A historic religious sanctuary may have preserved ancestral genetics of Japanese sika deer (Cervus nippon).

Deer have been a major resource for human populations for thousands of years. Anthropogenic activities, such as hunting, have influenced the genetic structure and distribution of deer populations. In Japan, wild Japanese sika deer (Cervus nippon) have been hunted since ancient times but have also been historically protected as sacred animals in several sanctuaries. Sika deer have been protected for over a thousand years in the religious sanctuary around the Kasuga Taisha Shrine on the Kii Peninsula, located in the center of Japan. Here, we used short sequence repeats (SSR) and mitochondrial DNA (mtDNA) to investigate the genetic diversity, population structure, and demography of Japanese sika deer inhabiting the Kii Peninsula, Japan, and discuss possible anthropogenic influences. Using SSR, three distinct genetic groups were distinguished on the Kii Peninsula: an Eastern genetic group, a Western genetic group, and an isolated genetic group with individuals in the religious sanctuary of Kasuga Taisha Shrine in Nara city. The isolated genetic sanctuary group had only the mtDNA haplotype S4. The SSR genotype data suggested a newer divergence time of the genetic groups of the religious sanctuary than would have occurred as a result of Late Quaternary climate change. This time scale coincided with the establishment of the sanctuary with Kasuga Taisha Shrine. Thus, the religious protection conserved genetic variation over a thousand years.

Complete mitochondrial genome and phylogenetic analysis of eight sika deer subspecies in northeast Asia.

Sika deer (Cervus nippon) are large ruminants distributed throughout northeastern Asia. The phylogenetic relationship of the sika deer subspecies remains unclear. The complete mitochondrial genomes of 287 sika deer from eight subspecies (C. n. hortulorum, C. n. sichuanicus, C. n. kopschi, C. n. taiouanus, C. n. yesoensis, C. n. centralis, C. n. nippon and C. n. yakushimae) were obtained. Haplotype network and development of a phylogenetic tree revealed China clusters and Japan clusters that were well separated based on the mitochondrial whole-genome level. Our studies indicate that China sika deer are genetically distinguishable from Japanese samples. Our findings increase the understanding of the phylogenetic relationships of sika deer and could provide useful information for sika deer conservation projects as well as for sika deer genomics, emergence and geographical distribution.

Introgression dynamics from invasive pigs into wild boar following the March 2011 natural and anthropogenic disasters at Fukushima.

Natural and anthropogenic disasters have the capability to cause sudden extrinsic environmental changes and long-lasting perturbations including invasive species, species expansion and influence evolution as selective pressures force adaption. Such disasters occurred on 11 March 2011, in Fukushima, Japan, when an earthquake, tsunami and meltdown of a nuclear power plant all drastically reformed anthropogenic land use. Using genetic data, we demonstrate how wild boar (Sus scrofa leucomystax) have persevered against these environmental changes, including an invasion of escaped domestic pigs (Sus scrofa domesticus). Concurrently, we show evidence of successful hybridization between pigs and native wild boar in this area; however in future offspring, the pig legacy has been diluted through time. We speculate that the range expansion dynamics inhibit long-term introgression and introgressed alleles will continue to decrease at each generation while only maternally inherited organelles will persist. Using the gene flow data among wild boar, we assume that offspring from hybrid lineages will continue dispersal north at low frequencies as climates warm. We conclude that future risks for wild boar in this area include intraspecies competition, revitalization of human-related disruptions and disease outbreaks.

Phylogeography of sika deer (Cervus nippon) inferred from mitochondrial cytochrome-b gene and microsatellite DNA.

The genetic diversity and phylogenetic relationships of sika deer of different subspecies are uncertain. In order to explore the phylogenetic relationship of different sika deer subspecies, this study used a wider sample collection to analyze mitochondrial sequences and nuclear microsatellites of sika deer. The full lengths of cytochrome-b gene of 134 sika deer were sequenced, and 16 haplotypes were obtained. Based on phylogenetic and haplotype networks analysis, the sika deer was not clustered according to subspecies but was divided into four lineages. Lineage I includes individuals from C.n.kopschi, C.n.sichuanicus, and C.n.hortulorum subspecies; Lineage II includes individuals from C.n.hortulorum subspecies; Lineage III includes individuals from C.n.centralis, C.n.yakushime, C.n.mageshimae, and C.n.keramae subspecies, namely southern Japanese population; Lineage IV includes individuals from C.n.centralis and C.n.yesoensis subspecies, namely northern Japanese population. The microsatellite analysis showed that the sika deer in China and Japan originated independently. The three subspecies of China have significant genetic differentiation, while the three subspecies of Japan have no significant differentiation. This study provides reference for the research of genetic diversity and phylogenetic relationship of sika deer, and also provides scientific data for the evaluation, protection, and utilization of sika deer resources.

Genetic Variation in Y-Chromosome Genes of Sika Deer (Cervus nippon) in Japan.

Sika deer (Cervus nippon) in Japan are classified into southern and northern groups. However, previous studies primarily relied on maternally inherited mitochondrial DNA (mtDNA). The paternally inherited Y-chromosome is useful for analyzing the contribution of males to the population genetic history of sika deer. In total, approximately 16 kb of partial sequences of four Y-chromosomal genes, Y-linked, sex-determining region Y, DEAD-box helicase 3 Y-linked, and Zinc finger protein Y-linked, were sequenced to investigate intraspecific variation. As a result, we identified nine intronic single nucleotide polymorphisms (SNPs) in 478 sika deer samples collected over the entire Japanese archipelago from Hokkaido to Kyushu. SNP genotyping revealed 10 distinct haplotypes (SYH1-SYH10). The most common haplotype (SYH1) was present in all populations and was the most abundant haplotype, identified in 80.3% of the sampled individuals. The remaining haplotypes were unique to a single locality. SYH1 was also central to all other haplotypes that diverged by a SNP, resulting in this haplotype being the core of a star-like cluster topography. We found that contrary to mtDNA patterns, there was no clear differentiation of Y-chromosome markers between the southern and the northern populations. Due to the female philopatry of sika deer, mtDNA may provide a highly structured differentiation of populations. On the other hand, the male-biased gene flow may provide a reduced differentiation of populations. Our findings revealed that the genetic structure of the Japanese sika deer is more complex than previously thought based on mtDNA-based phylogeographic studies.

Author Correction: Mating of escaped domestic pigs with wild boar and possibility of their offspring migration after the Fukushima Daiichi Nuclear Power Plant accident.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Mating of escaped domestic pigs with wild boar and possibility of their offspring migration after the Fukushima Daiichi Nuclear Power Plant accident.

The 2011 Tohoku earthquake drastically changed human activities in some regions of Fukushima Prefecture, Japan. The subsequent tsunami damage and radioactive pollution from the Fukushima Daiichi nuclear power plant resulted in the evacuation of humans, and abandonment of agricultural lands, allowing population expansion of wildlife into areas formally inhabited by domesticated livestock. Unintentional escape of domesticated pigs into wildlife inhabited environments also occurred. In this study, we tested the possibility of introgression between wild boar and domesticated pigs in Fukushima and neighboring prefectures. We analyzed mitochondrial DNA sequences of 338 wild boar collected from populations in the Tohoku region between 2006 and 2018. Although most boar exhibited Asian boar mitochondrial haplotypes, 18 boar, phenotypically identified as wild boar, had a European domesticated pig haplotype. Frequencies of this haplotype have remained stable since first detection in 2015. This result infers ongoing genetic pollution in wild boar populations from released domesticated pigs. In 2018, this haplotype was detected outside of evacuated areas, suggesting migration and successful adaptation. The natural and anthropocentric disasters at Fukushima gave us the rare opportunity to study introgression processes of domestic genes into populations of wild boar. The present findings suggest a need for additional genetic monitoring to document the dispersal of domestic genes within wild boar stock.

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.

Genome-wide study on genetic diversity and phylogeny of five species in the genus Cervus.

BACKGROUND: Previous investigations of phylogeny in Cervus recovered many clades without whole genomic support. METHODS: In this study, the genetic diversity and phylogeny of 5 species (21 subspecies/populations from C. unicolor, C. albirostris, C. nippon, C. elaphus and C. eldii) in the genus Cervus were analyzed using reduced-representation genome sequencing. RESULTS: A total of 197,543 SNPs were identified with an average sequencing depth of 16 x. A total of 21 SNP matrices for each subspecies/population and 1 matrix for individual analysis were constructed, respectively. Nucleotide diversity and heterozygosity analysis showed that all 21 subspecies/populations had different degrees of genetic diversity. C. eldii, C. unicolor and C. albirostris showed relatively high expected and observed heterozygosity, while observed heterozygosity in C. nippon was the lowest, indicating there was a certain degree of inbreeding rate in these subspecies/populations. Phylogenetic ML tree of all Cervus based on the 21 SNP matrices showed 5 robustly supported clades that clearly separate C. eldii, C. unicolor, C. albirostris, C. elaphus and C. nippon. Within C. elaphus clade, 4 subclades were well differentiated and statistically highly supported: C. elaphus (New Zealand), C. e. yarkandensis, C. c. canadensis and the other grouping the rest of C. canadensis from China. In the C. nippon clade, 2 well-distinct subclades corresponding to C. n. aplodontus and other C. nippon populations were separated. Phylogenetic reconstruction indicated that the first evolutionary event of the genus Cervus occurred approximately 7.4 millions of years ago. The split between C. elaphus and C. nippon could be estimated at around 3.6 millions of years ago. Phylogenetic ML tree of all samples based on individual SNP matrices, together with geographic distribution, have shown that there were 3 major subclades of C. elaphus and C. canadensis in China, namely C. e. yarkandensis (distributed in Tarim Basin), C. c. macneilli/C. c. kansuensis/C. c. alashanicus (distributed in middle west of China), and C. c. songaricus/C. c. sibiricus (distributed in northwest of China). Among them, C. e. yarkandensis was molecularly the most primitive subclade, with a differentiation dating back to 0.8-2.2 Myr ago. D statistical analysis showed that there was high probability of interspecific gene exchange between C. albirostris and C. eldii, C. albirostris and C. unicolor, C. nippon and C. unicolor, and there might be 2 migration events among 5 species in the genus Cervus. CONCLUSIONS: Our results provided new insight to the genetic diversity and phylogeny of Cervus deer. In view of the current status of these populations, their conservation category will need to be reassessed.

Japanese Macaques (Macaca fuscata) as Natural Reservoir of Bartonella quintana.

Bartonella quintana bacteremia was detected in 6 (13.3%) of 45 wild-caught Japanese macaques (Macaca fuscata). Multilocus sequence typing of the isolates revealed that Japanese macaques were infected with a new and specific B. quintana sequence type. Free-ranging Japanese macaques thus represent another natural reservoir of B. quintana.

Characterization of comparative genome-derived simple sequence repeats for acanthopterygian fishes.

Simple sequence repeats (SSRs) have become one of the most popular molecular markers for population genetic studies. The application of SSR markers has often been limited to source species because SSR loci are too labile to be maintained in even closely related species. However, a few extremely conserved SSR loci have been reported. Here, we tested for the presence of conserved SSR loci in acanthopterygian fishes, which include over 14 000 species, by comparing the genome sequences of four acanthopterygian fishes. We also examined the comparative genome-derived SSRs (CG-SSRs) for their transferability across acanthopterygian fishes and their applicability to population genetic analysis. Forty-six SSR loci with conserved flanking regions were detected and examined for their transferability among seven nonacanthopterygian and 27 acanthopterygian fishes. The PCR amplification success rate in nonacanthopterygian fishes was low, ranging from 2.2% to 21.7%, except for Lophius litulon (Lophiiformes; 80.4%). Conversely, the rate in most acanthopterygian fishes exceeded 70.0%. Sequencing of these 46 loci revealed the presence of SSRs suitable for scoring while fragment analysis of 20 loci revealed polymorphisms in most of the acanthopterygian fishes. Population genetic analysis of Cottus pollux (Scorpaeniformes) and Sphaeramia orbicularis (Perciformes) using CG-SSRs showed that these populations did not deviate from linkage equilibrium or Hardy-Weinberg equilibrium. Furthermore, almost no loci showed evidence of null alleles, suggesting that CG-SSRs have strong resolving power for population genetic analysis. Our findings will facilitate the use of these markers in species in which markers remain to be identified.

Phylogenetic characterization of three morphs of mussels (Bivalvia, Mytilidae) inhabiting isolated marine environments in Palau Islands.

Marine lakes in the Palau Islands are known to harbor unique marine fauna that have remained isolated since the formation of the lakes after the Last Glacial Maximum. We analyzed mussels from marine lakes located on different islands and conducted morphological, phylogenetic and population genetic characterization to clarify their evolutionary history. The mussels were morphologically classified into three differentiated morphs: NS, ON, and MC. Their common characteristics were consistent with the Brachidontes-Hormomya complex of the Mytilidae family. Phylogenetic analysis based on the nuclear 18S ribosomal RNA gene supported the taxonomic position of the mussels among the Mytilidae. In the mitochondrial cytochrome c oxidase subunit I (COI) gene lineage, NS-and MC-morphs were highly diverged from each other; their estimated time of divergence dates back to the mid-Pleistocene. ON-morph was more closely related to MC-morph, although the shell morphologies of ON- and MC-morphs were easily distinguishable. Population genetic analysis revealed the coexistence of highly diverged haplotypes within a population of ON-morph, indicating introgression of mtDNA among the morphs. Our data suggest that morphological differentiation of marine lake mussels can occur in a relatively short period under different environmental conditions. Thus, the marine lakes provide a unique site for the study of diversification in mussels.

Population genetic structure of the striped silverside, Atherinomorus endrachtensis (Atherinidae, Atheriniformes, Teleostei), inhabiting marine lakes and adjacent lagoons in Palau: marine lakes are "Islands" for marine species.

Although evidence for the evolution of terrestrial species on islands continues to rapidly accumulate, little is known about the evolution of marine species in geographically isolated environments such as islands as ocean currents often facilitate gene flow among populations. In this study, we focused on marine lakes of the Palau Islands, which are considered to be true analogues of terrestrial islands for marine species. To examine evolutionary processes in marine lakes, we conducted population genetic analyses on marine lake and lagoon populations of the striped silverside, Atherinomorus endrachtensis, using two mitochondrial DNA markers differing in evolutionary rate, the cytochrome b gene and the control region. The analyses revealed that the amount of genetic diversity of marine lake populations is much lower than that of lagoon populations and high levels of genetic differentiation occur among marine lake and lagoon populations. The present study has shown that marine lake populations have been completely isolated and have differentiated from lagoon populations, and each marine lake population is experiencing different evolutionary processes. These findings clearly demonstrate that marine lakes are excellent environments for the evolutionary study of marine species.

Mitochondrial phylogeography and subspecific variation in the red panda (Ailurus fulgens): implications for conservation.

The red panda (Ailurus fulgens) is an endangered species and its present distribution is restricted to isolated mountain ranges in western China (Sichuan, Yunnan, and Tibet provinces) and the Himalayan Mountains chain of Nepal, India, Bhutan, and Burma. To examine the evolutionary history across its current range, and to assess the genetic divergence among current subspecies and population structure among different geographic locations, we sequenced mitochondrial DNA from the control region (CR) and cytochrome (cyt) b gene for 41 individuals in Sichuan, Yunnan, Tibet of China, and Burma. 25 CR haplotypes (10 for cyt b) were identified from 11 geographic locations. Only three haplotypes were shared among sample localities, including one among current subspecies. Nine haplotypes were shared with the study of Su et al. [Mol. Biol. Evol. 18 (2001) 1070]. CR haplotype diversity was high (0.95+/-0.02) and nucleotide diversity among all haplotypes was relatively low (0.018+/-0.009). Phylogenetic confirmed trees show a shallow pattern with very little structure or statistical robustness. The application of two coalescent-based tests for population growth allowed us to interpret this phylogeny as the result of a recent population expansion. Analysis of molecular variance and nested clade analysis failed to detect significant geographic structure in both data sets. The lack of significant differentiation between subspecies does not indicate the presence of evolutionary significant units. We suggest that the present population structure has resulted from habitat fragmentation and expansion from glacial refugia. Due to its habitat requirements it is likely that the red panda has undergone bottlenecks and population expansions several times in the recent past. The present population may exhibit a pattern reminiscent of a relatively recent population expansion.

Nucleotide sequences of putative cDNAs for guinea-pig monoamine oxidase.

To study the molecular structure of guinea pig monoamine oxidase (MAO) and its phylogenetic relationship with other mammalian MAOs, we determined nucleotide sequences of putative MAO cDNAs isolated from guinea pig tissues. Both the 5'- and 3'-ends of the cDNAs were amplified using the RACE (rapid amplification of cDNA ends) method. The sequence (1924 bp) of a putative guinea-pig MAO-B cDNA covers a complete coding region that corresponds to 521 amino acids. We also analyzed a partial sequence of a putative guinea-pig MAO-A cDNA, which corresponds to 506 amino acids, but have left the region of 66 bp at the 3'-end undetermined. The nucleotide and deduced amino-acid sequences of the putative guinea-pig MAO cDNAs showed the highest homology with that of human MAO cDNAs among the known mammalian MAO sequences. These results suggest that guinea-pig MAOs are structurally similar to human MAOs. Our molecular phylogenetic data support the idea that guinea pigs and rodents diverged before the separation between rodents and other lineage leading to Primates and Artiodactyla.