﻿A new species of shrew moles, genus Uropsilus Milne-Edwards, 1871 (Mammalia, Eulipotyphla, Talpidae), from the Wuyi Mountains, Jiangxi Province, eastern China

﻿Abstract Asian shrew moles, genus Uropsilus, are the most primitive members of family Talpidae. They are distributed mainly in southwestern China and adjacent Bhutan, Myanmar, and Vietnam. In June 2022, we collected five specimens of Uropsilus from Mount Huanggang, Jiangxi Province, eastern China, which is the highest peak of the Wuyi Mountains. We sequenced two mitochondrial (CYT B and 12S rRNA) and three nuclear (PLCB4, RAG1, and RAG2) genes to estimate the phylogenetic relationship of the five shrew moles. We also compared their morphology with recognized species within the genus. Our results show that these specimens collected from Mount Huanggang differ from all named species in Uropsilus. We formally describe the species here as Uropsilushuanggangensissp. nov. Morphologically, the new species is distinguishable from the other Uropsilus species by the combination of dark chocolate-brown pelage, long snout, enlarged first upper incisor, similarly sized lacrimal and infraorbital foramens, and the curved and sickle-like coronoid process. The genetic distances of the cytochrome b (CYT B) gene between U.huanggangensis and other recognized Uropsilus species ranged between 9.3% and 16.4%. The new species is geographically distant from other species in the genus and is the easternmost record of the Uropsilus. The divergence time of U.huanggangensis was estimated to be the late Pliocene (1.92 Ma, 95% CI = 0.88–2.99).


Introduction
The shrew moles of the genus Uropsilus Milne-Edwards, 1871 are the sole living genus in the subfamily Uropsilinae in Talpidae (Mckenna et al. 1997;Hutterer 2005).These insectivores primarily inhabit the mountains of southwestern China, as well as adjacent areas in Bhutan and northeastern Myanmar, where they inhabit montane forests at 1,400-3,600 m elevation (IUCN 2015;Hoffmann ZooKeys 1186: 25-46 (2023), DOI: 10.3897/zookeys.1186.111592 Xueyang Ren et al.: A new species of Uropsilus from eastern China and Lunde 2008).Although the fossil record is sparse, the age of the associated fossils and the timing of the molecular evolution of mammals suggest that the subfamily Uropsilinae would have flourished and spread widely across Eurasia before the Late Miocene (Meredith et al. 2011).In contrast to other moles that have developed adaptive features such as broad front claws and reduced external ears, shrew moles exhibit shrew-like characteristics, including slender front claws, exposed external ears, and long tails almost equal in length to their bodies.All these morphological characteristics suggest that they have retained the terrestrial habits of primitive moles (Allen 1938).Phylogenetic relationships constructed using morphological methods (Motokawa 2004;Sánchez-Villagra et al. 2006) and molecular phylogenetic methods (Douady and Douzery 2003;Shinohara et al. 2003) consistently support that Uropsilus forms the basal branch in the phylogenetic tree of the family Talpidae.
Recently, Liu et al. (2013) described U. aequodonenia Liu et al., 2013 from Puge County, Sichuan, China, which has a dental formula of I 2/2, C1/1, P3 /3, and M 3 /3 = 36.Wan et al. (2013) suggested that U. nivatus and U. atronates are valid species based on molecular data.Furthermore, they identified seven recognized species and five putative species.Wan (2015) described six new species, but these designations were not recognized because they did not follow the International Code of Zoological Nomenclature (ICZN 2012).Wan et al. (2018) generated gene trees using additional specimens, which phylogenetic analyses revealed that species of Uropsilus could be sorted into three distinct lineages.One lineage includes U. investigator from western Yunnan and acts as the basal position of the genus; the second lineage includes U. aequodonenia, U. andersoni, and U. nivatus from Northern Yunnan and western Sichuan; and the third contains U. soricipes, U. gracilis, U. atronates.Additionally, Hu et al. (2021b) used multivariate analyses as well as phylogenetic analyses to describe a new species, U. dabieshanensis Hu et al., 2021, from the Dabie Mountains, Anhui Province, eastern China.The phylogenetic results indicate that the lineage of Uropsilus has two matrilines.More recently, Bui et al. (2023) described a new species, U. fansipanensis, from the northwestern Vietnam.Thus, nine taxa are currently recognized as full species under the genus Uropsilus: U. aequodonenia, U. andersoni, U. gracilis, U. investigator, U. soricipes, U. atronates, U. nivatus, U. dabieshanensis, and U. fansipanensis.Of all known species, U. dabieshanensis is the only one found in eastern China and is considered to have the easternmost distribution of the genus.
During a biodiversity study in June 2022, five shrew mole specimens were collected from the Wuyi Mountains, Jiangxi Province, eastern China (Fig. 1).Our molecular analysis reveals that the five specimens are genetically distinct from all recognized Uropsilus species and potentially represents a new species.In this study, we integrate genetic and morphometric approaches to elucidate the taxonomy and phylogeny of these specimens.

Sampling
In June 2022, five Uropsilus specimens were collected on Mount Huanggang in Wuyishan National Nature Reserve, Yanshan, Jiangxi Province, eastern China (Fig. 1).Specimens were collected using Sherman and pitfall (plastic buckets 15 cm in diameter and 28 cm in depth) traps.All specimens were euthanized, and muscle or liver tissue was extracted from each and preserved in pure alcohol for subsequent molecular studies.All specimens and tissues were deposited at the Biological Museum of Anhui Normal University (AHNU).Animals were handled in compliance with the animal care and use guidelines of the American Society of Mammologists (Sikes et al. 2016), following the guidelines and regulations approved by the internal review board of AHNU (approval no.AHNU-ET2021002), and with the permissions of local government authorities.

Phylogenetic analyses
Genomic DNA of the five specimens of Uropsilus from Mount Huanggang was extracted from the liver and muscle tissues using a DNA extraction kit (Tiangen DNeasy Blood and Tissue Kit, Beijing, China).Two mitochondrial genes (cytochrome b [CYT B], 12S rRNA [12S]) and three nuclear genes (phospholipase C beta 4 [PLCB4], recombination activating protein 1 [RAG1], and recombination activating protein 2 [RAG2]) were amplified using the primer pairs outlined in Suppl.material 1.The PCR products were purified and sequenced in both directions using the BigDye Terminator Cycle Kit v. 3.1 (Invitrogen, Waltham, MA, USA) on an ABI 3730xl sequencer (Applied Biosystems, Waltham, MA, USA).The obtained sequences were assembled using SeqMan (DNASTAR, Lasergene v. 7).Corresponding sequences of 38 specimens of nine recognized species and six unrecognized species of Uropsilus were downloaded from the GenBank (Suppl.material 2).We downloaded sequences of Talpa altaica and Sorex araneus as out-group taxa following Wan et al (2018).All sequences were then aligned in MEGA v. 11 (Tamura et al. 2021).
The uncorrected p-distance of the CYT B gene between species was calculated in MEGA v. 11 (Tamura et al. 2021).We used maximum likelihood (ML) and Bayesian inference (BI) methods to conduct phylogenetic analyses of mitochondrial-nuclear genes (mtDNA + nDNA, 4090 bp) concatenated datasets in Phylo-Suite (Zhang et al. 2020).The best-fit partitioning scheme and evolutionary models were selected using PartitionFinder v. 2.0 with the greedy algorithm under the Bayesian information criterion (BIC) (Suppl.material 3) (Lanfear et al. 2012).

Molecular dating
We used BEAST v. 2.6.6 (Bouckaert et al. 2019) to estimate divergence times based on the Birth-Death model as the tree prior and relaxed lognormal as the clock model prior.Evolutionary models or partition schemes were estimated based on the Bayesian Information Criterion (BIC) in PartitionFinder v. 2.0 (Lanfear et al. 2012).Two fossil calibrations were used following the guide of Wan et al. (2018): (1) the first division of Uropsilus at 6.18 Ma (95% HPD: 4.27-8.65Ma), with a lognormal distribution prior (mean: 6.20, SD: 0.215, offset: 0.02), so the median age was at 6.08 Ma and the 95% CI was 4.27-8.65Ma; (2) the earliest known U. soricipes from the Early Pleistocene 2.0-2.4Ma, with an exponential distribution prior (offset = 2.0, M = 0.67 [2.0 × 0.333]), so the median age was 2.46 Ma and the 95% CI was 2.03-4.01Ma.Each analysis was run for 100 million generations, sampling every 10000 generations.The first 10% of the samples were discarded as burn-in.Convergence was assessed using Tracer v. 1.7 (Rambaut et al. 2018).
The body weight (Wt) and four external measurements, including head and body length (HBL), tail length (TL), hindfoot length (HF), and ear length (EL), were taken from specimen labels or field notes.Twenty-one craniodental measurements were taken with digital calipers to the nearest 0.01 mm, following Yang et al. (2005Yang et al. ( , 2007)).All the craniodental measurements were taken by a single observer.The following measurements were taken: We compared morphology of the new species with other species of Uropsilus.Comparative morphological characters of these other species were obtained from Thomas (1912Thomas ( , 1922)), Allen et al. (1923), Hu et al. (2021b), andBui et al. (2023), and we followed these authors' terminologies in our morphological description of the new species.Meanwhile, to better distinguish between the different species, we compared the ratio of GNB to PL, as well as the ratio of TL to HBL.

PL
Overall similarities of skulls were assessed first through principal component analyses (PCA) based on the 21 log 10 -transformed craniodental variables.Groups of individuals sharing a comparable morphology were then discriminated through discriminant analysis (DA).To make the results more concise, we limited the PCA and DA analyses to the six taxa with the same dental for-mula, including: U. atronates, U. dabieshanensis, U. gracilis, U. investigator, U. nivatus, and the new species.The PCA and DA were conducted in SPSS v. 22.0 (SPSS Inc., USA).Furthermore, independent sample t-tests were conducted to test the variances of measurements highly correlated with PC1 and PC2 (i.e.LUTR, LBTR, GBSn, and GBUM; loading > 0.8) between the new species and the other species.
The ML and BI trees recovered similar topologies (Fig. 2).In all phylogenetic trees, sequences of the new species from Mount Huanggang formed a monophyletic clade with high support (SH-aLRT = 100, Utboot = 100, and PP = 1.00).
The new species has a sister relationship with the clade that is comprised of U. atronates, Uropsilus sp.6, U. fansipanensis, and U. gracilis; this clade is strongly supported in the BI tree (PP = 1.0), but this relationship only has moderate support in the ML tree (SH-aLRT = 89.6,Utboot = 76).BEAST divergence analyses show that the divergence of the new species from the others was estimated to be at the early Pleistocene (1.92 Ma, 95% CI = 0.88-2.99)(Fig. 3).

Uropsilus huanggangensis
Description.Uropsilus huanggangensis is a medium-sized species of Uropsilus (HBL = 72 ± 1 mm, PL = 21.44 ± 0.47 mm; Table 2).The dorsal pelage is dark chocolate-brown, consisting of brown fur with a light grey base; the ventral fur is slightly paler.The snout is very long, at about 12 mm, and is the longest in the genus.The tail is slim and relatively short (TL = 63 ± 3 mm, 57-65 mm), about 86% of the combined head and body length.The tail is black above and slightly paler below, with a sparse tuft of short hair at its tip.The hind foot is covered with short black hair; its length is 13-14 mm and constitutes approximately 18% of the combined head and body length.
The outlines of the skull are rounded, and there is a complete zygomatic arch.The rostrum is relatively long, the braincase is narrow, and the proportion of GNB and PL is 51.8%, which is the smallest of any species in the genus (GNB/ PL > 52.4% in other species).The zygomatic arches are stout and only slightly bow outward.The lacrimal foramen and infraorbital foramen are similar in size.
The dental formula is I 2/1, C1/1, P 4/4, M 3/3 = 38.I 1 is large and wide, causing the enlargement at the apex of the rostrum.I 1 is bigger than I 2 , and there is a visible gap between them.C 1 is almost equal to P 1 , while P 3 is smaller.P 2 is larger than P 1 and P 3 .The first upper molar M 1 and second upper molar M 2 are large, and have well-developed, W-shaped lateral cusps.In contrast, the third upper molars M 3 are reduced.The body of the mandible is long and slender.The coronoid process is high, pointed, and curved to the posterior, with an incisive tip pointing straight to the posterior, resembling the outline of a sickle.The angular process is long, rounded, and points downward at roughly 45°.The first lower premolar (P 1 ) is slightly smaller than the lower canine C 1 .P 1 and P 3 are similar in size.M 2 is W-shaped and larger than M 1 and M 3 .M 3 is slightly smaller than M 1 (Fig. 6).
Comparison.Among other Uropsilus species, U. huanggangensis is morphologically most similar to U. dabieshanensis and U. gracilis.However, the new species can be distinguished from them by many characteristics.
Compared to U. dabieshanensis, U. huanggangensis has darker fur, a relatively longer and slimmer tail, and a much larger hindfoot and ear, despite that the heads and body lengths of the two species are almost the same (Table 2; Fig. 5).The tail of U. huanggangensis (TL = 63 ± 3 mm) is relatively longer than U. dabieshanensis (TL = 56 ± 3 mm).Most individuals of U. huanggangensis (4 of 5) have a tail length of more than 63 mm, while most individuals of U. dabieshanensis (5 of 6) have a tail length less than 57 mm.The hairs on the tail (bristle hairs) of U. huanggangensis are shorter and sparser than those in U. dabieshanensis, and the tufts at the tail tip of U. dabieshanensis appear much longer.The skull of U. huanggangensis is much slenderer than in U. dabieshanensis (Fig. 6), and the proportion of GNB and PL (CB / GLS = 53.96%) in U. dabieshanensis is greater than that in U. huanggangensis (GNB / PL = 51.80%).The coronoid process is pointed and curved to the posterior in U. huanggangensis, while the coronoid process of U. dabieshanensis is high and straight, with a squared tip.
Compared to U. gracilis, the dorsal pelage of U. huanggangensis is much darker.The snout of U. huanggangensis is longer, and the incisor is larger than that of U. gracilis.The tail of U. huanggangensis (TL/HBL = 86%) is relatively shorter than U. gracilis (TL / HBL = 91%) in proportion, and the tufts at the tail tip of U. huanggangensis are much shorter than those in U. gracilis.In terms of body size, U. huanggangensis is relatively larger than U. gracilis for most external and craniomandibular measurements (Table 2).In particular, the range of Id-Gol (U. huanggangensis 12.90-13.03mm vs U. gracilis 11.46-12.68mm) between the two species does not overlap.The coronoid process of U. gracilis is high and squared, similar to that of U. dabieshanensis, but differs from that of U. huanggangensis.
Compared to U. atronates and U. nivatus, the dark chocolate-brown dorsal pelage of U. huanggangensis differs from the chestnut red of U. atronates and the black-gold pellage of U. nivatus.Meanwhile, U. huanggangensis is larger than both U. atronates and U. nivatus for most external and craniomandibular measurements (Table 2).
The pelage color of U. huanggangensis is dark chocolate-brown, which is much lighter than the black pelage of U. investigator.The ears of U. huanggangensis are relatively larger (EL = 9.20 ± 0.45 mm, range 9.00-10.00mm) than that of the U. investigator (EL = 7.95 ± 0.98 mm, range 6.50-9.50mm).The color of U. huanggangensis is uniform compared to the bicolored tail of U. investigator.Also, the P 1 of U. investigator is larger than P 3 , while P 1 and P 3 of U. huanggangensis are similar in size.
Compared to the upward orbital process of U. fansipanensis, the orbital process of U. huanggangensis is downward.The lacrimal foramen of U. fansipanensis is larger than infraorbital foramen, while the two are of similar size in U. huanggangensis.
Distribution and ecology.Uropsilus huanggangensis is currently known only from the type locality on Mount Huanggang, Wuyishan National Park, Jiangxi Province, eastern China, where pecimens were collected at elevations between 1830 and 2060 m a.s.l.Coniferous forests and shrub meadows, with abundant rocks on the ground, dominate the habitat in this area.

Discussion
For a long time, it was believed that the genus Uropsilus was only distributed in the mountains of southwestern China and adjacent Myanmar (Wan et al. 2013;Kryštufek and Motokawa 2018).Hu et al. (2021b) expanded the known distribution of the genus by reporting the presence of U. dabieshanensis on Dabie Mountain, Anhui, eastern China, which represents the easternmost distribution of genus.In the present study, through integrating morphological and molecular approaches, we demonstrate that the isolated population on Mount Huanggang is distinct from all nominal species of Uropsilus and recognize it as a new species, U. huanggangensis.
In line with previous studies (Wan et al. 2013;Hu et al. 2021b), our phylogenetic analyses reveal that Uropsilus species can be sorted into two distinct lineages.One lineage includes U. investigator and U. sp. 1, occupying the basal position of the genus, and the other lineage exhibits a widespread distribution throughout China.Although U. huanggangensis consistently forms a monophyletic group with strong support in all phylogenetic trees, it is worth noting that the phylogenetic trees of Uropsilus species display considerable instability, as previously reported in the literature.Additionally, eight putative new species (Uropsilus sp.1-8) have been identified but not yet officially described; there is a high level of cryptic diversity and extensive cryptic diversification within the genus.Broader sampling, in-depth gene sequencing, and morphological analysis are needed to improve the understanding of the genus.
As the easternmost occurring species of Uropsilus, our discovery of U. huanggangensis significantly expands our knowledge of the geographic distribution of the genus and contributes to our understanding of its macroevolution.The divergence of U. huanggangensis is estimated in the early Pleistocene (1.98 Ma, 95% CI = 0.88-2.99).Global cooling and drying events during this period (Qiu and Li 2005;Ge et al. 2013), as well as the isolation effects of Wuyi Mountain and Yangtze River, may have been critical in the divergence of U. huanggangensis, and Mount Huanggang may have provided a refuge for U. huanggangensis during the ice age.Recently, several new small mammal species have been described in eastern China, such as Chodsigoa dabieshanensis Chen et al., 2022, Crocidua dongyangjiangensis Liu et al., 2020, and Typhlomys huangshanensis Hu et al., 2021, indicating that biodiversity in the region is severely underexplored (Hu et al. 2021a;Chen et al. 2022).The description of U. huanggangensis in the Wuyi Mountains region highlights the overlooked biodiversity of the mountains of eastern China.It is therefore crucial to conduct further comprehensive investigations and taxonomic studies on small mammals in this region to gain a deeper understanding of the biodiversity of this region.

Figure 1 .
Figure 1.Sampling localities of specimens used in the phylogenetic analysis.

Figure 2 .
Figure 2. Molecular phylogenetic tree of Uropsilus based on mitochondrial-nuclear concatenated data and analyzed using maximum likelihood and Bayesian inference analyses.Numbers above branches refer to Bayesian posterior probabilities (PP).Numbers below branches indicate SH-like approximate likelihood ratio test supports (SH-aLRT)/ultrafast bootstrap supports (UFBoot).Scale bars represent substitutions per site.

Figure 3 .
Figure 3. Divergence times estimated using BEAST based on mitochondrial-nuclear concatenated data.Node numbers refer to divergence time in million years (Ma) and Bayesian posterior probabilities (PP).

Figure 4 .
Figure 4. Results of A principal component analysis (PCA) B discriminant function analysis (DA) for partial species with the same dental formula within the genus Uropsilus.

Figure 5 .
Figure 5. Dorsal and ventral views of three Uropsilus species A U. huanggangensis sp.nov.B U. gracilis C U. dabieshanensis.

Figure 6 .
Figure 6.Dorsal, ventral, and lateral views of the skull and lateral views of the mandible of three Uropsilus species A U. huanggangensis sp.nov.B U. gracilis C U. dabieshanensis.

Table 2 .
External and skull measurements (mm) used in morphometric analyses of the genus Uropsilus, including mean values, standard deviations, range, sample size and the ratio of partial measurements.ZooKeys 1186: 25-46 (2023), DOI: 10.3897/zookeys.1186.111592Xueyang Ren et al.: A new species of Uropsilus from eastern China

Table 3 .
Character loadings, eigenvalues, and percent variance explained on the first two components of a principal components analysis and the five canonical axes discriminant function analyses of the genus Uropsilus.