﻿A new species of Asiatic shrew of the genus Chodsigoa (Soricidae, Eulipotyphla, Mammalia) from the Dabie Mountains, Anhui Province, eastern China

﻿Abstract Asiatic shrews of the genus Chodsigoa (Soricidae, Eulipotyphla) currently comprise nine species, mostly occurring in southwest China. From May 2017 to August 2020, 11 specimens of Chodsigoa were collected from the Dabie Mountains in Anhui Province, eastern China. Their morphology was compared with other species within the genus and one mitochondrial (cytochrome b) and two nuclear (apolipoprotein B and breast cancer 1) genes were sequenced to estimate the phylogenetic relationships of these specimens. Based on morphological and molecular evidence, these specimens are recognized as a distinct species, Chodsigoadabieshanensissp. nov., which is formally described here. Morphologically, the new species is most similar to Chodsigoahypsibia, but it is distinguishable from all known congeners by the combination of dark brownish pelage, small size, and relatively short tail. Phylogenetic analyses revealed that C.dabieshanensissp. nov. forms a phylogenetic lineage sister to the clade containing C.parva + C.hypsibia. The-Kimura 2-parameter genetic distances of the cytochrome b (CYT B) gene between the new species and other nominal Chodsigoa species ranged between 8.6 and 17.6%. The new species is distributed at elevations from 750 to 1250 m in the Dabie Mountains and is geographically distant from other species in the genus.


Materials and methods
A total of 11 Chodsigoa specimens were collected from May 2017 to August 2020 from Yaoluoping National Nature Reserve (n = 1), Bancang Natural Reserve (n = 4), and Foziling Natural Reserve (n = 6), all located in the Dabie Mountains, Anhui province, eastern China (Fig. 1). Shrews were sampled using the pitfalls (plastic buckets 15 cm in diameter and 28 cm in depth). Specimens were euthanized and liver or muscle tissues were extracted and preserved in pure ethanol. Skulls were also extracted and cleaned. Specimens and tissues were deposited at the Biological Museum of Anhui University (BMAHU). Animals were handled consistent with the animal care and use guidelines of the American Society of Mammologists (Sikes et al. 2016), and also following the guidelines and regulations approved by the internal review board of Anhui Normal University, and with the permissions of local authorities.
To evaluate the morphological variation among populations of Chodsigoa, we performed a principal component analysis (PCA) in SPSS 19.0 (SPSS Inc., USA) using the log 10 -transformed craniodental measurements. We compared the morphology of the putative new species with other Chodsigoa species stored in Kunming Institute of Zoology (KIZ), the Sichuan Academy of Forestry (SAF), the Museum of Comparative Zoology, Harvard University (MCZ), and the American National Museum of Natural History (AMNH). The terminology for morphological descriptions followed Hoffman (1985), Lunde et al. (2003), and Chen et al. (2017).
Total genomic DNA of 10 C. dabieshanensis specimens were extracted using a DNA extraction kit (Qiagen DNeasy Blood and Tissue Kit, China). The complete CYT B gene and two nuclear gene segments [apolipoprotein B (APOB) and breast cancer 1 (BRCA1)] were amplified using primers and PCR conditions from Chen et al. (2021). The PCR products were purified and sequenced in both directions using the BigDye Terminator Cycle kit v. 3.1 (Invitrogen, USA) on an ABI 3730xl sequencer (Applied Biosystems, USA). Corresponding sequences of other Chodsigoa species were downloaded from GenBank (Table 1) and aligned with our new sequences using MUSCLE (Edgar 2004) and then checked manually by eye. Sequences of Episoriculus caudatus (Horsfield, 1851) and Neomys fodiens (Pennant, 1771) were included in the alignments as outgroup taxa. The Kimura-2-parameter (K2P) distances of the CYT B gene between species were calculated in MEGA 7 (Kumar et al. 2016).
Three datasets were used for the phylogenetic analyses: CYT B gene, concatenated nuclear genes, and concatenated mitochondrial and nuclear genes (Table 1). Maximum likelihood (ML) and Bayesian inference (BI) analyses were performed to reconstruct the phylogenetic relationships in PhyloSuite (Zhang et al. 2020) based on the best-fit partitioning schemes estimated using PartitionFinder v. 2.0 (Lanfear et al. 2012). The ultrafast bootstrap values (UFBoot) ≥ 95 and posterior probabilities (PP) ≥ 0.95 were considered as strong supports (Huelsenbeck and Rannala 2004;Minh et al. 2018).

Results
External and cranial measurements are summarized in Table 2. The PCA based on 128 intact skulls produced two axes with eigenvalues exceeding 1.0, which explained 94.2% of the variation (Table 3). The first axes (PC1) explained 86.2% of the variation and was strongly positively correlated with all variables, indicating it represented the overall skull size (Table 3). The second axis (PC2) explained 8.0% of the variation and was highly positively correlated with CH and BMF (loading > 0.67). A plot of PC1 and PC2 ( Fig. 2) showing that C. dabieshanensis are separated well from all named species. This new species occurs in the center of the morphospace, indicating its medium size in the genus. Morphologically, it is most similar to C. hypsibia, with which it occupies the upper left corner morphospace without overlap (Fig. 2), indicating its generally smaller size, larger BMF, and higher CH (Table 2).   Nine CYT B (1140 bp), nine APOB (513 bp), and nine BRCA1 (768 bp) sequences of C. dabieshanensis were obtained (GenBank accession numbers: OM200108-OM200133; Table 1). The ML and BI trees recovered very similar topologies, and therefore, only the ML gene trees are shown (Fig. 3). The phylogenetic analyses of all three datasets supported Chodsigoa clustered into two major clades (UFboot > 99, PP = 1.00). One clade was composed of C. parva, C. hypsibia, and C. dabieshanensis (Clade I), and the other clade was composed of C. caovansunga, C. furva, C. hoffmanni, C. parca, C. salenskii, C. smithii, and C. sodalis (Clade II). The C. dabieshanensis clade was strongly supported as a monophyletic lineage, sister to the clade containing C. parva and C. hypsibia (UFboot > 98, PP = 1.00). The K2P genetic distances of the CYT B gene between C. dabieshanensis and other nominal Chodsigoa species ranged from 8.6% (with C. hypsibia) to 17.6% (with C. sodalis) ( Table 4).
Based on the morphological, morphometric, and molecular evidence and the modern phylogenetic species concept (phylogenetic species concept based on both diagnosability and monophyly as operational criteria) (Mayden 1997;Gutierrez and Garbino 2018), we recognize the population from the Dabie Mountains as a new species of Chodsigoa, which is formally described below.
Paratypes. AHUDBS017001-005; AHU2008FZL001-004, 006. Ten specimens collected between May 2017 and August 2020 from the Dabie Mountains, Anhui   Etymology. The specific name dabieshanensis is derived from the Dabie Mountains, the type locality of the new species: -shan means mountain in Chinese, and the Latin adjectival suffix -ensis means "belonging to".
Diagnosis. The new species is assigned to the genus Chodsigoa for having three upper unicuspid teeth, with the tips of the teeth lightly pigmented (Fig. 4). Chodsigoa dabieshanensis sp. nov. can be distinguished from the other known species of Chodsigoa by the following combination of characters: small to medium in size (HB = 67.22 mm; CIL = 19.08 mm), dark brownish pelage; tail shorter than the HB, nearly similar ventral and dorsal pelage color, a small tuft of longer hairs at the tip of the tail (Fig. 5); markedly flattened braincase; and the foramen magnum is relatively wider than C. hypsibia. Phylogenetic analyses show that the new species is monotypic, sister to C. hypsibia and C. parva (Fig. 3).
Description. A small to medium-sized shrew (W = 5.24±0.36 g, range 4.67-5.89 g; HB = 67.22±3.23 mm, range 62.00-73.00 mm, Table 2) with dark brown dorsal pelage and slightly paler ventral pelage (Fig. 5). Tail is short (TL = 59.67±3.28 mm), about 90% of the head and body length, brown above, slightly paler below, and with a small tuft of longer hairs at the tip. External ears are prominent, rounded, and covered with very short dark hairs. Eyes are very small. The dorsal surfaces of hands and hind feet are covered with short brown hair, lighter at the margin. The thenar and hypothenar pads at the soles of the hindfeet are well separated.
The skull of C. dabieshanensis sp. nov. is short and broad, and the braincase is markedly flattened (Fig. 4). The skull is similar to C. hypsibia, but much shorter and broader. The rostrum is short, and the interorbital region is wide. From the ventral view, the rostrum gradually narrows in the premaxillary region. The palate is short, with an abrupt posterior edge. The basisoccipital is developed and the ridges are approximately parallel. The dentition is the same for the genus: 3.1.1.3/2.0.1.3 = 28. The first incisor is long, falciform; the apex straight downwards; the talon much lower than U 1 , approximately equal to U 3 . Three upper unicuspids are present. All unicuspids are crowded and overlap slightly at the base. U 1 -U 3 gradually decrease in size; U 3 is about half as high as U 1 , and in contact with P 4 , which is large and triangular in outline. The posterior borders of P 4 and M 1 are deeply excavated, appearing crescent, while the posterior borders of M 2 are much shallower. M 3 is reduced and much narrower with a single lobe. The tips of the anterior teeth have a lightly pigmented chestnut color except the molars.
The mandible is slender. The coronoid process is tall and squared, rising straight upward from the posterior of the toothrow. The condyloid process is weak and bifaceted, forming an angle at approximately 45° with the coronoid process. The angular process is long, straight, and very thin. The first lower incisor is long, with only a single basal cusplet. The incisor is slightly curved upwards, forming a hook at the tip. The first lower unicuspid is small and procumbent, crowded with a large incisor and the following premolar. The premolar has one forward-leaning cusp. The molar gradually decreases in size from M 1 to M 3 . Only the tips of I 1 , U 1 , P 1 , and M 1 are chestnutpigmented but not those of M 2 and M 3.

Comparison.
Among the species in the genus Chodsigoa, C. dabieshanensis sp. nov. is morphologically similar to the widely distributed C. hypsibia. However, the new species can be distinguished from C. hypsibia by many characters. In terms of body size, C. dabieshanensis sp. nov. is much smaller than C. hypsibia for most external and craniomandibular measurements (Table 2). In particular, the range of weight (4.67-5.89 g vs 6.40-14.00 g) and rostral length (6.48-6.81 mm vs 6.93-9.00 mm) between the two species does not overlap. The overall pelage of C. dabieshanensis sp. nov. is dark brown, almost black, which differs from the gray pelage of C. hypsibia. The skull of C. dabieshanensis sp. nov. is relatively shorter and broader than C. hypsibia, especially in the interorbital region, which appears much flatter (Fig. 4). The foramen magnum breadth is relatively larger than C. hypsibia. The posterior borders of M 2 in C. hypsibia are much more deeply excavated than in C. dabieshanensis sp. nov.. In C. dabieshanensis sp. nov., the basioccipital is well developed and the ridges are approximately parallel. By contrast, the basioccipital of C. hypsibia is narrow, so the ridges are nearly confluent in the middle.
Chodsigoa dabieshanensis sp. nov. (CIL = 19.08±0.22 mm) can be easily distinguished from C. parva (CIL = 15.79±0.27 mm) by its much larger size and the ranges of most of their external and cranial measurements do not overlap (Table 2). Furthermore, the tail of C. dabieshanensis sp. nov. (TL/HB = 80%) is relatively longer than C. parva (TL/HB = 88%). If the mean condyloincisive length is used as an indicator of overall size, C. dabieshanensis sp. nov. (CIL = 19.08±0.22 mm) is larger than C. sodalis (CIL = 17.97±0.12 mm), but smaller than C. furva (CIL = 20.63±0.39 mm), C. parca (CIL = 20.37±0.29 mm), and C. smithii (CIL = 22.23±0.54 mm) ( Table 2). The markedly flattened cranium of C. dabieshanensis sp. nov. is clearly distinguished from all other species in the genus, including C. caovansunga, C. furva, C. hoffmanni, C. parca, C. salenskii, C. smithii, and C. sodalis. The tail of C. dabieshanensis sp. nov. is shorter than head and body length, and it differs from C. sodalis (TL/HB ≈ 100%) and all other Chodsigoa species (TL/HB > 100%). The new species has a tuft of longer hair at the tip of the tail, in contrast to C. caovansunga, C. furva, and C. smithii. The thenar and hypothenar pads at the soles of the hindfeet are well separated and distinguishable from C. caovansunga, whose thenar and hypothenar pads of hindfeet are close together. Distribution and habits. Chodsigoa dabieshanensis sp. nov. is currently known from Yaoleping National Nature Reserve, Bancang Natural Reserve, and Foziling Natural Reserve, all located in the Dabie Mountains, Anhui province, eastern China. Most specimens were collected from deciduous broad-leaf forests at 750-1250 m a.s.l.

Discussion
Prior to this study, nine species were recognized in the genus Chodsigoa (Chen et al. 2017;Wilson and Mittermeier 2018). Our morphological and molecular results support that the specimens from the Dabie Mountains represent a new species of Chodsigoa, C. dabieshanensis sp. nov., based on the diagnosis-and-monophyly-based phylogenetic species concept (Mayden 1997;Gutierrez and Garbino 2018). Chodsigoa dabieshanensis sp. nov. is morphologically closely related to C. hypsibia and was previously considered as a marginal population of that taxon (Zhang et al. 2018). However, it can be distinguished from C. hypsibia by its dark brownish pelage and smaller size ( Table 2). The large genetic distance (8.6% by the CYT B gene) and phylogenetic analysis also strongly support they are two distinct species (UFboot > 98, PP = 1.00). As Chodsigoa are mainly distributed in southwest China and adjacent areas (Wilson and Mittermeier 2018), the distribution area of C. dabieshanensis sp. nov. is marginal. It is the only known species of Chodsigoa recorded in Anhui province, separated by at least 500 km from any other member of the genus, i.e., C. hypsibia from Luanxian, Henan Province (Zhou et al. 2020). The new species has no known congeners in Anhui Province; there are only two other soricid taxa recorded, Chimarrogale lender Tomas, 1902 and Crocidura spp. (Wang 1990;Jiang et al. 2015). The former is a large aquatic shrew (W > 20 g), and the latter has white, unpigmented dentition; these taxa are easily distinguishable from the new species.
The new species brings the number of Chodsigoa species to 10, sorted into two major clades; one including C. parva + C. hypsibia + C. dabieshanensis sp. nov. (Clade I), and the other (Clade II) comprised of the remaining species (Fig. 3). These results are also supported by morphology. Compared with the species in Clade II, the cranium of Clade I species is markedly flatter, and the tail of Clade I is relatively shorter (Clade I: TL/HB < 100%; Clade II: TL/HB ≥ 100%). All our gene trees showed C. dabieshanensis sp. nov. forms a subclade inside the main Clade I as the sister group of the subclade C. parva + C. hypsibia (UFboot > 98, PP = 1.00, Fig. 3).
As the most easterly distributed species of Chodsigoa, the discovery of C. dabieshanensis sp. nov. from the Dabie Mountains is important in understanding the macroevolution of the genus. Previous studies suggested that the tribe Nectogalini originated from Europe and migrated eastward to western Siberia and southward along northern China to southwest China (He et al. 2010). While the Hengduan Mountains are considered to serve as an important route for the southward migration (Zhang 2002;He et al. 2010), we have no knowledge of how this group migrated eastward. The oldest fossils of Chodsigoa are from the Early Pliocene in Gansu Provence, northern China (Zhang and Zheng 2001). Fossils of C. cf. hypsibia and C. cf. parva were discovered from the Early Pleistocene in Jianshi, Hubei and Wuhu, Anhui, both in eastern China, and more fossils were found in Wushan, Chongqing, southwest China in the Late Pleistocene (Qiu and Li 2005). These fossil records, together with our finding of C. dabieshanensis sp. nov., diverged earlier than C. hypsibia and C. parva, which suggests that the ancestor of Clade I arrived early in eastern China. Due to the present lack of broad geographic sampling, how the genus migrated to eastern China is still an open question. The Dabie Mountains are an extension of the Qinling fold belt and gradually stabilized by the end of the Tertiary (Feng 1976). Considering that the montane archipelagos always act as refugia and corridors to facilitate the dispersal of terrestrial small mammals (Chen et al. 2015;He and Jiang 2014;He et al. 2019), a parsimonious biogeographic scenario of the migration is via the Qinling and Dabie mountains. The ancestor of new species then became isolated due to climate change and following habitat turnover, resulting in a new species. Finer taxon sampling with additional sequence data is warranted to illustrate the migration patterns of the genus.