Bull Mar Sci. 92(1):17–31. 2016 research paper http://dx.doi.org/10.5343/bms.2015.1031
Connectivity of the squat lobsters Shinkaia crosnieri (Crustacea: Decapoda: Galatheidae) between cold seep and hydrothermal vent habitats
1 Institute of Marine Biology, Chien-Hui Yang 1 National Taiwan Ocean Shinji Tsuchida 2 University, 2 Pei-Ning Road, 2 Keelung 20224, Taiwan, ROC. Katsunori Fujikura 2 2 Japan Agency of Marine-Earth Yoshihiro Fujiwara Science and Technology, 2-15 Masaru Kawato 2 Natsushima-cho, Yokosuka, 3 * Kanagawa, 237-0061, Japan. Tin-Yam Chan 3 Institute of Marine Biology and Center of Excellence for the Oceans, National Taiwan Ocean ABSTRACT.—The deep-sea squat lobster, Shinkaia University, 2 Pei-Ning Road, crosnieri Baba and Williams, 1988, previously only observed Keelung 20224, Taiwan, ROC. in hydrothermal vents, was recently found in a cold-seep site * Corresponding author email: off the coast of southwestern Taiwan in the South China
Although both deep-sea hydrothermal vents and cold seeps are chemosynthetic ecosystems, they are generally composed of different communities Sibuet( and Olu 1998, Hourdez and Lallier 2007, Govenar 2010). More than 600 animal species have been reported in deep-sea vents and cold seeps (Van Dover et al. 2002, Desbruyères et al. 2006), including at least 125 species of decapod crustaceans in 33 families (Martin and Haney 2005). However, only a few decapod crustaceans (e.g., Alvinocaris longirostris Kikuchi and Ohta, 1995, Munidopsis acutispina Benedict, 1902, Munidopsis lauensis Baba and de Saint Laurent, 1992, Munidopsis naginata Cubelio, Tsuchida and Watanabe, 2007, and Shinkaia crosnieri Baba and Williams, 1988) have been reported in both deep-sea vents and cold seeps (Desbruyères et al. 2006, Fujikura et al. 2008, Baba et al. 2009, Lin et al. 2013, Li 2015). Among them, the galatheid S. crosnieri is often found to be particularly abundant (Fig. 1), except at the Minami-Ensei Knoll (Hashimoto et al. 1995). Moreover, this species is distinct from the other squat lobsters and was once placed in a separate subfamily, Shinkaiinae (Baba and Williams 1998). Shinkaia crosnieri was previously reported only in the deep-sea hydrothermal vents in the Okinawa Trough, northeast Taiwan, and the Bismarck Archipelago (Baba and Williams 1998, Chan et al. 2000). Recently, this species has also been determined to be a dominant species at a deep-sea cold seep on
Bulletin of Marine Science 17 © 2016 Rosenstiel School of Marine & Atmospheric Science of the University of Miami 18 Bulletin of Marine Science. Vol 92, No 1. 2016
Figure 1. Shinkaia crosnieri communities from (A) deep-sea hydrothermal vent and (B) cold seep. (A) is located at Okinawa Trough, Hatoma Knoll, 27°51.4’N 123°50.5’E, 1490 m, 27 April 2005; (B) is located at Formosa Ridge, South China Sea, 22°6.9’N 119°17.1’E, 1126 m, March 2007. the Formosa Ridge off the coast of southwestern Taiwan in the South China Sea Baba( et al. 2009, Li 2015; Fig. 1B). The geographic distance between the vents in the East China Sea and the cold seep in the South China Sea is not long (i.e., approximately 800 km; Fig. 2), and no noticeable morphological difference is observed between the materials in the two areas. Nevertheless, the two habitats represent distinct ecosystems (i.e., vent vs cold seep) and belong to different basins (i.e., the East and South China seas). Because hydrothermal vents and cold seeps are special chemosynthetic ecosystems with many endemic fauna, but are often geographically distant from each other, the connectivity of the various populations and the presence of cryptic species in these areas have become prominent research topic. Most of this population genetics research has examined vent animals. Some studies have found high gene flow among different populations (e.g.,Creasey et al. 1996 for the vent shrimp Rimicaris exoculata William and Rona, 1986; and Vrijenhoek 1997 for various vent animal groups), whereas some have not (e.g., Won et al. 2003 for Bathymodiolus mussels; Hurtado et al. 2004 for various annelids; and Johnson et al. 2006 for Lepetodrilus limpets). However, few population genetics studies have investigated species distributed in both hydrothermal vents and cold seeps (e.g., Kojima et al. 1995, Kyuno et al. 2009, both of which were conducted on bivalves). The present work attempted to elucidate the connectivity between northern and southern Taiwan, as well as between the vent and cold seep populations of S. crosnieri. Molecular analyses are now widely employed in population genetics studies, including those from chemosynthetic ecosystems (e.g., Craddock et al. 1995, Won et al. 2003, Hurtado et al. 2004, Smith et al. 2004, Johnson et al. 2006, Mateos et al. 2012). The present study used the mitochondrial cytochrome c oxidase (COI) and nuclear adenine nucleotide translocase (ANT) intron genes as genetic markers to investigate the connectivity between the hydrothermal vent and cold seep S. crosnieri populations. These two genetic markers have been successfully used to study the connectivity in decapod crustaceans, such as crabs and lobsters (e.g., Barber et al. 2012, Groeneveld et al. 2012, Tourinho et al. 2012, Yednock and Neigel 2014). Furthermore, principal component analysis (PCA) was used to identify any morphological differences among the cold seep and hydrothermal vent populations. The cold seep off the coast of southwestern Taiwan is now considered a potential site for deep-sea gas hydrate exploitation. More knowledge of the population characteristics of the dominant species at this site is crucial for exploitation management planning. Yang et al.: Connectivity of the squat lobster Shinkaia crosnieri 19
Figure 2. Location of sampling stations for Shinkaia crosnieri used for the molecular study. “r” cold seep; “q” hydrothermal vent; “Stn.” station; “spec.” number of specimens used; “HPD” collecting gear ROV Hyper-Dolphin of the JAMSTEC; “SONNE” collecting gear TV grabber of the RV SONNE. Photograph of S. crosnieri from the Formosa Ridge cold seep specimen labeled as “50_1_1” in Figure 4.
Materials and Methods
Molecular Analysis Sample Collection.—Sixty-five individual S. crosnieri samples were used in the pres- ent study, collected from the Okinawa Trough hydrothermal vents and the cold seep off the coast of southwestern Taiwan Fig.( 2). Specimens from the Okinawa Trough were from two different sites 300 km apart, with 12 samples from the Hatoma Knoll and 21 from the Izena Calderon. The Formosa Ridge cold seep site material had 32 specimens. Materials from the hydrothermal vents at the Okinawa Trough were col- lected using the remotely operated vehicle (ROV) Hyper-Dolphin (3000 m class) of the Japan Agency for Marine-Earth Science and Technology (JAMSTEC). Specimens from the cold seep off the coast of southwestern Taiwan were sampled using the Hyper-Dolphin and the TV grabber, which was deployed using the RV SONNE (Universität Bremen, Germany). The specimens were deposited at the JAMSTEC (Okinawa Trough, Izena Calderon, and Hatoma Knoll: 31 specimens; southwest Taiwan: 25 specimens) and the National Taiwan Ocean University (Okinawa Trough, Izena Calderon: 3 specimens; southwest Taiwan: 7 specimens). DNA Extraction, PCR Amplification, and Sequencing.—Genomic DNA was extracted from the chelae or abdomen muscle by using the QIAGEN® DNeasy Blood and Tissue Kit following the manufacturer’s protocol. COI and ANT genes 20 Bulletin of Marine Science. Vol 92, No 1. 2016 were amplified using the primer set from Folmer et al. (1994) (657 bp, LCO1490/ HCO2198) and Teske and Beheregaray (2009) (680 bp, DecapANT-F/DecapANT-R). PCR reactions were performed in 25 μl reactions with 50–250 ng of the DNA tem- plates, 2.5 μl of 10× polymerase buffer (TaKaRa TaqTM), 0.5 μl of 25 mM magnesium TM chloride (MgCl2, TaKaRa Taq ), 0.5 μl of 2.5 mM of deoxyribonucleotide triphos- phate (dNTPs) (TaKaRa Taq TM), 0.5 μl of 10 μM for each primer (MDBio Inc.), and 0.5 U of Taq polymerase (5 U μl−1, TaKaRa Taq TM). An additional 0.5 μL of 1% bovine serum albumin (stock concentration: 0.5 mg μl−1) was used only for the COI gene. The PCR cycling conditions were as follows: 5 min at 95 °C for initial denaturation, then 40 cycles of 30 s at 94 °C, 40 s at 48 °C (COI) or 52.5 °C (ANT), 30 s at 72 °C, and final extension for 7 min at 72 °C. After checking the size and quality of the PCR products by using 1% agarose gel electrophoresis, the remaining PCR products were transferred to the commercial biocompany for subsequent sequencing. Those PCR products were sequenced (forward and reverse) with the same PCR primer set on an ABI 3730 Genetic Analyzer (Applied Biosystems). SeqMan ProTM (LASERGENE®, DNASTAR) was used to clean and edit sequences for contig assembly. Data Analysis—A dataset of the COI sequence was translated into the correspond- ing amino acid by using EditSeq (LASERGENE®, DNASTAR) to determine whether pseudogene was included (Song et al. 2008). All COI and ANT sequences were depos- ited in GenBank (accession numbers: COI KU285501-KU285565, ANT KU285566- KU285603). MAFFT v7 (Katoh and Standley 2013) was used to align the generated sequences. The aligned data set was edited using BioEdit v7.1.3.0 Hall( 1999). An additional COI sequence of S. crosnieri was available from the GenBank (EU420129), and was included in the analysis. This sequence was from a specimen collected at the Izena Calderon (Dive 398) in the Okinawa Trough by the JAMSTEC (Yang et al. 2008). The uncorrected pairwise distance (p-distance) was estimated using MEGA 6.0 (Tamura et al. 2013) to compare the sequence similarity among the vent and seep specimens. The ANT sequence data set was treated as double genotype data by using DnaSP v5 (Librado and Rozas 2009) because of the existence of some heterozygous alleles. The sequence polymorphism information was also calculated using DnaSP v5, including the number of haplotypes (H), number of polymorphic sites (S), nucleo- tide diversity (π), and haplotype diversity (h) for each sampling locality (Table 1). The analysis of molecular variance (AMOVA) within and among the three sampling localities, as well as the index of FST between the vent and seep sites were calculated using Arlequin v3.5 (Excoffier and Lischer 2010), and 1000 replications were used to assess the significance of these statistics. The statistical parsimony network was constructed using TCS 1.21 (Clement et al. 2000) to demonstrate the genealogy of haplotypes at each collection site. An unrooted phylogenetic neighbor-joining (NJ) tree was constructed using MEGA v6 to evaluate whether the S. crosnieri from the hydrothermal vents and cold seep were from different clades. Only nodes with an estimated bootstrap value (BP) > 50 are displayed on the final NJ tree. Morphological Analysis Samples and Characters Selected.—Measurements from 63 specimens collected at the aforementioned three localities were used for morphological analysis. This in- cluded 13 specimens (5 males and 8 females) from the Izena Calderon, 18 specimens (6 males and 12 females) from the Hatoma Knoll, and 32 specimens (14 males and Yang et al.: Connectivity of the squat lobster Shinkaia crosnieri 21 h 0.496 (0.118) 0.292 (0.105) 0.442 (0.133) 0.350 (0.086) 0.398 (0.072) π ANT 0.001 (0.000) 0.001 (0.000) 0.001 (0.000) 0.001 (0.000) 0.001 (0.000) = number of polymorphic sites; S = number of polymorphic 8 4 6 S 10 15 6 5 5 H 10 14 n 16 10 26 76* 12 h 0.974 (0.022) 0.985 (0.040) 0.977 (0.017) 0.982 (0.008) 0.948 (0.028) π COI 0.007 (0.001) 0.006 (0.001) 0.007 (0.001) 0.019 (0.001) 0.005 (0.001) S n = number of sequences; H haplotypes; 31 17 37 57 22 H 11 17 27 48 21 n 22 12 34 66 32 Izena Calderon Hatoma Knoll Izena Calderon + Hatoma Knoll Formosa Ridge Locality h = haplotype diversity (standard deviation). 27°16.3´N, 127°04.8´E 27°51.4´N, 123°50.5´E 27°51.4´N, 123°50.5´E 22°6.9´N, 119°17.1´E Latitude/Longitude Hydrothermal vents Hydrothermal vents Hydrothermal vents Total Cold seep Habitat π = nucleotide diversity (standard deviation); analyzed. crosnieri genes of Shinkaia ANT 1. Sequence polymorphisms in the COI and Table sequences obtained. ANT sequences were phased from an original 38 ANT * 76 22 Bulletin of Marine Science. Vol 92, No 1. 2016
Figure 3. Morphometric characters measured for principal component analysis (PCA) in various populations of Shinkaia crosnieri, (CL) carapace length excluding rostrum, (CW) maximum carapace width, (RL) rostrum length from tip of rostrum to base of eyestalk, (EL) maximum eye- stalks length, (MRCL) maximum length of propodus of right chela, (MLCL) maximum length of propodus of left cheliped, (AB) middorsal length of abdomen excluding telson, (TL) middorsal telson length, (P2D) maximum length of dactylus of 2nd pereiopod, (P2P) maximum length of propodus of 2nd pereiopod, (P2C) maximum length of carpus of 2nd pereiopod, (P2I) maximum length of ischium of 2nd pereiopod, (P3D) maximum length of dactylus of 3rd pereiopod, (P3P) maximum length of propodus of 3rd pereiopod, (PLDL) maximum length of distal segment of 1st pleopod in males, (PLDW) maximum width of distal segment of 1st pleopod in males, (PLPL) maximum length of penultimate segment of first pleopod in males.
18 females) from the Formosa Ridge. The analysis encompassed 31 specimens [cara- pace length (CL) 6.81–42.95 mm] from the vents and 32 specimens (CL 10.37–46.29 mm) from the seeps. Some of the specimens used for molecular and morphometric analyses differed depending on the completeness of the specimens and success of DNA extraction. Character selection was mainly based on those discussed in the original descrip- tion of S. crosnieri by Baba and Williams (1998) as well as on the male first pleopod, which has been used to separate cryptic vent crab species (e.g., Guinot and Hurtado 2003). Seventeen morphometric characters were selected (Fig. 3): the CL excluding rostrum; maximum carapace width; rostrum length (from rostrum tip to eyestalk base); maximum eyestalk length; maximum length of the propodus of the right Yang et al.: Connectivity of the squat lobster Shinkaia crosnieri 23 chela; maximum length of the propodus of the left cheliped; middorsal length of the abdomen excluding the telson; middorsal telson length; maximum length of the dactylus, propodus, carpus, and ischium of the second pereiopod; maximum length of the dactylus and propodus of the third pereiopod; maximum length and width of the distal segment of the first pleopod in males; and maximum length of the penulti- mate segment of the first pleopod in males. All measurements were performed using a digital vernier caliper (±0.01 mm). Statistics.—CL was used as a reference dimension to standardize all other mea- surements corrected by size (Daniels et al. 2001, Mariappan and Balasundaram 2004,
Sun et al. 2013) as Ms/L0, where Ms is the length of the measured character and L0 is the CL of that particular specimen. Size-corrected morphometric measurements were then calculated using multivariate analysis. PCA is generally considered an or- dination technique for use in multivariate morphometrics (Polly et al. 2013), and it was employed in this study to evaluate the morphometric variations among the different populations by using the software XLSTAT (v2015.4.01.21058, Addinsoft, New York, USA).
Results
In total, 66 COI and 38 ANT sequences were obtained (Table 1). No stop codon was found in the COI data set. Ten samples, including one from a cold seep and nine from vents, were heterozygous at the ANT loci. The ANT sequences were phased, resulting in a final alignment with 76 sequences. The nucleotidep -distances among the cold seep specimens in the COI and ANT genes were 0%–1.2% and 0%–0.6%, respectively. Among the specimens from the hydrothermal vents, the COI and ANT sequence divergences were both 0%–1.7% within the two sites and 0%–0.6% between the two sites. However, the COI and ANT sequence divergences between the cold seep and vent materials were 2.1%–3.8% and 0%–0.7%, respectively. Overall, the COI gene nucleotide diversity was higher [0.019 (SD 0.001)] than that of the ANT gene [0.001 (SD 0.000)], and the specimens from the Izena Calderon hydrothermal vents had the highest values [0.007 (SD 0.001)]. The haplotype diversity of COI [0.982 (SD 0.008)] was also distinctly higher than that of the ANT data set [0.398 (SD 0.072), Table 1]. The hydrothermal vents at the Hatoma Knoll had the highest COI haplotype diversity [0.985 (SD 0.040)] among the three sites studied. Although the cold seep population had lower nucleotide and haplotype diversity in COI (0.005 ± 0.001 and 0.948 ± 0.028, respectively) than the hydrothermal vents, it exhibited higher haplo- type diversity in the ANT gene [h = 0.496 (SD 0.118)]. The AMOVA results Table( 2) showed that the variations in the COI haplotypes among the three populations (82.20%) were higher than they were within these pop- ulations (17.91%–17.94%), though the genetic differentiations did not reach a signifi- cant level (FCT = 0.822, P = 0.332). However, the variations between the seep and vent sites were high (82.09%) and these sites had a strongly significant genetic differentia- tion (FST = 0.821, P < 0.0001). Nevertheless, for the ANT gene, the variations within populations (98.01%–98.02%) were larger than those among populations (3.23%) and between the seep and vent sites (1.98%). Because no significant genetic differentia- tion between the hydrothermal vent and cold seep populations was observed n the
ANT gene (FST = 0.020, P = 0.124; FCT = 0.032, P = 0.313), only the NJ phylogenetic 24 Bulletin of Marine Science. Vol 92, No 1. 2016
Table 2. Analysis of molecular variance (AMOVA) on the COI and ANT sequence haplotypes of Shinkaia crosineri from three hydrothermal vent and cold seep sites. Two groups = cold seep and hydrothermal vents; Three populations = Formosa Ridge, Hatoma Knoll, and Izena Calderon.
AMOVA comparison Source of variation Variation (%) F-statistics P value COI Two groups Within populations 17.91
Among populations 82.09 FST = 0.821 0.000 Three populations Within populations 17.94
Among populations within groups −0.14 FSC = −0.008 0.693
Among groups 82.20 FCT = 0.822 0.332 ANT* Two groups Within populations 98.02
Among populations 1.98 FST = 0.020 0.124 Three populations Within populations 98.01
Among populations within groups −1.24 FSC = −0.013 0.727
Among groups 3.23 FCT = 0.032 0.313 * 76 ANT sequences were phased from an original 38 ANT sequences obtained. tree of the COI gene was presented (Fig. 4). The NJ tree revealed that the cold seep and hydrothermal vent materials were effectively separated and formed two distinct monophyletic groups (Bp = 100). In total, 14 haplotypes were found in the ANT gene, and most of them belonged to Haplotype “I” (Fig. 5B). However, the statistical par- simony network of COI revealed that the vent and seep populations separated into different clades Fig.( 5A). COI sequences from 32 specimens of the cold seep popula- tion yielded 21 haplotypes and 22 polymorphic sites [π = 0.005 (SD 0.001), h = 0.948 (SD 0.028)], and the dominant haplotype (among seven specimens) was radiated into the other haplotypes. The 34 COI sequences of the hydrothermal vent population revealed 27 haplotypes and 37 polymorphic sites [π = 0.007 (SD 0.001), h = 0.977 (SD 0.017)], and Haplotype “26” (containing five specimens) was shared by the two sites and was the most common haplotype. Moreover, PCA demonstrated only sexual di- morphism with no morphological differences among the three populations studied (Fig. 6). The 17 morphometric characters measured only accounted for 46.16% of the total variations in the first two principal components Table( 3).
Discussion
The present results demonstrate that the cold seep population of S. crosnieri o ff the coast of southwestern Taiwan has 2.1%–3.8% COI sequence divergence from the hydrothermal vent population in northeastern Taiwan (specifically, the Okinawa Trough). This difference in genetic divergence is high for conspecific taxa in che- mosynthesis-based fauna, which is generally <1% in COI sequence divergences (e.g., Creasey et al. 1996, Black et al. 1997, Kojima et al. 2001, Goffredi et al. 2003). Of the two other decapod crustaceans that occurred in the same cold seep site off the coast of southwestern Taiwan, the shrimp A. longirostris was also found at the Okinawa Trough hydrothermal vents, but no COI divergences existed between the two che- mosynthetic sites (Li 2015). For M. lauensis, the material in the southwestern Taiwan cold seep has only a 0.3% COI divergence from those in the southwest Pacific hy- drothermal vents (Lin et al. 2013). The mussel Bathymodiolus platifrons Hashimoto Yang et al.: Connectivity of the squat lobster Shinkaia crosnieri 25
Figure 4. Neighbor-joining phylogenetic tree based on the COI gene from 66 Shinkaia crosnieri specimens in the hydrothermal vents (red, 34 specimens) and cold seep (blue, 32 specimens). Only nodes with an estimated bootstrap value > 50 are displayed. 26 Bulletin of Marine Science. Vol 92, No 1. 2016
Figure 5. Statistical parsimony networks constructed from the haplotypes of Shinkaia crosnieri in the hydrothermal vent and cold seep populations. (A) 48 haplotypes in 66 COI sequences; (B) 14 haplotypes in 38 ANT sequences. and Okutani, 1994, which is also located in both the Okinawa Trough hydrothermal vents and southwestern Taiwan cold seep, has no significant genetic difference be- tween the vent and seep populations in Japan (Kyuno et al. 2009). However, chemo- synthesis-based fauna treated as different species generally have more than a 5% COI sequence divergence (e.g., Black et al. 1997, Guinot et al. 2002, Goffredi et al. 2003, Miyazaki et al. 2004, Smith et al. 2004). Moreover, no morphological distinction was detected between the cold seep and vent S. crosnieri materials, even by perform- ing PCA (Fig. 6). Thus, the genetic differences revealed in the present work suggest that the cold seep and vent materials are both S. crosnieri, but represent separate populations. The COI phylogenetic tree construction (Fig. 4) and haplotype network analysis (Fig. 5A) also strongly suggest that the vent and cold seep populations differ. Yang et al.: Connectivity of the squat lobster Shinkaia crosnieri 27
Table 3. Factor loadings of principal component analysis (PCA) for 16 morphometric characters corrected by size (carapace length excluding rostrum) on three populations of Shinkaia crosineri from hydrothermal vents and cold seep.
Factor F1 F2 Maximum carapace width 5.874 1.480 Rostrum length 0.201 0.609 Maximum eyestalks length 3.881 0.614 Maximum length of propodus of right chela 2.409 0.002 Maximum length of propodus of left cheliped 3.616 2.677 Mid-dorsal length of abdomen excluding telson 0.279 12.739 Mid-dorsal telson length 0.869 4.289 Maximum length of dactylus of 2nd pereiopod 14.335 5.116 Maximum length of propodus of 2nd pereiopod 15.327 5.159 Maximum length of carpus of 2nd pereiopod 13.099 4.920 Maximum length of ischium of 2nd pereiopod 8.435 0.022 Maximum length of dactylus of 3rd pereiopod 0.075 6.718 Maximum length of propodus of 3rd pereiopod 4.323 2.734 Maximum length of distal segment of 1st pleopod in males 8.995 17.634 Maximum width of distal segment of 1st pleopod in males 8.946 17.398 Maximum length of penultimate segment of first pleopod in males 9.336 17.890 Eigenvalue 4.610 2.780 Variability (%) 28.810 17.350 Cumulative (%) 28.810 46.160
Figure 6. Scatterplot of the principal component analysis (PCA) for 63 Shinkaia crosnieri speci- mens from hydrothermal vents and cold seep. 28 Bulletin of Marine Science. Vol 92, No 1. 2016
Although the ANT sequence has been useful in delineating population differences in decapod crustaceans (e.g., Barber et al. 2012), the present ANT results do not re- veal any significant distinction among the three populations of S. crosnieri. Including more samples and/or localities in the ANT analysis may yield a different result. Nevertheless, the COI gene analysis clearly demonstrates that cold seep and vent S. crosnieri are separate populations and hence may represent distinct evolutionarily significant units (ESU, Moritz 1994). This indicates that the conservation of the cold seep population will need to be considered during future gas hydrate exploitation at this site. Further study is necessary to explain whether the lack of connectivity between the two populations results from distinct environmental factors (i.e., vent vs seep) and/or geographical separation (northeast vs southwest Taiwan and/or East vs South China seas). Recent active surveys in the deep sea off the coast of south- western Taiwan have continually revealed new cold seep sites with biological com- munities (but without S. crosnieri; S Lin, National Taiwan University, pers comm). Determining whether S. crosnieri is restricted to the Formosa Ridge cold seep or can also be found in the other cold seeps of the South China Sea is an compelling avenue of future research.
Acknowledgments
We sincerely thank S Lin of National Taiwan University, Taipei and C Berndt of the GEOMAR Helmholtz Centre for Ocean Research, Kiel for providing us with the material collected by the RV SONNE. We also thank the captain of the RV Natsushima, the opera- tion team of the ROV Hyper-Dolphin, and the chief scientist H Machiyama on board the RV Natsushima, all of whom are from the JAMSTEC, for collecting the specimens; and LM Tseng of the National Taiwan Ocean University, Keelung for suggestions on the molecu- lar analysis. This work was supported by research grants from the Ministry of Science and Technology, Taiwan, ROC.
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