Taxonomic revision of the genus Mesechinus (Mammalia: Erinaceidae) with description of a new species

Hedgehogs in the genus Mesechinus (Family Erinaceidae), which include two currently recognized species (M. dauuricus and M. hughi), are distributed from northeast Mongolia to the upper Amur Basin in Russia and adjacent areas in northeast and northern China. In recent years, a population of Mesechinus hedgehogs was discovered from Mt. Gaoligong, southwestern Yunnan, China, far from the known distribution range of the genus. Furthermore, these hedgehogs are the only known population to be distributed at elevations higher than 2 100 m and in sympatry with gymnures. To evaluate the taxonomic status of these hedgehogs, we examined specimens representing Mesechinus taxa in China and further conducted morphometric and karyotypic analyses. Our results supported the existence of four species in China. Specifically, we identified the hedgehogs from Mt. Gaoligong as a new species, Mesechinus wangi sp. nov., and recognized M. miodon, previously considered as a synonym of either M. dauuricus or M. hughi, as a distinct species. Interestingly, we observed a supernumerary M4 on all specimens of Mesechinus wangi sp. nov., which is an extremely rare event in the evolution of mammalian dentition.


INTRODUCTION
Extant erinaceids, including spiny hedgehogs (Erinaceinae) and silky-skinned gymnures and moonrats (Galericinae), are found within the family Erinaceidae (Hutterer, 2005). The monophyly of each subfamily, as well as their sister-relationships, are well supported in various morphological and molecular studies (Corbet, 1988;Frost et al., 1991;He et al., 2012). These molecular studies also suggest that the living gymnures diverged from the ancestor of hedgehogs 40 million years ago (Ma), which is far older than the most recent common ancestor of living hedgehogs (Bannikova et al., 2014). Members in the two subfamilies are not only morphologically and genetically distinct but also characterized by different geographic distributions and habitats (Corbet, 1988). The living species in Galericinae are mainly distributed in humid montane forests of subtropical and tropical Southeast Asia (Echinosorex, Hylomys and Podogymnura), Southern China (Hylomys and Neotetracus), and Hainan Island (Neohylomys). With their most recent common ancestor considered to be in the late Miocene (Bannikova et al., 2014), living hedgehogs have adapted to diverse habitats and are widely distributed throughout Africa (Atelerix and Paraechinus) and Eurasia (Erinaceus, Hemiechinus, Mesechinus and Paraechinus) in deciduous woodland, coniferous forest, forest steppe, grasslands, savanna, dry steppes, semi-desert, and even arid desert (Corbet & Hill, 1992); until recently, however, they have never been found in tropical or subtropical rainforest. In 2003, Ai (2007) discovered a small population of hedgehogs from the southern-most edge of Mt. Gaoligong in Yunnan Province at approximately 2 200-2 600 m a.s.l., near the border between China and Myanmar. These hedgehogs are characterized by the absence of a spineless section on their head and by ears of similar length to the surrounding spines, suggesting that they are members of the genus Mesechinus (Figure 1). The discovery was unexpected and of interest because: (1) the location is at least 1 000 km from the known distribution of any other hedgehog species; (2) the elevations are higher than that of any known hedgehog habitat; (3) the habitat is subtropical montane evergreen broad-leaved forest, which is typical habitat of the gymnures but differs from any known hedgehog habitat; and (4) the animals are sympatrically distributed with gymnures (Neotetracus sinensis), which is also the first ever record. While these clues indicate that the population represents a distinct taxon, its taxonomic status has yet to be resolved.  (Hutterer, 2005).
After Sundevall described the type species Erinaceus (Mesechinus) dauuricus in 1842, another five forms were recognized, including przewalskii Satunin, 1907, hughi Thomas, 1908, miodon Thomas, 1908, manchuricus Mori, 1927, and sylvaticus Ma, 1964 Subsequently, however, manchuricus, przewalskii, and sibiricus were recognized as synonyms of M. dauuricus (Corbet, 1988), and miodon and sylvaticus as synonyms of M. hughi. The most debated species continues to be miodon, which was originally described together with hughi by Thomas (1908). Based on successive morphological research, some authors have included it in M. dauuricus (Corbet, 1978;Corbet & Hill, 1992), whereas others have included it in M. hughi (Hoffmann & Lunde, 2008;Hutterer, 2005). Furthermore, karyotypic study of miodon from its type locality demonstrated variable chromosomal numbers ranging from 2n=44 to 48 Kong et al., 2016a) due to the existence of B-chromosomes (Kong et al., 2016a), which has been interpreted as evidence of full species status (Kong et al., 2016a(Kong et al., , 2016b. However, B-chromosomes are rarely used for delimiting species and as its craniodental morphology has not yet been fully diagnosed, the species status of M. miodon remains suspicious.
In this study, we integrated morphometric and karyotypic approaches to revisit the taxonomy of Mesechinus. We examined whether M. miodon is distinguishable from other species and were particularly interested in the taxonomic status of the hedgehog population found from Mt. Gaoligong.

Specimens examined
We examined 59 specimens (skins and skulls) of Mesechinus deposited in the Institute of Zoology (IOZ) and Kunming Institute of Zoology (KIZ) of the Chinese Academy of Sciences (CAS), Shaanxi Institute of Zoology (SXIZ), Northwest University (NWU), and China West Normal University (CWNU) (see Supplementary Appendix I). These specimens included the M. hughi sylvaticus holotype. Photo images of the M. miodon holotype were also obtained for examining diagnosable characters and for morphological description and comparison with other named species. Morphology was examined and described following Corbet (1988), Frost et al. (1991), Gould (1995), and Thomas (1908). Based on our diagnosis and comparison of external and craniodental morphology, we recognized four species/putative species, including M. dauuricus (to include M. dauuricus dauuricus (n=8) and M. dauuricus manchuricus (n=5)), M. hughi (to include M. hughi hughi (n=28) and M. hughi sylvaticus (n=3)), and M. miodon (n=9). We recognized the animals from Mt. Gaoligong as a new species, which we name herein as Mesechinus wangi sp. nov. (n=6).

Morphological measurement and analysis
External measurements, including body weight (W), head and body length (HB), tail length (TL), length of hind foot (HF), and ear length (EL), were recorded from specimen tags. Spine length (SL) was measured from specimens. Twelve cranial characters were measured in millimeters with a digital caliper graduated to 0.01 mm (Table 1) following Pan et al. (2007): greatest length of skull (GLS), condylobasal length (CBL), basal length (BL), cranial height (CH), palatal length (PL), zygomatic breadth (ZMB), interorbital breadth (IOB), mastoid width (MTW), greatest width of nasal (GWN), breadth of first upper molar (BM 1 ), length of upper tooth row (LUTR), and length of below tooth row (LBTR). We extracted measurements from Allen (1938) for the eight specimens of M. miodon deposited in the Natural History Museum.
Morphometric variation was analyzed using principal component analysis (PCA) in SPSS v19.0 (SPSS Inc., Chicago, IL, USA). Only the 12 cranial measurements were used for PCA. All variables were log 10 -transformed before PCA. One-way analysis of variance (ANOVA) was used to test significant differences in external and cranial variables among species. *: Includes measurement of nine specimens measured by Allen (1938). Abbreviations are given in the Materials and Methods section.

Cell culture and cytological preparation
One specimen representing Mesechinus wangi sp. nov.
(museum catalog number: KIZ 034115) was used for cell cultures. We followed Hungerford (1965) for cell culture and metaphase preparation. The fibroblast cell cultures derived from skin fibroblasts and bone marrow are stored in the Kunming Cell Bank, Kunming, Yunnan, China. Images were captured using the Genus System (Applied Imaging Corp., USA) with a CCD camera mounted on a Zeiss Axioplan 2 microscope. Chromosomes of Mesechinus wangi sp. nov. were arranged based on their relative length in order from longest to shortest.

Morphological comparison and diagnosis
As mentioned previously, the hedgehogs from Mt. Gaoligong could be assigned to Mesechinus unambiguously based on external morphology. These animals lack a spineless area on their heads, which is distinct from Atelerix, Erinaceus, and Paraechinus (Figures 2, 3), and their ears are similar to the surrounding spines in length, which is distinguishable from Hemiechinus. The skull and teeth are also characterized by several typical Mesechinus features, including a robust jugal reaching the lacrimal (Figure 4), shallow suprameatal fossa, and narrowly separated anterior and posterior borders of the suprameatal fossa (Figure 4), which distinguish it from all other genera (see Frost et al., 1991 for discussion). We compared the external and craniodental morphology of our specimens. It is worth noting that the sample size for some species/subspecies was small and may not reflect intra-specific variation, especially that of teeth (see discussion in Frost et al., 1991;Gould, 2001), which needs to be verified in future study. All specimens examined in the current study showed few wholly white spines (Corbet, 1988; Figure 3). Spine lengths from longest to shortest were: M. miodon (~26 mm), M. dauuricus and Mesechinus wangi sp. nov. (~21-24 mm), and M. hughi (~21 mm). Spine color pattern was used by Thomas (1908) as a distinguishing feature for describing M. hughi and M. miodon ( Figure 3). We found that M. hughi from Shaanxi (including topotype of M. hughi hughi) and Shanxi (holotype of M. h. sylvaticus) and M. dauuricus shared similar characters: that is, white for two-thirds of length, followed by black ring, narrow light ring, and black tip ( Figure 3C, D). Mesechinus miodon was distinguished by spine light brown (rather than wholly white) for two-thirds of its length, followed by broad blackish-brown rings (rather than wholly black), light brown terminal (3-4 mm), and non-black tip ( Figure 3D). Mesechinus wangi sp. nov. was differentiated from M. hughi by dark ring extending to tip on most spines, with narrow white ring near tip ( Figure 3A). Based on skull morphology, frontals were relatively higher than parietals in M. hughi and Mesechinus wangi sp. nov., whereas parietals were higher than frontals in M. dauuricus and M. miodon (Figure 4). On the ventral side of the skulls, a posterior palatal shelf and well-developed spine were present on all specimens examined (Figure 4). Mesechinus miodon showed longer spines than other species ( Figure 4B). An epipterygoid process was present in all specimens (Figure 4; Frost et al., 1991). In M. miodon this process was well developed, extending labially ( Figure 4B), but only slightly or moderately developed in other taxa (Figure 4). The basisphenoid of M. dauuricus was previously considered to be uninflated, intermediate between the condition of Hemiechinus and that of Atelerix and Erinaceus (Frost et al., 1991). According to our examination, however, the basisphenoid was inflated in M. dauuricus, M. hughi, and M. miodon ( Figure 4B, C), similar to the condition observed in Hemiechinus auritus, whereas the basisphenoid was uninflated in Mesechinus wangi sp. nov. (Figure 4A), similar to that of Atelerix and Erinaceus.
On the dorsal side of the skull, the nasal-maxilla relationship was used in Corbet (1988) and Ma (1964), though Frost et al. (1991) determined that the relationship exhibited too much inter-specific variation. Nevertheless, nasal breadth was obviously and significantly (see below) different between Mesechinus wangi sp. nov. and other species. More specifically, Mesechinus wangi sp. nov. was characterized by: nasal broad, premaxilla extending only slightly posteriorly and frontal extending only slightly anteriorly on dorsal side, premaxilla not touching frontal, and nasal and maxilla sharing long common sutures ( Figure 4A). All other species exhibited much narrower nasal ( Figure 4B-D). Mesechinus hughi could be characterized by: premaxilla extending posteriorly, frontal extending anteriorly, not touching premaxilla, with nasal and maxilla sharing short sutures ( Figure 4C). Mesechinus d. manchuricus and M. miodon could be diagnosed by: premaxilla extending posteriorly, frontal extending anteriorly, premaxilla and frontal touching on dorsal side of skull (or nearly so), with nasal and maxilla not sharing common suture ( Figure 4B, D).
As M. miodon was named based on its small P 3 (triangular (equal-sided) in shape; Thomas, 1908)), examination of teeth was unavoidable here. Mesechinus dauuricus could be diagnosed by P 3 similar to P 2 in size; M. miodon could be diagnosed by P 3 smaller than P 2 (and smaller than that of M. dauuricus); Mesechinus wangi sp. nov. and M. hughi could be diagnosed by P 3 small, though similar to M. miodon ( Figure 4). All species showed reduced upper M 3 and small trigonid ( Figure  4). Most notably, Mesechinus wangi sp. nov. could be further distinguished by consistent presence of single-rooted M 4 on all specimens examined ( Figure 5), much smaller than M 3 .
As shown in Figure 6A, M. dauuricus, M. miodon, and Mesechinus wangi sp. nov. plotted closely in the positive region of PC1 and PC2, indicating that these taxa had larger skulls. Further, M. hughi plotted in the negative region of PC1, indicating this species had a smaller skull. In the PC1 and PC3 figure ( Figure 6B), Mesechinus wangi sp. nov. plotted in the positive region of PC3 against all other species, indicating this species had the widest nasal.
We employed one-way ANOVA for all external and cranial variables.
The results showed that all variables were significantly different among the four species (P<0.001), except for HB (F =2.080, P=0.134), HF (F =0.522, P=0.596), and PL (F =7.561, P=0.002).   Figure 7. The diploid number (2n) and autosomal fundamental number (FNa) were 48 and 92, respectively ( Figure 7A). The autosomes and X chromosomes were biarmed; however, we could not determine whether the Y chromosome was biarmed as it was too small. In total, 22 metacentric + 24 submetacentric autosomes were found in the karyotype. Both the X and Y chromosomes were metacentric, with the Y chromosome being smallest. G-banded karyotypic analysis identified homologous chromosomes ( Figure 7B). Compared with other species in the genus Mesechinus, Mesechinus wangi sp. nov. had the same 2n and FNa as M. dauuricus and M. hughi, but differed from the reported karyotype of M. miodon, which is characterized by the presence of 0-4 B-chromosomes (2n=44-48; FNa=82-92; Table 3). The numbers of metacentric chromosomes (M), submetacentric chromosomes (SM), and subtelocentric chromosomes (ST) also differed among species.

Taxonomic implications
We compared the morphology and karyotypes among Mesechinus taxa in China. Although sample sizes were small for several forms, the patterns detected in the morphological and karyotypic analyses helped clarify the taxonomy of this genus. It is worth noting, as well discussed in Gould (2001), that dental structures in hedgehogs can exhibit considerable intraspecific variation, and all dental characters should be treated with caution.

Figure 6 Plot of Mesechinus spp. for principal component factors 1 and 2 (A) and 1 and 3 (B)
Mesechinus miodon is still recognized as a subspecies of either M. dauuricus (Corbet & Hill, 1992) or M. hughi (Hutterer, 2005). Here, however, we recognized M. miodon as a distinct species based on morphometric and karyotypic analyses ( Figure 6; Table 3). Thomas (1908) named M. miodon and M. hughi but did not compare either with M. dauuricus.
In the current study, M. miodon was easily distinguished from M. hughi ( Figure 6A), with its obviously larger cranial measurements (Table 1). Mesechinus miodon and M. dauuricus exhibited similar overall skull shape and size (Table 1, Figure 6), but M. miodon was distinguishable based on different spine color pattern and smaller P 3 .
The implications of the karyotypic evidence are two-fold. On the one hand, M. miodon had a smallest number of metacentric chromosomes in the genus, and the numbers of submetacentric and subtelocentric chromosomes were also different from that of M. dauuricus, indicating that these two morphologically similar forms were distinct species. On the other hand, the existence of B chromosomes in the topotype of M. miodon (from Yulin), as reported in previous studies Kong et al., 2016a), should be treated with caution. Although B chromosomes are heterochromatic and can be verified easily using the C-banding karyotypic approach (e.g., Badenhorst et al., 2009), this was not adopted in the original studies mentioned above. The number of B-chromosomes is usually stable, rather than highly variable as reported for M. miodon (0-4), and is usually an odd number, rather than an even number (Table 3) as reported in other mammals (e.g., Badenhorst et al., 2009). Thus, reexamination of the C-banding karyotype using additional samples is warranted. Finally, B-chromosomes are considered adaptive characters that can vary between populations and may be a poor characteristic for distinguishing species. We recognized the hedgehogs from Mt. Gaoligong as a distinct new species due to their many unique features. This new species is the first known hedgehog to be found at elevations higher than 2 100 m (Erinaceus europaeus and M. hughi are distributed no higher than 2 100 m), while also inhabiting subtropical evergreen broad-leaved forests ( Figure 8) and co-occurring with gymnures. The color pattern of its spine is distinguishable from other Mesechinus species due to the lack of a narrow white ring ( Figure 3A). It has a broad nasal that shares a long common suture with the maxilla (Figure 4A), which differs from all other taxa. The presence of a supernumerary M 4 is also highly distinctive ( Figure 5A). We propose the name of Mesechinus wangi sp. nov. for the new species, in memory of the late Prof. Ying-Xiang Wang, a highly respected mammologist from the Kunming Institute of Zoology, Chinese Academy of Sciences. Animals were identified as M. dauuricus in the original article. Kong et al. (2016a) argued that the specimens should be M. miodon. 3 : Because of the existence of 0-4 B-chromosomes (M or SM; Kong et al., 2016a), the 2n could be 44-48 and NFa could be 84-92. 4 : Y chromosome most likely biarmed (see Figure 6B), but was too small to be confirmed.

Supernumerary upper molar
During the evolution of mammalian dentition, changes in tooth number are very common between taxa and within species, including in the eulipotyphlan mammals. Differences in number of teeth can be used as diagnostic characteristics in shrews (e.g., between Crocidura and Suncus) and talpid moles (e.g., between Euroscaptor Mogera, and Parascaptor ), especially the number of incisors and premolars (or unicuspids in Soricidae). These differences are often triggered by geographic isolation and speciation, such as observed in the Persian mole (Talpa davidiana; Kryštufek et al., 2001) and Japanese mole (Mogera wogura; Asahara et al., 2012).
Nevertheless, an increased number of molars is an extremely rare event observed in only a few taxa, such as the bat-eared fox (Otocyon megalotis), and can be impacted by the evolution of different feeding behaviors and explained using an inhibitory cascade model (Asahara, 2013;Asahara, 2016;Asahara et al., 2016). For example, adaptation toward an increased bite force in the hypercarnivorous bush dog (Speothos venaticus) resulted in an enlarged M 1 , thus prohibiting the development of M 3 dentition (Damasceno et al., 2013). Although erinaceids are characterized by highly variable dentition and tooth structure (Gould, 2001), M 4 and M 4 have never been observed in either living or fossil species. Loss of M 3 has been observed in short-faced hedgehogs, an extinct subfamily of gymnures (Brachyericinae), following remarkable shortening of the skull (Rich & Rich, 1971). Based on teeth features, Lopatin (2006) hypothesized that brachyericines from Asia underwent adaptation toward carnivory. If the inhibitory cascade model is valid in Erinaceidae, the presence of a supernumerary M 4 in Mesechinus wangi sp. nov. could be attributed to a combination of genetic bottleneck and isolation (hypothetically after long-distance dispersal from northern China) as well as adaptive selection from an omnivorous-insectivorous diet toward a highly omnivorous one resulting in reduced inhibition during the development of the upper molars (Asahara, 2016;Asahara et al., 2016).  Frost et al., 1991: 30. Hedgehog of genus Mesechinus (GLS=55.18 mm; Table 1). Length of ear similar to surrounding spines. Spines 21-23 mm in length, white for two-thirds of length, followed by black ring, light narrow ring, and black tip ( Figure 3); premaxilla extending posteriorly to frontal ( Figure 4); P 3 triangular (equal-sided) in shape, similar to P 2 in size (Figure 4). Comments: Because M. miodon has been recognized previously as a synonym or subspecies of this species, the distribution boundary between these two species is unclear and the specimens from the southern-most distributions (especially in northwestern China Ningxia and Shaanxi) need to be carefully re-examined.

Comments:
We recognized H. sylvaticus (Ma, 1964) as a synonym of M. hughi. To date, its taxonomic status has not been appropriately evaluated as it is only known from its holotype. Ma (1964) described sylvaticus as a new species but did not examine the specimens of M. hughi. Here, the characters used to define sylvaticus, such as spine color pattern and presence of sagittal ridge, were observed on all specimens of M. hughi examined. Therefore, we recognized sylvaticus as a synonym of M. hughi. This species inhabits mountainous broad-leaved forest, distinct from M. dauuricus and M. miodon, and its overall dark color may be an adaptation to such environments.
Ventral pelage pale white; spines 22-29 mm long, first two-thirds light brown (ivory white in other species), then broadly ringed blackish-brown, terminal 3-4 mm of spine light brown; parietal higher than frontal; well-developed spine on posterior palatal shelf; epipterygoid processes well developed; nasal narrow, premaxilla extending posteriorly, frontal extending anteriorly on dorsal side, touching premaxilla, nasal and maxilla without common suture; P 3 triangular (nearly equal-sided) in shape, smaller than P 2 . Distribution: Northern Shaanxi and eastern Ningxia, China (Figure 1).
Comments: Named as Erinaceus miodon based on small P 3 (Thomas, 1908). Corbet (1978) considered it as a synonym of Hemiechinus dauuricus, which was subsequently followed by many researchers (e.g., Corbet, 1978Corbet, , 1988Corbet & Hill, 1992). Hutterer (2005) assigned it as a synonym of M. hughi (perhaps following a comment in Frost et al. (1991)). However, both the skull size and shape were very distinct from M. hughi in our morphometric analyses ( Figure 6). Furthermore, it could be distinguished from M. dauuricus based on the different color patterns on the spine (Figure 3) and smaller P 3 . The epipterygoid processes were also longer than those in M.
dauuricus, though the sample size was small (Figure 4). We therefore recognized it as a valid species.
The karyotype of animals from the type locality varied from 2n=44-48 due of the presence of B chromosomes Kong et al., 2016a). Kong et al. (2016b) reported both M. miodon and M. dauuricus as distributed in Yuling in northern-most Shaanxi; however, the distribution boundary remains unknown. Etymology: Named in memory of Prof. Ying-Xiang Wang (1938Wang ( -2016, head of the mammal research group at the Kunming Institute of Zoology, Chinese Academy of Sciences. He undertook extensive research on the taxonomy, phylogeny, zoogeography, and conservation of mammals and made distinguished contributions to Mammalogy in China (Jiang, 2016). Diagnosis: Body size larger than M. hughi, but similar to M. dauuricus and M. miodon. Most spines (>80%) lack white ring, in contrast to other Mesechinus species. Frontal higher than parietal, differing from M. dauuricus and M. miodon but similar to M. hughi. Spine on posterior palatal shelf short, similar to M. hughi but different from M. dauuricus and M. miodon. Epipterygoid processes longer than that on M. dauuricus and M. hughi, but shorter than that on M. miodon.
Description: Large Mesechinus species (HB=202.40 mm; GLS=54.75 mm; CBL=54.55 mm; Table 1). Absence of spineless area on scalp; length of ear equal to surrounding spines; ventral pelage dark brown; spines 22-25 mm long, most spines (>80%) white for two-thirds of length and black for other one-third, small number of spines (<20%) white for two-thirds of length, then ringed in black, followed by narrow white ring, tip black; frontal higher than parietal; short spine (~1 mm) present on posterior palatal shelf, extending only slightly posteriorly; epipterygoid processes extend labially (2-3 mm); basisphenoids uninflated, two basisphenoids on both sides touch medially, excavated into shallow spherical fossa (namely, nasopharyngeal fossa; see Frost et al., 1991), breadth of nasopharyngeal fossa~1.5 mm, breadth of nasal~4.3 mm; premaxilla extending slightly posteriorly and frontal extending slightly anteriorly on dorsal side of skull, not meeting each other, nasal and maxilla sharing suture (~5-8 mm); jugal large, reaching lacrimal, lacrimal/maxilla suture unfused in adults; I 2 small, I 3 with two roots, larger than I 2 , P 2 rectangular shaped, similar to I 3 in size but larger than C 1 , P 3 smaller than P 2 , triangular (nearly equal-sided) in shape, M 3 heavily reduced, hypocone and metacone absent, M 4 single rooted, much smaller than M 3 ( Figure 5).

Comparisons: Mesechinus wangi sp.
nov. can be characterized by many unique features within Mesechinus, including unique color pattern on spine, uninflated basisphenoid, broad nasal, long common suture shared by nasal with maxilla, and presence of M 4 . It is similar to M. dauuricus and M. miodon in overall size (HB=202.40±26.10 mm) but is obviously larger than M. hughi (HB=189.71±24.20 mm; Table 1). Mesechinus wangi sp. nov. differs from M. dauuricus and M. miodon in relatively higher frontal than parietal in skull.

Distribution:
To date, this species is known only from three counties (Tengchong, Longling, and Longyang) of Baoshan in Yunnan, China, at elevations ranging from 2 200 m-2 680 m. The habitat is subtropical evergreen broad-leaved forest formed by a variety of vegetation, including Fagaceae, Lauraceae, Ericaceae, and Theaceae ( Figure 8)