Cryptic Marine Diversity in the Northern Arabian Gulf: An Integrative Approach Uncovers a New Species of Oyster (Bivalvia: Ostreidae), Ostrea oleomargarita
Animal biodiversity is greatly underestimated in nontemperate marine regions, especially for intertidal benthic organisms such as oysters. Recent surveys in the northern Arabian Gulf suggest the presence of numerous unidentified species, some of which form shallow reef ecosystems while others are cryptic and found under rocks. In this study, we focused on small oysters from Kuwait, which show typical characteristics in common with the genus Ostrea except for the presence of lophine chomata that would link it to the genera Lopha, Dendostrea, and Alectryonella. Phylogenetic analyses based on mitochondrial and nuclear markers unambiguously placed the Kuwait oyster within the Ostreinae as a sister to the Japanese species Ostrea futamiensis. The hypothesis that the Kuwait oyster represents a new species was assessed with phylogenetic and species delimitation methods combined with a morphological assessment. Results corroborated the Kuwait oysters as a new species herein described as Ostrea oleomargarita Oliver, Salvi, and Al-Kandari, sp. nov. The phylogeny of the Ostreinae shows extensive disagreement between morphology-based genera and phylogenetic clades. The genus Ostrea is polyphyletic, and the form and distribution of taxonomic characters such as chomata are not as definitive as suggested in previous studies. This study, along with other recent investigations, confirmed the Arabian Gulf as a key region for discovering marine animal diversity and suggested a possible biogeographic divide between the Eastern and Western Indo-Pacific. A pattern that has been documented in a growing number of taxa and that warrants further research attention.
Marine animal biodiversity is greatly underestimated, especially outside temperate regions . This lack of knowledge is due to a variety of taxonomic impediments, including the difficulty of sampling in marine ecosystems, the lower availability of systematists, and poorer support infrastructures for documenting biodiversity in tropical and subtropical regions . In addition, many intertidal benthic organisms, such as bivalve molluscs, show limited morphological complexity and high phenotypic plasticity driven by different environmental factors, making traditional taxonomic assessment based on morphology inherently challenging . This is particularly true for oysters (Ostreidae Rafinesque, 1815), a relatively small family of bivalves with 74 currently accepted species  that are widely distributed throughout world oceans and estuaries.
Despite the relatively low species diversity, oysters play an important role in marine ecosystems and are important fishery resources that support aquaculture industries worldwide. However, the diversity and distribution of living oysters are far from being fully understood (e.g., [5–7]) as focus has been given to commercially exploited species. The assessment of species diversity in oysters is hampered by the lack of reliable morphological characters for species identification as well as for their assignment to higher taxa . Historically, oysters have been described from few shell characters that are phenotypically plastic and phylogenetically unreliable [9–11]. Several molecular studies have shown how shell morphology is both unreliable and misleading for oyster classification up to the subfamily level [11–13]. Particularly emblematic in this respect are those cases where allopatric ecomorphs of a single species have been classified as distinct species in different subfamilies , or where syntopic populations of distinct species have long been considered as a single species due to their phenotypic convergence (e.g., [13, 14]). More recently, an integrative molecular and morphological approach has allowed a great leap forward in our knowledge of oyster diversity and phylogeny. This in turn has led to the characterization of local and endemic faunas (e.g., [15, 16]). However, while a molecular approach has great potential in the discovery of new species of oysters, these studies have been biased toward the clarification of the taxonomic status of cultured species and have been focused especially on the Eastern Indo-Pacific (e.g., [13, 17]). Far less is known on the oyster diversity of the Western Indo-Pacific, despite this region acting as a centre of origin for shallow water marine biota .
Recent surveys along the coast of Kuwait (northern Arabian Gulf) suggest that in these shallow waters, there are a number of unidentified oyster species including some that form prominent intertidal features. This is perhaps not surprising since serious biological investigation, in Kuwait, only dates from the 1980s . A recent survey of the intertidal macrofauna of Kuwait, based on a morphological assessment, reported five species of Ostreidae . These authors listed the ostreids Alectryonella cf. crenulifera (Sowerby, 1871) = Dendostrea sandvichensis (Sowerby, 1871), cf. Booneostrea subucula (Jousseaume in Lamy, 1925), cf. Nanostrea deformis (Lamarck, 1819), Crassostrea sp., and Saccostrea cuccullata (Born, 1778). It is evident from this listing that there was considerable doubt with many of the morphological identifications and that many of these oysters might represent undescribed species. One oyster listed by Al-Kandari et al.  as Crassostrea sp. has now been described as a new species Talonostrea salpinx Oliver Salvi Al-Kandari 2021 based on molecular and morphological data . That was the first record of the genus Talonostrea X.-X. Li and Z.-Y. Qi, 1994, in the Arabian region and the Indian Ocean as a whole.
In this study, we performed an integrative molecular and morphological assessment of specimens of a small cryptic, flat oyster (here referred to as “the Kuwait oyster”) that was tentatively referred to as the ostreine taxon Nanostrea deformis by Al-Kandari et al. . While all specimens possessed typical ostreine anachomata and chomata, a number also had small pustular (lophine chomata) that are characteristic of lophine genera [21, 22]. The subfamily Lophinae Vialov, 1936, is no longer accepted, and lophine genera are now included in the subfamily Ostreinae Rafinesque, 1815, because they form a subclade that is nested within the Ostreinae clade [10, 11, 23]. However, it is still unclear whether their shared morphological characteristics do represent synapomorphies of this subclade or instead lack any phylogenetic and systematic value within the Ostreidae [9, 21, 22, 24]. In this respect, the taxonomic and phylogenetic assessment of this small oyster from Kuwait might provide some insights on the phylogenetic significance of lophine chomata. Here, we used mitochondrial and nuclear markers to estimate phylogenetic relationships of this taxon within the Ostreidae and we used cytochrome oxidase I (COI) data to assess species delimitation, intraspecific variation, and phylogeographic relationships with its closest relative. Combined with a morphological assessment, these results support the recognition of Kuwaiti specimens as a new species that is described herein. The aim of this study is to identify independent evolutionary lineages or cryptic taxonomic units within northern Arabian Gulf flat oysters and to contribute to our understanding of the biogeographic and biodiversity patterns of intertidal fauna of this region.
2. Materials and Methods
2.1. Study Area
Details of the oceanography and marine biology of Kuwait can be found in Al-Yamani et al.  and more recently revised by Al-Yamani . These works contain an extensive synthesis of Kuwait’s marine environments, and we have extracted some pertinent characteristics relevant to the subject of this paper.
The intertidal of Kuwait features hard and soft substrate shores. In the inner Kuwait Bay and the northern regions, around Boubyan, soft sediment consisting of fine muds dominates. On the outer shores of Kuwait Bay, the soft sediments become sandier and this trend continues along the south coast. Hard substrate shores are present throughout the country and can consist of bedrock reefs or a mixture of boulders, and cobble intermixed with soft sediments. A similar assortment of substrates is also found on the outer islands of Failaka and Miskan.
The waters of Kuwait are shallow, much of the region being a flooded estuary rarely exceeding a few meters depth and with a maximum of 35 m offshore. The tidal range on average is 2 m but can reach 3.5 to 4 m during equinoxes. This, in combination with the shallow topography, results in extensive intertidal flats exposed for many hours. While sea water temperatures vary a little, air temperatures range from a possible 0°C on winter nights to 51°C at the height of summer. Consequently, the intertidal fauna is subjected to extreme variations when exposed at low tide. The shallowness of the water and predominance of fine muds offshore results in a high level of sediment resuspension especially in Kuwait Bay and around Boubyan. This is exacerbated around Boubyan through the input of riverine suspended solids from the Shatt el-Arab, such that the concentration can reach 510 mg/l. In Kuwait Bay, concentrations may reach 40 mg/l but drop off along the south coast to a recorded minimum of 0.3 mg/l.
The extremes and variation in temperature, salinity, and turbidity make the rocky intertidal a rather inhospitable environment for unprotected animals. On exposed surfaces, the molluscan fauna is limited to a small variety of gastropods, mainly thick-shelled Cerithiidae and Muricidae, and cemented bivalves such as larger oysters, spondylids, and plicatulids. However, in cryptic environments such as rock crevices and beneath rocks, there is a diverse fauna of smaller species .
Samples were initially collected during the KISR intertidal survey of 2014–2017 , and in 2019, further samples were collected specifically for tissue extraction for molecular studies. Fifty-six specimens of the morphotype listed in cf. Nanostrea deformis by Al-Kandari et al.  were collected from two localities (Figure 1; Table 1). Twenty-three specimens selected for molecular analyses were levered open, and the adductor muscle and mantle were excised whole or in part and fixed in 100% ethanol. All specimens used for the taxonomic assessment are deposited in the National Museum of Wales (NMW.Z).
2.3. Morphological Assessment
Morphometric assessments of oyster taxa are of little taxonomic utility because of the wide and overlapping intraspecific and interspecific variability in shell size and shape that are strongly determined by habitat type, growth stage, and settlement condition (e.g., [8, 10, 27–29]). Therefore, a morphological assessment of collected specimens was performed based on qualitative characters traditionally used to discriminate oyster taxa. Harry  and Littlewood  gave lists of characters used to define five groups of oysters: Saccostreinae, Striostreini, Crassostreinae (Crassostreini sensu; ), Ostreinae, “lophinae” (Lophinae sensu; ; now considered as part of Ostreinae; [4, 10, 11]), and Pycnodonteinae (Gryphaeidae Vialov, 1936). The shell characters listed were as follows: plication of the upper valve, hyote spines, lamellation of the upper valve, chalky deposits, commissural shelf, shape of adductor muscle, and colour of adductor scar. These characters were derived from Harry , and in that paper, the characters were further refined especially the form of the chomata, which are listed as vermicular, pustular, and ostreine (anachomata on the right valve and catachomata on the left valve). Vermicular chomata take the form of dense transverse ridges on either side of the ligament; ostreine take the form of single tubercles along the outer margin and where the anachomata and catachomata interlock; lophine or pustular chomata take the form of small raised pustules on the inner part of the margin and have no corresponding pits on the opposing valve (Figure 2). These characters will be used to compare species under consideration here.
2.4. Molecular Assessment
2.4.1. DNA Extraction, Amplification, and Sequencing
Total genomic DNA was extracted from alcohol-preserved tissues of the 23 specimens following standard high-salt protocols . Two mitochondrial gene fragments were amplified by polymerase chain reaction (PCR), the barcode fragment of the cytochrome oxidase subunit I (COI), and the 16S rRNA (16S), using primers and PCR protocols described in Salvi et al.  and Crocetta et al. . Additionally, for selected specimens, we amplified a fragment of the nuclear 28S rRNA (28S) using published primers slightly modified to better match the available 28S sequences of oysters: the primer F4ostrd-OS (5-TCCGATAGCAAACAAGTACCGTG-3) modified from 28S-F4-OSTRD by Mazón-Suástegui et al.  and the primer D6R-OS (5-GCTATCCTGAGGGAAACYTCAGAGG-3) modified from D6R by Park and Ó Foighil (2000). PCR protocol for the amplification of 28S followed Park and Ó Foighil (2000). Sequencing of PCR products was carried out by the company GENEWIZ (https://www.genewiz.com), using the forward and reverse primers employed for amplification.
The obtained chromatograms of each sequence were manually edited and assembled into a consensus sequence using Geneious v. 11.0.12 (Biomatters Ltd., Auckland, New Zealand). The COI chromatograms did not show any double peak, and the translated aminoacidic sequence did not have any stop codon, thus confirming that nuclear copies (pseudogenes) were not occurring in our mitochondrial COI sequence dataset. The 28S chromatograms did not show any heterozygote position. We used MEGA-X v.10.0.8  to calculate the number of variable sites of each alignment and to compute genetic distances between the Kuwait oyster and Ostrea futamiensis based on COI sequences.
We obtained 21 sequences of COI (600 base pair, bp), 23 sequences of 16S (446-451 bp), and two sequences of 28S (751 bp) of the Kuwait oyster. Details on sample data and GenBank accession numbers of sequences generated in this study are provided in Table 1 where we also indicated the GenSeq nomenclature for genetic sequences based on the taxonomic reliability of the source specimens following .
2.4.2. DNA-Barcoding and Phylogenetic Analyses
After verification that all the sequenced specimens belong to the same species (COI sequence ), we used one sequence (h1) as a query to perform a preliminary molecular species-level identification with the DNA-barcoding approach implemented in the BOLD Identification System (IDS; ). IDS analyses based on all COI barcode records with a minimum sequence length of 500 bp did not find either a species-level (less than 1% divergence) or a genus-level (less than 3% divergence) match for the query sequence. The top 20 matches were sequences of Ostrea futamiensis from Japan with a similarity score of 91.9-92.5%. Therefore, we performed phylogenetic analyses to assess the systematic relationships of the Kuwait oyster.
The phylogenetic assessment was performed in two steps. First, to establish the affiliation of the Kuwait oyster with extant subfamilies of Ostreidae, newly generated sequences of the three genes of two specimens were aligned with homologous sequences of 20 oyster species obtained from GenBank representing all currently accepted genera within the subfamilies Striostreinae, Saccostreinae, and Crassostreinae, and six out of nine currently accepted genera of Ostreinae . For this analysis, we used two species of Gryphaeidae as the outgroup based on previous phylogenetic studies [37–39]. Once the affiliation of the Kuwait oyster to the subfamily Ostreinae had been established, we built another dataset using 24 species of Ostreinae and two species of the subfamily Saccostreinae. The latter was used as the outgroup based on the results of the first analysis and previous phylogenetic studies [37, 39]. GenBank accession numbers and references for the DNA sequences used for phylogenetic analyses are reported in Table 2.
Multiple sequence alignments were performed with MAFFT v.7 on the web server (https://mafft.cbrc.jp/alignment/server/; ) using the E-INS-i iterative refinement algorithm. For each gene and dataset, details on alignment length and number of polymorphic sites and parsimony informative sites are provided in Supplementary Table S1. Single-gene alignments were concatenated in a single matrix that was used for downstream phylogenetic analyses using maximum likelihood (ML) and Bayesian Inference (BI) methods. ML analyses were performed in the W-IQ-TREE web server v.1.6.12 (http://iqtree.cibiv.univie.ac.at/; ) based on a partitioned model. For each gene partition, the best substitution model was determined by the ModelFinder module  using an edge-linked model and the cAIC criterion (the BIC criterion selected the same models). ML tree searches were based on the 20 best initial trees (out of 100 parsimony trees and the BIONJ generated) and until W-IQ-TREE cannot find a new best tree in the last 100 iterations (). Node support was assessed with 1,000 pseudoreplicates of ultrafast bootstrapping (uBS). Bayesian analyses were carried out with MrBayes v.3.2.7 , using for each gene partition the same substitution models selected by ModelFinder. We ran two Markov chains of five million generations each, sampled every 1,000 generations. Consensus trees (50% majority rule) and posterior probability values (PP) were calculated on trees sampled after a burn-in of 25%. Tracer v.1.7 (Rambaut et al., 2018) was used to check for run convergence and ESS . FigTree v.1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/) was used to visualize the tree.
The close phylogenetic relationship between the Kuwait oyster and Ostrea futamiensis recovered in ML and BI analyses was further investigated using a phylogenetic network approach based on COI sequence data. We used POPART v1.7  to construct phylogenetic networks based on the median-joining network  and the statistical parsimony  methods.
2.4.3. Species Delimitation
The BOLD Identification System (IDS) uses a standard conservative threshold of <1% sequence divergence for animal species identification as this method is designed to defend against “overdiagnosis” . Therefore, a lack of species-level match does not guarantee an interspecific divergence for the COI sequence of our unidentified oyster. To aid discrimination between intraspecific and interspecific variation, we applied species delimitation methods. For this purpose, we built a nonredundant database of COI of Ostreinae, mining sequences from the public repositories of GenBank and BOLD, with accepted and synonymized genus names as search queries (data updated to 23/11/2021). Retrieved sequences not identified to species level or shorter than 300 base pairs were eliminated. Duplicated () function  of RStudio v.1.4.1103  was used to dereplicate the dataset by removing sequences having identical GenBank accession numbers (see , for more details). This dataset was aligned with the newly generated COI sequences of oysters from Kuwait using the G-INS-i progressive method algorithm implemented in MAFFT. The alignment was trimmed to the standard COI barcoding fragment (5 portion of the gene) using Folmer’s primers as reference . The final alignment used for species delimitation analyses consisted of 456 sequences and 659 positions (among which 113 polymorphic sites and 93 parsimony informative sites).
We applied species delimitation (SD) on the generated dataset to infer species clusters using both distance-based and tree-based approaches. Assemble Species by Automatic Partitioning (ASAP) analyses were run using the ASAP web interface (https://bioinfo.mnhn.fr/abi/public/asap; ) with the Kimura-two parameter (K2P; ) substitution model and default parameters. ASAP delimitation was defined by evaluating both the partitions with the first and the second best ASAP scores according to Puillandre et al. . As a tree-based method, we used the multirate Poisson Tree Processes model (mPTP v.0.2.4; https://github.com/Pas-Kapli/mptp; ) applied on the maximum likelihood tree obtained using the W-IQ-TREE web interface. Saccostrea cuccullata (EU816078) was used as an outgroup and then removed from the SD analysis. mPTP analyses were performed using 10 runs of 100 million MCMC generations each, sampling every 10,000 (). Results from SD analyses were compared with the software LIMES v.1.3b  using the taxonomic index of congruence and the match ratios, which are two indexes that quantify the congruence between two partitions resulting from different SD approaches, and the relative taxonomic resolving power index , which quantifies the potential of an approach to capture a high number of species boundaries [55, 56].
3.1. Morphology and Patterns of Chomata
Using the characters listed above, the morphology of the Kuwait oyster is compared with that of the five major groups of extant Ostreoidea (Table 3).
Considering all characters, the Kuwait oyster has most characters in common with the Ostreinae, and only in the presence of lophine chomata does it differ (Figure 2). Of the 56 specimens collected for this study including the 23 used for the molecular study in only 7 shells was the presence of lophine-like pustules expressed. There was no difference in numbers between Failaka and Al Sha’ab material. Given the confined sampling sites, no association between habitats can be inferred. Pustules are most often seen in thinner (perhaps less mature) shells, and in some thicker shells, they can be seen below the nacreous layer suggesting that they can be overgrown as the shell thickens.
3.2. Phylogenetic Analyses
Maximum likelihood and Bayesian analyses resolved the phylogenetic position of the Kuwait oyster by placing it within the subfamily Ostreinae with a high statistical support (; ; Figure 3). Phylogenetic trees of Ostreinae show three main clades (Figure 3): the edulis group including Ostrea edulis, O. angasi, and O. chilensis (; ) which is sister to O. denselamellosa (although this relationship received low support); the stentina group including Ostrea stentina and nine allied Ostrea species (; ); and the algoensis group including the Kuwait oyster, four remaining Ostrea species, and species of the genera Nanostrea, Planostrea, Alectryonella, Dendostrea, and Lopha (; ). The Kuwait oyster has a sister relationship with O. futamiensis that is well supported (; ), and this sister-pair forms a clade with O. algoensis (; ). Species previously included in the Lophinae form a clade (genera Alectryonella, Dendostrea, and Lopha), nested within the algoensis lineage, but with low support (; ).
The median-joining network shows a clear phylogeographic separation between haplotypes of the Kuwait oyster and haplotypes of O. futamiensis (Figure 4). The haplogroups corresponding to these two species are recovered as two separate networks in TCS analyses (result not shown). Both species show a lack of geographic structure at the investigated geographical scale.
3.3. Molecular Species Delimitation
Results of species delimitation analyses based on ASAP and mPTP recovered the oyster from Kuwait as a species cluster distinct from the cluster of the closely related O. futamiensis (Figure 5). This result is confirmed in both partitions with the first (1.50) and second (4.50) best ASAP score and received maximum mPTP support (1.0). Overall, SD results by ASAP and the mPTP were highly congruent, with a value of 0.84 and a match ratio of 0.81. ASAP recovered 17 species groups, whereas mPTP recovered 20 groups. The latter results because of further splits in two of the groups recovered by ASAP: the group of the O. stentina splits into three clusters and the group of O. lurida/O. concaphila into two clusters. Both methods lumped O. permollis/O. puelcana and O. edulis/O. angasi sequences into a single cluster. Relative to the current taxonomy, as represented in MolluscaBase , the resolving power () of ASAP and mPTP was 0.84 and 1.0, respectively.
Based on morphological and molecular assessments, we assign the Kuwait oyster to a new species that is described in the following section.
Ostreoidea Rafinesque, 1815
Ostreidae Rafinesque, 1815
Ostreinae Rafinesque, 1815
Ostrea Linnaeus, 1758
Generic Definition. It is commonplace to give a generic definition when describing a new species. However, the molecular data presented here suggests that Ostrea is polyphyletic and that the new species described here and its sister taxon O. futamiensis do not cluster within the clade that includes Ostrea edulis, the type species of the genus Ostrea (see Discussion). While a systematic revision of the genus Ostrea is clearly needed, we have resorted to use Ostrea in its common understanding until a much more detailed phylogeny of the Ostreinae can be presented.
Ostrea oleomargarita. Oliver, Salvi, and Al-Kandari, sp. nov. (Figures 6–7)
Type Material. All type material is deposited in the National Museum of Wales (NMW.Z). Kuwait • holotype, 1 complete shell attached to a rock. Kuwait City, Al Sha’ab, 29.3675°N 48.0244°E. Low intertidal, attached under a rock on gravel. Coll. PG. Oliver, December 2019, not sequenced. NMW.Z.2021.009.008 (Figures 6(a), 6(b), and 7(a)). Paratypes, 11 spec. used for sequencing NMW.Z.2021.009.009/#1–#11.4 figured shells, data as holotype. NMW.Z.2021.009.010 (Figures 6(c)–6(f), 7(b), and 7(d)).
Kuwait • Paratypes, 12 spec. used for sequencing, NMW.Z.2021.009.011/#1–#12; #7 figured, Figures 6(g) and 7(c)). Ras Al Liwan, Failaka Island, 29.3902°N, 48.3988°E. Low intertidal, attached under rocks. Coll. PG Oliver, December 2019.5 shells +3 upper valves, not sequenced, NMW.Z.2021.009.012., as NMW.Z.2021.009.011.
Other Material Examined. Kuwait • 20 shells attached to a rock. Kuwait City, Al Sha’ab, 29.3675°N 48.0244°E. Low intertidal, attached under a rock on gravel. Coll. PG. Oliver, December 2019. NMW.Z.2021.009.013.
India • 1 shell +1v. Bombay (Mumbai), Bandra (approx. 19.06°N, 72.82°E). 5 April 1946. Annotated “Ranson 26.” 3v. Bombay (Mumbai), Juhu (approx. 19.10°N, 72.8°E). Coll. Winckworth, 23 October 1936. Annotated “Ranson 27.”
Shell Description. Small shells to 20 mm in diameter. Thin but robust. Roughly circular, oval to pyriform. Lower valve shallowly cupped to irregular; cemented for most or all of its attachment, margin smooth or if free then finely plicate. Nonnacreous margin, very narrow. Ligament area narrow, elongated in some. Prominent catachomata on both anterior and posterior dorsal margins. Adductor muscle large, reniform. Interior colour flushed olive-green, paler in some smaller shells, most with a distinct brown, narrow marginal band. Upper valve flat, irregular to domed. Anachomata corresponding to catachomata. Sparse elongate tubercles (lophine pustules) on posterior ventral edge in some. Internal colour as a lower valve. The outer surface is usually obscured by epifaunal growths, typically Bryozoa, calcareous algae, worm tubes, or other oysters. Sculpture mostly of flattened foliar scales, some areas finely tubercular, colour of radial bands of rust-brown to black on a grey-beige ground, some almost uniform ground colour. Thin chalky layers are present most visible in attached valves.
Derivation of Name. Derived from the combination oleo from olivarius (Latin) referring to the colour olive-green and margarita from margarites (Latin) referring to the pearly lustre of the inner surfaces of the valves. Thus, to the typical olive-green lustre of the nacreous inner surface of the valves.
Type Locality. Kuwait, Kuwait City, Al Sha’ab, 29.3675°N 48.0244°E. Intertidal, under rocks.
Distribution. In Kuwait, specimens from only two sites were verified as O. oleomargarita by molecular data. Similar shells were recorded from a number of other sites  under the name of Nanostrea deformis ranging from Khor Al-Subaya in the north of the country to other sites in Kuwait Bay and on Failaka. This species is probably widely distributed in the Northern Arabian Gulf but given the morphological plasticity, all records need confirmation by molecular data. Shells from the west coast of India were confirmed by shell morphology only.
Remarks. Comparison of the shells of O. oleomargarita with those of O. futamiensis can be made and distinctions listed, but the degree of variation seen in the former suggests that such differences may not be consistent. Perhaps, most significantly neither Seki  nor Hamaguchi et al.  note the presence of lophine pustules in O. futamiensis while these are present in some specimens of O. oleomargarita. Seki  gives the maximum size of O. futamiensis as 40 mm in height whereas O. oleomargarita reaches no more than 20 mm. Seki  states that O. futamiensis is circular or oval, and Hamaguchi states that it is circular while O. oleomargarita is much more variable. Seki  notes that the internal colour is greenish often tinged with dark blue, and this is seen in the photographs given by  (plate 8, Figures 1(b) and 1(c)); such blue colouration has not been observed in O. oleomargarita.
While these morphological differences may have been sufficient to differentiate the two species, they are not consistent enough, on their own, to warrant species recognition. Hamaguchi et al.  comment on the problematic shell-based taxonomy of five species of small Japanese flat oysters noting that molecular sequences were instrumental in separating them.
The northern Arabian Gulf is confirmed as a key region for the discovery of marine animal diversity. This study documented a new overlooked oyster species with phylogenetic affinities to an Indo-West Pacific species and a peculiar combination of morphological characters among oysters. This result echoes the recent discovery of another enigmatic oyster species in the same waters, Talonostrea salpinx, also related to the Indo-West Pacific taxa . The results are relevant to our comprehension of oysters’ diversity and distribution and the biogeography of the marine biota of the Pacific Ocean, as discussed in the following.
4.1. The New Ostreine from Kuwait: Insights into the Classification of Ostreinae and on the Taxonomic Value of Chomata
The Kuwait oyster, Ostrea oleomargarita sp. nov., shows an unprecedented combination of morphological characters typical of different groups of Ostreinae. The shell morphology is most similar to O. futamiensis, but in none of the descriptions or figures of that species is there mention of the presence of lophine pustules on the inner margin [57–59]. These pustules are not seen on all specimens from Kuwait, and in itself, this character may not be enough to justify the raising of the Kuwait populations to new species level. However, the molecular data presented here provides strong evidence that the Japanese and Kuwaiti populations do represent distinct species.
The combination of a phylogenetic approach with species delimitation methods strongly supports an interspecific divergence between O. oleomargarita sp. nov. and O. futamiensis (Figures 4 and 5). The average genetic divergence between these two species at the COI (-distance: 5.16%; K2P distance: 5.41%) is similar to, or higher than, values observed between closely related oysters (e.g., O. conchaphila/O. lurida, O. angasi/O. edulis, O. equestris/O. stentina, and O. puelchana/O. permollis; ([5, 60]; Raith et al., )). Remarkably, species delimitation methods, applied to oysters for the first time in this study, revealed a close match between current ostreine taxonomy  and species clusters inferred based on the barcoding marker COI (Figure 5). The only differences in species delimitations produced by distance-based and tree-based methods concern two well-known species complexes: the O. stentina complex and the O. lurida/O. concaphila pair. These complexes are lumped into two species clusters by the distance-based method, whereas they are split into more species by the tree-based approach. These complexes have undergone several taxonomic changes following various phylogenetic assessments [5, 21, 60, 61] and are currently considered as composed of distinct species of recent diversification [60, 62]. On the other hand, both SD methods lumped O. permollis/O. puelcana and O. edulis/O. angasi sequences into two clusters, an issue that will require further taxonomic assessments.
From a morphological point of view, features of Ostrea oleomargarita have most in common with the Ostreinae and only in the presence of lophine-like pustules is there affinity with lophine genera (e.g., Lopha and Dendostrea). The flattened form with only weak plications, lack of hyote spines, appressed lamella, and presence of chalky deposits are ostreine not lophine characters. The description and distribution of lophine pustular chomata are more variable than suggested by Stenzel  and Harry  who described them as “minute and numerous, they are located along margins and form from one to several lines.” This description fits well with Lopha cristagalli (Linnaeus, 1758) but not with many species assigned to the genus Dendostrea where the pustules are larger and variably present (see Figure 2). Interestingly, Harry  in Table 2 indicates that the ostreine genera Ostrea and Ostreola have lophine chomata but no ostreine chomata, but this must surely be an error and should be reversed. In that table, Harry also states that his ostreine Booneostrea lacks any chomata, but this is in error as in some shells, ostreine chomata are present on the posterior dorsal margin (pers. obs. PG Oliver). Therefore, there is an indication that the form and distribution of chomata are not as definitive as suggested in previous studies. The variable expression of pustules in O. oleomargarita appears to have no pattern of occurrence, although they are most often present in thinner (perhaps less mature) shells, and in some thicker shells, they can be seen below the surface suggesting they have been overgrown by later shell growth. The pattern and the dimension of these structures might drastically change within the life of an individual in lophine species too. The pustules as exhibited in O. oleomargarita are low and sparse unlike the dense pustules seen in L. cristagalli or some species of Dendostrea suggesting that they may not be analogous and a more considered review of ostreiod chomata is warranted.
Ambiguous classification based on morphological characters is commonplace in oysters (e.g., [16, 17, 62, 64, 65]), and in most cases, a molecular approach allowed a straightforward systematic assessment of debated species [10, 11]. Phylogenetic results unequivocally place O. oleomargarita within the subfamily Ostreinae. Ironically, also in the molecular phylogeny of Ostreinae, the position of O. oleomargarita and O. futamiensis is somehow in between the Nanostrea/Planostrea clade and the lophine clade. The phylogenetic trees based on ML and BI analyses of multilocus data (Figure 3) support three major clades within the Ostreinae, two of these consisting entirely of species currently recognised within the genus Ostrea (the edulis group and the stentina group), whereas in the third clade (the algoensis group), the lophine genera Alectryonella, Lopha, and Dendostrea cluster with Nanostrea and Planostrea and with four species of Ostrea (O. circumpicta, O. algoensis, O.futamiensis, and O. oleomargarita). The nonmonophyly of the genus Ostrea, as currently conceived, is not surprising and has been recovered in many molecular phylogenetic studies [5, 11, 15, 23, 58, 60, 66–69]. However, unlike other subfamilies such as the Crassostreinae for which the same main lineages have been recovered with high support in all phylogenetic studies (see the review by ), relationships among Ostreinae are poorly resolved and unstable across studies, especially among those using single genes. A better definition of the main lineages within Ostreinae is apparent in recent multilocus assessments based on mitochondrial and nuclear data and wider taxon sets ([11, 23, 67]; this study), suggesting that more data are needed to robustly resolve the phylogeny and systematics of this subfamily.
Phylogenetic trees estimated in this study and previous studies suggest that careful consideration of the criteria used to define Ostreinae genera is particularly needed. Many taxonomic incongruences observed are perhaps only so because a number of genera based on morphology have been retained (for example, in the lophine subclade) contrasting with the other clades where at least five other available generic names have been regarded as junior synonyms of Ostrea (e.g., Cryptostrea , Myrakeena , Ostreola Monterosato, 1884 and Undulostrea 3). Indeed, some of these available generic names might be useful to resolve the extensive polyphyly of Ostrea as currently conceived. This implies that O. oleomargarita, O. futamiensis, and O. algoensis could be assigned to a different genus in the future. However, the molecular dataset of Ostreinae is far from complete and certainly not sufficient to begin to alter their taxonomy and nomenclature. A further example of this complexity is the situation with species previously assigned to the genus Ostreola (the O. stentina complex) but where the available data was not used to modify the nomenclature and bring the genus Ostreola back into acceptance . This study and others such as Hu et al.  highlight the continuing difficulty of resolving the taxonomy of the Ostreidae, especially using morphological characters. Despite the increasing number of molecular studies, the current taxonomy remains based on morphological decisions. Firstly, the introduction of genera by Harry  was based entirely on morphology, and secondly, the current nomenclature used in MolluscaBase  resulted from the decisions made by Huber  again based on morphology. It is unfortunate that the decisions made by Huber are not supported by evidence so, for example, why have Nanostrea and Booneostrea been retained while other distinctive oysters such as Undulostrea have been synonymised. Of note here is that many of these more obscure genera, following synonymies by Huber  are now monospecific. It will probably become increasingly accepted to prioritise molecular data for oyster systematics; paradigmatic in this respect is the situation with Crassostrea and Magallana, a recent example where shell morphology is of no value in distinguishing these genera despite their remarkable evolutionary distinctiveness .
4.2. Potential Geographic Distribution and Focus on Underestimated Biodiversity of Persian Gulf and East/West Indian Ocean
The close phylogenetic relationship between O. oleomargarita and O. futamiensis and available sequences of these two species discloses a remarkably wide biogeographic gap. To date, we only have molecular data evidence for the presence of O. oleomargarita along the Kuwait coasts in the northern Persian Gulf. Shells in the NHMUK from the west coast of India agree morphologically with those from the Arabian Gulf but were identified as O. futamiensis by Ranson. (: 236) does not cite these specimens of O. futamiensis; instead, he lists material from the Bay of Bengal, Malaya, Singapore, Thailand, Philippines, and Japan. Ramakrishna and Dey  cite Saccostrea futamiensis from Gujerat (WIO) and Orissa (Bay of Bengal), and Subba Rao  does mention the species. The shell from Orissa is described by Subba Rao et al. , and in that, it states the interior is white, thus making the identification suspect. The distribution of O. futamiensis extends in a few scattered regions of the West Pacific Ocean  such as Hong Kong  and Korea . Therefore, it may be inferred that O. futamiensis is an East Indo-West Pacific species, whereas O. oleomargarita is a West Indian Ocean and Arabian species.
This distribution pattern could be supported by the fact that currents usually form an effective barrier for larval dispersal . In particular, Sivadas and Ingole  studied the biodiversity pattern of Indo-Pacific benthic organisms and found that the low similarity between the diversity patterns along the Western and Eastern Indian coasts is due to environmental factors such as habitat heterogeneity and spatiotemporal variability of coastal currents. Unfortunately, biogeographic patterns and genetic structures of marine species in the Western Indian Ocean are scarce, but studies start to highlight an effective allopatric distribution and cryptic diversity of benthic species between W and E Indian coasts [80–82]. This suggests that there may be many such examples to be discovered, especially if we consider that the biodiversity of the Western Indian Ocean is limited to some species or groups . The underestimated number of marine species of the Arabian/Persian Gulf is also based on a low current understanding of the species diversity of some countries . In addition, many intertidal benthonic organisms, such as bivalve molluscs, show high levels of phenotypic plasticity, challenging traditional taxonomic assessment based on morphology . Currently, two of the authors here are preparing an Atlas to the Bivalvia of Kuwait, and within the recognised 210 species, 23 are currently without verified species names (Oliver PG, pers. comm). Oysters, in particular, are engendered with very wide geographical ranges throughout the Indo-West Pacific, and it is likely that none of the species cited by Al-Kandari et al.  were correctly identified or have been properly described. The work of Taylor and Glover, on the Lucinidae, over the period of 1997–2021 revealed many examples where assumed pan Indo-Pacific species were complexes of species with more restricted ranges . Al-Kandari et al.  cited examples where some bivalve species (e.g., Congetia chesneyi; Oliver & Chesney, 1994) had disjunct distributions occurring in the northern Gulf and then on the Pakistan/North-West Indian coast, such species were also absent from Oman. Tsang et al.  using Tetraclita barnacles showed a pattern of regional species within the Western Ocean, and this pattern may be expected from a more detailed study of oysters. New efforts using a molecular approach are uncovering new marine groups and species in this region [15, 85, 86].
Given the inferred complexity of the malacofauna of the Western Indian Ocean fauna , a wide-ranging study of the Bivalvia is warranted. The Red Sea and Arabian upwelling regions have high levels of endemicity but also share species with the Gulf, Indo-Pakistan, and wider north WIO area . Not only are the relationships between the East and West Indian Oceans to be further elucidated but also biodiversity patterns within the Arabian region itself remain to be documented.
All genetic data analysed in this study, either original or from previous studies, is available on GenBank (http://www.ncbi.nlm.nih.gov/genbank/), with respective accession numbers listed in the manuscript (Table 1).
Conflicts of Interest
The authors declare that they have no conflict of interests.
The authors wish to thank Harriet Wood for the documentation of the voucher and type material into the collection of the National Museum of Wales and also thank Henk Dekker for his invaluable help in the collection of specimens in Kuwait in 2019. The Kuwait Institute for Scientific Research (KISR) provided financial support for this study.
Table S1: number of sequences, multiple sequence alignment length, and number of variable positions are reported for each gene and dataset (parsimony informative sites are variable sites with the less frequent nucleotide occurring in at least two sequences). (Supplementary Materials)
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