The Yap Trench is located on the southeastern boundary of the Philippine Sea Plate in the West Pacific Ocean. The trench forms the part of the Pacific Ring of Fire between the Palau Islands and the Mariana Trench. It is about 700 km long and nearly 9 000 m deep at its deepest point (Fujiwara et al., 2000). Studies on fauna of the Yap Trench are scarce.

Lepidopleuran chitons represent the earliest-diverging living polyplacophorans and are found predominantly in the deep sea, including sunken wood, cold seeps, other abyssal habitats, and occasionally in shallow water at temperate latitudes (Sigwart et al., 2011). Approximately 130 living species are known within the order Lepidopleurida, and all within the extant suborder Lepidopleurina (Sirenko, 2006).

Two chiton specimens were collected from sedimentary habitat by China’s manned Jiaolong submersible diving to a depth of 6 754 m in the north of the Yap Trench (9.867 5°N, 138.515 6°E) on May 18, 2016. This is a new locality record for chiton. As all specimens were collected and preserved as part of bulk samples in the field, the two chitons had suffered some post-mortem abrasion from overcrowding in the sample jars and transportation. The larger one was tightly curled (Fig. 1). Thus, characterization of radula and detailed gill arrangement was not applicable. The specimens were fixed in 100% ethanol and then preserved in 70% ethanol. The general morphology was examined focusing on shell and girdle. Their molecular phylogenetic positions in the order Lepidopleurida were reconstructed using mitochondrial cytochrome c oxidase I (COI) gene and nuclear 28S ribosomal RNA gene (28S rDNA).

The two whole chitons were surface dried, then morphological features were examined using a Zeiss Discovery V12 stereomicroscope and photographed using an AxioCam MRc digital camera (Carl Zeiss MicroImaging Inc., Germany). The preserved lengths were ~9.5 mm and 6 mm respectively. Morphologic description was according to published matrix of Sigwart (2009) and key in Sigwart and Sirenko (2012). Total genomic DNA was isolated from foot tissue using a CTAB (hexadecyltrimethylammonium bromide) protocol (Marko, 2002). Polymerase chain reaction (PCR) amplification of the mitochondrial COI gene was carried out using primers LCO1490 (5′-GGTCAACAAATCATAAAGATATTGG-3′) and HCO2198 (5′-TAAACTTCAGGGTGACCAAAAAATCA-3′) (Folmer et al., 1994). The 28S rDNA was amplified with primers 28SF1 (5′-ACCCGCTGAATTTAAGCATAT-3′) modified from the primer F63mod in Medina et al. (2001) and 28SR1 (5′-CCATTTAAAGTTTGAGAATAGGT-3′) which is the reverse complement of primer 28S rd4.8a in Giribet et al. (2006). The 25 µL volume reaction mixture contained 100 ng genomic DNA, 0.15 μmol/L of each primer, 12.5 µL 2×EasyTaq PCR SuperMix (-dye) (TransGen Biotech, Beijing). The reaction cycling profile involved an initial denaturation at 95°C for 5 min, followed by 30 cycles of denaturation at 94°C for 45 s, annealing at 50°C (for COI)/52°C (for 28S rDNA) for 45 s and extension at 72°C for 2 min, and a final extension at 72°C for 10 min. PCR products were purified using the E.Z.N.A.^® Gel Extraction Kit (Omega Bio-tek, Inc., GA, USA), and then were bidirectional sequenced using the PCR primers and the BigDye^® Terminator Cycle Sequencing Kit (v3.1) on an ABI PRISM^® 3 730 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). The forward and reverse sequences were assembled using SeqMan (Lasergene Version 7; DNASTAR, Inc., Madison, WI, USA), and the assembled files were checked by eye. Then homologous sequences were tested using NCBI BLAST (National Center for Biotechnology Information basic local alignment search tool) to confirm that they corresponded with known polyplacophoran sequences deposited in GenBank. To reconstruct the two chitons phylogenetic positions in the order Lepidopleurida, COI and 28S rDNA sequences used for molecular phylogenetic study (Sigwart et al., 2011) were included. Multiple alignments were performed using ClustalW (Thompson et al., 1994), then the shortest common length was used for phylogenetic reconstruction. Neighbour joining (NJ) trees of the two genes were reconstructed with the Kimura 2-parameter (K2P) model of nucleotide substitution. The nodal reliabilities were assessed using 1 000 non-parametric bootstrap replicates. Sequence alignments, data sets preparations and phylogenetic analyses were carried out using MEGA 6 (Tamura et al., 2013).

Class Polyplacophora Gray, 1821

Order Lepidopleurida Thiele, 1910

Family Leptochitonidae Dall, 1889

Genus Leptochiton Gray, 1847

Leptochiton sp. (Fig. 1).

External morphology. Animal small and elongate. Shell semicarinated, with low elevation (elevation ratio 0.33–0.35 in intermediate valves), valves slightly beaked. Head valve semicircular, of equal width to tail valve. Intermediate valves rectangular, lateral areas not raised, slightly convex anterior margin. Tail valve with median inflated mucro, postmucronal slope straight. Tegmentum densely sculptured with raised, round granules arranged in irregular quincunx. Apophyses subtriangular. Girdle narrow, dorsally and ventrally covered in elongate, bluntly pointed spicule-scales, distinctive long and sharp chitinous bristles in intersegmental areas. Gills arranged posteriorly.

Genetic sequences. Two COI gene sequences were 684 bp in length after exclusion of the ambiguous region, and there were three polymorphic sites between them (GenBank accession numbers: MG788319 and MG788320), with the genetic p distance of 0.004. A BLAST-n of COI resulted 89%, 88% and 87% identical to sequences of Leptochiton boucheti (GenBank accession number: JQ950261), Leptochiton vanbellei (HQ907871) and Leptochiton deforgesi (HQ907856), respectively. The assembled 28S rDNA fragments were 1 132 bp, and there were two variation sites between the two sequences (GenBank accession numbers: MG788321 and MG788322), with the p distance of 0.002. A BLAST-n of the two sequences resulted 98%–99% identical to sequences of L. boucheti (GenBank accession numbers: HQ907809 and HQ907810), 97%–98% identical to sequences of Leptochiton sp. n. 4 (HQ907809 and HQ907810) and 96% to sequence of L. vanbellei (HQ907831), respectively.

After alignment analyses, 32 COI and 29 28S rDNA sequences from 36 lepidopleuran taxa (Table 1) were included in NJ tree reconstruction, and the shortest common lengths of which were 554 bp and 993 bp respectively. NJ trees of both COI and 28S rDNA (Fig. 2) showed the two chiton specimens, Leptochiton sp._l (the larger one) and Leptochiton sp._s (the smaller one), falling within Clade II which is primarily made up of species found living in sunken wood deposits and from the tropical West Pacific in the phylogenetic study of Sigwart et al. (2011).

Remarks. Full species level description of these two specimens was not possible. Morphologically, this species strongly resembles L. vanbellei and L. deforgesi. Phylogenetically, it has a close evolutionary relationship with L. vanbellei, L. deforgesi and L. boucheti. This has been the third deepest record for deep-sea chitons so far (Sigwart and Sirenko, 2012).