﻿A new species of the genus Soriculus (Soricidae, Eulipotyphla, Mammalia) from Medog in the eastern Himalaya

﻿Abstract Himalayan shrews of the genus Soriculus (Soricidae, Eulipotyphla), currently represented by four nominal species, are endemic to the Himalayas and the Gaoligong Mountains. In April 2022 and April 2023, a total of 10 specimens of Soriculus were collected from Beibeng and Damu, Medog County, Tibet, China. The morphology of the specimens was compared with the four recognised species of the genus Soriculus. Additionally, two mitochondrial (Cyt b and 12S) and three nuclear (APOB, BRCAI and RAG2) genes were sequenced to test the phylogenetic relationships of these specimens with the other species. Our results indicate that these specimens represent a distinct species, Soriculusbeibengensissp. nov., which is formally described here. The new species is distinguished from the other Soriculus species by the combination of darker pelages, smaller size, the relatively stubby nasal and the widened posterior processes of incisors. Phylogenetic analyses revealed the new species is sister to S.minor. The p–distance of Cyt b gene between S.beibengensis sp. nov. and other nominal Soriculus species ranges from 9.1–16.3%. This new species has a known distribution at an elevation of 1,500–2,125 m in Medog County, Tibet, China. The discovery of this new species from Medog County has important implications for interpreting small mammal biogeographic patterns in the eastern Himalaya and the mountain chains of south-west China.


Introduction
The genus Soriculus Blyth, 1854 (Mammalia, Eulipotyphla, Soricidae) is an endemic genus in the Himalayas and Gaoligong Mountains and which is mainly distributed in countries and regions across the eastern Himalayas (Bhutan, Sikkim of India, Nepal, and Yunnan and Tibet of PR China) (Hutterer 2005;Motokawa et al. 2008;Li et al. 2024).The small size and preference to inhabit remote forested mountainous areas make it challenging to collect specimens, limiting further studies.

Materials and methods
A total of 10 Soriculus specimens of S. beibengensis sp.nov.were collected from Medog, Tibet, China in April 2022 (Chen et al. 2023) and April 2023 (Fig. 1).Animals were handled, based on the animal care and use guidelines of the American Society of Mammologists (Sikes and the Animal Care and Use Committee of the American Society of Mammalogists 2016) and following the guidelines and regulations approved by the internal review board of the Kunming Institute of Zoology (KIZ), and with the permission of local government authorities.All specimens were deposited at the Kunming Natural History Museum of Zoology, Kunming Institute of Zoology (KIZ), Chinese Academy of Sciences, Kunming, China.
Comparative morphological data of another 62 Soriculus specimens including S. nigrescens (11), S. nivatus (23), S. medogensis (7) and S. minor (21) were obtained from Chen et al. (2023).Principal component analysis (PCA) was employed to evaluate the morphological variation amongst populations of Soriculus.The PCA was implemented in SPSS 19.0 (SPSS Inc., USA) and all craniodental measurements were first log 10 -transformed.The morphological measurements of S. beibengensis sp.nov.was compared with other Soriculus species.The terminologies for morphological descriptions followed Motokawa and Lin (2005) and Chen et al. (2023).
We estimated divergence time using BEAST v.2.6.7 (Bouckaert et al. 2014) on the concatenated nuclear gene dataset for which also the best partition schemes and evolutionary models were estimated using PartitionFinder v.2.1.1 (Lanfear et al. 2017) with greedy algorithm and BIC criterion (Suppl.material 1).Divergence dates were calibrated, based on three secondary calibrations following Chen et al. ( 2023): (1) the split between Crocidurinae and Soricinae is estimated at about 36 Ma (95% confidence interval [CI] = 28.6-44.0Ma; Springer et al. ( 2018)).We established the prior using log-normal distribution with offset: 0, mean: 36, standard deviation: 0.135, which will specify a distribution centred at about 35.7 Ma and the 95% CI was 28.6-44.5 Ma. (2) The oldest Blarinellini was from the Early Middle Miocene (Harris 1998, Rzebik-Kowalska 1998) and the oldest Blarinini was in the Barstovian (Repenning 1967) at approximately 13.6-16.3Ma.We established the prior for the divergence of Blarinellini-Blarinini using log-normal distribution with an offset of 15 Ma, mean of 0 and standard deviation of 0.98, which will specify a distribution centred at about 16.0 Ma and the 95% CI was 15.2-20.0Ma. (3) The oldest fossil of Nectogalini was in the Late Miocene (MN10, 9.7-11.5 Ma;Fejfar and Sabol (2005)).We established the prior for the divergence of Nectogalini-Notiosoricini using log-normal distribution with an offset of 9.7 Ma, mean of 0 and standard deviation of 0.95, which will specify a distribution centred at about 10.7 Ma and the 95% CI was 9.91-14.5Ma.The analyses were implemented with 100 million generations, sampled every 10,000 generations.Each analysis used a random starting tree, a log-normal relaxed clock model and a birth-death tree prior.Finally, the convergence of the parameters was assessed using Tracer 1.7 (Rambaut et al. 2018) and the tree was annotated using FigTree v.1.4.4.

Morphological analyses
A summary of the external and cranial measurements of the five species under the genus Soriculus is given in Table 1.The S. beibengensis sp.nov.showed the highest morphometric similarity to S. minor, but is much smaller than S. medogensis, S. nigrescens and S. nivatus.The PCA, based on 55 intact skulls, produced two principal components with eigenvalues exceeding 1.0, which explained 88.17% of the variation (Table 2).The first principal component (PC1) explained 66.62% of the total variation and was strongly positively correlated with most of the variables (UTL, PIL, CIL, ML, LTR, BL, CH, P 4 M 3 , PPD, HAC, CB, HCP, IOB, M 2 M 2 , MB, DIF, BMF and HCV), indicating that it represented the overall skull size.The second principal component (PC2) explained 21.55% of the total variation and was strongly positively correlated with M 2 M 2 , MB, DIF, BMF and HCV (loading > 0.668), indicating that it represented the skull width.The plot of PC1 and PC2 (Fig. 2) showed that S. beibengensis sp.nov. is well-separated from other species, but positioned closer to S. minor.The two species plot on the negative axis of PC1, indicating the smaller skull in the genus.S. beibengensis sp.nov. is located in the upper right corner of S. minor, indicating it had a larger and wider skull compared to S. minor.

Phylogenetic relationship
In total, we obtained 3937 bp long sequences for five specimens of S. beibengensis, including 1140 bp Cyt b, 823 bp 12S, 507 bp APOB, 693bp RAG2 and 774 bp BRCA1.All new sequences have been deposited in the GenBank (Accession Numbers: PP213259-PP213263 and PP226949-PP226968, Suppl.material 2).The topologies of ML and BI trees of the three datasets were highly similar and only the BI gene trees are shown (Fig. 3).The phylogenetic trees generated from the datasets of nDNA and mtDNA + nDNA strongly supported the monophyly of Soriculus and clustered into two major clades: the first clade was composed of S. minor and S. beibengensis sp.nov.(Clade I) and the second clade was composed of S. medogensis, S. nigrescens and S. nivatus (Clade II) (PP = 1.00,UFboot ≥ 94).In contrast, the tree, based on concatenated mitochondrial genes, showed a closer phylogenetic relationship between Clade I and Chimarrogale himalyica, Nectogale elegans and Neomys fodiens, but this result was not significantly supported (PP = 0.79, UFboot = 62).A sister relationship of S. beibengensis and S. minor was strongly supported in all gene trees (PP = 1.00,UFboot = 100).The p-distance of the Cyt b gene between the S. beibengensis sp.nov.and other species ranged from 9.1% (with S. minor) to 15.9% (with S. medogensis) (Table 3).The results of divergence time estimates (Fig. 4) showed that S. beibengensis sp.nov.and S. minor diverged at the early Pleistocene (ca.2.06 Ma, 95% CI = 1.05 -3.40 Ma).
Etymology.The specific Latin name beibengensis named for Beibeng, the type locality, with the Latin adjectival suffix -ensis means "belonging to".
Description.Amongst five species of the genus Soriculus, S. beibengensis sp.nov. is the second smallest species.Its size is larger than S. minor, but smaller than S. nivatus, S. nigrescens and S. medogensis (Table 1).External morphology is similar to S. minor, with the dorsal pelage being dark grey to black and ventral pelage slightly paler.Tail is ambiguously bicoloured, dark above and pale below (Fig. 5).The tail is short (TL = 40.6 ± 1.8 mm,(38)(39)(40)(41)(42)(43)(44), averages 53% of the head and body length and 8 of 10 specimens examined have a tail length more than 40 mm.The foreclaws are enlarged, similar to other Soriculus species.The back of hands and feet are covered by light brown to black hairs.
The skull is distinctly smaller than S. nivatus, S. nigrescens and S. medogensis, but larger than S. minor.Braincase is low and relatively flattened and the posterior of the skull is rounded.The sagittal and lambdoidal crest are well-developed and clear, the latter is especially prominent.The nasal and rostrum are not clearly transitioned and are stubby.The posterior process of incisor is widened, forming a narrowed funnel-shaped channel between the processes of adjacent teeth.The basioccipital and basisphenoid are fused and narrowed markedly in the middle region, forming a spade-like structure (Fig. 6).
The coronoid process is high and straight, with a concave anterior surface and a spatulate tip.The condyloid process has a single slender point and is angled upward at roughly 45 degrees, with the tip sitting below the coronoid process (Fig. 6).The angular process is long, straight and very thin, the tip slightly expands and bends upwards.The condyloid process is double-faceted, having two projections.The dental formula of the S. beibengensis sp.nov.is the same as the genus is: I 3/2, C 1/0, P 2/1, M 3/3 (×2) = 30.The apex of the first upper incisor is straight downwards, the tip of the first upper incisor is slightly pigmented with orange.There are four upper unicuspids (U 1 -U 4 ); U 1 is the highest, followed by U 2 , U 3 and U 4 is the smallest.M 1 and M 2 are similar in size, while M 3 is reduced.The lower incisor (I 1 ) is long, with only a low cusp and the tips are pigmented with orange.The lower unicuspid (U 1 ) and P 4 are crowded.M 1 is larger than M 2 ; M 3 is the smallest.
Comparison.Amongst species of the genus Soriculus, S. beibengensis sp.nov. is morphologically similar to its sister species, the S. minor.Both of them have a darker pelage and smaller size than other species.However, the new species can be distinguished from S. minor by multiple features.S. beibengensis sp.nov. is larger than S. minor for most of the external and craniomandibular measurements (Table 1).Especially, the skull of S. beibengensis sp.nov. is significantly longer than that of the S. minor, the measurements of CIL (20.4-21.3mm vs. 19.2-20.2mm) and ML (11.3-11.9mm vs. 10.5-11.0mm) between the two species do not overlap.The nasal and rostrum of S. beibengensis sp.nov.are not clearly transitioned and seem to be stubby, while S. minor, as well as the other species, has a clear transition of the nasal and rostrum (Fig. 6).The posterior process of incisors in S. beibengensis sp.nov.are widened, forming a narrowed funnel-shaped channel between the processes, whereas they are not widened in S. minor (Fig. 6).
Soriculus beibengensis sp.nov.can be easily distinguished from S. nigrescens, S. nivatus and S. medogensis by its smaller size, the darker pelage colour and almost no pigmentation of the teeth (Fig. 5).Compared to S. nivatus, the measurements of CIL, PIL, BL, UTL, P 4 M 3 , IOB, CB, CH, PPD, ML and LTR of S. beibengensis sp.nov.are smaller, with no overlap and the teeth of S. nivatus

A B
10 mm are slender, appear to be the most delicate in the genus.Amongst S. beibengensis sp.nov., S. nigrescens and S. medogensis, the ranges of most of their external and craniodental measurements do not overlap (Table 1).The teeth of S. beibengensis sp.nov.are significantly smaller, but the teeth of S. medogensis are robust, with the broadest ramus region and the highest coronoid process in the genus.Compared to S. nivatus (TL/HBL = 63%) and S. medogensis (TL/ HBL = 60%), the tail of S. beibengensis sp.nov.(TL/HBL = 53%) is shorter and the tail length of S. nivatus more than 46 mm, the tail length of S. medogensis usually more than 50 mm (6 of 7 (species measurements?)),while S. beibengensis sp.nov.less than 44 mm.The size arrangement of the unicuspids of S. beibengensis sp.nov. is similar to S. minor, U 1 is the highest, followed by U 2 , U 3 and U 4 is the smallest, while other species usually have the largest U 2 , followed by U 1 , U 3 and U 4 .Distribution and habits.Soriculus beibengensis sp.nov. is known only from the type locality in Beibeng and Damu Town, Medog, Tibet, China at elevations from 1501 to 2123 m a.s.l.They were mainly distributed in mixed forest dominated by oak and a few individuals were distributed in conifer-broadleaf mixed forest.

Discussion
The genus Soriculus is one of the least-studied small mammals.Owing to the limited studies, several species were not described until recently and it was considered a monotypic genus for a long time (Motokawa 2003;Burgin and He 2018).With a series of recent surveys conducted by Chinese scientists in the Himalayan Region, the diversity of the Soriculus has gradually been discovered with four species recognised (Chen et al. 2023).Herein, we described the fifth species, S. beibengensis sp.nov, collected in Medog, Tibet, China.In the genus Soriculus, S. beibengensis sp.nov. is morphologically similar to S. minor, both having darker pelage and smaller body size, but the former has a significantly larger body and skull size than the latter.Moreover, the phylogenetic tree showed that the sequences of the S. beibengensis sp.nov.clustered as a single clade, sister to S. minor and the p-distance between the two clades is up to 9.1%.According to the diagnostic and monophyletic species concept (Mayden 1997;Gutiérrez and Garbino 2018), we recognise S. beibengensis sp.nov.as a distinct species under the genus Soriculus.
As research has progressed, the evolutionary relationships amongst species of the genus Soriculus have become clearer.The genus is mainly split into two clades, representing two different evolutionary processes.The fossil evidence of Nectogalini shows that different taxa in this family migrated southwards from the late Miocene to the early Pleistocene (He et al. 2010).Therefore, Soriculus also likely migrated southwards under the influence of global cooling and desiccation and settled in the Gaoligong Mountains and Himalayas in southwest China.Our results showed that both clades of Soriculus have extant species distributed in Medog, indicating that the genus Soriculus entered Medog at least twice and adopted Medog as a key refuge.However, the current evidence is not sufficient to determine the specific dispersal history of the genus Soriculus.Deeper biological and geological surveys will become the key for analysing the evolutionary history of the genus.

Figure 3 .
Figure 3.The phylogenetic tree inferred using Bayesian Inference (BI).The figure shows phylogenetic trees derived from A the mitochondrial genes B the nuclear genes, and C the mitochondrial and nuclear genes.The branch numbers indicate ultrafast bootstrap values (left) and BI posterior probabilities (right).

Figure 4 .
Figure 4.The divergence time of Soriculus species inferred from a time-calibrated phylogeny, based on the nuclear genes in BEAST v.2.6.7.The node numbers represent the median ages of the divergence times (upper) and posterior probabilities (below).The branch lengths represent divergence time, node bars indicate the 95% CI for each clade age and red bars indicate the calibration points.

Table 1 .
Summary statistics (mean, standard deviation, ranges and number of samples) of external and skull measurements (in millimetres) of Soriculus specimens used in the study; character abbreviations are detailed in the "Material and methods".

Table 2 .
Craniodental variation in Soriculus, based on principal component analysis (PCA).Character abbreviations are detailed in the "Material and methods".