Regional Studies in Marine Science 67 (2023) 103205 Contents lists available at ScienceDirect Regional Studies in Marine Science journal homepage: www.elsevier.com/locate/rsma Genetic evidence of the unique identity of the West African Mangrove Oyster (Crassostrea tulipa) from the Gulf of Guinea Rhoda Lims Diyie a,b,*, Samuel Addo c, Emmanuel Armah b, Charles Mario Boateng c, Mercy Oppong b, Mike Y. Osei-Atweneboana b a Council for Scientific and Industrial Research, Water Research Institute (CSIR-WRI), Fisheries and Aquaculture Department, P.O. Box AH 38, Accra, Ghana b Council for Scientific and Industrial Research, Water Research Institute, (CSIR-WRI), Biomedical and Public Health Research Unit, P.O. Box AH 38, Accra, Ghana c Department of Marine and Fisheries Sciences, School of Biological Sciences, University of Ghana, P.O. Box LG 99, Accra, Ghana A R T I C L E I N F O A B S T R A C T Keywords: This research enabled the genetic identification of the West African mangrove oysters, Crassostrea tulipa, as well COI gene as establishing the evolutionary relationship between it and other Crassostrea species. Essentially, the study Phylogenetic analysis assisted in clearing up a long-standing confusion over this species’ synonymy with C. gasar. Also, the population Haplotypes structure of 60 C. tulipa individuals, from three different ecotypes, was analyzed using the mitochondrial cyto- Population structure Crassostrea tulipa Ghanaian Coastal Waters chrome oxidase I (COI) genes as a marker. Results provided the first genetic sequences for C. tulipa and deposited in the GeneBank. Optimal and consensus bootstrap Neighbor-Joining trees distinctively differentiated C. tulipa from other Crassostrea species and consistently formed a different clade with C. gasar, with no bootstrap value from either NJ, MPT, or UPG trees supporting their similarity. C. tulipa sequences occurred as different haplo- types from other Crassostrea sp, with a mutation value as high as 288 and a haplotype diversity of 0.893 between C. tulipa and C. gasar sequences. High estimates of genetic distance (1.40–1.55) and patristic divergence were recorded between C. tulipa and C. gasar, in the same range as with seven other species. The study thus reveals the unique identity of C. tulipa as genetically distinct from C. gasar and other Crassostrea species. Based on the population structure analyses from the neutrality test, a low to high haplotype diversity h (0.000–0.963) and low nucleotide diversity π (0.00–0.378) were obtained. A negative mean Tajima’s D (− 0.65247), and a positive Fu’s Fs (3.194), suggest rare variations or low-frequency polymorphisms. In addition to serving as the basis for phylogeny, the identification of C. tulipa and the recent data on its population structure also serve as the basis for conservation efforts and hatchery-based aquaculture. 1. Introduction resource (Bay et al., 2017; Vargas et al., 2017). Identification of oysters is generally based on shell characteristics such as form, structure, color, Oysters are a group of bivalves adapted to the harsh environment in and muscle scar (Ignacio et al., 2000). However, the error margin for this intertidal zones characterized by strong variations in multiple abiotic mode of identification is very high, basically because of the lack of factors (Li et al., 2018; Gracey et al., 2008). They are an important distinctive morphological characters and disagreement as well as un- fishery resource distributed worldwide (Botta et al., 2020), and have certainty regarding identification, which makes it difficult to identify provided food for humans for at least 100,000 years (Baily and Milner, oysters. In addition, the plasticity in shell morphology, that is, being 2008). Oysters are constituted of diverse species of high commercial strongly influenced by environmental conditions contributes to this value, including the mangrove oysters. These species are essential in difficulty (Kenkel and Matz, 2016; Kenkel et al., 2013; Pfennig et al., marine ecosystems as well as in aquaculture programs. Accurate infor- 2010). Nevertheless, the principal species of oysters of economic interest mation on the specific identity and the understanding of oyster diversity based on a combination of reproductive data, the presence/absence of a and evolution are vital for conservation purposes and the effective promial chamber and the morphology of the adult shell hinge, have been management of this economically valuable and high-protein aquatic grouped into the genera Ostrea in the Western Atlantic Ocean (Ignacio * Corresponding author at: Council for Scientific and Industrial Research, Water Research Institute (CSIR-WRI), Fisheries and Aquaculture Department, P.O. Box AH 38, Accra, Ghana. E-mail address: rho_lims@yahoo.com (R.L. Diyie). https://doi.org/10.1016/j.rsma.2023.103205 Received 9 December 2022; Received in revised form 14 September 2023; Accepted 15 September 2023 Available online 23 September 2023 2352-4855/© 2023 Elsevier B.V. All rights reserved. R.L. Diyie et al. R e g i o n a l S t u d i e s in M a r i n e S c i e n c e 67 (2023) 103205 et al., 2000). However, within the genus Crassostrea, there is still much 2. Materials and methods debate as to the actual number of native species that occur on several coasts in South America and Africa (Brunetto et al., 2020; Osei et al., 2.1. Study area 2021; Morretes, 1949; Absher et al., 1989). A typical example is the tulipa species of Crassostrea which is believed to be the same as Cras- A total of 60 samples, twenty each, were collected from three estu- sostrea gasar. aries from three sites (coastal regions) in Ghana, namely the Densu, The confusion surrounding the identity of C. tulipa is so immense Nakwa and Whin (Fig. 1) in March, 2021. Sampling sites were selected that, some authors like Osei et al. (2021) in Ghana, and Brunetto et al. based on a two-stage sampling criterion which included geographical (2020) in Brazil, showed both C. tulipa and C. gasar as belonging to the isolation (Table 1), and the level of shell-fishing activities. Precisely, same species and made it clear in their statements captured” Crassostrea sampling locations are noted for oyster harvesting with fishing activities tulipa (= C. gasar) (Lamarck, 1819)”. This was to highlight the fact that, contributing over 50% as a primary occupation. they were referring to the same species under different names. Also, according to FAO (1980), the most prevalent oysters on the West African coast are the mangrove oysters, Crassostrea gasar and Crassostrea tulipa, 2.2. Sample collection and preparation (FAO, 1980). This report also noted that “it is not yet clear whether they are different species or belong to the same species, as they show only Oysters were taken in a variety of methods at various times at each of local variations in taxonomic characters”. Hence, it was advised to the sampling sites due to changes in the substrate’s characteristics and conduct research on the locally accessible C. gasar, which is likely tides (but often on sandy-mud sediments, attached to the mangrove equivalent to the C. tulipa described elsewhere on the West African roots or other hard objects). Basically, this was done either by diving and Coast. hand-picking individual oysters from the lagoon floor, by wading in and However, as of 2018, Guo et al. (2018) indicated that the Crassos- cutting the roots of mangrove trees to remove oyster clusters, or mostly treinae family contains roughly 26 species, some of which may be syn- during low tides. Oysters were randomly selected from each of the three onymous but have not yet undergone molecular confirmation. This sampling locations, properly washed to eliminate sand particles, and included Crassostrea tulipa, and three other species: Crassostrea cuttack- then transferred to the laboratory (CSIR-Water Research Institute, ensis, Crassostrea aequatorialis, and Crassostrea iredalei. Thus, despite its Biomedical and Public Health Research Unit) on ice. They were kept at exceptional palatability, commercial value, and ecological importance 4 ◦C while pending for further genetic testing. Samples were thawed as an aquatic resource, the West African mangrove oysters (Crassostrea. out in the lab, where the individual weight, shell height, and length were tulipa), which are endemic to the coast along the Gulf of Guinea and all measured. Shells were split open at the posterior end and shucked primarily found on the coast of Ghana and the West African subregion into a clean glass beaker using a stainless-steel knife that had been (Asare et al., 2019; Anyinla et al., 2011; Ansa and Bashir, 2007; Yank- cleaned and sanitized. The adductor muscles of each oyster were son, 2004a,b; Afinowi, 1985), have never been genetically described nor weighed, and approximately 10 g were taken for DNA extraction. confirmed as a species, as no data on this species from the West African coast has been reported. According to Obodai (1997), Ghana, a West African country 2.3. DNA extraction, PCR amplification and sequencing bordered on the south, by the Gulf of Guinea, with a coastal area stretching over 550 km, and covering four coastal regions namely Volta, Genomic DNA extracts were obtained from the adductor muscle Greater Accra, Central and Western Regions, thus representing the tissues of C. tulipa using the Quick-DNA Miniprep Plus kit 4068 (Zymo Southern part of Ghana, has 108 coastal water bodies. These comprises Research, USA). There were however slight modifications of the man- closed lagoons, open lagoons, and estuaries with their accompanied ufacturer’s protocol, to equally obtain higher yield and DNA quality, mangrove vegetation, mud/tidal flats, and marshes, which support the which was determined by a spectrophotometer. The mitochondrial cy- commercially important shellfisheries. To further tap into these locally tochrome c oxidase subunit I (COI) of C. tulipa was targeted for ampli- significant resident stocks of shellfish, mainly the oysters, with appro- fication and sequencing following standard protocols and using the ′ priate conservation measures and oyster aquaculture, which is forward primer, LCOC-CG-1490 (5 -TGTCAACAAATCATTTAGA-′ ′′ becoming imperative for food security for the region, accurate base in- CATTGG-3 ) and reverse HCOC-CG-2190 (5 TACTTGA CCAAAAACA- formation on species identity of local species of oysters, population TAAGACATGA-3′) described previously by de Melo et al. (2010), and structure, and species diversity is fundamental and crucial. Folmer et al. (1994). Each PCR was performed in 10 μl reaction volume Towards achieving this, molecular methods are highly suitable for containing 5 μl Syber green master mix, 0.2 μl of each of the forward and establishing such specific identity and status among oyster species reverse primer, 1.6 μl of Milli-Q water and 3 μl of template DNA. PCR (Wang et al., 2014; Gusmão et al., 2000). Molecular studies of living was performed in a peQlab thermal cycler using the following amplifi- oysters have revealed high genetic diversity at species, population, and cation conditions. An initial denaturing at 95 ◦C for 3 min; 35 cycles of 1 genome levels in recent studies (Guo et al., 2018). Genetic markers min at 95 ◦C, 1 min at 45 ◦C and 90 s at 72 ◦C, followed by a final developed have been useful for the rapid and effective identification of extension at 72 ◦C for 7 min. The PCR products were sequenced by oyster species (Klinbunga et al., 2003, 2005; Cordes et al., 2008), and Inquaba Company (Inquaba, South Africa) using Sanger sequencing. have contributed to the understanding of the true distribution of oysters. BioEdit was used to edit chromatographs of our sequencies to ensure Also, according to Hebert et al. (2003), from the “Consortium for the accuracy. The sequence results were analyzed using BLAST in the NCBI Barcode of Life” (Ratnasingham and Hebert, 2007), divergence in COI server, and other oyster species were identified and retrieved according sequences consistently facilitates the discrimination of closely allied to the BLAST results of individual COI sequences. species in all animal phyla, except the Cnidaria. Thus, this study was undertaken to obtain the molecular identity of C. tulipa found in the 2.4. Sequencing/Haplotype analysis coastal waters of Ghana using COI genes as a marker, to investigate the evolutionary history as well as establish the taxonomic relationship Sequencing of the COI gene of C. tulipa was to highlight the phylo- between it and other species of Crassostrea. The long-term objective is genetic relationship between it and the other Crassostrea species for the selection, breeding, and sustainable utilization of C. tulipa seed retrieved from GenBank. Inclusion of such sequences was based on their stocks. close identity to C. tulipa, and was set at a minimum identity of 95%. The scientific names and GenBank sequence accession numbers of oysters compared in the present study are described in Table 2. 2 R.L. Diyie et al. R e g i o n a l S t u d i e s in M a r i n e S c i e n c e 67 (2023) 103205 Fig. 1. Location of oyster sampling sites included in this study. and Crandall, 1997; Posada and Crandall, 1998). Estimates of average Table 1 evolutionary divergence over all sequence pairs were also estimated GPS locations of coastal sites where samples were collected. showing the number of base substitutions per site from averaging over Population Code Location Latitude Longitude all sequence pairs. Analyses were conducted using the Maximum Com- Densu Den Greater Accra 06◦36.984′N 000◦10.724′E posite Likelihood model. This analysis involved 27 nucleotide se- Nakwa Nak Central Region 06◦41.203′N 000◦17.651′E quences, with all ambiguous positions removed for each sequence pair Whin Whi Western Region 06◦05.918′N 000◦09.019′E (pairwise deletion option). The evolutionary analyses was inferred by using the Neighbor-Joining (NJ) method based on the Tamura 3-param- Two major sets of sequence analysis were carried out. For the anal- eter model (Tamura and Nei, 1993) and conducted in MEGA X (Kumar ysis 1, 22 COI sequence of Crassostrea species, and 2 Ostrea sp. sequence et al., 2018). Bootstrap consensus trees by the Maximum Likelihood Tree which served as an outgroup, in addition to our five sequences from this (ML), UPGMA Tree method and Maximum Parsimony analysis (MP) of study, were used. Thus, a total of twenty-seven sequences were taxa were also used to obtain supporting bootstrap values on the rela- analyzed. All sequences (apart from our C. tulipa sequences) were ob- tionship among sequences. Haplotype distribution pattern, neutrality tained from the NCBI GenBank platform. The 22 COI sequence of test (Tajima, 1989) and polymorphic patterns amongst the C. tulipa Crustacea species together with our sequences of C. tulipa, as well as that populations and the other species retrieved were also generated using of the outgroups with their accession numbers from GenBank can be the DNA sequence polymorphism software (DNAsp 4.) and the POPART seen under Table 2. Analysis 1 was carried to ascertain the difference V1.7 software (Bandelt et al., 1999), (http://popart.otago.ac.nz), to between our sequences and other important Crassostrea species from further confirm the interrelationship among species. Haplotype net- different sources and geographical locations. Analysis II was done to works were constructed based on minimum spanning network. The ascertain the genetic difference that exist between our sequences of network estimation was run at 95% probability limit. C. tulipa and several other sequences of C. gasar species. The C. gasar sequences used were of the following accession numbers; FJ717611.1, HM003520.1, HM003503.1, HM003499.1 and HM003524.1 (Table 2). 3. Results Alignment of the sequences was done by ClustalW (Thompson et al., 1994) using Molecular and Evolutional Genetic Analysis software MEGA 3.1. Morphological and general genetic information X (Kumar et al., 2018). Only distinct COI sequences of C. tulipa were aligned with sequences of other species obtained from the GenBank. The The sizes of the oysters used for this research work ranged from small same software was used to generate phylogenetic tree from the nucle- to big, but were matured in all cases. A mean maximum shell height of otide sequences. The best nucleotide substitutional method was deter- 123 ± 0.20 mm and a minimum of 20 ± 0.05 mm was recorded and that mined by computing the minimum theoretical Akaike information of the shell length had a maximum of 79 mm and a minimum of 16 mm. criterion (AIC), corrected minimum theoretical Akaikei information Morphologically, the shells of C. tulipa samples obtained were quite criterion (AICc), Bayesian information criterion (BIC), and using the variable depending on substrate, but mostly with roundish, or oval Hierarchical likelihood ratio test with a confidence level of 0.01 shape and elongated, with extensive fluting, and rough surface by (Schwarz, 1978; Sakamoto et al., 1986; Frati et al., 1997; Huelsenbeck irregular growth lamellae, as well as with irregular margins. Also, very 3 R.L. Diyie et al. R e g i o n a l S t u d i e s in M a r i n e S c i e n c e 67 (2023) 103205 Table 2 GenBank accession numbers of COI sequences of species used in the analyses. Group Species/description GeneBank accession number References Location Crassostrea Crassostrea rhizophorae JZ189401.1 Americo et al. (2013) Brazil Crassostrea rhizophorae JZ189412.1 Americo et al. (2013) Brazil Saccostrea_palmula FJ527304.1 Gutierrez-Rivera et al. (2016) Mexico Saccostrea_palmula KT317603.1 Raith et al. (2015a, b) California (USA) Crassostrea columbiensis KP455017.1 Pagenkopp Lohan et al. (2015) USA Crassostrea madrasensis MN583310.1 Suzana and Siti Azizah (2011) Malaysia Crassostrea iredalei JF915503.1 Suzana and Siti Azizah (2011) Malaysia Crassostrea nippona LC005437.1 Ozawa (2014) Japan/Philippines Crassostrea hongkongensis KP976208.1 Shen et al. (2015) China Crassostrea belcheri JF915511.1 Suzana and Siti Azizah (2011) Malaysia Crassostrea angulata KU933400.1 Chiesa et al. (2016) Portugal Crassostrea ariakensis FJ743527.1 Jung et al. (2009) South Korea Crassostrea dianbaiensis LC120781.1 Hamaguchi et al. (2016) Japan Crassostrea gryphoides FJ262985.1 Trivedi et al. (2008) India Crassostrea gigas KF644048.1 Layton et al. (2008) Canada Crassostrea virginica KF644323.1 Layton et al. (2014a, b) Canada(eastern oyster) Out group Ostrea sp. JF915514.1 Suzana and Siti Azizah (2011) Malaysia Ostrea edulis KX713488.1 Combosch et al. (2016) USA Our sequences Crassostrea tulipa_Densu_7 OM372500.1 Crassostrea tulipa_Nakwa_24 OM372501.1 Diyie and Armah (2022) Crassostrea tulipa_Nakwa_36 OM372502.1 Crassostrea tulipa_Whin _10 OM372503.1 Crassostrea tulipa_Whin_11 OM372504.1 similar to Crassostrea species, the adductor muscle impressions were as well mostly closer to the ventral margin than the hinge, with cupped lower valves. A total of five sequences were obtained from the samples collected 3.4. Molecular phylogenetics from COI gene sequences from three different geographical locations in Ghana along the Gulf of Guinea. Results from this study thus provide for the first time, genetic The evolutionary history inferred by the Neighbor-Joining (NJ) sequences for Crassostrea tulipa which has been deposited at the Gene- method generated a dendogram (Fig. 2a), which showed three main Bank under the accession numbers OM372500.1 (Crassostrea tulipa_- clusterings. A bootstrap consensus tree (Fig. 2b), clearly showed the Densu_7), OM372502.1 (Crassostrea tulipa_Nakwa_36), OM372503.1 several subclusters under it, with Ostrea sp as true outgroups. The 1st (Crassostrea tulipa_Whin _10), and OM372504.1 (Crassostrea tulipa_- cluster (Fig. 2a), at the top of the tree, consisted of almost all the other Whin_11). These C. tulipa sequences formed one haplotype. The 27 sequences and was further divided into four subclusters. The first sub- different sequences of Crassostrea used in the study generated 22 cluster was made of 7 species of Crassostrea, the second subcluster was haplotypes. made up of sequences; C. dianbiansis, C. iredalei, C. madresensis. The third subcluster consisted of two sequences of C.gasar, and C. brasiliana, on the same node with a bootstrap value of 83%, and together with 3.2. Multiple sequence alignment: nucleotide sequence analysis of C. columbiensis at 81% boostrap value. The fourth subcluster had 3 se- C. tulipa and other Crassostrea species quences; of the outgroups (Ostrea sp., Ostrea edulis, Saccostrea palmula KT317603.1), representing different genus from the true Crassostrea sp An alignment of our sequence together with twenty-two other COI and diverged from all the true Crassostrea species at 81% (Fig. 2a). sequences retrieved from the NCBI platform, showed a clear and distinct C. virginica also formed a divergent subcluster here at 63% bootstrap difference in the nucleotide alignments among same species and be- value. The mid or 2nd cluster consisted of our sequences; C. tulipa tween different species (Suppl. 1). Major similarities were found (Densu), C. tulipa (Nakwa 24), C. tulipa (Nakwa 36), C. tulipa (Whin 10) amongst the C. tulipa sequences. These similar sites were however well and C. tulipa (Whin 11), with C. gryphoides forming a divergent (79%) differentiated from the other sequences by the alignment pattern subclass under it. The other Crassostrea species sequences at the top observed (Suppl. 1). cluster diverged from the C. tulipa sequences with a bootstrap value of 90%. The third cluster consisted of the 2 sequences of C. rhizophora sp. 3.3. Estimates of evolutionary divergence between sequences and Saccostrea palmula FJ527304.1. Sequences of C. tulipa from the various locations in Ghana, Nakwa, Densu and Whin forming a separate The genetic distances among all the samples ranged from 0.00 to cluster from the other species suggests that the C. tulipa sequences are 3.45, with an overall mean value of 1.08 (Table 3). Amongst the se- genetically similar to each other than they are to the GenBank down- quences for C. tulipa, no distances were observed at 0.00. Likewise, there loaded sequences. Again, a booststrap concensus tree generated from was no or little divergence between C. gasar and C. brasillinia at 0.01 and three different evolutionary analysis, ML, UPGA and MP consistently 0.00. However, relatively high genetic distances were found between differentiated between C. tulipa sequences and C. gasar as well as the our sequences and the other sequences retrieved, ranging from 1.400 other species, by concisely placing them on different clades, with no and 3.918. The genetic distances amongst the other sequences were also bootstrap values supporting their similarity (Supplementary 2). Among relatively shorter, between 0.02 and 0.358 than they were to the C. tulipa the C. tulipa sequences however, Densu and Whin 11 populations were sequences (Table 3). Genetic distance between C. tulipa sequences and more identical to each other, whereas Nakwa 36 and 24 were also close the ‘supposedly’ synonymous species C. gasar sequences ranged between to Whin 10, than they were to Densu population. This was evident on the 1.40–1.55, in the same range as with 7 other species (C. madrasensis, phylogenetic tree as the first two were on the same node, as a subcluster, C. nippona, C. dianbaiensis, C. angulata, C. virginica, C. iredalei, and with a bootstrap value of 87%, whiles Nakwa populations were on a C. brasiliana). The species with lowest genetic distance (0.28) from different node with a divergent bootstrap value of 86% (Fig. 2a). C. tulipa species was C. gryphoides 4 R.L. Diyie et al. R e g i o n a l S t u d i e s in M a r i n e S c i e n c e 67 (2023) 103205 5 Table 3 Pairwise patristic distances between sequences of 27 COI sequences of oyster species. 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 Crassostrea_Tulipa_Densu_7 0.00 Crassostrea_Tulipa_Nakwa_24 0.00 Crassostrea_Tulipa_Nakwa_36 0.00 Crassostrea_Tulipa_Whin_10 0.00 0.00 Crassostrea_Tulipa_Whin_11 0.00 0.00 0.01 Crassostrea_Gasar_FJ717611.1 1.55 1.55 1.53 1.55 Crassostrea_Brasiliana_FJ7176 1.55 1.55 1.53 1.55 0.00 Saccostrea_Palmula_KT317603. 1.85 1.85 1.85 1.85 1.71 1.71 Saccostrea_Palmula_KT317603. 1.77 1.76 1.75 1.75 0.31 0.30 1.41 Crassostrea_Sikamea_EU816012 1.56 1.56 1.56 1.56 0.23 0.23 1.50 0.32 Crassostrea_Columbiensis_KP45 1.63 1.61 1.61 1.60 0.18 0.19 1.56 0.30 0.26 Crassostrea_Rhizophorae_JZ18 2.47 2.51 2.54 2.48 2.28 2.28 1.76 2.09 2.19 2.24 Crassostrea_Rhizophorae_JZ18 2.63 2.63 2.63 2.63 3.37 3.37 1.32 2.85 3.45 3.74 2.86 Crassostrea_Madrasensis_MN58 1.48 1.47 1.47 1.46 0.26 0.26 1.48 0.30 0.19 0.27 2.06 3.02 Crassostrea_Iredalei_JF915503 1.51 1.50 1.50 1.50 0.26 0.26 1.43 0.29 0.18 0.27 2.03 3.07 0.02 Crassostrea_Nipona_LC005437 1.55 1.55 1.55 1.52 0.26 0.26 1.32 0.32 0.16 0.27 2.19 3.07 0.17 0.17 Crassostrea_Gasar_HM00352 1.40 1.40 1.40 1.40 0.01 0.00 1.71 0.33 0.22 0.19 2.24 3.41 0.26 0.26 0.27 Crassostrea_Hongkongensis_K 1.67 1.66 1.66 1.63 0.26 0.26 1.56 0.29 0.15 0.27 2.06 2.92 0.18 0.17 0.12 0.27 Crassostrea_Belcheri_JF915511 1.63 1.61 1.61 1.61 0.28 0.28 1.56 0.30 0.16 0.28 2.30 3.26 0.17 0.18 0.18 0.29 0.18 Crassostrea_Angulata_KU93340 1.53 1.53 1.53 1.53 0.26 0.26 1.43 0.29 0.11 0.29 2.14 3.12 0.18 0.18 0.17 0.27 0.14 0.18 Crassostrea_Irakensis_FJ7435 1.45 1.41 1.41 1.40 0.26 0.26 1.32 0.35 0.25 0.26 2.03 3.45 0.25 0.27 0.24 0.25 0.26 0.26 0.25 Crassostrea_Dianbaiensis_LC12 1.55 1.54 1.54 1.52 0.27 0.27 1.48 0.32 0.16 0.27 2.28 3.02 0.18 0.17 0.15 0.27 0.14 0.17 0.17 0.29 Crassostrea_Gryphoides_FJ262 1.56 1.54 1.54 1.54 0.26 0.26 1.53 0.30 0.18 0.25 2.04 3.00 0.13 0.13 0.19 0.26 0.17 0.17 0.18 0.26 0.16 Crassostrea_Gigas_KF644048.1 0.28 0.28 0.28 0.28 1.62 1.61 2.17 1.73 1.76 1.72 2.54 2.26 1.68 1.74 1.72 1.58 1.78 1.71 1.62 1.49 1.73 1.78 Crassostrea_Virginica_KF6443 1.62 1.61 1.60 1.60 0.26 0.26 1.41 0.30 0.11 0.29 2.18 3.13 0.18 0.18 0.16 0.27 0.13 0.17 0.03 0.25 0.15 0.19 1.72 Ostrea_sp._JF915514 1.55 1.51 1.51 1.49 0.30 0.31 1.48 0.26 0.29 0.30 1.81 3.69 0.30 0.31 0.28 0.30 0.31 0.32 0.32 0.34 0.30 0.30 1.60 0.30 Ostrea_Edulis_KX713488.1 1.49 1.49 1.49 1.49 0.34 0.34 1.54 0.24 0.30 0.33 2.04 3.09 0.28 0.29 0.29 0.34 0.30 0.29 0.32 0.31 0.30 0.27 1.78 0.31 0.10 R.L. Diyie et al. R e g i o n a l S t u d i e s in M a r i n e S c i e n c e 67 (2023) 103205 Fig. 2a. A Neighbor-Joining (NJ) phylogenetic tree of oysters constructed with 31 mitochondrion COI sequences to establish the evolutionary relationships. (Saitou and Nei, 1987). 3.5. Haplotype analysis 3.6. Analysis II: Haplotype analysis of C. tulipa and C. gasar sequences 3.5.1. Analysis 1: Haplotype network of C. tulipa and other Crassostrea A total of five more other COI nucleotide sequences of C. gasar; species (C. gasar FJ717611.1 C. gasar HM003520.1 C. gasar HM003503.1 A haplotype network using minimum spanning network and Epsilon C. gasar HM003499.1 and C. gasar HM003524.1) were obtained from of 0 in Popart 1.7 (Bandelt et al., 1999), was produced from these NCBI platform and aligned with 3 of our sequences, representing each analysis with each circle in the haplotype network (Fig. 3) correspond- sampling site; C. tulipa Den 1(OM372500.1), C. tulipa Nar2 ing to one haplotype and the size, proportional to its frequency among (OM372502.1) and C. tulipa Whi1(OM372503.1), to ascertain if they the samples. Colors of the circles correspond to each individual oyster could conceivably align with any of the published sequences of C. gasar sequence or sampling locations. Haplotypes 1 and 2 were shared by our from different geographical locations. The resulting haplotype network five C. tulipa sequences whiles each of the other 22 sequences harbored (Fig. 4), produced a total of six haplotypes with a haplotype diversity h one haplotype each. The number of haplotypes generated in this study of 0.893 and a nucleotide diversity π, of 0.298. The Tajima’s D and Fu’s F was thus 21. The haplotype diversity h, recorded a mean value of 0.963. values were 2.099 and 7.708 respectively. The total number of muta- The mean of the Tajima’s D analysis was − 0.65247, whiles the Fu’s F tions, Eta was 291 whiles the number of nucleotide sites were 762. Of had a mean value of 3.194 (Table 4). The nucleotide diversity parame- the six haplotypes generated, one haplotype, Hap 6, was harbored solely ters recorded a total of 797 nucleotide sites, out of which 201 sites were by our sequences (C. tupila Densu, C. tulipa Nakwa and C. tupia Whin). viable (Table 5). The nucleotide diversity (Pi) π, and the total number of Phylogenetic analysis showed few distinctions among all C. tulipa se- mutations (Eta) were 0.379 and 402 respectively (Table 5). The standard quences, although not significant per AMOVA and Fst value. Since all deviation and standard error of the nucleotide diversity were 0.04208 sequences were not used in the 2nd analysis, those included formed one and 0.0017706 respectively, whiles the average number of nucleotide haplotype, thus, confirming Fst results of no significant genetic differ- difference, k, was 87.111. Whereas the invariable monomorphic sites entiation. The other five haplotypes, Haps 1 to 5 where each occupied by were very low recording a value of 3, with 20 Singleton variable sites, one of the five C. gasar sequences. Specifically, Haplotypes 1, 2, 3 4 and 5 the Parsimony informative sites were very high at 181 and 179 from was shared solely by C. gasar (FJ717611.1), C. gasar (HM003520), both DNAsp and popArt analysis respectively (Table 6). AMOVA showed C. gasar (HM03503.1), C. gasar (HM0034991-.1) and C. gasar high between population variations than within populations for C. tulipa (HM003524.1) respectively (Fig. 4). No single haplotype was shared by populations, with Fst value showing no significant differentiation. our sequences and the other C. gasar sequences. The mutations amongst the haplotypes were 1 and 288. The highest mutations of 288, were observed between Hap 6 (C. tulipa sequences) and Hap 1 (C. gasar FJ717611.1), and also between our sequences and Hap 2 (C. gasar 6 R.L. Diyie et al. R e g i o n a l S t u d i e s in M a r i n e S c i e n c e 67 (2023) 103205 Fig. 2b. Bootstrap consensus tree by Neighbor-Joining method. The bootstrap consensus tree inferred from 500 replicates is taken to represent the evolutionary history of the taxa analyzed (Felsenstein, 1985). Table 4 Table 5 Haplotype neutrality tests of COI sequences of oysters. Nucleotide tests. Haplotype parameters All populations C. tulipa populations Haplotype parameter Mean Mean (All (C. tulipa Number of haplotypes (h) 21 1 populations) populations) Haplotype diversity (Hd) 0.9630 0.00 (P < 0.05) Number of viable sites 201 Variance of haplotype diversity 0.00068 Number of total nucleotide sites 797 Standard deviation of haplotype diversity 0.026 Nucleotide diversity (Pi) from popart 0.379 0.00 Fu’s F statistic 3.194 Nucleotide diversity (Pi) from DNAsp 0.427 Tajima’s D − 0.65247 Total number of mutations, Eta 402 (P < 0.05) Total number of singleton mutations, 124 Strobeck’s S statistic 0.106 Eta(s) Standard deviation of nucleotide 0.04208 diversity HM003520). However, among all the other C. gasar sequences, only a Standard variance of nucleotide 0.0017706 mutation value of one was recorded between them, suggesting strong diversity similarity. Average number of nucleotide 87.111 difference, k 4. Discusion 4.1. Genetic identity and diversity Table 6 Polymorphic sites. It is well recognized that environmental factors have a significant Sites with alignment gaps or missing data 593 role in the difficulty of morphologically identifying Crassostrea species Invariable (monomorphic) sites 3 (Lam and Morton, 2003; Sheng et al., 2021). This, together with the Singleton variable sites 20 enormous diversity and complex history of introduction of oysters and Parsimony informative sites from DNAsp 181 Parsimony informative sites from popArt 179 their seedlings, has led to numerous disputes regarding the identity of AMOVA each species (Liu et al., 2021; Reece et al., 2008). The dominant and Fixation index (Fst) 0 (p > 0.05) indigenous species are unsure if they are different or the same in certain areas. Therefore, molecular analysis was required to determine the 7 R.L. Diyie et al. R e g i o n a l S t u d i e s in M a r i n e S c i e n c e 67 (2023) 103205 Fig. 3. Haplotype network of cytochrome oxidase 1 gene sequences. (Each circle in the network (Fig. 3), corresponds to one haplotype, and the size is proportional to its frequency among the samples. Colors of the circles correspond to oyster sampling locations and sequences downloaded from the NCBI platform). (For inter- pretation of the references to color in this figure legend, the reader is referred to the web version of this article.) precise identity of the Ghanaian stocks of Crassostrea species and to NCBI database, sequence analysis also assisted with the financial and compare them with other species of the same genus, in particular, to time benefits of having to collect samples from multiple geographic confirm the possibility of C. tulipa and C. gasar being different or syno- regions to incorporate in the analysis. nyms due to their similar morphological characteristics. Thus, this study Blast analysis using sequenced results also helped in the discovery genetically describes the West African Mangrove Oysters, C. tulipa, that sequences of the tulipa species of Crassostrea are not yet available endemic to the southern coast of Ghana. on any genetic database and have no 100% identity to any other species Given that DNA sequencing is ideal for establishing species identity bearing the same name. The lack of genetic information on these species and phylogenetic analysis, it was the most reliable method utilized in along the West African Coast was therefore confirmed by this study. As a this investigation (Wang and Guo, 2008), especially for the uncharac- result, the sequences from this study have been registered and deposited terized genetic C. tulipa species found on Ghana’s coast. Additionally, on the NCBI website and could be retrieved under the accession since the cytochrome oxidase C subunit I (COI) gene is a highly- numbers; OM372500.1, OM372501.1 OM372502.1 OM372503.1 for preferred genetic marker with highly conserved protein-coding genes references. in the mitochondrial genome of mammals, targeting it for sequencing ClustalW alignment of our sequences showed significantly distinct was suitable (Folmer et al., 1994). They are frequently used to identify patterns in comparison with the other species of Crassostrea (Suppl. 1), taxa and analyze the genetic diversity of mollusks (Hsiao et al., 2016; retrieved from GenBank and selected for analysis based on their Sekino et al., 2016; In et al., 2017; Nowland et al., 2018; Özcan Gökçek morphological similarity, economic importance, and geographical dis- et al., 2020; Tan et al., 2020; Melo et al., 2021). With the already tributions. Particularly, the nucleotide alignment patterns of C. tulipa existing sequences from earlier research that had been deposited at the sequences were very different to those of C. gasar which is recorded as 8 R.L. Diyie et al. R e g i o n a l S t u d i e s in M a r i n e S c i e n c e 67 (2023) 103205 Fig. 4. Haplotype network of cytochrome oxidase 1 gene sequences. (Each circle in the haplotype network corresponds to one haplotype, and the size is proportional to its frequency among the samples. The number of perpendicular bars on branches represents the number of mutations between haplotypes. Popart 1.7 Bandelt et al., 1999). the only mangrove oyster identified on the west coast of Africa, and a confirm the relatedness of C. tulipa and C. gasar synonym of C. tulipa, Also, in further phylogenetic analysis, sequences of Haplotype analysis, that is, the study of a pattern of descent of a set of C. tulipa from this study formed a different clade (Figs. 2a and 2b) from linked alleles occurring on the same chromosome and either preserved other similar mangrove oysters from the same Crassostrea genus and 2 intact or separated by recombination over time, was also utilized as a sequences from outgroup genus. The inclusion of the outgroup se- critically important analytical tool in this study for the opportunities it quences, and their occurrence on a different clade in the phylogenetic offers in understanding the inheritance of polymorphic traits and their tree from the reference C. tulipa species, with relatively lower bootstrap regulation. Generally, comparative analyses of haplotype sequences, values of 80% and 26% (Figs. 2a and 2b) was to confirm the credibility allow many efficiencies in genetic studies (Lloyd et al., 2016), with the of the analysis, as this is expected under ideal analytical conditions, utmost being its potential in identifying identical-by-descent (IBD) re- where sequences aligned are diverse. This again indicates that C. tulipa gions that are shared between pairs of individuals (Gusev et al., 2009). has a unique identity and distinct from all known species of Crassostrea. Thus, in a haplotype analysis, only the sequences of C. tulipa oysters Although C. tulipa is said to be synonymous with C. gasar, the topology of from this study shared one haplotype (Whin, Nakwa, Densu), while each our trees showed that C. gasar, C. brasiliana, C. columbiensis and of the other sequences from the NCBI database was found on a different C. virginica are rather closely related in the broader clustering or haplotype (Figs. 3 and 4). This indicates that sequences of C. tulipa from grouping (Fig. 2a) forming a monophyletic group. The closeness of the various ecological zones sampled are identical to each other but C. sikemia, C. gigas and C. angulate at the same node, and also that of different from all the other species of the same genus. Most significantly, C. iredalei and C. madresensis. in this study, is in agreement with the trees the haplotypes of C. gasar were rather much closer to that of C. brasiliana generated by de Melo et al. (2010), Lapègue et al. (2002), and Boudry with just three (3) mutations and also, much closer to the other Cras- et al. (2003). Crassostrea gigas in Japan (Kawamura et al., 2017), and the sostrea species (Figs. 3 and 4), than they were to C. tulipa. The inconsis- highly divergent species like, C. virginica in America as indicated in other tency concerning C. gasar and C. brasiliana was clarified when a studies (Thongda et al., 2018), was as well confirmed in this present C. brasiliana rRNA 16S sequence deposited in the GenBank (DQ839413) study, as it formed a single divergent cluster in the phylogenetic was compared with that of C. gasar (AJ312937) by Lapègue et al. (2002) analysis. and found that they were identical, an indication that they are synonyms In a further analysis where the evolutionary divergence over all and belong to the same species. Thus, results from this study also sequence pairs was estimated, the different sequences of C. tulipa species confirm that C. gasar is a synonym of C. brasiliana as they occurred on the and on the other hand, the C. gasar and C. brasiliana sequences, recorded same haplotype. Accordingly, it was expected that other equally syn- 0.00 genetic distance among themselves to confirm their similarity. onymous species would share the same haplotype with them however, However, a genetic distance as high as 1.40 to 1.55 was recorded be- the resulting haplotype network with only C. gasar and C. tulipa se- tween C. tulipa and C. gasar in a similar range as with some of the other quences (Fig. 4), produced a total of six different haplotypes instead of Crassostrea species. The closest species to C. tulipa was rather C gry- the expected one haplotype as was recorded among C. tulipa sequences phoides with a genetic distance of 0.28, whiles the highest genetic dis- or with a low mutation value between them, like the mutation of 1 tance was between C. tulipa and C. rhizophora sequences. Generally, recorded between the different C. gasar sequences. However, a mutation populations with many similar alleles have small genetic distances, value as high as 288 with a haplotype diversity of 0.893 was recorded which indicates that they are closely related and have a recent common between C. tulipa and C. gasar sequences. ancestor. Genetic distances as recorded in this study (Table 4) could not Lapègue et al. (2002), in their study on trans-Atlantuc distribution of 9 R.L. Diyie et al. R e g i o n a l S t u d i e s in M a r i n e S c i e n c e 67 (2023) 103205 Crassostrea, where a phylogenetic tree was built with seven 16S se- together with anthropogenic impacts and historical movements which quences from Crassostrea and Saccostrea species, showed that C. gasar is tend to affect species adaptation and eventual modification and shaping intermediate between the American Crassostrea species (C. virginica and of the genetic structure. This genetic information thus far suggests that C. rhizophorae) and the Asian species (C. gigas and C. ariakensis). The balancing selection to include both subclusters could be essential to study indicated among its conclusions that C. gasar was transported from increase diversity and variability in genetic traits. Guo et al. (2018) Africa to America. Hence, against the background of C. gasar coming highlighted on the importance of balancing selection as a major force in from Africa, it was expected that it would fall in the same group of COI shaping genetic variation among oyster populations. High levels of ge- sequence of C. tulipa which did not, although quite close, with low netic diversity can be achieved by strong balancing selection, which nucleotide diversity (0.289). This then suggests a unique identity for results from different life stages, long-distance dispersal potential, and C. tulipa as a species. fluctuating environmental conditions (Guo et al., 2018). The similarity between C. tulipa and C. gasar could therefore not be confirmed with any of the series of analyses, as they formed distinctly 5. Conclusion different nucleotide alignment patterns, consistently occurred on different clades and nodes either with Neighbor-Joining, ML UPGA, and In this study, Crassostrea tulipa is genetically confirmed as occurring MP phylogenetic analysis (Supl 2), in addition to the fact that bootstrap on the coastal waters of Ghana. It is also confirmed as genetically values did not support that similarity. They also occurred as different distinct from C. gasar and other Crassostrea species, as no single analysis haplotypes and with relatively high estimates of genetic distance and undertaken in the present study confirmed their similarity. They divergence. consistently formed well-differentiated clades in the molecular phylog- enies and also occurred as different haplotypes, and with a relatively 4.2. Genetic structure of C. tulipa in the coastal waters of Ghana high estimate of genetic distance and divergence. The overall genetic structure of the Mangrove oyster, C. tulipa The study also investigated the population structure of 60 samples analyzed in this study also suggests that it is an indigenous species to from three different ecotypes of C. tulipa, utilizing mitochondrial cyto- Ghanaian coastal waters that has undergone population expansion, chrome oxidase I (COI) genes as markers, for its importance in conser- through adaptations to variable environmental conditions and with low vation and selective breeding programs. A low to high haplotype to high haplotype diversity that informs a greater opportunity for diversity (Table 4) was recorded amongst C. tulipa sequences and also resiliency to climate future change, and sustainable aquaculture devel- with the other sequences in this study. The pattern of genetic variability opment. The molecular information obtained from this study has im- with high haplotype diversity, but relatively low nucleotide diversity as plications for the proper management of wild-harvested oysters in recorded in this study suggests that the C. tulipa population analyzed Ghana. from the various ecological zones has undergone population expansion. High genetic diversity often represents a greater opportunity for resil- CRediT authorship contribution statement iency to climatechange, thus the genetic diversity in the present study is a likely adaptation to variable environments. However, the variations in Rhoda Lims Diyie: Conception and design of study, Acquisition of the diversity indices indicate the need for conservation and positive data, Analysis and/or interpretation of data, Writing – original draft, traits for hatchery-based aquaculture development through selective Writing – review & editing. Samuel Addo: Acquisition of data, Writing – breeding (Gjedrem, 2012; McAndrew and Napier, 2010). Analyses of all review & editing. Emmanuel Armah: Acquisition of data, Analysis and/ locations together from the haplotype neutrality test resulted in a or interpretation of data, Writing – original draft. Charles Mario negative mean Tajima’s D that was significant and a positive Fu’s Fs Boateng: Acquisition of data. Mercy Oppong: Writing – original draft. (Table 3). A negative Tajima’s D indicates an excess of rare variations or Mike Y. Osei-Atweneboana: Writing – review & editing. low-frequency polymorphisms, a characteristic that is consistent with population growth. The positive Fu’s Fs however indicates no excess Declaration of competing interest alleles, expected from recent population growth bottleneck or direc- tional selection under culture settings (Zainal et al., 2016; Ray et al., The authors declare that they have no known competing financial 2003). The overall negative and significant Tajima’s D value obtained interests or personal relationships that could have appeared to influence from the present analysis, is however, a distinctive characteristic of the work reported in this paper. indigenous species implying that C. tulipa is expectedly an indigenous species. Data availability Therefore, the development of hatchery protocols for this species could be regarded as a prerequisite for advancements and progress to Data will be made available on request. commercial-scale hatchery productions as suggested by Southgate and Lee (1998). For a country like Ghana that is interested in the aquaculture Acknowledgments of this species, especially in its hatchery production, the genetic confirmation of the geographic range of C. tulipa is important as shown The authors are grateful to the Biomedical and Public Health by Utting and Spencer (1991), and Lucas (2012). Research Unit of CSIR, Water Research Institute, Accra for permitting This study also drew further distinctions, thus mild structuring be- the use of the molecular laboratory, and supporting research with lo- tween all the sequences from the three ecological populations of gistics. Technical support by Judith Wayo is duly acknowledged. All C. tulipa, based on the phylogenetic and haplotype analysis, as two authors approved the version of the manuscript to be published. haplotypes (Hap 1 and 2), were shared among the five C. tulipa pop- ulations from analysis 1. However, Fst showed minimal and non- Funding significant (p > 0.05) population differentiation which was also consistent with analysis 2. Nonetheless, this has implications for the This research did not receive any specific grant from funding proper management of wild harvested oysters in Ghana. Among the agencies in the public, commercial, or not-for-profit sectors. C. tulipa sequences, Densu and Whin, as well as Nakwa and Whin pop- ulations were more identical to each other, forming two subclusters. Ethics approval and consent to participate This could possibly be attributed to variations in water depth, salinity, and rate of tidal influence both from the sea and freshwater discharge, The work described herein has been carried out (where appropriate) 10 R.L. Diyie et al. R e g i o n a l S t u d i e s in M a r i n e S c i e n c e 67 (2023) 103205 in accordance with the Code of Ethics of the World Medical Association Guo, Li, C., Wang, H., Xu, Z., 2018. Diversity and evolution of living oysters. J. Shellfish (Declaration of Helsinki) for animal experiments. All oysters were Res. 37 (4), 755–771. https://doi.org/10.2983/035.037.0407. Published Version: https://doi.org/10.2983/035.037.0407. handled in accordance with the Northern Territory Governments animal Gusev, A., Lowe, J.K., Stoffel, M., Daly, M.J., Altshuler, D., Breslow, J.L., Friedman, J.M., ethics requirements and guidelines. Peer, I., 2009. Whole population, genome-wide mapping of hidden relatedness. Genome Res. 19, 318–326. Gusmão, J., Lazoski, C., Solé-Cava, A.M., 2000. A new species of Penaeus (Crustacea: Appendix A. Supplementary data Penaeidae) revealed by allozyme and cytochrome oxidase I analyses. Mar. Biol. 137 (3), 435–446. Supplementary material related to this article can be found online at Gutierrez-Rivera, J.N., Camacho-Jimenez, L., Tovar-Ramirez, D., Garcia-Gasca, A., 2016. https://doi.org/10.1016/j.rsma.2023.103205. Gene expression of heat shock protein 70 (HSP70) and glutathione S-transferase mu class (GST mu) in the oyster saccostrea palmula transplanted in a polluted estuary. Unpublished. 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