Mitochondrial and nuclear gene-trees discordance
Discordance between mtDNA and nuDNA gene-trees could be attributed to incomplete lineage sorting [21], introgression [22] and explosive speciation [23]. Explosive speciation (i.e., rapid radiation) offers an explanation for the discrepancy owing to the very short branches that have poor support. Our time-calibrated Bayesian analysis indicates rapid cladogenesis from the late Pliocene to the Early Pleistocene period (Fig. 3), where most diversification occurred. Such rapid speciation can preclude resolution of the precise sequence of events because phylogenetically informative DNA substitutions will not have time to accumulate (e.g., [24, 25]). Another explanation for the discrepancy is an old introgression event. For example, there is strong support for the clade C. lasuria + C. kurodai in mtDNA analyses and a small genetic distance between these two species. However, the two species are very different and do not cluster together in nDNA analyses.
Phylogeny and rapid radiation among China’s Crocidura species
Our fine-scale sampling of Crocidura from China reveals two well-supported major groups of Crocidura (Fig. 2). The group 1 seemingly represents north-western species of Crocidura, and the group 2 represents species from south-eastern China. However, the phylogenetic relationships among manyspecies in the group 2 remain unresolved (Fig. 2). Previous phylogenetic studies of Philippine Crocidura using multilocus data also yielded many poorly supported branches [13,14]. Even using hundreds of ultraconserved elements and whole mitogenomes, Giarla and Esselstyn [26] could not fully resolve the phylogenetic relationships among Philippine Crocidura. Perhaps only analyses of whole genomes may resolve the phylogenetic relationships in the group.
Climate change and periodic orogenesis may have driven the diversification of China’s Crocidura. According to our estimation, China Crocidura specieshave rapidly diversified from the late Pliocene to the early Pleistocene period, followed by a series of diversifications through the Pleistocene period. Prevailing trends towards cooling and desiccation at the Pliocene/Pleistocene boundary [27] have led to the diversification of mesophilic taxa (e.g., [16, 25]). A cooling event resulted in habitat changes and the consequent rapidly diversification among most species of Crocidura, as did rapid uplifting of the QTP at 3.6 Ma, 1.8 Ma and 1.1 Ma [28].
Taxonomic implications and diversity of Crocidura in China
Our data and analyses confirm the taxonomic status of the six well-recognized species: C. shantungensis, C. dracula, C. suaveolens, C. vorax, C. lasiura, and C. tanakae (Fig. 2; Additional file 4: Table S4). The mitochondrial and nuclear gene trees resolve each species as a well-demarcated clade, with substantial genetic differences between them (Table 3). Species delimitation analyses also identify each of them as a full species (Additional file 4: Table S4).
Jameson and Jones [29] originally described C. horsfieldii kurodai. Subsequently, Jiang and Hoffmann [2] revised the genus Crocidura in southern China and placed C. tadae and C. kurodai from Taiwan as junior synonyms of C. rapax. Hutterer [1] recognized three subspecies, C. rapax tadae, C. r. kurodai, and C. r. lutaoensis following Fang and Lee [30]. This view was followed by Hoffmann and Lunde [31], and Jenkins et al. [32]. Our analyses suggested the two subspecies, C. rapax rapax and C. rapax kurodai, should be elevated to full species status based on a high level of divergence (Table 3) and GMYC and BPP analyses results (Fig. 3 and Additional file 4: Table S4).
Interestingly, When the C. attenuate samples from Vietnam were added, the phylogenetic tree showedthat C. r. rapax was embedded within C. attenuate, making the latter a paraphyletic group (Fig. 3). Although the genetic distance between the two species is only 0.049, this distance is greater than the genetic distance ( = 0.039) between C. kurodai and C. lasiura. GMYC and BPP analysis strongly supported that they were two different species.We also examined a number of specimens including near type site specimens (Baoxing of Sichuan), and found that they were completely different in individual size, skull size and altitude distribution (unpublished data). In addition, C. attenuata population from Vietnam is supported as a separate species in GMYC. Therefore, further research will be needed to clarify the taxonomic status of the C. attenuate complex in the future.
Crocidura wuchihensis was originally described from Hainan by Shou et al. [33]. Subsequently, Lunde et al. [34] identified one specimen from Vietnam as C. wuchihensis. Some specimens of Crocidura from Guangxi and Vietnam also have been referred to as C. wuchihensis [13]. Jenkins et al. [32] stated that C. wuchihensis was widely distributed across Vietnam, including the provinces Lao Cai, Ha Giang and Lang Son in the north and Ha Thinh and Quang Nam in Central Vietnam. Bannikova et al. [3] considered the distribution of C. wuchihensis to be restricted to areas east and north of the Red River. However, our analyses contradicted these assertions. We have tested and compared the holotype from the Institute of Zoology of the Chinese Academy of Sciences. Our analyses suggested that the populationof C. wuchihensis_HN from Hainanand those of C. wuchihensis_GX from Guangxi and Vietnamare valid species, which implies that the true C. wuchihensis may only occur on Hainan Island. Our results call for the reappraisal of the taxonomic status of shrews previously referred to as C. wuchihensis from Guangxi and Vietnam.
Crocidura indochinesis were previously considered a subspecies of C. horsfieldii [35]. Subsequent, Lunde et al. [36] considered C. indochinesis from Ke Go Nature Reserve, Vietnam as a full species. Our GMYC analysis strongly supported it as a species. Whether there is a distribution of C. indochinesis in China is still unknown, because we don’t know if the population in southern China are the conspecific with those in Vietnam.
Crocidura suaveolens is not known in China [2]. However, Dubey et al. [11] and Bannikova et al. [37] considered C. sibirica distributed in Xinjiang, China as Crocidura suaveolens, based on nuclear genes and mitochondria. Subsequently, specimens of C. aff. suaveolens have been reported from southern Gansu, China [38]. These specimens form the sister-group of C. suaveolens. Our GMYC analysis supported the population as a potential speciesFig. 3). In addition, Jiang and Hoffmann [2] listed C. gmelini from Central Asia, including Xinjiang, China. However, Ohdachi et al. [9] stated “It is possible that C. sibirica ( = C. suaveolens) might be a synonym of C. gmelini”.. All Crocidura from seven sample sites of Xinjiang appear to be C. suaveolens (Fig. 1). Therefore, it is necessary to sample C. gmelini to clarify the phylogenetic relationships and taxonomy between itand C. suaveolens. The occurrence of C. gmelini in China remains uncertain.
Crocidura dracula Thomas, 1912 [39] was first described from Mengzi of Yunnan, China. Subsequently, the taxonomic design was followed by Allen [40] and Ellerman and Morrison-Scott [41]. However, Jenkins [42] made C. dracula a subspecies of C. fuliginosa. Others have followed the arrangement [1, 2]. However, chromosomal and recently genetics studies suggested that these two are different species [3, 4, 12, 15, 43]. Burgin and He [4] considered the two forms as full species. Our analyses also supported three clades ofC. fuliginosa groups that represent three different species: C. dracula from Northern Vietnam and southern China, C. fuliginosa from southern Vietnam, Cambodia and Malaysia, and C. sp. 3 from Motuo, Xizang. It is worth noting that whether C. fuliginosa from Southern Vietnam, Cambodia and Malaysia were conspecific with those from the type locality in Burma has still not been tested. In addition, a population was also reported as C. dracula grisescens in Zhejiang [44]. Jiang and Hoffmann [2] considered the population to probably represented an undescribed taxon.
Our analyses also resolved another cryptic species, Crocidura sp. 1, despite only one specimen being available from Zada, Xizang. This species is closely related to the Zarudny’s rock shrew (Crocidura zarudnyi) from Iran based on the cytb gene. It is the sister-group of C. shantungensis and C. suaveolens based on nucleargenes. The condyloincisive length of the cryptic white-toothed shrew (19.55 mm; our unpublished data) is close in size to C. attenuata in China. Despite considerable effort, only one specimen was collected. The dearth of specimens precludes an adequate assessment of its taxonomic status. The continuous discovery of new species such as Bufo zamdaensis [45] and Laudakia papenfussi [46] shows a strong need for further exploration in the region.
Another cryptic species C. sp. 2 from Xuefengshan, Hunan and Dongyang, Zhejiang was also strongly supported. It was not identified as any known species based on our morphological data. Genetically, it has a high level of divergence from all other members of the group (10.2%–14.7%) (Table 3) and appears as a monophyletic group in the molecular phylogenetic tree (Fig. 2; Fig. 3). Thus, C. sp.2 is another likely new species. Molecular analyses offer important insights but extensive sampling and comprehensive morphological and morphometric comparisons are necessary to reach a final conclusion.