Adding the Molecular Diversity Information of the Common Fouling Barnacle Amphibalanus amphitrite (Darwin, 1854) (Crustacea: Cirripedia) from the Persian Gulf and Gulf of Oman to the Global Diversity Pattern

© 2023 Academia Sinica, Taiwan Open Access Adding the Molecular Diversity Information of the Common Fouling Barnacle Amphibalanus amphitrite (Darwin, 1854) (Crustacea: Cirripedia) from the Persian Gulf and Gulf of Oman to the Global Diversity Pattern Asma Moeinadini1, Alireza Sari1,*, Adnan Shahdadi2, Ahmad-Reza Katouzian1,3 , Alimorad Sarafrazi4, and Elahe Elahi5 1School of Biology and Centre of Excellence in Phylogeny of Living Organisms, University of Tehran, Tehran, Iran. *Correspondence: E-mail: [email protected] (Sari). E-mail: [email protected] (Moeinadini) 2Department of Marine Biology, Faculty of Marine Sciences and Technology, University of Hormozgan, Bandar Abbas, Iran. E-mail: [email protected] (Shahdadi) 3Alberta Society of Professional Biologists, 370, 105 12 Ave SE, Calgary AB T2G 1A1. E-mail: [email protected] (Katouzian) 4Taxonomy Research Department, Iranian Research Institute of Plant Protection, Tehran, Iran. E-mail: [email protected] (Sarafrazi) 5School of Biology, University of Tehran, Tehran, Iran. E-mail: [email protected] (Elahi) Received 8 March 2022 / Accepted 18 January 2023 / Published 25 April 2023 Communicated by Benny K.K. Chan The balanid barnacle, Amphibalanus amphitrite, is known as one of the most common fouling species in the world. A phylogenetic study using material from around the world recovered three distinct clades for this species. Material from the Persian Gulf (PG) and the Gulf of Oman (GO) were not included in that survey. In the present study, we aimed to assess the genetic diversity of the balanid barnacles of these two gulfs and to evaluate their phylogeography. In total, 94 COI DNA sequences were obtained from the PG and the GO material. Most of these sequences clustered into a single clade, corresponding to clade I of the previous global study. However, two sequences, one from the PG and one from the GO, fell into a separate clade corresponding to clade III of the previous study. These two gulfs share some common haplotypes, but host several unique ones that are separated from the most common haplotype mainly by a single mutation. Based on various indices, the genetic diversity of the PG material was higher than that of the GO. Low values of ΦST show a regular gene flow among the stations and the two gulfs. The Bayesian skyline plots and the mismatch distribution analyses both showed signs of a recent population expansion in the PG and the GO. We also modeled the potential distribution areas for A. amphitrite to reveal the separate suitable habitats for the clades. The current phylogeographic status and genetic diversity of A. amphitrite in the PG and GO appears to have been shaped by both historical events and recent human activities. Key words: Barnacle, Amphibalanus amphitrite, Phylogeography, Population Genetic, Gene flow. Citation: Moeinadini A, Sari A, Shahdadi A, Katouzian A, Sarafrazi A, Elahi E. 2023. Adding the molecular diversity information of the common fouling barnacle Amphibalanus amphitrite (Darwin, 1854) (Crustacea: Cirripedia) from the Persian Gulf and Gulf of Oman to the global diversity pattern. Zool Stud 62:16. doi:10.6620/ZS.2023.62-16. Zoological Studies 62:16 (2023) doi:10.6620/ZS.2023.62-16 1 © 2023 Academia Sinica, Taiwan BACKGROUND Knowledge of the distribution patterns of marine species is important for understanding their ecology and biogeography (Robinson et al. 2011). These patterns may be changed by human-mediated dispersal on a global scale (Gallardo et al. 2015), leading to the possible homogenization of the world’s biota, including those associated with plate tectonics or glacial cycles (Brown and Lomolino 1998; Bank et al. 2015). In coastal and estuarine habitats, most structural and functional modifications of communities in the recent decades were caused by the introduction and establishment of non-native species (Ruiz et al. 1999; Levin and Crooks 2011). By creating movement corridors, transport networks have promoted the dispersal of non-native species to new regions and thus caused stress to native species by altering their habitats (Hulme 2009). International marine shipping networks, which account for 90% of world trade, can catalyze the spreading of many marine organisms and the establishment of their populations far beyond their native home range (Hulme 2009; IMO 2021). Biofouling significantly promotes the dispersal of organisms, including organisms with low inherent dispersal capacity, and the consequence is increased gene flow between populations, making a homogenized genetic structure (Olden et al. 2004; Crispo et al. 2011). For instance, despite limited larval dispersal of polychaetes Hydroides elegans (Haswell 1883), microsatellites studies revealed a high level of genetic similarity between seven subpopulations from the Atlantic, Pacific, and Indian Oceans and the Mediterranean Sea (Pettengill et al. 2007). Although this implies that few migrants may be sufficient to homogenize local genetic variation (Hartl and Clark 2007; Hellberg 2009; Pannacciulli et al. 2009 2017), genetic differentiation sometimes remains even for species with high dispersal capacity over long distances. The maintenance of distinct genetic profiles of the cosmopolitan marine planktonic diatom Pseudo-nitzschia pungens (Casteleyn et al. 2010), the pedunculate and acorn barnacles Pollicipes sp. (Quinteiro et al. 2007) and Tetraclita sp. (Tsang et al. 2012; Reynolds et al. 2014), may have been due to patterns of oceanic currents, natural selection, geographic distance or historical events, respectively. Barnacles are highly conspicuous for having a wide distribution caused largely by ship hull fouling or ballast water (Carlton et al. 2011; Gollasch and David 2011). These animals settle on and colonize diverse types of hard substrates including rocks, manmade marine structures and sailing vessels. Some species are “sessile voyagers” on turtles, sea snakes, or whales (Kim et al. 2020; Dreyer et al. 2020) and can be found on floating and drifting objects such as timber, cuttlefish bones, bottles, cans, and light plastic sheets. Barnacles will affect economic loss by causing increased fuel consumption of vessels and by damaging submarine structures (Holm 2012). The widespread barnacle species, Amphibalanus (= Balanus) amphitrite (Darwin 1854) from the family Balanidae (Pitombo 2004; see Chan et al. 2021 for the latest barnacle classification) is commonly used as a model organism for ecological studies (Clare et al. 1994; Holm et al. 2000; Khandeparker et al. 2002; Lagersson and Høeg 2002; Leslie et al. 2005; Wong et al. 2011; Ip et al. 2021; Campanati et al. 2016), and antifouling assays (Rittschof et al. 1992; Hirota et al. 1996; Hellio et al. 2004; Maréchal and Hellio 2011). This barnacle is a common intertidal fouling species in tropical and warm temperate waters worldwide (Henry and McLaughlin 1975; Chen et al. 2014), including the Persian Gulf (PG) and the Gulf of Oman (GO) (Shahdadi et al. 2014). Its cosmopolitan distribution seems to be largely related to anthropogenic activities, especially recently increased shipping traffic affecting transmission of larvae and adult barnacles globally (Seebens et al. 2013; Banks et al. 2015). This transmission causes changes in the genetic diversity of populations. The changes in genetic diversity can be investigated by various approaches including DNA barcoding. DNA barcoding is a useful approach in uncovering genetic diversity, population structures and phylogenetic patterns (DeSalle and Goldstein 2019; Kim et al. 2019). This method can be used to reveal the genetic structure of non-indigenous species and their evolutionary potential and to assist with the management of introduced/invasive species (Strayer et al. 2006). Using the mitochondrial COI marker, Chen et al. (2014) investigated genetic differentiation in A. amphitrite in various regions of the world. They found three clades for the examined specimens including two widely distributed clades and one with only two representatives from North Carolina (USA) and Singapore (see Table 1 in Chen et al. 2014). While most sampling sites hosted members of only one clade, some localities harbored members of two clades and had higher genetic diversity (see Fig. 1 and Table 1 in Chen et al. 2014). This sympatry was assigned to both historical events and recent human activities (Chen et al. 2014). However, a very important part of the species global distribution, the PG and the GO, were not included in their study. The GO is the northwestern extension of the Arabian Sea (Indian Ocean) and expectedly shares common biota with that sea (Owfi et al. 2016). In contrast, the PG is a semi-closed and relatively young sea connected to the GO and Arabian Sea through the page 2 of 17Zoological Studies 62:16 (2023) © 2023 Academia Sinica, Taiwan Strait of Hormoz (Lambeck 1996). Until the early Holocene, the PG basin was almost dry (Sheppard et al. 2010). Its current marine community started to form only around 15 ka BP, receiving its biota from the northwestern part of the Indian Ocean (Teller et al. 2000). The PG reached its current sea level about 6–8 ka BP (Lambeck 1996). Currently, the PG is among the major destinations of oil tankers from all over the world Fig. 1. Map of the sampling localities. Table 1. Sampling localities and summary of statistics of genetic variability for Amphibalanus amphitrite # Location Latitude Longitude N Nh Np h π 1 Bushehr Province, Bandar Genaveh (GV) 29°33'17"N 50°29'08"E 3 - - - - 2 Hormozgan Province, Bandar Charak (BC) 26°43'32"N 54°16'51"E 2 - - - - 3 Qeshm Island, Toola and Hamoon harbors (TH) 26°55'01"N 55°56'13"E 15 11 17 0.9 0.0047 4 Qeshm Island, Messen and Kandaloo (MK) 26°41'53"N 55°54'49"E 14 10 22 0.89 0.0091 5 Hormozgan Province, Hormoz Island (HR) 27°02'33"N 56°29'38"E 2 - - - - Persian Gulf (PG) (total) 36 24 46 0.93 0.0077 6 Hormozgan Province, Bandar Jask (JS) 25°42'18"N 57°47'07"E 10 7 8 0.87 0.0037 7 Sistan and Baluchestan Province, Jod (JD) 25°27'04"N 59°30'17"E 5 - - - - 8 Sistan and Baluchestan Province, Chabahar, Tis, Chabahar (CH) 25°21'12"N 60°36'08"E 30 19 25 0.89 0.0058 9 Sistan and Baluchestan Province, Gwatr Bay (GU) 25°10'34"N 61°36'14"E 13 6 35 0.92 0.0102 Gulf of Oman (GO) (total) 58 35 63 0.87 0.0066 N, sample size; Nh, number of haplotypes; Np, number of polymorphic sites; h, haplotype diversity; π, nucleotide diversity. page 3 of 17Zoological Studies 62:16 (2023) © 2023 Academia Sinica, Taiwan with about 53,000 visits annually by oil transporting ships (Al-Yamani et al. 2015). Therefore, the present biota of the PG and its genetic composition are expected to reflect natural historic events as well as anthropogenic activities (Spalding et al. 2007). To test this hypothesis and to fill the void of knowledge pertaining to the PG and the GO, the present work aimed to study the genetic variation within the populations of A. amphitrite from these two gulfs using the mitochondrial COI gene. The study also investigated the genetic diversity within each Gulf and compared the diversity indices with those of other populations globally. MATERIALS AND METHODS Sample collection Specimens of Amphibalanus amphitrite were collected from nine locations along the PG and the GO (Table 1, Fig. 1). In total, 94 individuals were collected from both artificial substrata (e.g., humanmade structures, piers, small vessel hulls and floating objects) and natural habitats (e.g., intertidal rocks, mollusk shells, crab carapaces and mangrove trunks). The specimens were kept in 96% or absolute ethanol immediately upon collection and transferred to the Molecular Systematics Laboratory at the University of Tehran for molecular analysis. DNA extraction, amplification and sequencing Total genomic DNA was extracted from muscles using the salt precipitation method (Katouzian et al. 2016). A 576-bp fragment of the cytochrome c oxidase subunit I gene (COI) was amplified by the polymerase chain reaction using primer pairs LCO1490-JJ and HCO2198-JJ (Astrin and Stüben 2008) as described in Chen et al. (2014). The PCR products were outsourced for sequencing to LGC Genomics GmbH (Berlin, Germany) and Macrogen Europe, Amsterdam using the same forward primers. Sequences were proofread via Chromas Lite (v. 2.1.1) (Technelysium Pty Ltd, Queensland, Australia). Sequences of all unique haplotypes/genotypes were submitted to GenBank (http://www.ncbi.nlm.nih.gov) and are available under accession numbers (OQ119797–OQ119890). COI sequences of A. amphitrite from previous study (Chen et al. 2014) were also retrieved from GenBank and included in the analyses (accession numbers KC138445, KM211362–KM211497). Amphibalanus reticulatus, A. variegatus and Balanus glandula were used as outgroups (GenBank accession numbers JQ035518.1, JQ035522.1 and KU204282.1, respectively). Sequence data analyses Sequences were aligned using Clustal W (Thompson et al. 1994; Villesen 2007) implemented in BioEdit 7.0.5 (Hall 1999). The ML tree was obtained using raxmlGUI, v. 1.3 (Silvestro and Michalak 2012) with 1000 bootstrap pseudoreplicates. The selected evolutionary model was GTR+G (Rodriguez et al. 1990). Two maximum parsimony haplotype networks (Templeton et al. 1992) were constructed with PopART (Leigh and Bryant 2015), one for clade I at the global level (Chen et al. 2014) taken from the tree, and one for the sequences of the present study. We calculated the distribution of pairwise differences (i.e., mismatch distribution; Rogers and Harpending 1992) to trace population size change in DnaSP v.5.10 (Librado and Rozas 2009). To assess how mtDNA effective population size changed through time, we analyzed historical demography using coalescent based Bayesian Skyline Plot (BSP) in BEAST 2.4.7 (Bouckaert et al. 2014). We selected GTR+G as the best model of nucleotide substitution and adopted a substitution rate of 3.1% per MY for COI (according to Tsang et al. 2008). We set a strict clock model as prior and ran three independent MCMC analyses with 60 million generations, sampling every 6,000 steps, to verify the consistency of the results. The initial 25% of the samples were discarded as burn-in. The convergence of all parameters was tested and BSP produced in Tracer 1.6 (Rambaut et al. 2014). Standard genetic indices were calculated to determine the genetic diversity within the species based on COI sequences. The haplotype diversity (h), nucleotide diversity (π), number of polymorphic sites (Np) and number of haplotypes (Nh) were calculated using DnaSP v.5 (Librado and Rozas 2009) for each local population and for the complete dataset of each Gulf. Overall mean p‐distance was analyzed with MEGA v.6 (Tamura et al. 2013). We computed pairwise ΦST with 1,000 permutations in Arlequin v.3.5.2.2 (Excoffier and Lischer 2010) to investigate population differentiation patterns among local populations (only populations with n ≥ 8 were included). Species distribution modelling To construct the distribution modelling of A. amphitrite, we included localities of specimens of clade I and III used in worldwide phylogenetic analysis compiled from field observations and available data based on Chan et al. (2014). The ocean climate layers were downloaded from Bio-ORACLE (Ocean Rasters for Analysis of Climate and Environment) data set page 4 of 17Zoological Studies 62:16 (2023) © 2023 Academia Sinica, Taiwan (Tyberghein et al. 2012). Variables were selected based on their ecological meaningfulness and contribution rate species in distribution model. To avoid the effect of high correlation among layers, we first examined all layers using OpenModeller 1.0.7 (De Souza Muñoz et al. 2011) and then used the Pearson correlation method to obtain higher correlative layers (> 0.7). Layers with lower correlation (< 0.7) were selected for further analyses: Temperature (Mean and Range), Phytoplankton Mean, Salinity (Mean and Range), Current Velocity Mean, Min., Max., Ltmax, dissolved oxygen range and Phosphate Mean. All the data were downloaded as raster format with a 5-arc-minute from Bio-ORACLE data set (Tyberghein et al. 2012). RESULTS Five out of the nine sampling localities were located within the PG, and four were located in the GO (Fig. 1, Table 1). Here, we postulated two main populations, namely the PG and GO populations. 36 specimens from the PG and 58 specimens from the GO were studied. Each of the 94 COI sequences contained approximately 576 base pairs (bp), and none of these contained a stop codon. In total, 53 polymorphic sites were found. 136 additional sequences of previous studies on the species were obtained from GenBank and included into the dataset for tree and haplotype network construction. Sequence data analyses In the ML tree, three distinct and well separated clades were recovered (Fig. 2). Sequences of the present study (green circles) are well distributed in the tree, with representatives in two clades. Most sequences were placed in clade I and only two specimens of the present study were significantly different from the others (Fig. 2) namely one from the PG (Kandaloo) and one from the GO (Gwatr) (Table 1). The phylogenetic tree showed 186 sequences, including 92 sequences of the present study placed in clade I. The haplotype network constructed for the clade I recovered 101 haplotypes, with 36 unique haplotypes for the PG and GO (Fig. 3). In the haplotype network (Fig. 4) of the Iranian samples (clade I), some haplotypes were present in only one of the two gulfs, while other haplotypes were found in both gulfs. Except for some outlying haplotypes, the network is nearly star-shaped and most of the haplotypes remained close to the main haplotype with only one mutation step. However, the haplotype network (Fig. 4) showed no clear patterns of isolation between specimens from the two gulfs. The mismatch distributions showed a clear unimodal pattern in the populations of the PG and the GO. The distribution of pairwise haplotype differences was skewed to the left (Fig. 5). An exponential growth in effective population size, shown in Bayesian Skyline Plots (Fig. 6), was consistent with the results of the mismatch distribution analysis. However, the BSP inferred slightly different timings of expansion from the mismatch analyses. The two populations shared a broadly similar timing of demographic growth, which began at 200 ka BP (Fig. 6). The number of sequences available for the GO produced a plot showing a tendency to demographic expansion, truncated at 200 ka BP. Diversity indices were calculated for material from the sampling localities with 10 or more sequences, as well as for each gulf in total. The analyses showed that the specimens from both gulfs had high genetic diversity, and that the diversity within the PG was higher than the diversity within the GO (i.e., haplotype diversity in the PG = 0.93; the GO = 0.87). The individual sampling localities of both gulfs also showed high genetic diversity (Table 1). The ΦST value was calculated only between populations with a sample size > 10 (Table 2). Most pairwise ΦST values between populations were small, and only two pairwise comparisons were significant (p < 0.05) (Table 2). Species distribution modelling All models were run in ten replicates for the current time. The potential distribution models of A. amphitrite showed perfect Area Under Curve (AUC) test values, with an index of 0.89 ± 0.03 and 0.96 ± 0.03 for clade I and clade III, respectively. This showed significance for the binomial omission test, hence the maps were evaluated as very good (more than 0.850). The contribution performance of layers for each period is presented in table 3. Based on the results, temperature mean and range for clade I and temperature mean and current velocity min. for clade III made the largest contributions to current habitat suitability in the predictions modeling. Accordingly, the suitable habitats for A. amphitrite were the tropical and subtropical areas (Fig. 7). DISCUSSION In the framework of biogeography, barnacles are among the most interesting invertebrates. During their pelagic larval stages, they are able to disperse under the influence of oceanic currents. By the end of page 5 of 17 Zoological Studies 62:16 (2023) © 2023 Academia Sinica, Taiwan the larval phase and after dramatic changes through metamorphosis, they become sessile which is expected to limit their active distribution, gene flow among populations, and cause decreased genetic homogeneity. Their fouling behavior, however, has greatly counteracted their natural dispersal ecology in recent decades (Holm 2012). It seems that ships have played a great role in their dispersal throughout the open waters (Yamaguchi et al. 2009). The fouling behavior increases gene flow among distant populations and therefore Fig. 2. Phylogenetic tree constructed with Maximum Likelihood (ML) based on the COI gene for selected sequences of A. amphitrite, focusing on Iranian populations (green circles). Amphibalanus reticulatus (JQ035518.1), A. variegatus (JQ035522.1) and Balanus glandula (KU204282.1) were used as outgroups. Numbers show the bootstrap values after 1000 pseudo-replicates. Clades I, II and III are corresponding to those in Chen et al. (2014). page 6 of 17Zoological Studies 62:16 (2023) © 2023 Academia Sinica, Taiwan Fig. 3. Maximum-parsimony mitochondrial DNA haplotype networks for material from the PG and the GO, and the retrieved sequences from the GenBank (only sequences of the clade I in Chen et al. (2014) phylogenetic tree are included). Fig. 4. Maximum-parsimony of mitochondrial DNA haplotype networks for the PG and the GO populations constructed in PapArt. Hatch marks represent mutations; numbers in the figure legend refer to sampling sites in table 1 and figure 1. page 7 of 17Zoological Studies 62:16 (2023) © 2023 Academia Sinica, Taiwan Fig. 5. Frequency distribution of the number of pairwise nucleotide differences (mismatch) between COI haplotypes in the two populations of Amphibalanus amphitrite. The solid line shows the theoretical distribution under the model of demographic expansion. Fig. 6. Bayesian skyline plots of effective population size through time in Amphibalanus amphitrite from two biogeographical areas (A: PG and B: GO), based on the 576 bp sequences of COI and a nucleotide substitution rate of 3.1%/MY. The bold black curve is the median of the parameter NeT, which is proportional to the effective population size; the blue lines delimit the 95% highest posterior density. For comparison, all x-axes have the same scale. The plots are truncated to the median estimate of each area’s TMRCA. page 8 of 17 Zoological Studies 62:16 (2023) © 2023 Academia Sinica, Taiwan Table 2. Pairwise estimates of COI genetic divergence (ΦST) for Amphibalanus amphitrite among five biogeographical areas. The significance of ΦST values was tested by a permutation test with 1000 replicates MK(PG) TH(PG) GU(GO) CH(GO) JS(GO) MK(PG) 0 TH(PG) 0.00748 0 GU(GO) 0.00728 0.03051* 0 CH(GO) 0.03196* 0.00921 0.01027 0 JS(GO) 0.01838 0.00420 0.00528 0.02062 0 * p < 0.05. Table 3. The frequency of contribution of environmental variables in predicting the clades geographic distribution models Environmental variable (unit) Clade IClade III Temperature Mean 39.8 56.8 Temperature Range 25.7 4.9 Phytoplankton Mean 15.3 5.9 Salinity Mean 5.5 1.4 Salinity Range 3.9 6.2 Current Velocity Mean 2.7 3.7 Current Velocity Ltmax 2 0 Dissolved oxygen Range 1.9 1.6 Phosphate Mean 1.6 5.3 Current Velocity Min 1 14.2 Current Velocity Max 0.6 0 Fig. 7. MAXENT reconstruction for clade I (A) and clade III (B) of Amphibalanus amphitrite in the world representing current distribution models. page 9 of 17 Zoological Studies 62:16 (2023) © 2023 Academia Sinica, Taiwan Owfi F, Braulik GT, Rabbaniha M. 2016. Species diversity and distribution pattern of marine mammals of the Persian Gulf and Gulf of Oman. Iran J Fish Sci 15(2):927–944. Pannacciulli FG, Manetti G, Maltagliati F. 2009. 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