer populations in the Japanese islands were estimated to have occurred less than 0.5 million years before present. Our results provide an invaluable insight into better understanding the evolutionary history, phylogeny, taxonomy, and population genetics of the sika deer.     

Phylogenetics and population genetics of the louse fly,Lipoptena mazamae,from Arkansas,U.S.A.

Louse flies, also known as deer keds (Lipoptena mazamae Rondani), infest cervids such as white‐tailed deer, Odocoileus virginianus and vector pathogens such as Anaplasma and Bartonella schoenbuchensis to cattle and humans, respectively. The population genetic structure of 30 L. mazamae collected from white‐tailed deer in four regions of Arkansas, U.S.A., designated by county boundaries, was examined using DNA sequences of a 259‐bp region of the mitochondrial DNA rRNA 16S gene. Of the 259 nucleotide characters, 33 were variable and 6 haplotypes were identified. Two haplotypes occurred only once (haplotype 3 and 4), whereas two other haplotypes occurred in 43% (haplotype 1 in two regions) and 40% (haplotype 6 in three regions) of the samples. Phylogenetic relationships of the six L. mazamae haplotypes were constructed with other Hippoboscid and Glossinid samples and two clades resulted. Clade 1 was located in the north and western Ozarks whereas clade 2 was found in the northern and eastern Ozarks. Results from the present study indicate that Lipoptena may be a polyphyletic genus; consequently, more research into genetic variation within this genus is necessary.     

Evaluation of introgressive hybridization among Cervidae in Japan's Kinki District via two novel genetic markers developed from public NGS data

Hybridization and backcrossing of native populations with introduced species can lead to introgression and genetic alteration. In this study, we evaluated introgression in 43 deer from a potential hybrid zone around Okinoshima Island, Kinki District, Japan. This region witnessed the migration of a hybrid population (cross between the Formosan sika deer [Cervus nippon taiouanus] and other deer species) that could potentially breed with the native Japanese sika deer (C. n. centralis). We used an existing genetic marker for the mitochondrial cytochrome b gene and two novel markers for nuclear DNA, developed using publicly available next‐generation sequencing data. We identified one mainland deer with a mitochondrial haplotype identical to that of the Formosan sika deer as well as nuclear heterozygous sequences identical to those of Formosan and Japanese sika deer. This suggests that the mainland deer is a hybrid offspring of the Okinoshima population and native deer. However, only Japanese sika deer sequences were found in the other 42 samples, indicating limited introgression. Nevertheless, hybridization pre‐ and postintroduction in the Okinoshima population could cause multispecies introgression among Japanese sika deer, negatively affecting genetic integrity. We developed a simple test based on polymerase chain reaction–restriction fragment length polymorphism to detect introgression in natural populations. Our method can accelerate genetic monitoring of Japanese sika deer in Kinki District. In conclusion, to prevent further introgression and maintain genetic integrity of Japanese sika deer, we recommend establishing fences around Okinoshima Island to limit migration, besides a continued genetic monitoring of the native deer.     

Tracing the genetic roots of the sika deer Cervus nippon naturalized in Germany and Austria

To determine the geographical origin of the sika deer (Cervus nippon) naturalized in Germany and Austria, we sequenced the mitochondrial control region for 214 individuals. Adding these sequences to previously published data from native sika deer across its natural geographic range, the total comes to 245, extending what is already known about the geographical variation in this sequence in Cervus nippon. From these sequences, a neighbour-joining tree was constructed. This tree showed that the 49 different mitochondrial (mt)DNA types are grouped into three distinct phylogenetic clusters, which correspond to different geographic areas. Similarities between sequences of the naturalized sika deer and those described from native sika deer from both southern Honshu, Kyushu with associated islands, and northern Honshu suggest that the ancestors of the sika deer populations in Germany and Austria originated from the Japanese archipelago. In contrast, there is no evidence that female sika deer of Chinese, Taiwanese or north Vietnamese origin were involved in the ancestry of the present sika population in Germany and Austria.     

The impact of habitat fragmentation on genetic structure of the Japanese sika deer (<Emphasis Type="Italic">Cervus nippon</Emphasis>) in southern Kantoh,revealed by mitochondrial D-loop sequences

In southern Kantoh, Japanese sika deer (Cervus nippon) are distributed discontinuously due to large urban areas and developed road networks. To assess the impact of habitat fragmentation on sika deer subpopulations, we examined mitochondrial D-loop sequences from 435 individuals throughout southern Kantoh. About 13 haplotypes were detected, and their distributions revealed spatial genetic structure. Significant genetic differentiation was observed among seven of eight subpopulations. We found no significant correlation between pairwise F _ST and geographical distance among subpopulations. Genetic diversity indices suggested that seven of eight subpopulations had probably experienced population bottlenecks in the recent past. Therefore, and in the light of the results of a nested clade analysis of these haplotypes, we conclude that recent fluctuations in population size and the interruption of gene flow due to past and present habitat fragmentation have played major roles influencing the spatial genetic structure of the sika deer population. This is the first evidence of spatial genetic population structure in the highly fragmented sika deer population in Honshu, Japan.     

Food habits of sika deer in the Shiranuka Hills,eastern Hokkaido: a northern example from the north–south variations in food habits in sika deer

Seasonal changes in the composition of the diet of the sika deer population in the Shiranuka Hills, eastern Hokkaido, in 1998 were determined by fecal analyses. The deer were dependent on Sasa nipponica, a dwarf bamboo, throughout the year, particularly in winter when it accounted for as much as 77.7% of the diet. It accounted for 33.1% and 45.6% in spring and summer, respectively, and this decreased to 12.2% in autumn. Besides S. nipponica, all the graminoid categories accounted for large amounts (66–96.7%), while dicotyledonous plants accounted for little (3–8%) except in autumn when they accounted for 31%. The strong dependence of the Shiranuka population on graminoids was different from other Hokkaido deer populations, for example the population from Ashoro/Onbetsu and the extremely high density population on Nakanoshima Island. In spite of these differences, food for all Hokkaido sika deer was poor in winter. Along the north–south geographical cline in the food composition of sika deer along the Japanese archipelago, the Shiranuka population was positioned as a grazer type, in contrast to the southern populations. However, it is important to note that variations are great among local populations in Hokkaido.     

Adaptive management of sika deer populations in Hokkaido, Japan: theory and practice

We investigated the utility of adaptive management (AM) in wildlife management, reviewing our experiences in applying AM to overabundant sika deer (Cervus nippon) populations in Hokkaido, Japan. The management goals of our program were: (1) to maintain the population at moderate density levels preventing population irruption, (2) to reduce damage to crops and forests, and (3) to sustain a moderate yield of hunting without endangering the population. Because of significant uncertainty in biological and environmental parameters, we designed a “feedback” management program based on controlling hunting pressure. Three threshold levels of relative population size and four levels of hunting pressure were configured, with a choice of four corresponding management actions. Under this program, the Hokkaido Government has been promoting aggressive female culling to reduce the sika deer population since 1998. We devised a harvest-based estimation for population size using relative population size and the number of deer harvested, and found that the 1993 population size (originally estimated by extrapolation of aerial surveys) had been underestimated. To reduce observation errors, a harvest-based Bayesian estimation was developed and the 1993 population estimate was again revised. Analyses of population trends and harvest data demonstrate that hunting is an important large-scale experiment to obtain reliable estimation of population size. A serious side effect of hunting on sika deer was inadvertent lead poisoning of large birds of prey. The prohibition of the use of lead bullets by the Hokkaido Government was successful in reducing the lead poisoning, but the problem still remains. Two case studies on sika population irruption show that the densities set by maximum sustainable yield may be too high to prevent damage to agriculture, forestry, and/or ecosystems. Threshold management based on feedback control is better for ecosystem management. Since volunteer hunters favor higher hunting efficiency in resource management (e.g., venison), it is necessary to support the development of professional hunters for culling operations for ecosystem management, where lower densities of deer should be set for target areas. Hunting as resource management and culling for ecosystem management should be synergistically combined under AM.     

Increased brown bear predation on sika deer fawns following a deer population irruption in eastern Hokkaido, Japan

By the 1970s, brown bears (Ursus arctos) in Hokkaido, northern Japan, were opportunistic omnivores that mainly depended on plant materials. Because the sika deer (Cervus nippon) population irrupted in eastern Hokkaido in the 1990s, we expected that brown bears might prey on sika deer fawns. First, we developed a simple and cost-effective method of monitoring possible bear predation on deer fawns by analyzing the widths of deer hairs remained in bear scats. Based on hair thickness standards, we distinguished the brown bear consumption of deer fawns from adults by analyzing bear scats (n?=?108) collected during the deer birthing season (late May?Clate July) in 1999?C2008. To evaluate the importance of fawns to bears, we compared the occurrence of fawn and adult deer hairs in bear scats among three periods (I, 1999?C2000; II, 2003?C2005; III, 2006?C2008) in eastern Hokkaido. The occurrence of fawn hairs in bear scats increased from 12.5 to 27.3?% in volume and from 6.3 to 33.6?% in frequency from period I to period III, whereas adult hairs in scats decreased from 42.8 to 26.1?% in volume and from 34.4 to 22.7?% in frequency during the same time. These data suggest that bears increasingly preyed on deer fawns after the deer population irruption and decreasingly used adult carcasses because of the enforcement of deer carcass treatment by the Hokkaido government.     

Phylogenetic relationships among european red deer, wapiti, and sika deer inferred from mitochondrial DNA sequences

We determined the mitochondrial cytochrome b gene sequences (1140 bp) of one subspecies of the European red deer (Cervus elaphus in Europe), three subspecies of the wapiti (C. elaphus in Asia and North America), and six subspecies of the sika deer (C. nippon in Japan). Our phylogenetic analysis revealed the monophyly of the European red deer, that of the wapiti, and that of the sika deer. The wapiti, however, was shown to be more closely related to the sika deer than to the European red deer. This is in conflict with traditional morphological results, which suggest a close sister group relationship between the wapiti and the European red deer. The divergence time between the European red deer and the wapiti plus the sika deer was estimated to be approximately 0.80 Ma, and that between the wapiti and the sika deer was estimated to be 0.57 Ma. The sika deer was subdivided into two subspecies groups, and the wapiti was also found to consist of an Asian group and a North American group.     

Population genetic structure and the effect of founder events on the genetic variability of introduced sika deer, Cervus nippon, in Germany and Austria

The relationships among 214 wild-living sika deer from five locations in Germany and two in Lower Austria were examined using mitochondrial DNA (mtDNA) control region sequence. A total of 18 haplotypes are grouped consistently into two major divergent clades, A and B, which differ by a mean of 8.4% sequence divergence. Recently introduced sika deer showed a complex pattern of population structuring, which probably results from historical vicariance in at least two unknown source populations from southeastern Asia (as previously described by morphological and mtDNA findings), and subsequent population admixture as a result of human-mediated restocking. A strong genetic differentiation among populations was indicated by a global _ST value of 0.78 reflecting mainly the differential distribution of clades A and B haplotypes. There was no association between related haplotypes and their distribution among local populations. These indicate that genealogy is a better predictor of the genetic affinity among most sika deer populations than their present-day locations. The abundant mitochondrial divergence we observed, may reflect a subspecies differentiation and could be associated with phenotypic differences among the introduced sika deer.An erratum to this article can be found at     

Species dilemma of musk deer (Moschus spp) in India: molecular data on cytochrome c oxidase I suggests distinct genetic lineage in Uttarakhand compared to other Moschus species

Musk deer are of high conservation priority owing to poaching pressure because of its musk pod. Representation of musk deer status using genetics is poorly documented in India, and it is not confirmed as to how many species of musk deer are present. We characterize for the first time, the genetic diversity of musk deer from Uttarakhand using Cytochrome Oxidase sub-unit (COI) gene (486?bp) and compared with the data available for other species. Results revealed the presence of six haplotypes in the Uttarakhand population amongst 17 sequences. Of these, 12 sequences shared the single haplotype. The intra-species sequences divergence was 0.003–0.017, whereas divergence with other species of musk deer was 0.071–0.081. Bayesian phylogenetic tree revealed that samples from Uttarakhand formed a separate clade with respect to other species of musk deer, whereas three species distributed in China clustered in the same clade and showed low sequences divergence, i.e., 0.002–0.061. Because of different ecomorph reported, we suggest using the barcoding based approach for inter and intra-species distinction and delineating species boundaries across the range for effective conservation. Besides, systematic classification, DNA barcoding would also help in dealing wildlife offence cases for disposal of the legal report in court.     

江西桃红岭国家级自然保护区梅花鹿生境适宜性评价

华南梅花鹿(Cervus nippon)被IUCN列入濒危物种,也是我国国家Ⅰ级重点保护动物。目前种群仅分布于江西、浙江、安徽等狭窄的区域内,形成多个孤立种群,生境破碎和丧失被认为是限制梅花鹿种群增长的主要原因。于2011年3月至2013年3月采用样线法和样方法对桃红岭国家级自然保护区梅花鹿栖息地进行了野外调查,利用空间模拟方法,结合地理信息系统(GIS)技术的空间分析功能,以植被类型、坡度、坡向、海拔和人类干扰活动作为评价因子进行了生境适宜性评价。结果表明,桃红岭地区以森林为主,各类林地面积约9 488.15 hm~2,占75.90%,植被类型分为落叶阔叶林、针叶林、常绿阔叶林、针阔混交林、竹林、灌丛、草丛和芭茅丛,面积分别为1664.57、1638.63、3438.21、1247.15、87.85、1143.88、60.92 hm~2和206.94 hm~2。在不考虑人类活动影响时,梅花鹿的适宜生境和次适宜生境面积分别是2233.99 hm~2和2980.24 hm~2,分别占保护区总面积的18.61%和24.83%;而考虑人类活动影响时,梅花鹿的适宜生境和次适宜生境面积分别是1224.04 hm~2和2164.70 hm~2,分别占保护区总面积的10.20%和18.04%。由于梅花鹿的生境受到居民点、主要道路、农田耕作、森林采伐等人类活动的强烈影响,导致大量适宜和次适宜生境丧失、隔离,景观破碎度指数由0.4345增加到0.5898。以潜在可利用生境面积计算,保护区梅花鹿环境容纳量为(568±160)只,而以实际可利用生境面积计算,则只能容纳(368±105)只。适宜生境的丧失和破碎可能是限制桃红岭梅花鹿国家级自然保护区梅花鹿种群恢复的重要因素,在此基础上,通过实际调查提出了管理措施。     

Food habits of sika deer and nutritional value of sika deer diets in eastern Hokkaido, Japan

Rumen content analysis and field observations were used to investigate the food habits and diet quality of sika deer (Cervus nippon yesoensis Heude) from 1991 to 1993 in eastern Hokkaido, Japan. Diets varied seasonally, with deer consuming graminoids and browse in winter, forbs and agricultural crops in spring and summer and all of these plant foods in autumn. Eighty-four plant species with sika deer bite marks were identified and their use also varied seasonally. The diversity of food resources available provided both critical protein and digestible energy, allowing for physiological maintenance and seasonal growth. With these high-quality diets, deer maintained good body condition in eastern Hokkaido, where the population density was relatively low.     

Seasonal fluctuations of body condition in northern sika deer on Hokkaido Island, Japan

We analyzed seasonal and sexual fluctuations in kidney mass (KM) and kidney fat mass (KFM) as indices of condition in Hokkaido sika deerCervus nippon yesoensis Heude, 1884. For 76 male and 132 female sika deer, seasonal fluctuations in KM and KFM were given by fitted sine wave growth curves. Although the kidney fat index (KFI) is used frequently to evaluate animal condition, we reject it because it is based on the assumption that kidney mass is proportional to body mass in all seasons. Our data did not support this assumption. KFM is a better indicator of Hokkaido sika deer condition than KFI. Although sex-based differences in cervid KFM are said to reflect differences in reproductive cycles, the seasonal similarities in sika deer KFM levels may represent adaptations to the long severe Hokkaido winter. Because in our study deer populations were at low densities and had high pregnancy rates, our sine wave growth models can be regarded as reference for fat level fluctuations in Hokkaido sika deer.     

Genetic structure of,and hybridisation between,red (Cervus elaphus) and sika (Cervus nippon) deer in Ireland

This study investigated the levels of genetic diversity and variation exhibited by red and sika deer in Ireland, along with the extent and regional location of hybridisation between these two species. Bi-parental (microsatellites) and maternally-inherited (mitochondrial DNA) genetic markers were utilised that allowed comparisons between 85 red deer from six localities and 47 sika deer from 3 localities in Ireland. Population genetic structure was assessed using Bayesian analysis, indicating the existence of two genetic clusters in sika deer and three clusters in red deer. Levels of genetic diversity were low in both red and sika deer. These genetic data presented herein indicate a recent introduction of sika deer and subsequent translocations in agreement with historical data. The origins of the current red deer populations found in Ireland, based on genetic data presented in this study, still remain obscure. All hybrid deer (red/sika) found in this study were found in Wicklow, Galway and Mayo where the ‘red-like’ deer exhibited sika deer alleles/haplotypes, and vice versa in the case of Wicklow. Molecular methods proved invaluable in the identification of the hybrid deer because identification of hybrids based on phenotypic external appearances (pelage and body proportions) can be misleading. Areas where red and sika deer are sympatric need to be assessed for the level and extent of hybridisation occurring and thus need to be managed in order to protect the genetic integrity of ‘pure’ red deer populations.