The Effectiveness of Molecular, Karyotype and Morphological Methods in the Identification of Morphologically Conservative Sibling Species: An Integrative Taxonomic Case of the Crocidura attenuata Species Complex in Mainland China.

The conservation of morphology has resulted in considerable issues in the taxonomy of small mammals, especially for the identification of sibling species. Moreover, it is often difficult to completely solve such taxonomic problems by relying only on a single research method. The genus Crocidura is one of the genera with a conservative morphology and high species diversity. Among them, Crocidura attenuata has been considered in the field as the most widely distributed and common species. In fact, it is a species complex containing multiple species, and the classification and distribution of this species is controversial. In this study, the species and distribution of the Crocidura attenuata species complex experienced an integrated revision using three different levels of research methods: molecular, karyotype and morphology. The results show that (1) the C. attenuata species complex contains four known species (C. attenuata, C. tanakae, C. anhuiensis and C. dongyangjiangensis) and a cryptic species distributed in Guangxi, which may be the same undescribed species as the "C. attenuata" distributed in Vietnam. (2) C. attenuata is only distributed around the Sichuan Basin, C. tanakae is the most widely distributed throughout Southern China, and C. anhuiensis and C. dongyangjiangensis are almost sympatric in Southeast China. Furthermore, (3) although the molecular method lacks a unified threshold for species classification, it can rapidly and effectively identify the species of the C. attenuata species complex. Although karyotype and morphology methods cannot completely solve the species classification issues in respect of the C. attenuata species complex, they can provide supplemental information for taxonomic purposes. Therefore, the integrated taxonomic method can present the advantages of different methodological levels, and will provide further evidence for the taxonomy of sibling species with a conservative morphology.

A new shrew mole species of the genus Uropsilus (Eulipotyphla: Talpidae) from northwestern Vietnam.

A new medium-sized shrew mole species of the genus Uropsilus from Mount Fansipan, Hoang Lien National Park, Lao Cai Province, northwestern Vietnam is described based on morphological and molecular differences. Uropsilus fansipanensis sp. nov. is distinguished from the other Uropsilus species by the combination of the following features: the dorsum is lightly reddish-brown and venter is dark gray; the dark gray tail is long and slender, with a scattered white base and short bristle hairs; orbital process is oriented upwards posteriorly; lacrimal foramen is well developed and much larger than infraorbital foramen; the lower first premolar is approximately the same size as the lower third premolar. Genetic distances in terms of mitochondrial cytochrome b from other Uropsilus species presented pairwise divergences from 8.63 to 20.70%. To date, the new species is known to exist only in the type locality of Mt. Fansipan, a wet and cold temperate climate area with an upper montane forest at an elevation of approximately 2900 m, forming the southernmost distribution of the genus Uropsilus.

Osteological and vascular morphology and electrolyte homeostasis of sea turtles.

Sea turtles have well developed lacrimal glands for their electrolyte homeostasis. In turtles, stapedial artery and palatine artery send branches to supply orbital region, but supply artery for lacrimal glands was not identified. Micro-CT scans showed dorsoventrally large lacrimal glands of sea turtle are supplied by both stapedial artery and palatine artery. The circulatory pattern in cranial region was reconstructed based on the micro-CT scans, showing that sea turtle has basically similar pattern with the common snapping turtle: stapedial artery supplies orbital region and mandibular artery is ramified from stapedial artery. We also investigate the foramen stapedio-temporalis in turtles using osteological specimens. The foramen stapedio-temporalis, where the stapedial artery passes through, has different size among four families of turtles. We compared the sum of cross sections of left and right foramen stapedio-temporalis since homeostasis of one individual is maintained by a pair of lacrimal glands. The size difference may reflect primarily the share of stapedial artery against palatine artery in cranial circulation pattern and blood supply of orbital regions. Our observations confirmed a significantly larger cross-section in the foramen stapedio-temporalis of sea turtles than other freshwater/terrestrial turtles. Since the circulatory pattern is shared, the size difference of foramen stapedio-temporalis reflects the amount of arterial blood supply to lacrimal glands. Therefore, the size of the foramen stapedio-temporalis may indicate marine adaptation of turtles and are applicable to both fossil and osteological specimens.

Comparative morphological study of skeletal muscle weight among the red jungle fowl (Gallus gallus) and various fowl breeds (Gallus domesticus).

We examined the weight distribution of skeletal muscles of the red jungle fowl, then compared these values with those of domesticated populations to determine how muscle distribution has changed by selecting breeding. Sonia, Fayoumi, and Rhode Island Red were selected for comparison from livestock breeds, while Japanese Shamo and Thai fighting cocks were selected from cockfighting groups. Principal component analysis was applied using body size-free data. The mass distribution of muscles clearly differed between the wild, livestock, and cockfighting groups, demonstrating that muscle distribution has changed after selecting breeding, coupled with functional demands of each group. The red jungle fowl, which has the ability to fly, could be clearly distinguished from the flightless domesticated populations due to differences in flight pectoral muscle size. The cervical muscles in the wild population were smaller than in the domesticated groups; these do not contribute to flight. The gluteal muscles were larger in the fighting cock group, functionally coupled to their traditionally preferred upright posture. Wild bird populations typically exhibit reduced weight of their hind limbs, associated with flight, but as the red jungle fowl displays largely terrestrial behavior, these muscles are similar in arrangement and relative size to those of the livestock groups. We showed that the mass distribution pattern of skeletal muscles expresses selecting breeding strategy and clearly reflects the specific traits for each group.

Rodent-Borne Orthohantaviruses in Vietnam, Madagascar and Japan.

Hantaviruses are harbored by multiple small mammal species in Asia, Europe, Africa, and the Americas. To ascertain the geographic distribution and virus-host relationships of rodent-borne hantaviruses in Japan, Vietnam, Myanmar, and Madagascar, RNAlater™-preserved lung tissues of 981 rodents representing 40 species, collected in 2011-2017, were analyzed for hantavirus RNA by RT-PCR. Our data showed Hantaan orthohantavirus Da Bie Shan strain in the Chinese white-bellied rat (Niviventer confucianus) in Vietnam, Thailand; orthohantavirus Anjo strain in the black rat (Rattus rattus) in Madagascar; and Puumala orthohantavirus Hokkaido strain in the grey-sided vole (Myodes rufocanus) in Japan. The Hokkaido strain of Puumala virus was also detected in the large Japanese field mouse (Apodemus speciosus) and small Japanese field mouse (Apodemus argenteus), with evidence of host-switching as determined by co-phylogeny mapping.

Zoogeographical barriers causing discontinuous osteometrical variations in the northern treeshrew skulls.

Morphological variation of the skull was examined in the northern treeshrew (Tupaia belangeri) from various localities across Southeast Asia. Through a multivariate analysis, the treeshrews from South Vietnam exhibited distinct morphological characteristics compared to other populations from Thailand and Laos, and Malaysia. The plots of the specimens of North Vietnam are not randomly mixed with Thailand plots segregation in the scatteregrams of canonical discriminant analysis. Since the skulls of the population from North Vietnam were morphologically similar to those form central Laos and northern and northeastern Thailand, the zoogeographical barrier effect of Mekong River was not clearly confirmed. The population of the Kanchanaburi in western Thailand is clearly smaller in size compared to the other populations. The southern border of the distribution of this species is determined by the Isthmus of Kra or Kangar-Pattani Line. In the northern treeshrew, which is distributed from southern China to Bangladesh and southern Thailand, we have detected osteometrical geographical variation driven by geography. These results indicate that the skull morphology in the Tupaia glis-belangeri complex distinctively differs in South Vietnam, western Thailand, and southern Thailand. The zoogeographical barrier and factor separating these districts are expected to clarify in the future.

Complex Historical Biogeography of the Eastern Japanese Skink, Plestiodon finitimus (Scincidae, Squamata), Revealed by Geographic Variation in Molecular and Morphological Characters.

We investigated the geographic diversification of Plestiodon finitimus, which occurs in the central to northern parts of the Japanese Islands, based on a time-calibrated mitochondrial DNA (mtDNA) phylogeny and external morphological characters. The mtDNA phylogeny suggests that P. finitimus diverged from its sister species Plestiodon japonicus in western Japan 2.82-4.63 million years ago (MYA), which can be explained by geographic isolation due to the spread of sedimentary basins in the Pliocene. The primary intraspecific divergence was that between P. finitimus lineages in central and northeastern Japan 1.58-2.76 MYA, which could have been caused by the upliftings of major mountain ranges. In the northeastern lineage, mtDNA and morphological characters suggest a geographic differentiation between sub-lineages of the northwestern Tohoku District (α) and other areas (ß). Although the sub-lineage ß occurs in a disjunct geographic range, consisting of Hokkaido and the central to south of Tohoku, these areas are bridged by populations with intermediate characteristics along the Pacific side of northern Tohoku. Overall, the geographic variation in P. finitimus in northern Japan can be explained by an initial allopatric divergence of the sub-lineages α and ß at 0.71-1.39 MYA, a recent northward expansion of the sub-lineage ß, and subsequent secondary introgressive hybridization between the sub-lineages.

Phylogeography of the Japanese greater horseshoe bat Rhinolophus nippon (Mammalia: Chiroptera) in Northeast Asia: New insight into the monophyly of the Japanese populations.

The Japanese greater horseshoe bat (Rhinolophus nippon) is distributed widely in East Asia. Within the species, R. nippon in Northeast Asia is regarded as the lineage that diverged most recently. However, the monophyly of the Japanese populations is unclear due to insufficient data about phylogenetic relationship of the western Japanese populations. To test the monophyly of the Japanese populations of R. nippon, we sampled R. nippon from western Japan and performed a phylogeographic analysis based on mitochondrial DNA cytochrome b and the D-loop. The Northeast Asian lineage consisted of three main clades in eastern Japan (clade I), western Japan (clade II), and the continent as well as the Kumamoto population in westernmost Japan (clade III). The results of this study do not support the monophyly of the Japanese population. The findings suggest the "reverse colonization" of R. nippon from the Japanese Archipelago to the Eurasian continent, and provide important insight into the role of the island system in creation and supply of diversity to the continent.

Genetic Diversity and Phylogeography of Thottapalayam thottimvirus (Hantaviridae) in Asian House Shrew (Suncus murinus) in Eurasia.

Murid and cricetid rodents were previously believed to be the principal reservoir hosts of hantaviruses. Recently, however, multiple newfound hantaviruses have been discovered in shrews, moles, and bats, suggesting a complex evolutionary history. Little is known about the genetic diversity and geographic distribution of the prototype shrew-borne hantavirus, Thottapalayam thottimvirus (TPMV), carried by the Asian house shrew (Suncus murinus), which is widespread in Asia, Africa, and the Middle East. Comparison of TPMV genomic sequences from two Asian house shrews captured in Myanmar and Pakistan with TPMV strains in GenBank revealed that the Myanmar TPMV strain (H2763) was closely related to the prototype TPMV strain (VRC66412) from India. In the L-segment tree, on the other hand, the Pakistan TPMV strain (PK3629) appeared to be the most divergent, followed by TPMV strains from Nepal, then the Indian-Myanmar strains, and finally TPMV strains from China. The Myanmar strain of TPMV showed sequence similarity of 79.3-96.1% at the nucleotide level, but the deduced amino acid sequences showed a high degree of conservation of more than 94% with TPMV strains from Nepal, India, Pakistan, and China. Cophylogenetic analysis of host cytochrome b and TPMV strains suggested that the Pakistan TPMV strain was mismatched. Phylogenetic trees, based on host cytochrome b and cytochrome c oxidase subunit I genes of mitochondrial DNA, and on host recombination activating gene 1 of nuclear DNA, suggested that the Asian house shrew and Asian highland shrew (Suncus montanus) comprised a species complex. Overall, the geographic-specific clustering of TPMV strains in Asian countries suggested local host-specific adaptation. Additional in-depth studies are warranted to ascertain if TPMV originated in Asian house shrews on the Indian subcontinent.

A revision of the geographical distributions of the shrews Crocidura tanakae and C. attenuata based on genetic species identification in the mainland of China.

The Taiwanese gray shrew (Crocidura tanakae) and Asian gray shrew (C. attenuata) are so similar in size and morphology that the taxonomic status of the former has changed several times since its description; C. tanakae has also been regarded as an endemic species of Taiwan Island. In recent years, molecular identification has led to several reports of C. tanakae being distributed in the mainland of China. In this study, we determine the geographical distribution of C. attenuata and C. tanakae based on more than one hundred specimens collected during 2000 to 2018 over a wide area covering the traditional ranges of the two species in the mainland of China, and show a substantial revision of their distributions. Among 110 individuals, 33 C. attenuata and 77 C. tanakae were identified by Cytb gene and morphologies. Our results show, (1) C. attenuata and C. tanakae are distributed sympatrically in the mainland of China; (2) contrary to the previous reports, the distribution range of C. attenuata is restricted and much smaller than that of C. tanakae in the mainland of China; (3) Hainan Island, like Taiwan Island, is inhabited by C. tanakae only according to the present data.

Dental integration and modularity in pinnipeds.

Morphological integration and modularity are important for understanding phenotypic evolution because they constrain variation subjected to selection and enable independent evolution of functional and developmental units. We report dental integration and modularity in representative otariid (Eumetopias jubatus, Callorhinus ursinus) and phocid (Phoca largha, Histriophoca fasciata) species of Pinnipedia. This is the first study of integration and modularity in a secondarily simplified dentition with simple occlusion. Integration was stronger in both otariid species than in either phocid species and related positively to dental occlusion and negatively to both modularity and tooth-size variability across all the species. The canines and third upper incisor were most strongly integrated, comprising a module that likely serves as occlusal guides for the postcanines. There was no or weak modularity among tooth classes. The reported integration is stronger than or similar to that in mammals with complex dentition and refined occlusion. We hypothesise that this strong integration is driven by dental occlusion, and that it is enabled by reduction of modularity that constrains overall integration in complex dentitions. We propose that modularity was reduced in pinnipeds during the transition to aquatic life in association with the origin of pierce-feeding and loss of mastication caused by underwater feeding.

Molecular phylogeographic analyses and species delimitations reveal that Leopoldamys edwardsi (Rodentia: Muridae) is a species complex.

Leopoldamys edwardsi is a species with wide distribution ranges in southern China but is not discussed in studies on geographic variation and species differentiation. We used 2 mitochondrial (Cytb, CO1) and 3 nuclear (GHR, IRBP and RAG1) genes to clarify species phylogeography and geographical differentiation. Maximum likelihood (ML) and Bayesian phylogenetic inference (BI) trees consistently indicated that L. edwardsi is a species complex containing 3 main lineages with high Kimura-2-parameter (K2P) divergences (i.e. lineages LN , LS and LHN ) found in the northern and southern China and Hainan Island, respectively. The 3 species delimitation methods, automated barcoding gap discovery, Bayesian poisson tree process analysis and Bayesian phylogenetics and phylogeography, consistently supported the existence of cryptic species. Divergence times among the main lineages were inferred to be during the Pleistocene, with LHN /LS split at 1.33 Ma and LN /(LHN +LS ) at 2.61 Ma; the diversifications of L. edwardsi complex might be caused by the rapid uplifts of Tibetan Plateau, paleoclimate change and complex topography. The divergence between LHN and LS was probably related to the separation of Hainan Island from the mainland via the formation of the Qiongzhou Strait. Lineages LN and (LS +LHN ) likely diverged due to the Wuyi-Nanling mountain range forming a dispersal barrier. Our results suggested that L. edwardsi complex contains at least 3 distinct species: LHN represents L. hainanensis, endemic to Hainan Island and previously considered as a subspecies L. e. hainanensis; LS represents a cryptic species distributed throughout the southern Chinese continent; and LN represents the nominotypical species L. edwardsi.

Karyotypes of field mice of the genus Apodemus (Mammalia: Rodentia) from China.

Karyotypes of four Chinese species of field mice of the genus Apodemus were examined, including Apodemus chevrieri (diploid chromosome number, 2n=48, fundamental number of autosomal arms, FNa=56), A. draco (2n=48, FNa=48), A. ilex (2n=48, FNa=48), and A. latronum (2n=48, FNa=48). Karyotypes of A. chevrieri, A. draco, and A. ilex are reported here for the first time, providing useful information for their species taxonomy. Determining the karyotypes of all species of Apodemus in Asia, both in this and previous studies, provides a solid overview of the chromosome evolution and species differentiation of the genus in East Asia. In addition to allopatric speciation, chromosome rearrangements likely played an important role in the formation of the four Apodemus species groups as well as speciation within each group in East Asia. For example, increased centromeric heterochromatin in A. latronum may have contributed to the post-mating reproductive isolation from the A. draco-A. ilex-A. semotus clade.

Complete mitochondrial genome of maritime striped squirrel Tamiops maritimus (Rodentia: Sciuridae).

The complete mitochondrial genome of the maritime striped squirrel (Tamiops maritimus) was first sequenced and characterized. The genome was 16 523 bp in length, and the composition and the arrangement of genes were analogous to other rodents. The sequence of T. maritimus was used to construct phylogenetic tree with additional mitochondrial genomes of seven sciurid species available on GenBank. Phylogenetic result indicated that T. maritimus has a close relationship with T. swinhoei. Our mitochondrial genome data may provide information for species identification, diversity evaluation, and other studies about this genus.

Effects of Usag-1 and Bmp7 deficiencies on murine tooth morphogenesis.

BACKGROUND: Wnt5a and Mrfzb1 genes are involved in the regulation of tooth size, and their expression levels are similar to that of Bmp7 during morphogenesis, including during the cap and early bell stages of tooth formation. We previously reported that Usag-1-deficient mice form supernumerary maxillary incisors. Thus, we hypothesized that BMP7 and USAG-1 signaling molecules may play important roles in tooth morphogenesis. In this study, we established double genetically modified mice to examine the in vivo inter-relationships between Bmp7 and Usag-1. RESULTS: We measured the volume and cross-sectional areas of the mandibular incisors using micro-computed tomography (micro-CT) in adult Bmp7- and Usag-1-LacZ knock-in mice and their F2 generation upon interbreeding. The mandibular incisors of adult Bmp7+/- mice were significantly larger than those of wild-type (WT) mice. The mandibular incisors of adult Usag-1-/- mice were the largest of all genotypes examined. In the F2 generation, the effects of these genes were additive; Bmp7+/- was most strongly associated with the increase in tooth size using generalized linear models, and the total area of mandibular supernumerary incisors of Usag-1-/-Bmp7+/- mice was significantly larger than that of Usag-1-/-Bmp7 +/+ mice. At embryonic day 15 (E15), BrdU assays demonstrated that the labeling index of Bmp7+/- embryos was significantly higher than that of WT embryos in the cervical loop. Additionally, the labeling index of Usag-1-/- embryos was significantly the highest of all genotypes examined in dental papilla. CONCLUSIONS: Bmp7 heterozygous mice exhibited significantly increased tooth sizes, suggesting that tooth size was controlled by specific gene expression. Our findings may be useful in applications of regenerative medicine and dentistry.

Geographic Variation in Skull Morphology of the Large Japanese Field Mice, Apodemus speciosus (Rodentia: Muridae) Revealed by Geometric Morphometric Analysis.

We analyzed geographic variation in skull morphology of the large Japanese field mouse (Apodemus speciosus) and determined changes in skull morphology that occurred during the evolutionary history of A. speciosus in relation to the estimated distribution range in the last glacial maximum (LGM). We analyzed 1,416 specimens from 78 localities using geometric morphometric techniques applied to the dorsal side of the cranium and mandible. While large variations within and among the populations in Honshu, Shikoku, and Kyushu were observed, geographic patterns were not observed. Hokkaido and peripheral island populations showed shared differentiation from the Honshu, Shikoku, and Kyushu populations with a larger skull and distinct mandible shape. In addition, these two groups also differed from each other in accumulated random shape variation. Common characteristics found in Hokkaido and peripheral island populations were considered to be the ancestral states, which were retained by geographic isolation from the main islands. Random variations in Hokkaido and the peripheral island populations were formed through stochastic processes in relation to their isolation. Characteristic morphologies widely found in the populations of Honshu, Shikoku, and Kyushu were considered to be derived states that expanded after separation from the peripheral islands. Complex geomorphology and a shift in distribution range related to climate change and altitudinal distribution are suggested to have formed the complex geographic variation in this species.

Complete mitochondrial genome of the mandarin vole Lasiopodomys mandarinus (Rodentia: Cricetidae).

The complete sequence of the mitochondrial genome of the mandarin vole (Lasiopodomys mandarinus) was completed and annotated in this study. The circular genome is 16,375 bp in length and contains the typical 37 genes that are arranged in the same order as that of the putative ancestor of vertebrate, 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes and 2 non-coding regions. This study will provide genetic resource to clarify the taxonomic position of genus Lasiopodomys.

The nearly complete mitochondrial genome of Chinese pygmy dormouse Typhlomys cinereus (Rodentia: Platacanthomyidae).

In this study, we report the nearly complete mitochondrial genome of a Chinese pygmy dormouse Typhlomys cinereus (Rodentia: Platacanthomyidae). The 15,011 bp genome is consisted of 13 protein-coding genes, 16S rRNA, 21 tRNAs, partial 12S rRNA and control region. A phylogenetic tree was built using 12 protein-coding genes of 19 species from Dipodoidea and Muroidea. Our result shows that T. cinereus represents the earliest split within Muroidea. The genome would contribute to further study of phylogeny in Muroidea and Rodentia.

Complete mitochondrial genome of Perny's long-nosed squirrel Dremomys pernyi (Rodentia: Sciuridae).

The complete mitochondrial genome of Perny's long-nosed squirrel (Dremomys pernyi) was firstly sequenced and characterized. The genome was 16,573 bp in length, and its composition and arrangement of genes were analogous to other rodents. The sequences of 13 protein-coding genes were used to construct phylogenetic tree for D. pernyi and other 13 sciurid species available on GenBank. To date, this is the first species whose complete mitochondrial genome sequence was sequenced in genus Dremomys. Our results will provide information for further molecular studies.

A morphological analysis of the skull size and shape of Kerivoulinae (Chiroptera: Vespertilionidae) from Vietnam.

Southeast Asia is a region of high biodiversity, containing species of plants and animals that are yet to be discovered. In this region, bats of the subfamily Kerivoulinae are widespread and diverse with six species recorded in Vietnam. However, the taxonomy of the Kerivoulinae in Asia is complicated. In our study, we used diagnostic characters and multivariate analysis to determine morphological differences between the genera Kerivoula and Phoniscus. We showed that the two genera are distinguishable by the size of second upper incisors, the shape of skull, nasal sinus, canines, second upper and lower premolars. In addition, the two genera can be osteometrically separated by measurements of the braincase height, interorbital width and shape of anterior palatal emargination. Our data clearly revealed the morphological variations in the skull shape of Kerivoula hardwickii in Vietnam. This suggests a possible separation into three morphotypes, representing cryptic species supported by statistical differences with wide variation in skull shape, size and teeth. These results demonstrated Kerivoula hardwickii can be separated three subspecies, and the result will serve as the basis for the future assessment and classification of this group in Southeast Asia.