New alien species in the Atlantic Ocean?

Marine Biodiversity Records, page 1 of 6. # Marine Biological Association of the United Kingdom, 2010 doi:10.1017/s1755267210000357; Vol. 3; e39; 2010 Published online New alien species in the Atlantic Ocean? tomoyuki nakano 1 and free espinosa 2 1 Department of Geology and Palaeontology, National Museum of Nature and Science, 3-23-1 Hyakunin-cho, Shinjuku-ku, Tokyo 169-0073, Japan, 2 Laboratorio de Biología Marina, Universidad de Sevilla, Avenida Reina Mercedes, 6, 41012, Sevilla, Spain The family Patellidae has been recognized as a complex taxonomic group for many years and its poor fossil record has complicated the study of the group. One of these limpet species, Cymbula nigra has its distributional range from Namibia to the Mediterranean and two varieties of Cymbula nigra (formerly named Patella nigra) have been reported: Patella nigra plumbea (typical form widely spread) and Patella nigra ghananis (variety from Ghana). The results of the present study clearly show that some specimens from Ghana reported as Cymbula nigra belong to a different species. In fact, this species is closely related to Cellana toreuma from Java (Indonesia), whereas specimens from Java appear as a distinct and cryptic species of Cellana toreuma distributed in Japan to Hong Kong. The specimens from Ghana could have been recently introduced in West Africa from the Indian Ocean. It represents the first record of the genus Cellana in the Atlantic. Furthermore, the variety described as Patella nigra ghananis in the previous literature probably were specimens of this new Cellana species. Keywords: Cellana, limpets, West Africa, alien species, Cymbula nigra, Patella nigra Submitted 4 September 2009; accepted 8 March 2010 INTRODUCTION The family Patellidae has been recognized as a complex taxonomic group for many years (Fisher-Piette, 1938), and its poor fossil record has complicated the study of the group (Ridgway et al., 1998). Nevertheless, there are many recent studies on taxonomic and phylogenetic topics (Corte-Real et al., 1996; Ridgway et al., 1998; Koufopanou et al., 1999; Weber & Hawkins, 2002, 2003, 2005; Nakano & Ozawa, 2004, 2007; Sá-Pinto et al., 2005; Espinosa & Ozawa, 2006; Nakano et al., 2009). Although limpet fauna has been extensively studied in the north-east Atlantic, South Africa, North America, Australia New Zealand and the Far East, poor attention has been conferred to West Africa. There is a lack of knowledge on ecological, taxonomic and genetic studies of West African limpets. One of these limpet species, Cymbula nigra (da Costa, 1771), has its distributional range from Namibia to the Mediterranean (Hodgson et al., 1996; Koufopanou et al., 1999). Christiaens (1973) mentioned two varieties of Cymbula nigra (formerly named Patella nigra): Patella nigra plumbea (typical form from Senegal) and Patella nigra ghananis (variety from Ghana). This author synonymized Patella plumbea Lamarck, 1819 and P. safiana Lamarck, 1819 into P. nigra da Costa, 1777, although Powell (1973) considered both taxa as a different species. The pictures included in Christiaens (1973) are clearly different, with a typical shell form of C. nigra in the former variety (identical to those observed in Morocco and the Mediterranean, personal observation), and a smaller shell in the latter. Morphological (Hodgson et al., 1996) and genetic studies (Koufopanou et al., 1999; Nakano & Ozawa, 2007) have pointed out that specimens from Angola and the Mediterranean are the same species (named Cymbula Corresponding author: F. Espinosa Email: free@us.es safiana by these authors) and belong to the genus Cymbula instead of the genus Patella. However, following the International Code of Zoological Nomenclature (Article 23) the specific name has to be the oldest name nigra (Moreno & Arroyo, 2008), therefore, the valid name for the taxon would be Cymbula nigra. The aim of the present study was to confirm if the varieties from West Africa belong to the taxon Cymbula nigra. MATERIALS AND METHODS Sampling collection The species collected in the present study are shown in Table 1. Several specimens of Cymbula nigra from different localities of the Strait of Gibraltar were collected and their morphological characters were studied. After these observations were done, one specimen from Ceuta (North Africa, Strait of Gibraltar) was selected taking into account that all the specimens were morphologically similar. On the other hand, two specimens labelled as Cymbula nigra from Old Ningo, Ghana (West Africa) were collected by Dr David Reid (Natural History Museum, London). Additionally, several specimens of Cymbula nigra (collected in Ghana by Dr José Templado and deposited in the National Museum of Natural Sciences, Madrid, Spain) were studied morphologically. The genetic study on those samples was not possible due to their formalin preservation. DNA extraction, PCR amplification and DNA sequencing Living specimens were preserved in 95% ethanol. Total DNA was extracted from a fragment of the mantle or foot muscle tissue, either using High Pure PCR Template Preparation 1

2 tomoyuki nakano and free espinosa Table 1. Sampling sites and species collected. Species Locations DNA code GenBank accession number Cellana toreuma Yamada-cho, Iwate Prefecture, Japan L6 AB445019 Akashouzaki, Fukui Prefecture, Japan L9 AB445020 Cape d Aguilar, Hong Kong L307 AB445021 Cape d Aguilar, Hong Kong L308 AB445022 Cape d Aguilar, Hong Kong L309 AB445023 Morozaki, Aichi Prefecture, Japan L730 AB445024 Yukinoura, Mie Prefecture, Japan L731 AB445025 Hayama, Kanagawa, Prefecture, Japan L732 AB445026 Ayukawa, Miyagi Prefecture, Japan L733 AB445027 Tappizaki, Aomori Prefecture, Japan L734 AB445028 Takanabe, Miyazaki Prefecture, Japan L735 AB445029 Shoudoshima, Kagawa Prefecture, Japan L736 AB445030 Mihama-cho, Wakayama Prefecture, Japan L737 AB445031 Reihoku, Kumamoto Prefecture, Japan L738 AB445032 Ping Lang Qiaq, Taiwan L832 AB445033 Shitiping, Taiwan L833 AB445034 Yeliu, Taiwan L834 AB445035 Cymbula nigra var. ghananis Old Ningo, Ghana GH1 AB445036 Old Ningo, Ghana GH2 AB445037 Cymbula nigra Ceuta, Spain BE1 AB445038 Kit (Roche) or a standard phenol/chloroform extraction method. In the latter procedure, a small amount of tissue was treated with 200 ml of TEN buffer (10 mm Tris, ph 8.0, 10 mm EDTA and 10 mm NaCl), 20 ml of 10% SDS, and 40 ml of proteinase K (20 mg/ml) at 378C for 1 2 hours with gentle rotation, followed by phenol/chloroform and chloroform extractions, and ethanol precipitation. Amplification of a part of the cytochrome-c oxidase I (COI) was achieved with the LCO1490 (5 GGTCAACAAATCAT AAAGATATTGG 3 ) and HCO2198 (5 TAAACTT CAGGGTGACCAAAAAATCA 3 ) primer pair described by Folmer et al. (1994). PCR amplification was performed in 25 ml of reaction volume containing 10 mm Tris HCl ph 8.3, 50 mm KCL, 1.5 mm MgCl 2,200mM dntps,0.2mm each primer, 0.5 mg/ml BSA (Sigma), 2 units of Taq polymerase (Takara), and 1 ml of template DNA solution. The cycling parameters for amplification consisted of an initial denaturation for 3minutesat948C; followed by 30 cycles of denaturation for 45 seconds at 948C, annealing for 90 seconds at 548C, and extension for120secondsat728c; and ended with a 5 minutes extension at 728C. Amplification products were purified using High Pure PCR Product Purification Kit (Roche). Direct double-stranded cycle sequencing of 25 to 30 ng of COI PCR product was performed in both directions using the Applied Biosystems BigDye vr.3 dye terminator cycle sequencing kit. Cycle sequencing was performed using an Applied Biosystems GeneAmp PCR System 9700. The cycling parameters were 25 cycles of 10 seconds at 968C, 5 seconds at 508C, and 4 minutes at 608C. Sequencing reaction products were purified using ethanol precipitation and analysed on an ABI PRISM 377 DNA sequencer. Sequence analysis and phylogeny reconstruction We determined the nucleotide sequences of a fragment of COI exactly 658bp. Sequence of COI was aligned using MacClade 4.03 (Maddison & Maddison, 2002) referring to translated amino acid sequence. Third codon positions of COI sequences were retained. Distance analysis was carried out using the neighbour-joinning (NJ) algorithm (Saito & Nei, 1987) with Kimura 2-p model and 1000 bootstrap replicates to assess the stability of each node. Haplotype analysis based on the sequence of a fragment of COI was undertaken. The haplotypes were defined using the Redundant Taxa option in MacClade 4.03 (Maddison & Maddison, 2002), and the minimum spanning tree (MST) (Kruskal, 1956; Prim, 1957) was constructed using ARLEQUIN version 2.0 (Schneider et al., 2000). The MST is computed from the matrix of pairwise distances calculated between all pairs of haplotypes using a modification of the algorithm described in Rohlf (1973). The MST is convertible to a minimum spanning network (MSN). RESULTS The specimens of Cymbula nigra from Ghana showed the same haplotype (Figures 1 & 2) but both belong to a different species than those specimens from Ceuta because of the great genetic distance found (186 substitutions). To establish the species identity of these sequences, a NJ tree (Figure 1) was constructed with some sequences from Nakano & Ozawa (2007) and Nakano et al. (2009) (the codes used by these authors have been maintained. Note that individuals L3, L687, L688 and L496 had been previously sequenced in Nakano & Ozawa, 2004, 2007). We have included three sequences of C. nigra in the NJ tree: the individual BE1 (from Ceuta, Spain) and the individuals GH1 GH2 (from Old Ningo, Ghana), showing a very different sequence of the COI. The sample from Benzú (Ceuta, Spain) was grouped within the genus Cymbula as it was expected. Furthermore, the specimen was grouped with a sample of Cymbula nigra also from Ceuta sequenced previously by Nakano & Ozawa (2007). However, the sample from Old Ningo (Ghana) was grouped closely with the species Cellana toreuma, indicating that both specimens from Ghana belong to the genus Cellana instead of Cymbula.

new alien species in the atlantic ocean? 3 Fig. 1. Neighbour-joining tree based on sequences of 658 bp obtained by Nakano & Ozawa (2007) for COI fragment (the codes for each sequence have been maintained like in the original paper) with the inclusion of new sequences obtained for Cellana species and C. nigra from Benzú (Ceuta, Spain) and Old Ningo (Ghana) of individuals BE1 and GH1 GH2 respectively. Arrows indicate the position of each sequence in the tree. Note the position of the specimens GH1 and GH2 from Ghana. Corresponding haplotypes are indicated (see also Figure 2). Numbers on each node indicate bootstrap values. In order to clarify the taxonomic status of these individuals, a MST analysis was carried out with several sequences of Cellana species (from Nakano & Ozawa, 2004, 2007, Nakano et al., 2009), including those of Cellana toreuma sequenced during the present study (Figures 1 & 2). Specimens GH1 and GH2 from Ghana were grouped closely with C. toreuma from Java (Indonesia) and, subsequently, the MSN showed that specimens from Ghana are the same species as those from Java (only two substitutions of difference between haplotypes 4 and 34; see Figure 2). Conversely, C. toreuma from Japan Taiwan Hong Kong belong to a different species. Morphological characters of different specimens studied in the present study are shown in Figure 3. DISCUSSION The samples from Ghana belong to the genus Cellana, and would represent the first record of this genus in the Atlantic. Morphologically, the specimens are similar to C. toreuma species according to the pictures and description included in Powell (1973). However, this species is distributed in the Far East (Japan, China, Taiwan and Philippines) according to this author. In the same way, we have also identified the specimen from Java (Indonesia), which is closely related with the samples from Ghana, as C. toreuma based on morphological characters of the shell. Nevertheless, the genetic data obtained do not support this contention. The data indicate clearly that specimens from

4 tomoyuki nakano and free espinosa Fig. 2. Minimum spanning network of the haplotypes found for Cellana species (same sequences as for phylogenetic tree of Figure 1). Dotted lines indicate the specimens identified as Cellana toreuma. Haplotypes 1 3 and 5 7 come from Japan, Taiwan and Hong Kong, whereas haplotype 4 comes from Java, Indonesia and haplotype 34 are the specimens GH1 and GH2 from Ghana. Fig. 3. (A) Cymbula nigra, specimen BE1 (Ceuta); (B) C. nigra, specimen from the National Museum of Natural Sciences, Madrid, Spain (Ghana); (C & D) Cellana sp., specimens from the Natural History Museum, London, UK (Ghana); (E) Cellana sp. (Java, Indonesia); (F) Cellana toreuma (Japan). Scale bars 1 cm.

new alien species in the atlantic ocean? 5 Java and Ghana belong to a different and cryptic species of C. toreuma. There are many works that have revealed cryptic species using molecular techniques within molluscan groups (Scutellastra, Paulay & Meyer, 2002; Patelloida, Kirkendale & Meyer, 2004, Nakano & Ozawa, 2005; Monodonta, Donaldet al., 2005; Astraliun, Meyer et al., 2005; Tricolia, Williams & Ozawa, 2006 among others). In this context, it would be plausible to surmise, although our results are preliminary, that the two varieties mentioned by Christiaens (1973) of Cymbula nigra: Patella nigra plumbea (typical form from Senegal) and Patella nigra ghananis (variety from Ghana) were different species. The pictures included in this work are clearly different, with a typical shell form of C. nigra in the former variety (identical to those collected in the present study), and a smaller and more similar to Cellana toreuma shell in the latter (similar to both individuals sequenced from Ghana; see Figure 3). We cannot discard the fact that the variety from Ghana was really a Cellana species that could be broadly spread in West Africa, taking into account that Christiaens (1973) referred that he studied a great number of limpets from Ghana and all of those belong to the var. nov. Patella nigra ghananis. Additionally, the proximity between specimens GH1 GH2 from Ghana and L496 from Java (see Figures 1 & 2) revealed that this fact can be due to a recent introduction of this species in the Atlantic Ocean; otherwise, the genetic differences should be greater. In this sense, limpets with two different and distant distributional areas due to introduction events are not a new record (see Voss, 1959; Baker et al., 2004 for the case of Siphonaria pectinata in the Gulf of Mexico/Florida and Southern Europe/Western Africa). Nakano & Ozawa (2007) reported the specimens of C. toreuma from Japan and Indonesia as very close in their phylogenetic analyses and, apparently, belonging to the same species. This could be due to the fact that those analyses did not include the new sequence of Cellana radiata enneagona from Ogasawara (Bonin) Island, south-eastern Japan (see Figures 1 & 2, specimen L687; haplotype 30) that is inserted between the two C. toreuma groups (Nakano et al., 2009). The similar tropical distribution of the specimens from Indonesia and Ghana could have facilitated the introduction of the species in West Africa from Indonesia as fouling or as larval stage in ballast waters. 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