The Haima cold-seep field, covering 618 km², was recently discovered on the northwestern slope of the South China Sea. Within this field, an area of 350 km² has had cold seep activity and developed into cold-seep carbonate rocks with crusts and nodules that harbor a distinct biological community (Liang et al., 2017; Zhao et al., 2020). To date, more than 80 species of macrobenthic organisms have been sampled from the Haima cold seeps, including mostly mollusks, polychaetes, and crustaceans, a few species of echinoderms, and other animals. Since the discovery of the Haima cold seeps, many new molluskan species and the first recorded occurrences of species in the South China Sea have been reported while exploring the benthic communities associated with the cold seeps (Zhang et al., 2016; Zhang and Zhang, 2017; Chen et al., 2018; Sun et al., 2018; Jiang et al., 2019; Xu et al., 2019; Dong et al., 2021). Among them, primarily gastropods and bivalves have been found, with only one reported species of the class Polyplacophora (Dong et al., 2021).

Most polyplacophorans, also called chitons, live in shallow waters, usually in the intertidal zones on rocky substrates. Only a small fraction of chitons have been found in abyssal or bathyal habitats, especially in chemosynthetic environments such as sunken wood, cold seeps, or hydrothermal vents. Until 2010, only 18 chiton species had been found in chemosynthetic areas, of which 16 species were found in cold seeps (Schwabe and Sellanes, 2010). However, new deep-sea species have been discovered with the improvement of exploration technology (Sigwart et al., 2010; Sigwart and Sirenko, 2012; Sirenko and Sellanes, 2016; Sigwart and Chen, 2018; Sirenko, 2020). Saito and Okutani (1990) described a chiton of the order Chitonida from hydrothermal vents in the central Okinawa Trough, East China Sea. They established the genus Thermochiton for Thermochiton undocostatus Saito & Okutani, 1990 and placed the genus in the family Ischnochitondae. The second member of the genus, Thermochiton papuaensis, was reported in deep waters near Papua New Guinea, likely inhabiting communities with chemical activity (Sirenko, 2020). However, both species were described merely by their morphology without any genetic information. Their relationship within the family Ischnochitondae has not been determined by molecular analysis.

Ischnochitonidae is the largest family of Polyplacophora. Kaas and Van Belle (1980) classified the family into 18 genera, comprising approximately 400 species, which contained almost half of the known chitons at the time. Although the taxa were later rearranged (Sirenko, 2006), this large family currently includes 10 genera and approximately 228 species (World Register of Marine Species). Chitons of this family have distinct apophyses, well-developed slits on all valves, insertion plates that are not pectinated, and dorsal surfaces that lack ocelli. Their adanal gills do not extend to the anus. Additionally, all members of the family Ischnochitonidae have a scaly dorsal girdle and varying tegmental sculptures (Kaas and Van Belle, 1987). The inherent paraphyly and lack of well-defined diagnostic morphological features of this family have led to taxonomic controversy. Some molecular studies suggest that the family Ischnochitonidae is not monophyletic (Okusu et al., 2003; Owada, 2018; Irisarri et al., 2020). Even the monophyly of its type genus, Ischnochiton, remains doubtful (Owada, 2018; Irisarri et al., 2020). However, most ischnochitonids have only been described by their morphology, and molecular information is scarce. Indeed, the sequences of mitochondrial cytochrome c oxidase subunit I (COI), the most widely used effective DNA barcoding gene marker, have only been published for 24 species of Ischnochitonidae in the NCBI database.

The evolutionary history of geographically separated but closely related species is tangled but fascinating. Chemoautotrophic environments are ideal natural laboratories for studying the spread, connectivity, and evolution. Hydrothermal vents or cold seeps, are essentially isolated islands in the deep sea. As most vent-endemic species are benthic with limited mobility at their adult stage, the larval stage is vital for dispersal and colonization of species obligately living in chemoautotrophic environments (Tyler and Young, 1999; Tyler and Young, 2003). The wide geographic range of distributed species and long-distance dispersal or high connectivity of panmictic populations are achieved via long periods of planktonic larval duration (PLD) for swimming larvae (Adams et al., 2012; Fukumori and Kano, 2014; Yahagi et al., 2019). Some vent or seep mollusks, such as Shinkailepas myojinensis (Yahagi et al., 2017), Shinkailepas briandi (Yahagi et al., 2019), Bathymodiolus childressi, and Thalassonerita naticoidea (= Bathynerita naticoidea) (Arellano et al., 2014), develop long-lived planktotrophic larvae (Warén and Bouchet, 2001; Fukumori and Kano, 2014), which can vertically migrate hundreds of meters to photic and even euphotic zones to hunt for abundant food supplies (Bouchet and Warén, 1994; Arellano et al., 2014). These larvae were considered to float for at least 7–12 months in the pelagic water column before settling, based on the sizes of planktonic and settled larvae, as well as post-settlement juveniles (Arellano et al., 2014; Yahagi et al., 2017). The longer PLD of these species and stronger currents at the sea surface presumably fuel broad-ranged distribution and genetic homogeneity (Bouchet and Warén, 1994; Yahagi et al., 2017). Several crustaceans have developed the same strategy (Teixeira et al., 2011; Beedessee et al., 2013). However, the strategies employed by vent-associated chitons to colonize new territories and expand their distribution have not yet been reported, partly because of their rare occurrence.

During an exploratory cruise of the Haima cold seeps, two specimens of deep-sea chiton were collected and reported as Leptochiton tenuidontus Saito & Okutani, 1990 by Dong et al. (2021). This species was misidentified and should be classified as a new species of the genus Thermochiton. In this study, we clarify the identification of this new species. Our account is based on analyzing the valve, girdle, and radula morphological characteristics of the chiton, as well as DNA sequencing of the mitochondrial COI, 16S ribosomal DNA (16S), and nuclear 28S ribosomal DNA (28S) gene regions. DNA sequences were used to confirm the arrangement of this Thermochiton species within the family Ischnochitondae and to explore its evolutionary relationship with its shallow-water relatives. Further, a numerical model was used to simulate the dispersal range and estimate the potential dispersal ability, thus suggesting the evolutionary pathway of Thermochiton species.

The discovery of T. xui sp. nov. increased the number of known members of the genus Thermochiton. Currently, the three species of the genus likely all live in areas of chemosynthetic activity. The genus Thermochiton was established for T. undocostatus Saito & Okutani, 1990, a chiton found in hydrothermal vents at the central Okinawa Trough, East China Sea (Saito and Okutani, 1990). The second member of the genus, T. papuaensis, was reported near Papua New Guinea and is likely to inhabit communities with chemical activity (Sirenko, 2020). Our new species was sampled from the Haima cold seeps in the South China Sea. The distribution areas of the genus are geographically far removed, located in the southwest and northwest Pacific. The type locality of T. papuaensis is approximately 4,637 km away from this sampling site and approximately 4,217 km away from the type locality of T. undocostatus ( Figure 1 ).

The new species is the only species with molecular information in the genus. It is impossible to analyze the phylogenetic relationship between the three Thermochiton species or infer how their ancestors spread based merely on morphological or molecular information. The modeling results in this study indicated that one Thermochiton species is unlikely to have crossed the complex Pacific western boundary currents to reach the three localities in which the genus has been reported. The western Pacific vents in distant basins are potentially connected by the Kuroshio Current (Mitarai et al., 2016). In addition, the South Equatorial Current can distribute vent species more than 4,000 km within the southwest Pacific. Nevertheless, the results of our numerical model suggested that at least 15 months were required for larvae to spread from Papua New Guinea to the Okinawa Trough via surface currents. Northward evolutionary migration is highly more likely than southward migration for Pacific deep-sea chitons. The upper prediction of the model reported by Yearsley and Sigwart (2011) estimated no more than 240 days (i.e., 8 months) for the PLD of deep-sea chitons. The deep-sea mollusk with the longest PLD is the cold seep mussel B. naticoidea, whose larvae reportedly disperse for 7–12 months at the surface before settling (Arellano et al., 2014). The larvae of the seastar Mediaster aequalis can reportedly persist for approximately 13 months before settling (Birkeland et al., 1971). Nevertheless, it was estimated that even the longest PLD could not be expected to exceed 400 days (O’Connor et al., 2007). Thus, one Thermochiton species is unlikely to have a sufficiently long PLD and dispersal ability to cross the complex Pacific western boundary currents, even under ideal conditions and with extremely strong surface currents. It is more likely that currents carried the pelagic larvae of their ancestors to spread and radiate into different species along the evolutionary pathway during colonization. We hypothesize that (1) ‘stepping-stones’ facilitated their colonization, i.e., suitable habitats for chitons between the southwest and northwest Pacific, such as seeps, vents, sunken wood, or carcasses, and/or (2) evolutionary ‘bridge species’ existed during their spread, i.e., chitons distributed at ‘stepping-stone’ sites that have not yet been found or became extinct with the disappearance of these ephemeral habitats. Therefore, additional expeditions are needed to further explore the deep-sea fauna and collect more data in order to reconstruct the evolutionary history of this genus.

Most deep-water chitons belong to the order Lepidopleurida, a basal clade of the class Polyplacophora. Sigwart (2017) suggested that this clade originated at a depth of approximately 500 m and radiated to either deep-sea or coastal areas. Most of the radiation to sunken wood occurred in the Jurassic. Many lepidopleuran chitons diversified during the Cenozoic and invaded the deep sea and different chemosynthetic habitats. Many other mollusks in hydrothermal vents, seeps, and organic falls have the pattern of Cenozoic radiation in the deep sea (Johnson et al., 2010; Lorion et al., 2013; Taylor et al., 2014). Indeed, our molecular clock analysis revealed that the genus Thermochiton originated from a shallow-sea ancestor around 5.10 to 10.07 MYA in the Late Miocene. The genus follows the “onshore to offshore” evolutionary pattern (Jablonski et al., 1983; Vrijenhoek, 2013), but occurred independently of the invasion of lepidopleuran chitons into chemosynthetic habitats.

A total of 23 species of deep-sea chitons have been identified in the bathyal and abyssal zones of the East and South China Seas to date (Kaas, 1979; Saito and Okutani, 1990; Xu, 1990; Saito, 2005; Saito, 2011; Saito, 2013; Sirenko, 2016b; Sigwart and Chen, 2018; Sirenko, 2018). Among them, 15 species live in hydrothermal vents, cold seeps, or sunken wood habitats ( Table 2 ). Chemosynthetic areas are the main habitats of deep-sea chitons. Sirenko (2018) discovered 16 chiton species off Taiwan, 13 of which were new occurrences and two were new species, based on two exploration missions in 2000 and 2001. These data significantly increased the deep-sea chiton biodiversity in the East and South China Seas. To date, only four species of deep-sea chitons have been found in the South China Sea (Leptochiton vietnamensis Sirenko, 1998; Parachiton laevisquamatus Sirenko, 2015; Loricella. scissurata (Xu, 1990); Lepidozona acostata Sirenko, 2016; and Thermochiton xui sp. nov.). Sirenko and Zhang (2019) reported 30 chiton species from the vicinity of Hainan Island. However, the deep sea and numerous islands of the South China Sea have not been adequately sampled. The number of chitons currently recorded in the deep and shallow waters of the South China Sea is far from exhaustive compared with the nearly 100 species of chiton recorded in Japan (Slieker, 2000; Saito, 2001b; Saito, 2017) and the 68 species of chiton found in Vietnam (Sirenko, 2016b). The abundance of chiton species found in other locations suggests that the South China Sea has a high degree of biodiversity waiting to be discovered.

Our phylogenetic analysis based on the concatenated sequences from three gene regions supported the non-monophyly of the family Ischnochitonidae, as well as that of the genera Ischnochiton, Lepidozona, and Callistochiton ( Figure 7 ). The outgroups were nested within the clade of the family Ischnochitonidae, similar to the findings reported by Owada (2018). The results suggest that the molecular data are not consistent with the assignment of species to genera and families based on plesiomorphic similarities. Both morphologic and genetic data indicated the complexity of the family Ischnochitonidae. Members of the genus Connexochiton Kaas, 1979 resemble those of the genus Thermochiton. Members of both genera have rounded granules at the distal end of the dorsal girdle scales, and the heads of the major lateral teeth of the radulae are unicuspidal, bent, and sickle-shaped. However, members of the genus Thermochiton have pores of aesthetes distributed throughout the tegmentum with concentric, undulating costae, whereas the pores of aesthetes of members of the genus Connexochiton are only distributed in granules. The genus Subterenochiton Iredale & Hull, 1924 is similar to members of the genus Thermochiton but differs by the presence of flattened, closely set, and same-sized tegmentum granules, as well as a single slit per side in the intermediate valves. The genus Thermochiton can be clearly distinguished from most Ischnochiton species by the following characteristics: small body size vs. medium to large body size; tegmentum granules raised vs. flat granules; intermediate valves with 2 slits per side vs. 1 slit per side; dorsal scales with granules vs. no granules; head of the major lateral tooth of the radula is unicuspidal vs. bicuspidal. Meanwhile, Ischnochiton luteoroseus Suter, 1907, Ischnochiton granulifer Thiele, 1909, and Ischnochiton albinus Thiele, 1911 also share similarities with members of the genus Thermochiton, including rounded granules on the dorsal girdle scales, unicuspidal head of the major lateral tooth of the radula, and a distinct protuberance on the major lateral tooth head. Interestingly, several species of the genus Lepidozona, Ischnochiton, Subterenochiton, and Callistochiton have dorsal scales with granules and/or longitudinal ribs, which is similar to members of the genus Thermochiton. Thus, these taxa need to be re-examined and more taxon sampling is needed. The current classification system of chitons was established based mainly on the morphology of valves, spicules, and girdle processes. Few taxonomic studies have combined morphology with molecular biology (Sigwart et al., 2010; Irisarri et al., 2020). However, as additional molecular data become available, classification uncertainty will undoubtedly arise. Emerging new data will provide insights into phylogenetic relationships and improve the stepwise systematics approach. For example, the previous morphological classification placed Ischnochiton. hakodadensis in the genus Ischnochiton, but molecular analysis revealed that the species was nested within the genus Lepidozona [(present study and findings reported by Owada (2018)]. The current information is insufficient to suggest any rearrangement of the family Ischnochitonidae; thus, this family remains poorly defined. Integrative taxonomy incorporating molecular data and morphological evidence will help solve this problem (Zhang et al., 2020). More evidence is needed to clarify phylogenetic relationships and improve stepwise systematics of chitons.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ Supplementary Material.

JZ conceived and designed this project. HW, JZ, and BS performed morphological examination and description. HL and JZ conducted molecular analyses. XW and CL preformed the numerical modeling. DD and XL collected the samples. All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.

This work was supported by the Marine S&T Fund of Shandong Province for Pilot National Laboratory for Marine Science and Technology (Qingdao) (No. 2022QNLM050102-2), the National Key Research and Development Program of China (2021YFE0193700), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB42000000, and XDA22050203), the National Natural Science Foundation of China (31772422, and 42176114), the International Science Partnership Program of the Chinese Academy of Sciences (No. 133137KYSB20200002), and the Senior User Project of RV KEXUE (KEXUE2020GZ01).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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