The genus Crocidura, commonly known as white-toothed shrews for the pale outer layer of their teeth, is the largest genus of mammals, comprising approximately 205 species spread across the Palearctic, Oriental, and Afrotropical regions. Members of the genus look so similar externally, differing mainly in body size, relative tail length, and tail hairiness, that even seasoned taxonomists struggle to separate them. Morphology alone has repeatedly failed to resolve which populations deserve species rank. Classic chromosomal analysis, long overshadowed by DNA sequencing, offers an independent line of evidence, and the new study demonstrates just how decisive it can be when applied to a contested group.

The focal point of the research is the small-sized Crocidura horsfieldii species complex, a cluster of shrews that has long been taxonomically confused in the literature. Five taxa, Crocidura watasei, C. tadae, C. kurodai, C. wuchihensis, and C. indochinensis, have at various times been suggested to be junior synonyms or subspecies of C. horsfieldii. Earlier karyological work already showed that C. watasei, with a diploid number of 2n = 26, and C. tadae and C. kurodai, both with 2n = 40, differ considerably from C. horsfieldii at 2n = 38, supporting their recognition as valid species. But no karyological information had ever been reported for C. wuchihensis or C. indochinensis, leaving two members of the complex in chromosomal limbo.

To fill that gap, the team established primary fibroblast cell lines from ear and lung tissue of the captured female, culturing the cells in DMEM medium supplemented with 15% fetal bovine serum and antibiotics for three to four weeks. Metaphase chromosome preparations were obtained using a standard technique: cells were treated with bromodeoxyuridine for 12 hours, then with colchicine for about an hour to arrest division, exposed to a hypotonic potassium chloride solution, and fixed in methanol and glacial acetic acid. Chromosome spreads were air-dried, baked at 65 °C, and stained with conventional Giemsa. For finer resolution, the researchers applied GTG-banding, the trypsin/Giemsa method that produces the characteristic dark and light stripes allowing each chromosome pair to be identified unambiguously.

The results are unambiguous. Crocidura indochinensis has a diploid number of 2n = 38 and a fundamental number of FN = 56. Its autosomes consist of four metacentric or submetacentric pairs, four subtelocentric pairs, and ten acrocentric pairs, while the X chromosome is a medium-sized submetacentric. Each chromosome pair displays a distinct G-banding pattern, and homologous pairs are clearly identifiable. Historically, C. indochinensis was regarded as a subspecies of C. horsfieldii, a classification adopted by several influential authors. But the two taxa occupy markedly different biogeographical ranges: C. indochinensis occurs across northern and central Thailand, northern and southern Vietnam, northern and eastern Myanmar, northern and central Lao PDR, and southern China in Guizhou, Sichuan, and Yunnan provinces, whereas C. horsfieldii appears to be restricted to Sri Lanka and peninsular India.

The chromosomal comparison drives the point home. The karyotype of C. horsfieldii from peninsular India, though it shares the same diploid number of 2n = 38, has a fundamental number of only FN = 48, comprising two meta- or submetacentric pairs, two subtelocentric pairs, and fourteen acrocentric pairs. The difference in FN between the two species, combined with their disjoint distributions, supports the recognition of C. indochinensis as a distinct and valid species rather than a subspecies. Within the broader complex, the new karyotype also differs substantially from that of C. watasei from Japan and from the shared karyotypes of C. tadae and C. kurodai from Taiwan and its offshore islands, each of which carries its own distinctive chromosomal complement and sex chromosome morphology.

Beyond species delimitation, the study offers a window into karyotype evolution within the genus. Using the G-banded chromosomes of Crocidura dsinezumi as a reference, a species previously shown to retain extensive chromosomal homology across Palearctic and Oriental Crocidura, the researchers compared banding patterns chromosome by chromosome. Sixteen of the eighteen autosomal pairs in C. indochinensis appear to have similar morphology and G-banding pattern to the corresponding pairs in C. dsinezumi. The differences that do exist are remarkably few: a single fusion/fission event involving chromosome 7, whose large distal p-arm region most likely corresponds to chromosome 19 of C. dsinezumi, and a centromeric shift or a pericentric inversion involving chromosome 8, which is subtelocentric in C. indochinensis but acrocentric in C. dsinezumi. The X chromosomes of both species exhibited identical GTG-banding patterns.

The new karyotype closely resembles the presumed ancestral karyotype of the genus Crocidura, thought to consist of four pairs of metacentric chromosomes, three pairs of subtelocentric chromosomes, eleven pairs of acrocentric chromosomes, a metacentric X chromosome, and an acrocentric Y chromosome. Tracing deeper ancestry, comparative analysis showed that twelve of the eighteen autosomes of C. indochinensis have retained their ancestral state relative to the putative ancestral karyotype of the genera Crocidura and Suncus, proposed to have had a diploid number of 2n = 44. Chromosomes 1, 4, and 14 of C. indochinensis likely evolved through fusion events involving ancestral chromosomal segments, while the biarmed chromosome 7 was formed through a single break in an ancestral chromosome followed by fusion with another ancestral element.

Less than one-third of Crocidura species have been karyotyped to date, with diploid numbers ranging from 2n = 22 to 2n = 68. Considerable karyotypic variation exists among species, whereas little or no variation has been observed at the intraspecific level, a pattern suggesting that karyotypes can serve as reliable distinguishing characteristics for classification and for resolving taxonomic problems within the genus. Although phylogenetic analyses based on genetic sequences remain the most reliable tools for species identification, the new findings reinforce the argument that chromosomal data provide valuable independent evidence, particularly in groups where morphology has repeatedly misled taxonomists.

For C. indochinensis, the first cytogenetic characterization closes a long-standing gap and cements its status as a valid species within a complex that has puzzled researchers for decades. The high degree of chromosomal homology with C. dsinezumi, differing in only one fusion-fission event and one change in centromeric position, enhances understanding of karyotype evolution within Crocidura and provides a foundation for future cytogenetic, genomic, and evolutionary studies of this remarkably diverse group. As chromosome-level analyses become increasingly accessible, specimens like the shrew from Yongde Snow Mountain will continue to play an outsized role in untangling the hidden diversity of the world’s smallest mammals.

Subject of Research: Karyotype description of the Indochinese shrew Crocidura indochinensis from Yunnan, China

Article Title: First karyotype description of the Indochinese shrew Crocidura indochinensis Robinson et Kloss, 1922 ( Mammalia , Eulipotyphla , Soricidae ) from Yunnan, China

Article References: Jin, J., Dai, J., & Ye, J. (2026). First karyotype description of the Indochinese shrew Crocidura indochinensis Robinson et Kloss, 1922 (Mammalia, Eulipotyphla, Soricidae) from Yunnan, China. Comparative Cytogenetics, 20, 195-205. https://doi.org/10.3897/compcytogen.20.191955

Image Credits: AI Generated

DOI: 10.3897/compcytogen.20.191955

Keywords: Crocidura indochinensis, karyotype, GTG-banding, Yunnan, shrew taxonomy, chromosome evolution, Crocidura horsfieldii complex, cytogenetics, First, description, Indochinese, shrew