Iranian Journal of Fisheries Sciences 20(1) 96-108 2021 DOI: 10.22092/ijfs.2021.123480 Research Article Molecular phylogeny of bivalve families (Arcidae, Chamidae, Margaritidae, Ostreidae, ) in the Persian Gulf

Masaeli Sh.1; Ghavam Mostafavi P.2*; Hosseinzadeh Sahafi H.1; Tamadoni Jahromi S.3; Nabinejad A.4 ; Noaman V.5

Received: March 2016 Accepted: July 2016

Abstract is one of the least studied classes of the Persian Gulf. A survey and molecular analysis was conducted to determine bivalve diversity in the Persian Gulf. To the best of our knowledge, this is the first report of barcoding data on bivalves of the Persian Gulf. We examined 40 individuals representing 8 species, 6 genera and 5 families. We collected samples from Hengam, Larak and Qeshm Islands and Bandar Lengeh in Persian Gulf, Iran. After DNA extraction, mitochondrial 16S ribosomal DNA (16SrDNA) and Cytochrome Oxidase subunit I were amplified by polymerase chain reaction (PCR). Based on obtained 16SrDNA and COI gene sequences and maximum parsimony, neighbor joining and maximum likelihood trees of these genes there was no overlap between maximum Kimura 2- parameter distance among conspecifics. Most species formed agglutinate sequence units with a small amount of changes. Eventually, comparison of the 8 selected studied species with metadata from India, Brazil, Japan, China, and America exposed that these species in Persian Gulf are classified in sister clades with high bootstraps except Pinctada. Since there is not much

Downloaded from jifro.ir at 10:19 +0330 on Sunday September 26th 2021 work on bivalves identification in the Persian Gulf, larger sampling and more research is needed to investigate mollusc diversity in this area.

Keywords: Bivalvia, Persian Gulf, 16SrDNA, DNA sequencing, COI

1-Iranian Fisheries Science Research Institute, Agricultural Research, Education and Extension Organization, Tehran, Iran. 2-Department of Marine Biology,Faculty of Natural Science and Environment ,Science and Research Branch, Islamic Azad University,Tehran,Iran 3-Persian Gulf and Oman Sea Ecology Research Center, Iranian Fisheries Science Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Bandar Abass, Iran. 4-Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization, (AREEO), Karaj, Iran . 5-Department of Parasitic Disease, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), karaj, Iran *Corresponding author's Email: [email protected] 97 Masaeli et al., Molecular phylogeny of bivalve families in the Persian Gulf

Introduction continued non-stop throughout the One powerful use of molecular Phanerozoic, with relatively small characters in systematics is to reductions during the end-Permian examine basal divergences for the and end-Cretaceous extinction groups which morphology could incidents. Gradually diversified and not identify in species level. Many expanded molluscs species became scientists have used sequence data dominant in most marine to investigate the relationships ecosystems (Waller, 1998). between animal phyla and the Approximately, 20,000 species classes within them (Philippe et al., of marine bivalves exist worldwide, 1994; Boore et al., 1995; Halanych so this class offers a rich diversity et al., 1995; Winnepenninckx et al., of life (Pearse et al., 1987). 1994). Of the protostome phyla, Following that, studies in the are among the most Persian Gulf continued by Melvill ancient and diverse classes. Living (1904), Smythe (1972) and representatives of this phylum are Hosseinzadeh Sahafi et al. (2001). usually divided into seven classes. Most of these studies have focused Bivalves and gastropods are the on identification of bivalves. most common classes in which According to the available most species are placed in the literature, in the Arabian side of the modern fauna (Halanych et al., Persian Gulf some of the molluscs 1995). To date, molecular including bivalves are studied by a investigations of the molluscs have small number of conchologists focused primarily on relationships (Basson et al., 1977). of other phyla to Mollusca, Mollusca with almost 100,000

Downloaded from jifro.ir at 10:19 +0330 on Sunday September 26th 2021 demonstrated by the classes species organize the second largest Gastropoda, Bivalvia, and the animal phylum (Barnes et al., Polyplacophora, the latter being 2009). Many mollusc species are considered as basic (Canapa et al., used as bioindicators (Astani et al., 2000). Bivalvia is a class of marine 2012; Saeedi, 2012). In terms of and freshwater molluscs that have biodiversity and ecology, regardless laterally compressed bodies of great variety in Persian Gulf enclosed by a shell consisting of molluscs, little is known about two hinged parts. The first bivalve subtidal species. Some studies on fossils are found in Lower molluscs of the Iranian coasts of Cambrian sediments. But changes Persian Gulf are conducted but are were not found in the fossil records mostly centralized on specimens both taxonomically and greater than about 5 mm in size, ecologically until the Lower therefore small species are often Ordovician. These changes neglected (Tadjallipour, 1974; Iranian Journal of Fisheries Sciences 20(1) 2021 98

Rezai Marnani et al., 1994; systematics, such protocols can Hosseinzadeh Sahafi et al., 2001; potentially be applied to important Nassaj et al., 2010; Asgari et al., subjects in ecology, conservation 2012). Morphological studies are and issues related to economy sometimes unable to perform (Armstrong and Ball, 2005; reliable species identification; Markmann and Tautz, 2005; therefore molecular studies are Savolainen et al., 2005; Smith and necessary to be done (Ardura et al., Fisher, 2009). The phylogenetic 2010). reconstruction of a bivalve family In the past, species were (Pectinidae) based on mitochondrial identified primarily on the basis of 12S and 16SrRNA and nuclear morphology, a main problem here histone H3 sequence study was that the border line between (Puslednik and Serb, 2008), was intra-specific variation and inter- quite in contrast with the then specific morphological similarities morphological hypothesis of were sometimes unclear (Kyle and Pectinid evolution (Waller, 2006). Wilson, 2007). Accurate and An important difficulty in relatively simple identification of Pectinidae systematics remains the species is based on the nucleotide evolutionary relationships of the sequence of widely used species- subfamilies and major tribes level, usually a short DNA (Puslednik and Serb, 2008). fragment. DNA barcoding is Various tools, allowing introduced in a bold decision to phylogenetic investigation of RNA overcome some of these and protein sequences at the shortcomings (Hebert et al., 2003). sequence structure level, are

Downloaded from jifro.ir at 10:19 +0330 on Sunday September 26th 2021 Sometimes, short sequences of developed (Jow et al., 2002; Smith different markers such as a et al., 2004; Seibel et al., 2006). mitochondrial or nuclear target DNA barcoding is introduced as a gene confirm the COI sequence faster and more accessible method data (Monaghan et al., 2006; for species identification (Hebert et Sonnenberg et al., 2007). These al., 2004; Blaxter et al., 2005; findings allow barcoding tools to be Kress et al., 2005; Saunders, 2005). used not only for species The feasibility of identifying identification, but also for species by DNA barcodes is related biodiversity related issues. After to the sequence variation among two hundred years of morphology living groups. Bivalvia is a great based taxonomic studies, species and diverse class among , are now identified in a more but little is known about molecular effective molecular method level variations within this class of (Waugh, 2007). In addition to molluscs (Smith et al., 2004). It is 99 Masaeli et al., Molecular phylogeny of bivalve families in the Persian Gulf

estimated that only half of the Phenol chloroform isoamyl alcohol existing species of molluscs are (25:24:1) was added into the tube and studied (Coleman, 2007). inverted 5-6 times (Selig et al., 2008). Additionally, dominance of mollusc After centrifuge, the same steps were species is grossly underestimated in repeated two times. NaCl and cold ecological surveys (Bouchet et al., absolute ethanol was added and was 2008). The incidence of secret kept in -20°C overnight. After spin and species also makes identification pouring out the ethanol, the DNA more difficult. A quicker and easier pellets stayed at the bottom of the tube way for species recognition would under the following thermal profile: therefore be useful for molecular 94°C for 10 min; five cycles of 94°C level identification of bivalves for 45 s, 45°C for 45 s, 72°C for 45 s; (Schander and Willassen, 2005). 40 cycles of 94°C for 45 s, 51°C for 45 s, 72°C for 45 s; 72°C for 10 min (Gil, Materials and methods 2007). Each reaction mixture contained Marine molluscs were sampled from 25 µL of 10% Trehalose, 5 µL of several sites in Bandar Abbas, Bandar 10·PCR buffer, 2 µL of 50 mM MgCl2, Lengeh, Qeshm, Larak and Hengam 1 µL of 10mM dNTP mix, 0.5 µL of Islands in Persian Gulf, during May and each primer (10pmol), 1–2 µL of DNA July 2015 by wading and scuba diving and 1–2 U SmarTaq DNA polymerase (Fig. 1). The stations and number of (Cinnagen Tehran, Iran). Those were replicates are shown in Table 1. also identified in our study. Deionized All specimens were kept in water was added to obtain a reaction refrigerator of agriculture research volume of 50 µL (Gil, 2007). PCR center. Species were identified based on products were visualized on 1% agarose

Downloaded from jifro.ir at 10:19 +0330 on Sunday September 26th 2021 conchology. DNA was extracted from gels. Sequencing was performed on living bivalves or frozen somatic tissue samples that produced a single band. (Barber et al., 2006). Three DNA extraction methods were used for DNA extracting. DNA was extracted with a DNeasy tissue extraction kit from CINAGEN and MBST kit, using the tissue protocol as recommended by the manufacture. A piece of tissue (150- 200mg) was removed and placed in 850μl extraction buffer (Tris–HCl, EDTA, SDS, NaCl) and 5 μl proteinase K was added into a micro centrifuge tube, then incubated in water bath at 55°C for 12 hr (overnight). Then Iranian Journal of Fisheries Sciences 20(1) 2021 100

Figure 1: Map of sampling locations in the Persian Gulf.

Table1: Sample sites, location and molecular identification of each specimen. Code Sampling Location Map Coordinates Sampling Species Molecular Date Identification

Downloaded from jifro.ir at 10:19 +0330 on Sunday September 26th 2021 A Hengam Island 26⁰40′19.67⁰N- 24.06.2015 Saccostrea cuccullata 55⁰51′42.16⁰E B Larak Island 26⁰53′16.36⁰N- 15.07.2015 Crassostrea virginica 56⁰22′11.25⁰E C Qeshm Island 26⁰54′38.85⁰N- 25.07.2015 undulatus 56⁰13′44.08⁰E D Bandar Lengeh 26⁰33′26.95⁰N- 14.08.2015 Pinctada imbricata radiata 54⁰54′11.83⁰E E Bandar Lengeh 26⁰33′26.95⁰N- 14.08.2015 Pinctada imbricata fucata 54⁰54′11.83⁰E F Qeshm Island 26⁰54′38.85⁰N- 25.07.2015 Barbatia obliquata 56⁰13′44.08⁰E G Larak Island 26⁰53′16.36⁰N- 15.07.2015 sp. 56⁰22′11.25⁰E H Hengam Island 26⁰40′19.67⁰N- 24.06.2015 Saccostrea scyphophilla 55⁰51′42.16⁰E

101 Masaeli et al., Molecular phylogeny of bivalve families in the Persian Gulf

A band of almost 500 bp in addition to China and South Africa. Two types of the band of expected size was observed dividing values were calculated. A in a few cases. In this condition, DNA common global divergence value was in the 1000 bp band was removed using calculated for each species which was a gel extraction kit (Fermentas the average of all pairwise intervals of International Inc., ON, Canada) and sequences depending to the identical sequenced. Sequencing was carried out species regardless of location of origin. using the ABI Big Dye terminator Territorial divergences were computed chemistry and an ABI prism 3700 as mean interval of all con special instrument, Applied Biosystems, Korea sequences from the same position (Wolf et al., 2008). To further query (Selig et al., 2008). A number of existence of cryptic species flagged by experimental molecular operational COI sequences, 16srDNA fragments taxonomic units (MOTUs) were derived were used. For amplification of the 500 based on patterns of sequence bp fragment encoding 16srRNA, the clustering and grade of variegate for 16Sar-5 and 16Sbr-3 primers were used each nominal species. NJ tree covering (Ivanova et al., 2007). for all sequences was built. Finally degrees of observed divergence at Barcode sequence analysis different taxonomic levels of samples Chromatograms were first examined of the present study were compared visually to avoid reading mistakes, with those of BOLD project sequencer software (Technelysium, encompassing broader geographical Australia) was used. Sequences were ranges (Blaxter et al., 2005). then aligned in Mega 6.0 (Tamura et al., 2007). Fasta alignment was checked Results

Downloaded from jifro.ir at 10:19 +0330 on Sunday September 26th 2021 onto the NCBI website. Computing of In this research 5 bivalve families Kimura 2-parameter (K2P) distances including Arcidae, Chamidae, (Kimura, 1980) and output of neighbor- Margaritidae, Ostreidae and Veneridae joining (NJ) tree (Blaxter et al., 2005) were studied. Also intra specific of K2P distances were done using the variation and inter specific divergence BOLD Management and Analysis in six genera of marine bivalves was System. Computing K2P intervals and investigated. 16srRNA from species output of NJ tree of 16srDNA Pinctada imbricata radiata, Pinctada sequences were done using MEGA 6.0 imbricata fucata (family Margaritidae), (Nassaj et al., 2010). (family Meta data analysis were done using Veneridae), Barbatia obliquata (family BOLD management and analysis Arcidae), Chama sp. (family system to contrast 16s sequences of Chamidae), Saccostrea cuccullata, selected species of the present study Saccostrea scyphophilla, Crassostrea with conspecifics from Australia, India, virginica (family Ostreidae) was Iranian Journal of Fisheries Sciences 20(1) 2021 102

sequenced to compare divergence aligned mitochondrial 16s ribosomal within and between the species. The DNA sequences is shown in "Figure 2". resulting phylogenetic tree for the

Figure 2: Neighbor joining, maximum parsimony and maximum likelihood tree for specimens in the Persian Gulf from mitochondrial 16S ribosomal DNA sequences. First numbers on left show maximum likelihood, second numbers in middle show neighbor joining, third numbers on right show maximum parsimony.

Downloaded from jifro.ir at 10:19 +0330 on Sunday September 26th 2021 In the current study maximum high bootstrap from Pinctada clade and parsimony (MP), neighbor joining (NJ) classified in almost similar sister and maximum likelihood (ML) trees groups. Iranian Pinctada imbricata based on 16srRNA data were used for fucata from Bandar Lengeh were totally the families of bivalvia. In this study different from Pinctada imbricata Paratapes undulatus from Persian Gulf fucata from Australia and Japan, so with Paratapes undulatus from they were classified in two different Northern Atlantic Ocean were classified clades. In this research Crassostrea in sister groups (MP:77 ,NJ:94,ML:91). virginica and Saccostrea cuccullata of The Iranian species of Chama sp. with Persian Gulf (MP: 70, NJ: 100, ML: 99) (MP: 100 bootstrap, NJ: 96, ML: 99) were classified in one sister clade. They was similar to the same species from were similar to Saccostrea scyphophilla the Western North Atlantic and both from Persian Gulf with MP: 100, NJ: were classified in the same sister group 98, ML: 99. These species were similar and classified in separate clade with to the same species from Brazil and 103 Masaeli et al., Molecular phylogeny of bivalve families in the Persian Gulf

were relatively close to each other; so West Pacific were classified in a sister they were located in the same sister groups (MP: 96, NJ: 98, ML: 98). High clade. In current study, Saccostrea quality photographs of bivalves are cuccullata from South China Sea with shown in Figure 3. Saccostrea scyphophilla from the Indo-

Figure 3: In situ images of bivalve specimens collected in this study; A: S. cuccullata, B: C. virginica, C: P. undulatus, D: P. imbricata radiata, E: P. imbricata fucata, F: Barbatia obliquata, G: Chama sp., H: S. scyphophilla.

Discussion The reason for this difference can be Reduction in aquatic resources in many special condition of the geographical parts of the world stresses the area, i.e. existence of estuaries and importance of fisheries and aquaculture mangrove forests. These factors can resources management. Before any kind affect genetic diversity of the area. For Downloaded from jifro.ir at 10:19 +0330 on Sunday September 26th 2021 of action, study and determining the example mangrove forests are genetic structure of valuable species via important nursery and feeding areas for molecular methods is necessary. This is larva of crustaceans and molluscs very important in sustainable utilization (Barber et al., 2006). One of the main programs of marine reserves, aquatic causes of isolation of species is industry and breeding programs (Lin et geographical distance which can affect al., 2002). The data related to DNA the genetic distance due to physical and sequence is used to determine ancestral natural barriers which can reduce relations in development of most genetic transfer (Bisby et al., 2010). It animals. These data are less influenced is reported that animal immigration by selection index and phylogenetic behavior is an important factor on the relations; as a result the real genetic transfer of gene and change in structure is demonstrated (Saavedra and population structure. Mollusca often Peňa, 2006). select nests in different areas in their life cycle and for the completion of Iranian Journal of Fisheries Sciences 20(1) 2021 104

their life cycle have to move to Acknowledgement different areas (Lin et al., 2002). The authors are grateful to Dr. Geographic movement of bivalves into Asgharian, University of Southern each area is related to the California for his useful advice and his environmental conditions, especially answers to all our questions during this due to variability of salinity, or other study and we also thank Majid Afkhami important factors such as the seabed and Hossein Rameshi for collecting the and hydrological conditions, existence samples. of estuaries and also dispersion of the bivalves which can have an effect on References gene transfer (Weersing and Toonen, Ardura, A., Linde, A.R., Moreira, 2009). J.C. and Garcia-Vazquez, E., In conclusion, results obtained from 2010. DNA barcoding for the present study, as the first study on conservation and management of diversity and genetic structure of Amazonian commercial fish. bivalvia in coastal waters of Biological Conservation, 143(6), Hormozgan using mt DNA sequencing 1438-1443. 16srRNA, demonstrated that molecular Armstrong, K.F. and Ball, S.L., 2005. markers, especially mt marker, can be a DNA barcodes for biosecurity: suitable parameter for separation of invasive species identification. aquatic populations such as bivalvia. Philosophical Transactions of the These kinds of markers can be used in Royal Society of London B: the study of species and related Biological Sciences, 360(1462), common ancestor (Puslednik and Serb, 1813-1823. 2008). In this study haplotype diversity, Asgari, M., Yekta, F.A. and Izadi, S.,

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