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.     

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.     

ALLOZYME DIFFERENTIATION AND GENETIC STRUCTURE IN ISLAND AND MAINLAND JAPANESE POPULATIONS OF CAMPANULA PUNCTATA (CAMPANULACEAE)

Allozyme variation was investigated in 17 Japanese populations of Campanula punctata, ten from the Izu Islands and seven in the mainland (Honshu). The data indicate that there are two groups, a mainland group and an island one, and that the systematically problematic Oshima Island (northernmost Izu island) populations are closely related to those of the other islands. Nei's genetic identity values among islands and among mainland populations were 0.95 and 0.97, respectively, while the value between island and mainland populations was 0.84, suggesting that the island populations are an independent species. Total genetic variation was nearly the same among island and mainland populations. However, the apportionment of variation within and among populations was considerably different; 14% of gene diversity exists among mainland populations, while 31% of the diversity exists among island populations. Mean outcrossing rates of self-incompatible mainland and Oshima populations are 0.62–0.79, supporting xenogamy; those in self-compatible island populations are 0.37–0.57 in the northern islands, indicating a mixed mating system, and 0.16–0.25 in southern ones, indicating dominant inbreeding. Total genetic diversity in each island population decreased with distance from the mainland. Genetic and geological data suggest that the ancestors of insular populations were founded on northern islands in a relatively ancient period and that they dispersed progressively to the southern ones. Chromosome number (2n = 34) and isozyme numbers indicate gene duplications in this species, which suggests it is an ancient polyploid.     

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.     

Correlations between feeding type and mandibular morphology in the sika deer

Morphological comparisons of the sika deer Cervus nippon mandible and molars were conducted between two (northern and southern) Japanese subspecific lineages and among local populations of different ('grazer' or 'intermediate feeder') feeding types. The northern lineage showed greater M₁ breadth, M₃ hypsodonty and mandibular corpus height than the southern lineage. Such differences were not observed between the 'grazer' and 'intermediate feeder' populations of the northern lineage. However, a northern population, which inhabits a particularly harsh environment (Kinkazan Island), had the largest values of relative molar size and hypsodonty, although this was not statistically significant. These results imply that, in the Japanese sika deer, the selective pressures acting on the current 'grazer' populations are not strong enough to bring out noticeable adaptive change in molar size and hypsodonty, but adaptive change in these traits may occur in an environment that promotes excessive molar wear, more than that seen in the current sika deer habitats of Japan. Combined with what is known of the Pleistocene history of the sika deer, we infer that the ancestral population of the northern Japanese lineage likely acquired their relatively larger and more hypsodont molars in an extremely harsh environment during the last or previous glacial periods.     

Bottlenecks, drift and differentiation: the population structure and demographic history of sika deer (Cervus nippon) in the Japanese archipelago

We assessed genetic differentiation and diversity in 14 populations of sika deer (Cervus nippon) from Japan and four populations of sika deer introduced to the UK, using nine microsatellite loci. We observed extreme levels of differentiation and significant differences in diversity between populations. Our results do not support morphological subspecies designations, but are consistent with previous mitochondrial DNA analyses which suggest the existence of two genetically distinct lineages of sika deer in Japan. The source of sika introduced to the UK was identified as Kyushu. The underlying structure of Japanese populations probably derives from drift in separate glacial refugia and male dispersal limited by distance. This structure has been perturbed by bottlenecks and habitat fragmentation, resulting from human activity from the mid-nineteenth century. Most current genetic differentiation and differences in diversity among populations probably result from recent drift. Coalescent model analysis suggests sika on each of the main Japanese islands have experienced different recent population histories. Hokkaido, which has large areas of continuous habitat, has maintained high levels of gene flow. In Honshu the population is highly fragmented and is likely to have been evolving by drift alone. In Kyushu there has been a balance between gene flow and drift but all the populations have experienced high levels of drift. Habitat fragment size was not significantly associated with genetic diversity in populations but there was a significant correlation between habitat fragment size and effective population size.     

Phenotypic and genetic divergence among island populations of sika deer (<Emphasis Type="Italic">Cervus nippon</Emphasis>) in southern Japan: a test of the local adaptation hypothesis

Transplant and common garden experiments have been used in studies on local adaptation, but are difficult to be conducted for large animals with long life span. A previous study on the southern Japanese islands demonstrated that relative limb lengths of sika deer (Cervus nippon) were short on islands with steep slopes. We hypothesized that this morphological variation was evidence for local adaptation, and tested this hypothesis by comparing phenotypic divergence with neutral genetic divergence among eight populations of the sika deer in the southern Japanese islands. Divergence patterns differed between the phenotypic and neutral genetic features. Genetic similarity was high among individuals on Kyushu (OI, KGS, and KGK). Individuals on Tanegashima (TN) and Yakushima (YK) also constituted a group, whereas individuals on Tsushima (TS), Wakamatsujima (WM), and Kuchinoerabujima (KE) formed a genetically distinct group. Phenotypic data indicated that individuals from TS, OI, KGS, and KGK exhibited similarity, whereas individuals on YK formed an isolated group that was separated from the other populations. The degree of phenotypic divergence was larger than that of neutral genetic divergence between TN and YK. These results suggest that divergent selection worked between two of the eight island populations (TN and YK). The morphological trait of captive-bred individuals from TN and YK, which had never experienced their original environments, retained their original morphological features. By combining the results of multiple analyses, we found that the difference in relative limb length between the two populations was consistent with local adaptation hypothesis, although conclusive results were not obtained for the other populations.     

Historical effects on local variation in walnut-feeding behavior by the Japanese wood mouse, Apodemus speciosus

The Japanese wood mouse Apodemus speciosus eats large, hard-walled walnuts of Juglans ailanthifolia immediately after finding or after hoarding them. However, not all individuals can efficiently eat the nuts. In this study, to examine local variation in the ability to eat walnuts, feeding behavior was compared among nine wood mouse populations, four from mainland Honshu, where the walnut tree is distributed, and five from the Izu Islands (30-100 km south of Honshu), which lack the tree species. To avoid the effects of pre-capture experience with walnuts, mice from areas lacking the walnut trees were used for testing, even in the Honshu sites. Most mice from Honshu were able to eat walnuts after a 14-day training period, whereas most insular mice could not, with the exception of mice on Kouzushima Island. An analysis of the population genetic structures of these mice based on sequences of the mitochondrial control (D-loop) region revealed that the four insular populations are genetically distinct from the mainland populations, whereas the Kouzushima population remains genetically similar to the mainland populations. The relatively recent colonization of Kouzushima may explain why mice from this island were able to feed on walnuts despite the lack of walnut trees on the island. Thus, walnut-feeding ability appears to have some innate basis in the Japanese wood mouse, and this trait would be selected for in a walnut-available environment as it would better enable mice to survive during food shortages.     

A comparison of food habits of two sympatric ruminants of Mt. Yatsugatake,central Japan: sika deer and Japanese serow

Comparison of food habits of sympatric animals provides understanding of interspecific relations. Previous studies of food habits of the two ruminants of Japan, sika deer (Cervus nippon) and Japanese serows (Capricornis crispus), have shown that sika deer are variable, and they are gazers in northern Japan, but browsers in southern Japan, whereas Japanese serows are browsers. However, these studies described the food habits of each species separately, and no study has compared the food habits of these species living in sympatry. Therefore, we examined these species on Mt. Yatsugatake, central Japan, using microhistological analyses and nutritional analyses of feces. Sasa nipponica, a dwarf bamboo, predominated in the fecal composition of sika deer, whereas both S. nipponica and dicotyledonous plants were found in the feces of Japanese serows. Crude protein levels of serow feces were higher than those of deer. The particle sizes of plant fragments in serow feces were smaller than those in deer feces, suggesting that serow fed more selectively on digestible plants than deer did. These results support the suggestions of previous studies conducted in different habitats and show that sika deer are less selective grazers, and Japanese serows are more selective browsers. Thus, food differences are likely explained by the feeding ecophysiology of the animals and not habitat differences.     

Impact of geographical isolation on genetic differentiation in insular and mainland populations of Weigela coraeensis (Caprifoliaceae) on Honshu and the Izu Islands

Aim To provide insights into genetic differentiation between insular endemic Weigela coraeensis var. fragrans and its progenitor variety W. coraeensis var. coraeensis, the population genetic structure of both varieties was examined, and factors promoting genetic differentiation between the two taxa were explored. Location The natural range of W. coraeensis (sensu lato) throughout mainland Japan (Honshu) and the Izu Islands. Methods The analysis included 349 and 504 individuals across the mainland (Honshu) and the Izu Islands, respectively, using 10 allozyme and 10 microsatellite loci. The population genetic structure of W. coraeensis was assessed by analysing genetic diversity indices for each population, genetic differentiation among populations, model‐based Bayesian clustering or distance‐based clustering, and bottleneck tests. Results The level of genetic diversity in each of the populations on the Izu Islands was negatively correlated with geographical distance between each island and the mainland. The populations on the mainland and on the Izu Islands were genetically differentiated to a certain extent; however, the microsatellite analyses suggested that gene flow also occurred between the mainland and the islands, and among individual islands. These microsatellite analyses also suggested recent bottlenecks in several populations in both areas. Main conclusions The decrease in genetic diversity throughout the Izu Islands, which correlated with distance to the mainland, Honshu, may be the result of a repeated founder effect occurring at a series of inter‐island colonizations from north to south. The stepping stone‐like configuration of the islands may have played a role in the dispersal of the species. Geographical isolation by sea would effectively result in genetic differentiation of W. coraeensis between mainland Honshu and the Izu Islands, although some gene flow may still occur between Honshu and the northern Izu Islands. The differentiation process of the endemic plants on the Izu Islands is anagenetic but not completed, and the study of these plants will provide insightful knowledge concerning the evolution of insular endemics.     

A record of acorn eating by sika deer in western Japan

The rumen contents of 14 sika deer (Cervus nippon) collected in November 1996 in Yamaguchi Prefecture, the western end of Honshu, Japan, were analyzed by the point-frame method. The contents were composed of high-quality foods like deciduous and evergreen browse and acorns. The composition, which consisted of a significant amount of acorns (27.8 ± 22.5%, mean ± SD), was in contrast to that of northern sika deer populations which exclusively eat graminoid leaves including dwarf bamboos. This is the first record of acorns being a major contributor to diet for wild sika deer. By the point-frame method, acorns were underestimated, and the weight contribution was much greater.     

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.     

ANALYSIS OF MITOCHONDRIAL DNA POLYMORPHISMS AMONG CHANNEL ISLAND DEER MICE

Mitochondrial DNA (mtDNA) from 131 deer mice, Peromyscus maniculatus, collected on the eight California Channel Islands and from seven southern California mainland locations, was isolated and analyzed for restriction endonuclease fragment polymorphisms. A total of 26 mtDNA genotypes were distinguishable among the deer mice sampled. All of the island samples had mtDNA restriction-fragment patterns not found among the mainland samples. Distributions of specific restriction-fragment patterns provide evidence for at least four separate colonization events to the Channel Islands. The estimated percentage of sequence divergence between all mtDNA's in this study was less than 1%, suggesting that colonization of the islands occurred fairly recently, probably within the last 500,000 years. Levels of mtDNA heterogeneity were much lower within island populations than within mainland populations.     

Seasonal food habits of a sika deer population in the warm temperate forest of the westernmost part of Honshu, Japan

The rumen contents of 63 sika deer (Cervus nippon Temminck) shot in the westernmost part of Honshu were analyzed by the point-frame method. The rumen contents were dominated by forbs and the majority of these were evergreen monocotyledonous plants, probably Ophiopogon spp. Evergreen browse leaves (woody leaves), such as Eurya japonica Thunb., Ilex crenata Thunb. and Trachelospermum asiaticum Nakai, were also important, constituting 20–40% of the rumen contents. Other categories accounted for only small proportions, although in summer, graminoids increased but twigs and bark decreased in importance and in fall and winter, fruits and seeds increased in importance. The diet of the deer was characterized by high-quality foods even in winter and by a stability or less-pronounced seasonality throughout the year than found in the northern deer. These results were compared with those from other populations, including a northern sika deer population in Japan.     

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。     

The genus Cervus in eastern Eurasia

In 2004, Christian Pitra and co-workers published the first molecular phylogeny of Old World deer which advanced our understanding of the Cervinae immeasurably by demonstrating the non-monophyletic status of the red deer/wapiti group, the chital/hog deer group and the swamp deer/Eld's deer group. Therefore, many conspicuous external features—antler complexity, mane and rump-patch development—turned out to be related not to phylogeny as much as to climatic-related lifestyle factors. At a lower level, molecular genetics has reinforced some conclusions drawn on the basis of morphology or behaviour. Striking examples are the divisions between mainland and Japanese sika and between northern and southern forms of Japanese sika. In this paper, I will look at the species living in what Pitra et al. (Evolution and phylogeny of old world deer. Mol Phylogenet Evol 33:880–895, 2004) identified as the heartland of cervine evolution: eastern Eurasia. I will consider the two species groups in this region that seem to crystallize both the problems of cervine classification and the ways in which the new sources of evidence have opened up new avenues of inquiry.     

Evolution of Mating Systems in Island Populations of Campanula microdonta: Pollinator Availability Hypothesis

Abstract Island populations of Campanula microdonta and mainland Honshu populations of C. punctata have several different mating systems: self-incompatible (SI) and therefore obligately outcrossing in mainland Honshu and Oshima Island; self-compatible (SC) and largely outcrossing in the northern islands of Toshima and Niijima; and SC and predominantly inbreeding southern ones of Miyake and Hachijo. Several features possibly associated with the mating systems are described. A hypothesis is proposed that the mean and variance of pollinator availability together with inbreeding depression could explain the observed patterns of mating system as the results of evolution.     

Phylogeography of the endangered tideland snail Batillaria zonalis in the Japanese and Ryukyu Islands

The phylogeography of the endangered tideland snail Batillaria zonalis in the Japanese and Ryukyu Islands was analyzed on the basis of nucleotide sequences of a mitochondrial gene for cytochrome oxidase c subunit I (COI). Extremely low genetic diversity was found in populations at both the northern and southern boundaries of the geographic distribution of this species in Japan, i.e., Sendai Bay and Iriomotejima Island, respectively, which might be attributed to the population bottleneck due to historical environmental variations and/or the recent foundation of populations in the marginal part of the inhabitable range. Most populations contained unique rare haplotypes, and significant genetic differentiation on the whole was shown, while no clear geographic genetic structure was detected between the Japanese and Ryukyu Islands or over the distribution area of B. zonalis in Japan, with the exception of significant genetic divergences in Ago Bay in the central part of Honshu and the southern part of Okinawajima Island.