The taxonomy of Saccostrea oysters is problematic because of their highly variable shells. Molecular studies have revealed diverse sequence lineages that do not correspond to valid species names including some Saccostrea mordax–like oysters. The S. mordax–like oysters (n = 202) were collected from southern China and classified into three lineages, A, B, and C, based on both shell morphology and mitochondrial 16S ribosomal RNA (16S) and cytochrome oxidase I (COI) sequences. Molecular and morphological analyses indicate lineages A and B are S. mordax, and lineage C is a new species Saccostrea mordoides sp. nov. The shell morphology of the new species is variable, as in other Saccostrea oysters, but distinctive in that shell height is similar to shell length with the right valve much smaller than the left valve and having undulate laminae. The average Kimura's two-parameter distance between S. mordoides sp. nov. and other Saccostrea oysters is 0.046–0.120 for 16S and 0.106–0.240 for COI, which are significantly higher than typical distances among closely related oyster species. This study shows that S. mordoides is a new species different from S. mordax, highlighting rich species diversity of Saccostrea oysters and the need for molecular taxonomy.
INTRODUCTION
Oysters are highly variable in shell morphology. Different authors identified species based on different ecotypes and created considerable confusion in oyster classification (Harry 1985, Guo et al. 2018). Among ostreid species, the taxonomy of rock oysters Saccostrea (Dollfus & Dautzenberg 1920) is most problematic because Saccostrea species have greater plasticity in shell morphology than species of other genera because of their close attachment to substrates (Hamaguchi et al. 2014).
Zhang and Lou (1956) described four Saccostrea species from rocky shores of China based on shell morphology: Saccostrea glomerata, Saccostrea cucullata, Saccostrea mordax, and Saccostrea echinata. Based on Mayr's (1963) definition of superspecies, Stenzel (1971) and Harry (1985) considered S. cucullata as the only valid Saccostrea species or superspecies in Indo-Pacific. Using both shell morphology and anatomy, Li and Qi (1994) recognized only two Saccostrea species, S. cucullata and S. echinata, in China, and regarded S. glomerata, Saccostrea kegaki, and Saccostrea malabonensis as synonyms of S. echinata, and S. mordax as synonym of S. cucullata. Xu and Zhang (2008) recorded five Saccostrea species (S. glomerata, S. mytiloides, S. cucullata, S. mordax, and S. echinata) in China. Lam and Morton (2009) described the shell characters of S. cucullata and S. mordax from Malaysia and Singapore. Huber (2010) suggested that S. cucullata was restricted to Central Western Africa, Natal, Red Sea, and to Arabia, and that the specimens from India, Andaman Sea, Gulf of Thailand, the Philippines, Australia, and Japan should be the small rock oyster S. mordax. Amaral and Simone (2016) described anatomical differences of five Saccostrea species (S. cucullata, S. glomerata, S. echinata, Saccostrea palmula, and S. mordax) from the Pacific Ocean. The taxonomy of Saccostrea remains confusing and unresolved, as many authors relied solely on shell characters which are highly variable depending on life stages and the environment (Lam & Morton 2006, Sekino & Yamashita 2016).
Analysis of DNA sequence data has contributed to resolving some questions about oyster identifications and taxonomic relationships (Guo et al. 2018). Multiplex genus- and species-specific polymerase chain reaction (PCR) markers were developed for the identification of oysters from China (Wang & Guo 2008), and Crassostrea gigas angulata was identified as a subspecies of C. gigas (Wang et al. 2010). Several new species of oysters have been identified based on mitochondrial DNA and morphology analysis (Wu et al. 2013, Xia et al. 2014, Li et al. 2017a, Hu et al. 2019). Lam and Morton (2006) using partial mitochondrial 16S classified Saccostrea species of Indo-West Pacific into two clades, one consisted of Saccostrea cucullata A–G, Saccostrea kegaki, and Saccostrea glomerata, and the other consisted of Saccostrea mordax A and B. Hamaguchi et al. (2014) discovered the Indo-West Pacific rock oyster S. cucullata F in Kagoshima Bay, S. cucullata C and S. cucullata F in Wakayama Prefecture. These were the first records of Indo-West Pacific rock oysters in mainland Japan. According to the original description and type locality of S. cucullata, Sekino and Yamashita (2016) considered S. cucullata should not represent the Indo-West Pacific Saccostrea oysters, and refer to Lam and Morton's (2006) “S. cucullata” superspecies as “non-mordax” oysters. Furthermore, they identified additional Saccostrea lineages that were not reported by Lam and Morton (2006), including non-mordax lineage H, I, and J. Based on the original species description and type locality, Sekino and Yamashita (2016) considered that Saccostrea malabonensis might correspond to lineage F and Saccostrea echinata correspond to lineage H. Li et al. (2017a) identified S. cucullata lineage F and specimens from Myanmar as S. malabonensis. The species Saccostrea palmula, which is distinct from other species of Saccostrea, has variable morphologies within the Gulf of California but little sequence divergence (Raith et al. 2016). Guo et al. (2018) reviewed genetic data and recognized 21 Saccostrea species, including three possible species under S. mordax.
The species Saccostrea mordax is a widely distributed species and morphologically well recognized by its solid shells, with strong tooth-like margins, raised purple or black muscle scar, and purple interior margins (Huber 2010). The S. mordax and S. mordax–like oysters can be separated from the non-mordax oysters based on their mtDNA sequences and shell morphology, and further grouped into three lineages A, B, and C (Lam & Morton 2006, Sekino & Yamashita 2013). This study sampled and analyzed 202 S. mordax–like oysters from southern China. Morphological and phylogenetic analyses indicate that S. mordax lineages A and B correspond to the valid species S. mordax, and lineage C is a new species of Saccostrea.
MATERIALS AND METHODS
Sample Collection
Unknown oysters were collected at different times from Hainan, Guangdong, and Guangxi, China (Table 1 and Fig. 1). The samples were fixed in greater than 99% ethanol for subsequent DNA extraction.
Non-mordax oysters were identified based on mtDNA sequences and shell morphology (Lam & Morton 2006), and excluded from this study (n = 1,571). Only Saccostrea mordax–like oysters were subjected for further analyses (n = 202).
TABLE 1.
Sampling sites and sample size (n) for oysters collected and analyzed.
DNA Extraction, PCR Amplification, and Sequencing
DNA was extracted from adductor muscle using the TIANamp Marine Animals DNA Kit (Tiangen, Beijing). Using primers 16SF (5′-GCCTGTTTATCAAAAACAT-3′) and 16SR (5′-CCGGTC TGAACTCAGATCACG-3′), a fragment of mitochondrial 16S was amplified by PCR. A fragment of cytochrome oxidase I (COI) was amplified with primer pairs of LCO1490 (5′-GGTCAACA-AATCATAAAGATATTGG-3′) and HCO2198 (5′-TAAACT-TCAGGGTGACCAAAAAATCA-3′) (Folmer et al. 1994). Polymerase chain reaction was performed in 25 µL containing 9.5 µL ddH2O, 12.5 µL 2 × TSINGKE Master Mix, 0.5 µL of each primer (10 µM), and 2 µL template DNA, on a BIO-RAD thermal cycler with the following parameters: pre-denaturation at 94°C for 5 min, followed by 35 cycles of 94°C for 30 sec, 48–50°C for 50 sec, extension at 72°C for 1 min, and a final extension step at 72°C for 10 min. The PCR products were checked with electrophoresis on a 1.0% agarose gel. Sequencing was performed on an ABI 3730xl DNA Analyzer. Sequences obtained in this study were submitted to the NCBI ( http://www.ncbi.nlm.nih.gov/) under gene accession numbers MT298862–MT298891 (16S) and MT293806–MT293857 (COI).
Molecular Identification and Phylogenetic Analysis
DNA sequences were aligned using MEGA v. 7.0.26 (Kumar et al. 2016). Haplotypes were identified by DNASP v. 5.10.01 (Librado & Rozas 2009). Neighbor-joining (NJ) (Saitou & Nei 1987) and Bayesian inference (BI) approaches were used for phylogenetic analysis. Neighbor-joining analyses were performed with MEGA v. 7.0.26 using Kimura's two-parameter (K2P) distances (Kimura 1980). Before BI analyses, jModel Test v. 2.1.10 (Darriba et al. 2012) was used for searching the best-fit substitution model. MrBayes v. 3.2 (Ronquist et al. 2012) was used to calculate the BI tree. During BI analysis, Markov chain Monte Carlo simulation was run for 2 × 106 generations. Sequence divergence among groups was calculated with MEGA v. 7.0.26 using Kimura's 2-parameter model.
Description of Shell Characteristics
Conchological characteristics used to describe and identify species (Littlewood 1994) included plication of the margin, ligament extent, the presence of chalky deposits on the inner surface of the valves, shape and attachment area of the left valve, the presence and pattern of chomata, and the color and shape of the adductor muscle scar.
RESULTS
Mitochondrial 16S Ribosomal RNA Gene
The 16S fragment was sequenced for a total of 202 oysters (Table 1). Among the 202 sequences, 30 haplotypes were observed, and the common haplotype was found in 55.1% of the oysters and at all but two locations (Table 2). BLASTn was performed to search for related sequences in GenBank for each sequence examined in this study, identifying 28 closely related sequences (Table 3), representing all Saccostrea lineages or species proposed in previous studies. The length of the 16S used for phylogenetic analysis was 495 bp. The Hyotissa, Ostrea, and Crassostrea species were used as out-groups.
The topological structures of the NJ and BI trees are congruent, although there were inconsistencies in the clustering patterns within major clades. Only the Bayesian tree is presented (Fig. 2). The Saccostrea oysters fell into mordax and non-mordax clades. All specimens from this study were found in three lineages (A, B, and C) of the mordax clade. Lineages A and B clustered together, although the position of some haplotypes varied within the cluster. Fifteen haplotypes representing 66.3% of all oysters studied and at all locations were clustered with Saccostrea mordax A. Lineage B contained 13 haplotypes representing 30.3% of oysters from 78.6% locations sampled. The mean sequence divergence between lineages A and B was 1.0% (Table 4). The pairwise sequence divergence between the haplotypes of S. mordax A and B was 0.4%–2.1%, suggesting that variation in both lineages is high and the relationship between the two lineages is complex. The other two haplotypes grouped with lineage C and were only found in Haitang Bay in Hainan Province. The mean sequence divergence between lineage C and lineages A and B were 4.2% and 4.1%, respectively, which are significantly higher than intraspecific divergence (Wang et al. 2010). Lineage C separated from other lineages with high supportive values.
TABLE 2.
Distribution of haplotypes of the 16S rRNA gene observed in this study.
Mitochondrial COI Gene
A 561-bp fragment of the mitochondrial COI was sequenced for 79 of the 202 oysters, yielding 52 haplotypes. Phylogenetic analysis was conducted using all COI haplotypes obtained in this study and closely related sequences from GenBank (Table 3), including two Crassostrea species, two Ostrea species, and two Hyotissa species that were used as out-groups.
TABLE 3.
Details of 16S rRNA and cytochrome oxidase I nucleotide sequences retrieved from GenBank.
The BI and NJ trees from COI have the same topology that is consistent with the 16S trees, except that lineages A and B were separated more clearly in the 16S tree. Only the BI tree for COI is presented (Fig. 3). Most haplotypes are clustered with Saccostrea mordax A or B, whereas five haplotypes were clustered with the reference S. mordax C (AB748839) and clearly separated from other lineages. The COI sequences are more variable than the 16S sequences. The mean sequence divergence between lineages A and B was 2.0% (Table 5), with the pairwise sequence divergence between the haplotypes of A and B ranging from 0.9% to 3.0%. The mean sequence divergence between lineage C and other lineages was 10.4%–27.7%, which is well above the divergence between two oyster species (Guo et al. 2018). The high divergence in both genes suggests that lineage C is a new species, independent of the other two S. mordax lineages.
Figure 2.
Bayesian phylogenetic tree of Saccostrea oysters reconstructed based on partial 16S rRNA sequence. Numbers at nodes are posterior probability/bootstrap values (≥70).
TABLE 4.
Mean sequence divergence of 16S rRNA between species and lineages of Saccostrea.
Figure 3.
Bayesian phylogenetic tree of Saccostrea oysters based on partial cytochrome oxidase I DNA sequences. Numbers on nodes are posterior probability/bootstrap values (≥70).
TABLE 5.
Mean sequence divergence of cytochrome oxidase I gene between species and lineages of Saccostrea.
Systematics of the New Species
Order Ostreida Férussac (1822)
Superfamily Ostreoidea Rafinesque (1815)
Family Ostreidae Rafinesque (1815)
Subfamily Saccostreinae Li and Wang (2013)
Saccostrea mordoides sp. nov. new species
Type Measurements and Deposition
The holotype and two paratypes comprising empty dry shells and ethanol-preserved tissues have been deposited in the Marine Biological Museum of the Chinese Academy of Sciences, Institute of Oceanology, Qingdao. Shell measurements of the type materials are given in Table 6.
Description of the Holotype
The shell of the holotype is moderately sized with triangular outline (Fig. 4A). The shell height is about the same as the shell length, whereas the shell height is usually much longer than the shell length in most Saccostrea mordax (Fig. 5). The left valve is slightly cupped and completely attached. The margin of the left valve is thick and steep with evenly spaced ribs ending as marginal crenulations. The right valve is slightly convex with brownish patches and much smaller than the left valve. The right valve is only slightly smaller than the left valve in S. mordax (Fig. 5). Concentric growth lines spreading from the umbo of the right valve give rise to brown lamellae. The hinge line is straight and short. The interior of the shell is mostly glossy and white (Fig. 4A). The right valve has purplish-brown margin with elongated chomata distributed unevenly in a single line. The adductor muscle scar on the right valve is located toward the posterior ventral half of the pallial area, circular and deep purple with light purple bands. The muscle scar on the left valve is white with a brownish patch.
Variability in Shell Characters
Like other Saccostrea species, shells of Saccostrea mordoides sp. nov. are highly variable as demonstrated by the holotype and two paratypes (Fig. 4). The outline is triangular or elongated oval, and the attachment area is variable, depending on substratum and space. The left valve of juveniles is thin and cupped. The inner surface of the right valve is usually white or dark purple (Fig. 4). The adductor muscle scar varied in shape from circular to elongated oval and in color from light to dark purple. Shell characteristics and muscle scars are highly variable in Saccostrea mordax (Fig. 5).
Habitat
All five specimens of Saccostrea mordoides sp. nov. were collected from rocky shores in Haitang Bay, Hainan Province, China (Fig. 1). The salinity was 25.9, and the water temperature was 29°C at the time of collection on May 22, 2013. The five specimens of S. mordoides sp. nov. were among 47 oysters collected from the Haitang Bay site, and the rest were Saccostrea mordax A (29) and B (13). Within the sampling range, S. mordax A and B are also sympatric (Table 2).
Etymology
Shell morphology of the new species is similar to that of Saccostrea mordax, and hence named Saccostrea mordoides sp. nov.
Shell Morphology of Saccostrea mordax A and B
Shells of Saccostrea mordax A and B are variable and mostly found in three ecotypes: elongated D-shape, cup shape, and triangular (Fig. 5). The shell morphology of the elongated D-shape ecotype (Fig. 5, D1–D4) is consistent with the syntype of S. mordax (Fig. 5, G), which also has elongated D-shaped shells with rigid tooth-like crenulations (Gould 1850). The syntype has a right valve that is smaller than the left valve and has a dark purple band on the interior along plicated shell margin. The surface of the right valve is white and smooth. The inner edge of the right valve has round chomatas producing corresponding pits on the left valve. The adductor muscle scar is round and deep purple on both shells, whereas it is only purple on the right shell on the specimens from this study. The right valve fits tightly into the left valve with tooth-like crenulations, making it nearly impossible to separate the shells without damage. The syntype of S. mordax was collected from Fiji Islands and also found in Japan, Hainan of China, and Australia (Lam & Morton 2006).
The cup-shaped ecotype has a well-developed umbonal cavity and mostly white adductor muscle scar. The size of the right valve is small with purple patches along the interior margin. The exterior surface of the right valve has radial ribs and purple patches (Fig. 5, E1–E4). The triangular ecotype is similar to Saccostrea mordax described by Lam and Morton (2004). It has thin and fragile shells. The left valve is completely attached to the substrate with margin steeply raised. The right valve is slightly convex, with parallel grooves extending from halfway along the dorsoventral axis to the ventral shell margin. The margin of the right valve is round and has evenly spaced crenulations (Lam & Morton 2004). The interior of the shells is white with dark purple patches along the margin. The adductor muscle scar is oval and white, with faint purple stripes (Fig. 5, F1–F4).
TABLE 6.
Shell measurements and characteristics of the type materials of Saccostrea mordoides sp. nov.
Figure 4.
Shell morphology of Saccostrea mordoides sp. nov. from Haitang Bay, Hainan, China. The holotype (A): external view of the right (A1) and left valves (A2), internal view of the right (A3) and left valves (A4); Paratype 1 (B): external view of the right (B1) and left (B2) valves, and internal view of the right (B3) and left (B4) valves; Paratype 2 (C): external view of the right (C1) and left (C2) valves, and internal view of the right (C3) and left (C4) valves.
Figure 5.
Shell morphology of Saccostrea mordax from southern China and syntype. D1–D4, elongated D-shape of S. mordax A; E1–E4, cup shape of S. mordax A; F1–F4, triangular shape of S. mordax B; and G, syntype of S. mordax Gould (1850) from Fiji (Smithsonian National Museum of Natural History Collections USNM 5958).
DISCUSSION
The New Species Saccostrea mordoides sp. Nov.
Oysters of genus Saccostrea are generally grouped into Saccostrea mordax and non-mordax superspecies based on shell morphology. The taxonomy of each superspecies remains problematic. Genetic analysis indicates that each superspecies contains multiple genetic lineages that have not been properly assigned to species. Lam and Morton (2006) discovered two groups of S. mordax. Sekino and Yamashita (2013) suggested that at least two S. mordax lineages inhabited the coasts of Okinawa Island including lineage C.
This study shows that Saccostrea mordax lineage C is a new species with high genetic divergence from S. mordax lineages A and B. The new species status of Saccostrea mordoides sp. nov. is supported by phylogenetic analyses of both mitochondrial 16S and COI. Whereas S. mordoides sp. nov. is clustered with S. mordax A and B on the phylogenetic trees, the K2P sequence divergence between the haplotypes of S. mordoides sp. nov. and mordax A/B is 4.1%–5.3% in 16S and 8.9%–12.3% in COI, which are higher than most closely related oyster species (Guo et al. 2018). For example, the sequence divergence between Crassostrea gigas and C. sikamea, two well-recognized species, is 2.1%–2.4% in 16S and 10% in COI (Wang et al. 2010). As a unique taxonomic unit, S. mordoides sp. nov. is separated from other lineages of Saccostrea in both 16S and COI trees with high support values (Figs. 2 and 3).
Despite variations, Saccostrea mordoides sp. nov. has some morphological characteristics that are different from those of sympatric Saccostrea mordax and other species of Saccostrea. According to the original species description and the photo of the syntype (Fig. 5, G), S. mordax is a medium-sized oyster (SH 50 mm) with concave left valve and the brown margin erected and profoundly plicated (Gould 1850). Compared with S. mordax, S. mordoides sp. nov. does not have strongly plicated margin, and its hinge line is much shorter.
To confirm Saccostrea mordoides sp. nov. is not a synonym of other Saccostrea species, the new species is compared with all species of this genus listed in the World Register of Marine Species ( http://www.marinespecies.org/), Encyclopedia of Life ( http://eol.org/), Worldwide Mollusc Species Database ( http://www.bagniliggia.it/WMSD/WMSDhome.htm), Inaba and Torigoe (2004) and Huber (2010) in morphological description, habitat, and distribution. The S. mordoides sp. nov. is clearly different from known Saccostrea species.
This study indicates that Saccostrea mordoides sp. nov. is distributed in Hainan Province of China and Yakata of Japan (Sekino & Yamashita 2013). Data on the new species remain limited, and further studies are needed to determine its full geographic distribution. Furthermore, most sequence lineages of Saccostrea do not correspond to valid species names yet, highlighting abundant challenges in the taxonomy of Saccostrea. More studies are needed to study and assign the lineages to specific species.
The Saccostrea mordax Lineages A and B
The average K2P sequence divergence between Saccostrea mordax lineages A and B is about 1.0% in 16S and 2.0% in COI, similar to that observed between two subspecies Crassostrea gigas gigas and C. gigas angulata, 1.05%–1.32% in 16S and 2.22%–3.37% in COI (Wang et al. 2010). It is higher than intraspecific divergence in most species (Wang et al. 2010, Guo et al. 2018). The pairwise sequence divergence between some haplotypes of S. mordax A and B was even higher or close to interspecific level, although haplotypes of 16S and COI were not consistently separated into two clades in the phylogenetic trees. These results indicate that considerable genetic divergence exists between some S. mordax A and B oysters, but the level of divergence seems to be at intraspecific or subspecies levels.
Although the shell morphology of Saccostrea mordax A and B is variable and appears in three different ecotypes, one of the ecotypes, the elongated D-type, closely resembles the syntype of S. mordax (Gould 1850), suggesting that the species identification is correct. Although the other two ecotypes show different shell characteristics, they are genetically similar and should also be classified as S. mordax. There is no clear association between genetic lineages (S. mordax A and B) and ecotypes, suggesting that the variation in shell morphology is mostly due to environmental factors. The two genetic lineages are sympatric in southern China, as expected for the same species S. mordax (Table 2). Huber (2010) considered S. mordax was synonym of Saccostrea scyphophilla. Without molecular data and detailed morphological analyses, it is prudent to retain S. mordax as the species name. Although the type locality of S. mordax is Fiji Islands, S. mordax A and B have a wide distribution in the tropical and subtropical Western Pacific and in both Northern and Southern Hemispheres. Wide distributions have been reported for some other oyster species such as Crassostrea talonata (Li et al. 2017b), Ostrea equestris, and Ostrea neostentina (Hu et al. 2019). A comprehensive survey of all mordax-like oysters across its reported range of distribution may reveal additional variation or species.
ACKNOWLEDGMENTS
We are grateful to our colleagues Tao Zhang, Huilian Liu, Yaohua Shi, Peizhen Ma, Xiaolong Wang, and Bingqin Liu for helping with sampling. This work was partly supported by the National Key R&D Program of China (2019YFD0901303), National Science Foundation of China (Nos. 41776179, 42076092), Strategic Priority Research Program of CAS (XDB42000000 and XDA23050404), the earmarked fund for Modern Agro-industry Technology Research System (CARS-47), “Taishan Overseas Scholar” project of Shandong, U.S. NOAA CBO Non-native Oyster Research Program (NA04NMF4570424), and USDA NIFA Hatch (NJ32920).
