Mitogenomic exploration supports the historical hypothesis of anthropogenic diffusion of a zoonotic parasite Echinococcus multilocularis.

Animal movement across regions owing to human activity can lead to the introduction of pathogens, resulting in disease epidemics with medical and socioeconomic significance. Here, we validated the hypothesis that human activity, such as the transportation of infected animals, has played a significant role in introducing the zoonotic parasite Echinococcus multilocularis into Hokkaido, Japan, by synthesizing and evaluating parasite genetic data in light of historical records. Our analysis indicates that a major genetic group in Hokkaido originated from St. Lawrence Island, USA, which is in accordance with the route suggested by historical descriptions. Moreover, we identified a minor genetic group closely related to parasites found in Sichuan, China. This fact implies that parasite invasion in Japan may result from complex and inadvertent animal translocations. These findings emphasize the anthropogenic impacts on zoonotic parasite spread and provide a crucial perspective for preventing future potential epidemics.

Applications of Blocker Nucleic Acids and Non-Metazoan PCR Improves the Discovery of the Eukaryotic Microbiome in Ticks.

Ticks serve as important vectors of a variety of pathogens. Recently, the viral and prokaryotic microbiomes in ticks have been explored using next-generation sequencing to understand the physiology of ticks and their interactions with pathogens. However, analyses of eukaryotic communities in ticks are limited, owing to the lack of suitable methods. In this study, we developed new methods to selectively amplify microeukaryote genes in tick-derived DNA by blocking the amplification of the 18S rRNA gene of ticks using artificial nucleic acids: peptide nucleic acids (PNAs) and locked nucleic acids (LNAs). In addition, another PCR using non-metazoan primers, referred to as UNonMet-PCR, was performed for comparison. We performed each PCR using tick-derived DNA and sequenced the amplicons using the Illumina MiSeq platform. Almost all sequences obtained by conventional PCR were derived from ticks, whereas the proportion of microeukaryotic reads and alpha diversity increased upon using the newly developed method. Additionally, the PNA- or LNA-based methods were suitable for paneukaryotic analyses, whereas the UNonMet-PCR method was particularly sensitive to fungi. The newly described methods enable analyses of the eukaryotic microbiome in ticks. We expect the application of these methods to improve our understanding of the tick microbiome.

Amblyomma testudinarium infestation on a brown bear (Ursus arctos yesoensis) captured in Hokkaido, a northern island of Japan.

The tick Amblyomma testudinarium Koch, 1844 (Acari: Ixodidae) is known as a vector of several pathogens such as Rickettsia tamurae and severe fever with thrombocytopenia syndrome (SFTS) virus. This tick species is present in many Asian countries, including Japan, where its distribution is limited to the warm areas of Kanto region and the southwestern region. The present study reports the recovery of a partially engorged A. testudinarium from a wild brown bear captured in Shari town, Hokkaido. In addition to morphological identification, the specimen was genetically characterized by the complete mitochondrial genome sequencing. The results showed that the length of the obtained mitogenome is 14,835 bp that encodes 13 protein-coding, two ribosomal RNA (rRNA) (12S and 16S), and 22 transfer RNA genes with two non-coding control regions. The phylogenetic analysis indicated that our sample clustered with A. testudinarium from Nara, Japan, but separated from A. testudinarium from China. Although the introduction of the tick through livestock transportation cannot be ruled out, the detection of A. testudinarium in Hokkaido prefecture, which is separated from the main island where A. testudinarium is present in the south, may suggest the introduction by migratory birds. This study provides important insights on the distribution and host range of A. testudinarium. This will be useful for the future taxonomic analysis of ticks based on the complete mitogenome sequencing. To our knowledge, this is the northernmost detection point of the tropical tick A. testudinarium.

Mouse Hair Significantly Lightened Through Replacement of the Cysteine Residue in the N-Terminal Domain of Mc1r Using the CRISPR/Cas9 System.

A loss-of-function mutation in the melanocortin 1 receptor gene (MC1R), which switches off the eumelanin production, causes yellowish coat color variants in mammals. In a wild population of sables (Martes zibellina) in Hokkaido, Japan, the mutation responsible for a bright yellow coat color variant was inferred to be a cysteine replacement at codon 35 of the N-terminal extracellular domain of the Mc1r receptor. In the present study, we validated these findings by applying genome editing on Mc1r in mouse strains C3H/HeJ and C57BL/6N, altering the codon for cysteine (Cys33Phe). The resulting single amino acid substitution (Cys33Phe) and unintentionally generated frameshift mutations yielded a color variant exhibiting substantially brighter body color, indicating that the Cys35 replacement produced sufficient MC1R loss of function to confirm that this mutation is responsible for producing the Hokkaido sable yellow color variant. Notably, the yellowish mutant mouse phenotype exhibited brown coloration in subapical hair on the dorsal side in both the C3H/HeJ and C57BL/6N strains, despite the inability of the latter to produce the agouti signaling protein (Asip). This darker hair and body coloration was not apparent in the Hokkaido sable variant, implying the presence of an additional genetic system shaping yellowish hair variability.

Author Correction: Dakrông virus, a novel mobatvirus (Hantaviridae) harbored by the Stoliczka's Asian trident bat (Aselliscus stoliczkanus) in Vietnam.

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

Late Pleistocene climate change and population dynamics of Japanese Myodes voles inferred from mitochondrial cytochrome b sequences.

The Japanese archipelago is comprised of four main islands-Hokkaido, Honshu, Shikoku, and Kyushu-which contain high mountainous areas that likely allowed for lineage differentiation and population genetic structuring during the climatic changes of the late Pleistocene. Here, we assess the historical background of the evolutionary dynamics of herbivorous red-backed voles (Myodes) in Japan, examining the evolutionary trends of mitochondrial cytochrome b gene (Cytb) sequence variation. Four apparent signals from rapid expansion events were detected in three species, M. rufocanus and M. rutilus from Hokkaido and M. smithii from central Honshu. Taken together with results from previous studies on Japanese wood mice (Apodemus spp.), three of the expansion events were considered to be associated with predicted bottleneck events at the marine isotope stage (MIS) 4 period, in which glaciers are thought to have expanded extensively, especially at higher elevations. In the late Pleistocene, the possible candidates are transitions MIS 6/5, MIS 4/3, and MIS 2/1, which can be characterized by the cold periods of the penultimate glacial maximum, MIS 4, and the last glacial maximum, respectively. Our data further reveal the genetic footprints of repeated range expansion and contraction in the northern and southern lineages of the vole species currently found in central Honshu, namely M. andersoni and M. smithii, in response to climatic oscillation during the late Pleistocene. The time-dependent evolutionary rates of the mitochondrial Cytb presented here would provide a possible way for assessing population dynamics of cricetid rodents responding to the late Pleistocene environmental fluctuation.

Endangered island endemic plants have vulnerable genomes.

Loss of genetic diversity is known to decrease the fitness of species and is a critical factor that increases extinction risk. However, there is little evidence for higher vulnerability and extinction risk in endangered species based on genomic differences between endangered and non-endangered species. This is true even in the case of functional loci, which are more likely to relate to the fitness of species than neutral loci. Here, we compared the genome-wide genetic diversity, proportion of duplicated genes (PD), and accumulation of deleterious variations of endangered island endemic (EIE) plants from four genera with those of their non-endangered (NE) widespread congeners. We focused on exhaustive sequences of expressed genes obtained by RNA sequencing. Most EIE species exhibited significantly lower genetic diversity and PD than NE species. Additionally, all endangered species accumulated deleterious variations. Our findings provide new insights into the genomic traits of EIE species.

Dakrông virus, a novel mobatvirus (Hantaviridae) harbored by the Stoliczka's Asian trident bat (Aselliscus stoliczkanus) in Vietnam.

The recent discovery of genetically distinct shrew- and mole-borne viruses belonging to the newly defined family Hantaviridae (order Bunyavirales) has spurred an extended search for hantaviruses in RNAlater®-preserved lung tissues from 215 bats (order Chiroptera) representing five families (Hipposideridae, Megadermatidae, Pteropodidae, Rhinolophidae and Vespertilionidae), collected in Vietnam during 2012 to 2014. A newly identified hantavirus, designated Dakrông virus (DKGV), was detected in one of two Stoliczka's Asian trident bats (Aselliscus stoliczkanus), from Dakrông Nature Reserve in Quang Tri Province. Using maximum-likelihood and Bayesian methods, phylogenetic trees based on the full-length S, M and L segments showed that DKGV occupied a basal position with other mobatviruses, suggesting that primordial hantaviruses may have been hosted by ancestral bats.

Environmental DNA Collected from Snow Tracks is Useful for Identification of Mammalian Species.

Noninvasive genetic analysis is being used increasingly in field surveys. However, detecting large and middle-sized mammals, such as Carnivora species, using noninvasive samples, such as scat or hair, is time- and labor-intensive due to their low densities and elusive behaviors. As snow tracks are the most frequently encountered natural signs of terrestrial mammals in winter, we employed several methods to recover environmental DNA (eDNA) from snow tracks. We performed both DNA metabarcoding and Sanger sequence analyses, in combination with universal primers on the mitochondrial 12S rRNA gene for mammals and taxon-specific primers on the mitochondrial NADH dehydrogenase subunit 2 gene for Martes species (martens and sables in Mustelidae). Snow samples of four Martes melampus tracks, one Cervus nippon track, one Vulpes vulpes track, and the track of an unidentified Carnivora species were collected from a snowfall area in Kyoto, Japan, in February 2018. Regarding DNA metabarcoding analyses, the sequences of three Carnivora species (M. melampus, V. vulpes, and Canis lupus familiaris) and a deer (C. nippon) were obtained from their respective snow tracks. Using Sanger sequencing, eDNA on snow tracks was recovered at the species level except for M. melampus using universal primers, while eDNA of M. melampus was sequenced using Martes-specific primers. Snow track surveys in combination with eDNA techniques could dramatically improve the efficiency of monitoring and conservation of mammals.

Contrasting phylogeographic histories between the continent and islands of East Asia: Massive mitochondrial introgression and long-term isolation of hares (Lagomorpha: Lepus).

Hares of the genus Lepus are distributed worldwide, and introgressive hybridization is thought to be pervasive among species, leading to reticulate evolution and taxonomic confusion. Here, we performed phylogeographic analyses of the following species of hare across East Asia: L. timidus, L. mandshuricus, L. coreanus, and L. brachyurus collected from far-eastern Russia, South Korea, and Japan. Nucleotide sequences of one mitochondrial DNA and eight nuclear gene loci were examined, adding sequences of hares in China from databases. All nuclear DNA analyses supported the clear separation of three phylogroups: L. timidus, L. brachyurus, and the L. mandshuricus complex containing L. coreanus. On the other hand, massive mitochondrial introgression from two L. timidus lineages to the L. mandshuricus complex was suggested in continental East Asia. The northern population of the L. mandshuricus complex was mainly associated with introgression from the continental lineage of L. timidus, possibly since the last glacial period, whereas the southern population of the L. mandshuricus complex experienced introgression from another L. timidus lineage related to the Hokkaido population, possibly before the last glacial period. In contrast to continental hares, no evidence of introgression was found in L. brachyurus in the Japanese Archipelago, which showed the oldest divergence amongst East Asian hare lineages. Our findings suggest that glacial-interglacial climate changes in the circum-Japan Sea region promoted distribution shifts and introgressive hybridization among continental hare species, while the geographic structure of the region contributed to long-term isolation of hares on the islands, preventing inter-species gene flow.

Estimation of Evolutionary Rates of Mitochondrial DNA in Two Japanese Wood Mouse Species Based on Calibrations with Quaternary Environmental Changes.

Reliable estimates of evolutionary rates of mitochondrial DNA might allow us to build realistic evolutionary scenarios covering broad time scales based on phylogenetic inferences. In the present study, we sought to obtain estimates of evolutionary rates in murine rodents using calibrations against historical biogeographic events. We first assumed that land-bridge-like structures that appeared intermittently at glacial maxima with 100,000-year intervals shaped the divergence patterns of cytochrome b (Cytb) sequences (1140 bp) of the larger Japanese wood mouse Apodemus speciosus. The comparison of sequences from peripheral remote islands that are separated from one another by deep straits allowed us to estimate mitochondrial DNA evolutionary rates (substitutions/site/million years) to be 0.027 to 0.036, with presumed calibrations from 140,000, 250,000, 350,000, and 440,000 years ago. Second, we addressed rapid expansion events inferred from analyses of the Cytb sequences of the lesser Japanese wood mouse A. argenteus. We detected five expansion signals in the dataset and established three categories based on the expansion parameter tau values: 3.9, 5.6-5.7, and 7.8-8.1. Considering that the climate became warmer 15,000, 53,000, and 115,000 years ago after preceding periods of rapid cooling, we calculated evolutionary rates to be 0.114, 0.047, and 0.031, respectively. This preliminary concept of the evolutionary rates on a time scale from 15,000 to 440,000 years ago for the wood mouse should be refined and tested in other species of murine rodents, including mice and rats.

Ancient colonization and within-island vicariance revealed by mitochondrial DNA phylogeography of the mountain hare (Lepus timidus) in Hokkaido, Japan.

We examined the phylogenetic status and history of the mountain hare Lepus timidus in and around Hokkaido using mitochondrial cytochrome b (cyt b) sequences from 158 samples from Hokkaido and 14 from Sakhalin, as well as four samples from the Korean hare, L. coreanus. The phylogenetic analysis of the cyt b sequences generated in this study and obtained from DNA databases showed the clear genetic specificity of the Hokkaido lineage as a clade. The Hokkaido lineage was estimated to have diverged from the other conspecific and L. coreanus lineages 0.46 and 0.30 million years ago (Mya), respectively. These results suggest that the common ancestor of the mitochondrial lineage in Hokkaido and Korea inhabited Far East Asia before colonization by the present continental lineages of L. timidus, including the Sakhalin population. We estimated the time of the most recent common ancestor of the Hokkaido population to be 0.17 Mya, and found two distinct haplogroups within the island. One group had greater genetic diversity (mean number of pairwise differences: π = 0.0188 ± 0.0108) and appears to have expanded from the west to the entire island of Hokkaido. The other had lower genetic diversity (π = 0.0038 ± 0.0037) and its distribution was concentrated in the east. These contrasting west/east trends indicate that the Hokkaido population was fragmented in the past, and then subsequently expanded. Our study suggests that Hokkaido was an important refugium for boreal species in the far eastern region, and allowed the formation of various population genetic structures within the island.

The influence of Pleistocene refugia on the evolutionary history of the Japanese hare, Lepus brachyurus.

We performed a phylogeographic analysis of the Japanese hare, Lepus brachyurus, using the mitochondrial cytochrome b gene (1140 bp). In total, 119 haplotypes were recovered from 197 samples isolated from 82 localities on three main islands of the Japanese archipelago: Honshu, Sikoku, Kyushu, Sado Island and the Oki Islands. Results showed two distinct clades at a genetic distance of 3.5%, equivalent to an estimated 1.2 million years. The two clades, encompassing seven subclades, showed an apparent geographic affinity to Kyushu, Shikoku and the nearby area of Honshu (southern group) by one clade, whereas the other clade covered the remaining area of Honshu (northern group). The landscape shape interpolation analysis exhibited a higher genetic diversity in the southern parts of central Honshu (northern group) and Shikoku and Kyushu regions (southern group), suggesting the existence of multiple geographical origins of population expansion in each clade. The Bayesian skyline plot analysis showed that lineage diversifications occurred about 0.35, 0.20 and 0.05 million years ago (Mya), which coincide closely with the glacial-interglacial cycles during the Pleistocene. Therefore, we suggest that the Japanese hare population once inhabited northern and southern refugia, and subsequently developed several populations through local demographic fluctuations. The present day demarcation in the northern and southern geographic groups is considered to be a temporal remnant of Pleistocene population dynamics and the geographic boundary between them could move or fade away in time.