Discovery of a new mammal species (Soricidae: Eulipotyphla) from Narcondam volcanic island, India

We discovered a new Crocidura species of shrew (Soricidae: Eulipotyphla) from Narcondam Island, India by using both morphological and molecular approaches. The new species, Crocidura narcondamica sp. nov. is of medium size (head and body lengths) and has a distinct external morphology (darker grey dense fur with a thick, darker tail) and craniodental characters (braincase is rounded and elevated with weak lambdoidal ridges) in comparison to other close congeners. This is the first discovery of a shrew from this volcanic island and increases the total number of Crocidura species catalogued in the Indian checklist of mammals to 12. The newly discovered species shows substantial genetic distances (12.02% to 16.61%) to other Crocidura species known from the Indian mainland, the Andaman and Nicobar Archipelago, Myanmar, and from Sumatra. Both Maximum-Likelihood and Bayesian phylogenetic inferences, based on mitochondrial (cytochrome b) gene sequences showed distinct clustering of all included soricid species and exhibit congruence with the previous evolutionary hypothesis on this mammalian group. The present phylogenetic analyses also furnished the evolutionary placement of the newly discovered species within the genus Crocidura.

www.nature.com/scientificreports/ also consecutively used to discriminate shrew species, detect cryptic diversity, or study phylogenetic evolution, biogeographic origin and radiation, and phylogeography 8,[24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40][41][42][43] . Only a single study aimed to assess the genetic signature of two endemic species, C. andamanensis and C. nicobarica from the AN Archipelago 44 . Therefore, an understanding of the species diversity on this group of islands remains incomplete. The present study is based on the assumption that a few hitherto unreported shrew species exist beyond the known biogeographic distribution of the group in the AN Archipelago, which warrants further investigation through integrative approaches. We performed both morphological and molecular assessments to confirm a new shrew species from the volcanic Narcondam Island in the AN Archipelago, which is herein described as Crocidura narcondamica sp. nov. (Fig. 1). The newly discovered species is validated by a morphometric and molecular comparison with 13 and 15 species, respectively, distributed in the AN Archipelago, on the mainland of India, and the close biogeographic realms of Myanmar and Sumatra. We estimated the genetic divergence from related species and performed phylogenetic analyses to corroborate the taxonomic identity and evolutionary relationships of this novel species.

Materials and methods
Study area. Narcondam Island (13.45° N 94.27° E; Fig. 2) is located about 130 km east of North Andaman, and about 446 km off the west coast of Myanmar [45][46][47] . The island covers an area of 6.8 km 2 and the highest peak (volcanic cone) is 710 m above sea level; however, the base lies approximately 1500 m beneath the sea 45 . This isolated island is part of a volcanic arc that continues northward from Sumatra to Myanmar 48 . The climatic condition of this small, conical island can be defined as a humid, tropical, and coastal. The island is thickly vegetated, bordered by cliffs on the southern side and crested by three peaks. The forest types can broadly be categorized as three zones: wet evergreen on the slopes and highest zones of the volcano, moist deciduous or semi-evergreen at lower elevations, and littoral forest along the coastline 47 .
Ethics statement, sampling, and morphological examination. To  www.nature.com/scientificreports/ E) by the pitfall method. Their external measurements were taken in the field and include head and body length (HB), tail length (TL), ear length (E), and hindfoot length including claw (HF). Photographs of fresh specimens were taken by the fourth author in the field. The collected specimens were preserved in 70% molecular grade ethanol for further investigation. Skulls were extracted later, cleaned, and prepared in the museum (  Table 1, Supplementary Table S2). Only adult specimens were included, as determined by fully erupted molars and fused basioccipital suture 5,51 . Photographs of cranial and dental views were taken by the first author using a Nikon D7000 camera. The collected specimens of the possible new species of shrew were compared with the morphometric data of 13 congeners reported from the AN Archipelago, the mainland of India and from Myanmar (Supplementary Table S2). The external and craniodental characters were also compared with the archival specimens of C. attenuata and C. jenkinsi available in the NZC of ZSI, Kolkata (  Table S2). The morphology and craniodental measurements along with other collateral information of the museum and other comparative species are given in Supplementary Table S2. Sequence quality control and dataset preparation. The study obtained both forward and reverse chromatograms from the holotype and paratype samples. The noisy parts of each chromatogram were trimmed at both ends and quality value (> 40) was used to screen for making the consensus sequences through the SeqScanner Version 1.0 (Applied Biosystems). The sequences were translated through the online platform ORF finder (http:// www. ncbi. nlm. nih. gov/ gorf/ gorf. html) to validate the sequence by comparison to the vertebrate consensus. The annotated sequences were contributed to GenBank. A total of 54 mtCytb sequences were acquired from GenBank (Supplementary Table S3). A total of 58 sequences were aligned by ClustalX software 58 to form a combined dataset (473 bp) for further genetic distance and phylogenetic analysis. The sequences (accession no. KP061993 and KP062007) of Crocidura monax endemic to Tanzania, East Africa were used as an out-group in the present phylogenetic analyses.
Genetic distance and phylogenetic analysis. The Kimura-2-parameter (K2P) genetic distance was calculated in MEGAX 59 . The most suitable model for the present dataset was estimated by using JModelTest v2 with the lowest BIC (Bayesian Information Criterion) score 60 . The maximum-Likelihood (ML) phylogenetic tree was constructed using the IQ-Tree web server (http:// iqtree. cibiv. univie. ac. at.) with General Time Reversible (GTR) model including a proportion of invariable sites (+ I) and rate of variation across sites and 1000 bootstrap support 61 . The Bayesian (BA) tree was constructed in Mr. Bayes 3.1.2 by selecting nst = 6 for the GTR + G + I with one cold and three hot chains of metropolis-coupled Markov Chain Monte Carlo (MCMC); it was run for 1,000,000 generations with 25% burn-in with trees saving at every 100 generations 62 . The MCMC analysis was  Distribution and habitat. The new species is presently known only from its type locality, Narcondam Island, Andaman and Nicobar Archipelago, in the Bay of Bengal, India. It was collected from a littoral forest along the coastline at 11 m elevation. No anthropogenic disturbances were observed in the habitat except for a security post (Fig. 2).

Diagnosis and description.
The new species C. narcondamica sp. nov. (Fig. 1) is assigned to the genus Crocidura by the presence of three upper unicuspids and clearly distinguished from all other AN Archipelago shrews by its body size and tail length, which are considerably shorter (Supplementary Table S2). The new species possesses a darker-grey dense fur dorsally and a thick and darker tail (Fig. 1a), whereas the species known from the AN Archipelago possesses a different dorsal pelage and tail; C. jenkinsi ( Supplementary Fig. S3a) and C. hispida have a spiny dorsal fur with a slender tail, C. andamanensis has a bluish-grey dorsal fur washed with brown and a darker brown tail, C. nicobarica has a bristly sooty brown dorsal fur with a slender tail, and C. attenuata has a soft brownish-grey dorsal fur with a slender tail 2,51 ( Supplementary Fig. S2a).
The new species also differs from other congeners occurring in the mainland of India, and in Myanmar ( Table 1, Supplementary Table S2). The head and body length of the new species (holotype: 67 and paratype: 63 mm) is considerably smaller, than C. fuliginosa and C. pullata, but overlapping with other species such as, C. attenuata (60- (41-51 mm), and shorter than C. cranbrooki (65-88 mm), C. fuliginosa (62-89 mm). Although the HB and TL of the newly discovered species overlap with C. attenuata, the morphological characters are significantly different (soft brownish-grey dorsal pelage with a brownish slender tail; Supplementary Fig S2a).
The length of the hindfoot of C. narcondamica sp. nov. (holotype: 13.4 and paratype: 12.4 mm) also differs from three species, C. pullata (14-16 mm), C. cranbrooki (14-16 mm), and C. fuliginosa (15-19 mm), which are distributed in India and Myanmar. However, the rest of the congeners known from the same biogeographic region show an overlapping length of the hindfoot (Supplementary Table S2).

Molecular identification and phylogenetic interpretation.
Partial mitochondrial mtCytb gene sequences (Accession Nos. MW417367 and MW417368) were generated from both the holotype and paratype of the new species and submitted to GenBank. The BLAST search results showed a 90% similarity with the available sequence of a specimen Crocidura sp. (MN691031) collected from Xizang, China. The next closest results of the similarity search revealed a 89.57% similarity with a specimen of Crocidura sp. (MN691019) collected from Yunnan, China and with C. attenuata (MK765768) collected from Jiangxi, China. The present dataset of 21 Crocidura species, including the new one shows an overall mean inter-species genetic distance of 11.7%. The new species revealed a substantial mean genetic distance (12.02% to 16.61%) to other Crocidura species (Supplementary Table S4). The new species is genetically distant to C. andamanensis (16.61%) and C. nicobarica (15.09%) distributed in the same group of islands, AN archipelago. The new species also genetically distant (12.02% to 16.57%) from other congeners known from the mainland of India and Myanmar. The new species also show substantial genetic distance (13.13% to 16.44%) to other congeners distributed in Sumatra. Both ML and BA phylogenetic trees showed similar topologies with high posterior probabilities and bootstrap supports (Fig. 4,  Supplementary Fig. S4) and showed that the three Crocidura species from the AN archipelago included in the analysis do not form a monophyletic group but are related to entirely different branches of the evolutionary tree. www.nature.com/scientificreports/ www.nature.com/scientificreports/

Discussion
Island ecosystems are regarded as discrete biogeographic units and as a significant model for evolutionary studies 8,32,64 . In the Miocene-Pliocene, volcanic eruption produced many new islands and their sporadic land connections during the Pleistocene, allowed both geographic and temporal processes of species diversification in Southeast Asia 32 . The Indian plate separated from Africa-Madagascar-Seychelles and drifted towards the Eurasian plate, which also allowed multiple opportunities for animal oversea dispersal and biological connections between the India mainland and Southeast Asia 65 . Due to the remoteness and inaccessibility throughout the year, the smaller islands of the AN archipelago and other tropical islands are little explored in comparison to larger islands. The political boundaries of India include a number of islands in both the Arabian Sea and the Bay of Bengal. The islands of the Arabian Sea are primarily built up by coral reefs, whereas the Bay of Bengal islands are distinguished by habitable submarine mountains 45 . Narcondam Island is one of 836 islands of the AN Archipelago, and is a small dormant volcanic island with almost 80% forest cover 45,66 (Fig. 2). It is believed that the volcano was active during Holocene eruptions 66,67 . However, bathymetric study revealed that a number of seamounts have been well-developed on the Andaman seafloor, which changes the trend of the volcanic arc from northeast to southeast to Sumatra Island. Hence, it is evidenced that there is a direct connection between the submerged volcanic arc-chain of the Andaman Sea from Sumatra to Barren and Narcondam volcanic islands 68,69 . The island has been recognised by UNESCO as a World Heritage site due to its sensitive ecosystem and the occurrence of an endangered species, the Narcondam hornbill (Rhyticeros narcondami). Further, this island has been also designated as Wildlife Sanctuary under the provisions of the Indian Wildlife (Protection) Act, 1972.
The unparalleled biogeography of oceanic islands provides a suitable habitat for many Crocidura species discovered in the recent past. The known distribution of the new shrew species is restricted to Narcondam Island, a very small island. Isolated islands appear to provide a suitable habitat for many endemic Crocidura species, such as C. canariensis, which is restricted to the Canary Islands, C. fingui and C. thomensis, restricted to the Sao Tome and Principe Islands, C. orii, restricted to the Amamioshima group of the Ryukyu Islands, and C. trichura, restricted to Christmas Island 4,70 . The present discovery of a new shrew from isolated Narcondam Island adds an interesting detail to this pattern.
Considering the molecular-based species identification, the new species is clearly genetically distinct. Previous studies already showed that the mtCytb gene can often be effectively used to discriminate shrew species and to detect cryptic diversity in different geographical regions 7,42,43,57 . The genetic assessment of Crocidura species also facilitates the description of their radiation and diversification in Southeast Asian countries 32 . The estimated K2P genetic distance, ML and BA phylogenies clearly discriminate all the studied shrew species with sufficient genetic distances and distinct clustering. The new species C. narcondamica sp. nov. shows a substantial genetic distance (12.02%) to C. rapax (distributed in China, India, Myanmar, and Taiwan), and even larger distances in comparison with other AN archipelago species such as C. andamanensis with 16.61%, and C. nicobarica with 15.09%. The initial phylogenetic analysis indicates that the numerous endemic Crocidura species are not the result of a local radiation, as could be initially expected, but appear to be derived from at least three independent colonization events. To understand this surprising pattern, we recommend generating more molecular data of this group of mammals from different geographical regions to clarify their in-depth phylogenetic relationships, provide estimates of the divergence events, and allow a better alignment with the biogeographical history of the Indo-Malayan and Sundaic realms.
So far, Narcondam Island is popularly known by the occurrence of the endemic Narcondam hornbill. The ecological study of animals on this island was initially restricted only to this bird. Later on, researchers focused on a faunal expedition and recorded 17 fishes, 8 reptiles, 28 birds, 2 mammals (Chinese Forest Rat, Rattus andamanensis and Island Flying Fox, Pteropus hypomelanus), 13 spiders, 8 butterflies, and 2 sea cucumbers from this isolated island 47 . However, the known diversity of the mammalian fauna is very sparse on Narcondam Island; especially because no data were reported on soricid fauna (shrews). The discovery of C. jenkinsi on South Andaman Island by Chakraborty 12 was the latest discovery of a Crocidura species from India. With this new description of the Narcondam shrew, altogether twelve species of Crocidura are now known from India including the AN Archipelago; viz., C. andamanensis, C. attenuata, C. fuliginosa, C. hispida, C. horsfieldii, C. jenkinsi, C. nicobarica, C. pergrisea, C. pullata, C. rapax, C. vorax, and C. narcondamica sp. nov. Among the Indian Crocidura shrews, four species known from the AN Archipelago have been categorized as threatened by the IUCN Red List of Threatened Species (2020-3) due to their remarkable endemism 70 . Habitat loss due to selective logging, anthropogenic activities, and natural disasters have been identified as the major threats for this group of animals in the AN Archipelago 71,72 . As the Narcondam Island is uninhabited, the new species C. narcondamica sp. nov. may not face the anthropogenic disturbances, but the extremely restricted insular habitat and the associated limited population size will automatically result in increased vulnerability of the species 73 . Raptors may be assumed to be potential natural predators of this species, but this threat is probably negligible compared to the generally precarious position of any small-island endemic with respect to natural disasters and stochastic population fluctuations. Further studies on the taxonomy, ecology, and distribution of the new species will help to understand its present status in more detail.

Data availability
The following information was supplied regarding the accessibility of DNA sequences: The generated partial fragment of mitochondrial Cytochrome b gene sequences are deposited in GenBank of NCBI under accession number MW417367 and MW417368. www.nature.com/scientificreports/