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New data reinforcing the taxonomic status of Lepidion eques as synonym of Lepidion lepidion (Teleostei, Gadiformes)
Biochemical Systematics and Ecology 68 (2016) 6e10
Contents lists available at ScienceDirect
Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco
New data reinforcing the taxonomic status of Lepidion eques as synonym of Lepidion lepidion (Teleostei, Gadiformes) ~o n b, Juan-Carlos Arronte c, Alejandro de David Barros-García a, *, Rafael Ban a Carlos a
Department of Biochemistry, Genetics and Immunology, University of Vigo, C/ Fonte das Abelleiras s/n, 36310 Vigo, Spain Instituto de Investigaciones Marinas, Consejo Superior de Investigaciones Científicas (IIM-CSIC), C/ Eduardo Cabello, 6, 36208 Vigo, Spain c tico Rodrigo Uría s/n, 33071 Oviedo, Spain Department of Biology of Organisms and Systems, University of Oviedo, C/ Catedra b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 February 2016 Received in revised form 10 June 2016 Accepted 26 June 2016
The Mediterranean morid codling Lepidion lepidion is thought to be endemic, yet its taxonomic distinctiveness from the morphologically similar and more wide-ranging Atlantic Lepidion eques is unresolved, and has been controversial since the beginning of the twentieth century. Despite the abundant taxonomic literature questioning the interspecific relationship between these taxa, their current status remains unchanged. To elucidate the differentiation of the specimens identified as L. lepidion and L. eques collected across much of their geographic ranges, the sequence divergence of the cytochrome oxidase I “DNA barcode” gene of the mitochondrial genome was evaluated. A network analysis indicates that the most observed haplotypes are common to both species throughout their Mediterranean and North Atlantic distribution areas. This molecular evidence suggests the absence of biogeographical barriers and is insufficient to support the different species designations, giving L. eques the taxonomic status of junior synonym of L. lepidion. © 2016 Elsevier Ltd. All rights reserved.
Keywords: COI haplotype Morid codling Mitochondrial DNA Systematics
1. Introduction The Mediterranean Lepidion lepidion (Risso, 1810) and the Atlantic Lepidion eques (Günther, 1887) are two codling fish species included in the family Moridae. Their taxonomic relationship has been controversial since the beginning of the twentieth century. The first known species was the former, described in one specimen from Nice, France, in the northwestern Mediterranean Sea (Risso, 1810). Afterwards, Lepidion eques was described in the northeastern Atlantic, following the examination of specimens captured in the Faroe Channel; a larger eye and shorter head appearing to be the key distinctive traits (Günther, 1887). However, the taxonomic similarity between these two species was soon pointed out by several authors (Collett, 1905; Roule, 1919; Norman, 1935; Grey, 1956; Raimbault, 1963). A more recent and comprehensive revision of the morphology of these two Lepidion species concluded that both could be considered to have a sub specific rather than a specific
Abbreviations: COI, cytochrome c oxidase I. * Corresponding author. ~o n), [email protected] (J.-C. Arronte), [email protected] E-mail addresses: [email protected] (D. Barros-García), [email protected] (R. Ban (A. de Carlos). http://dx.doi.org/10.1016/j.bse.2016.06.017 0305-1978/© 2016 Elsevier Ltd. All rights reserved.
D. Barros-García et al. / Biochemical Systematics and Ecology 68 (2016) 6e10
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relationship, although it would be unwise to make L. eques a synonym of L. lepidion (Templeman, 1970). This criterion remained unchanged until recently, when COI DNA sequencing along with morphological and meristic analysis of a number of Lepidion specimens from the eastern and western coasts of the Iberian Peninsula found no specific differences between the Mediterranean Lepidion lepidion and the Atlantic Lepidion eques, suggesting that the latter is a junior synonym of the former ~o n et al., 2013). The aim of this investigation was to carry on a molecular analysis on specimens captured in their dis(Ban tribution areas in order to confirm the synonymy status of these two Lepidion species. 2. Materials and methods 2.1. Sample collection and specimen identification Between 2007 and 2015, 72 specimens, 20 identified as L. lepidion and 52 as L. eques, were collected by commercial and research vessels at diverse locations (Fig. 1). The specimens of L. lepidion have a western Mediterranean origin, at the Balearic basin, off Barcelona. The L. eques specimens were captured in several locations in the eastern Atlantic, such as the Galicia Bank s Canyon, in the Bay of Biscay, (10 samples) and Porcupine seamount, in the northwestern Spanish waters, (26 samples), Avile Sea Bight, in the southwest of Ireland, (13 samples). Three specimens have a northwestern Atlantic origin and were captured in Canadian waters. The COI barcode of one sample from Greenland was mined from BOLD (GLF150-14). The specimens were immediately frozen on board and, once in the laboratory, muscle samples were removed from thawed individuals and stored in 95% ethanol. The DNA barcodes included in this study are deposited in GenBank under accession numbers JX437971eJX437986, JX437989eJX437998, KT351924eKT351953 and KT989513eKT989528. 2.2. DNA extraction, PCR amplification and sequencing DNA was extracted by means of the E.Z.N.A Tissue DNA Kit from OMEGA bio-tek. The standard 50 barcoding region of COI (ca. 650 bp) was amplified using the primers cocktail COI-3 (Ivanova et al., 2007), with Thermo Scientific Phire Green Hot Start II PCR Master Mix and reaction conditions as follows: 98 C for 30 s followed by 35 cycles of 98 C for 5 s, annealing at 52 C for 5 s and 72 C for 10 s, with a final extension at 72 C for 1 min. COI amplicon bands were visualised on 1.2% agarose gels (Seakem LEAgarose), stained with ethidium bromide and purified with ExoSAP-IT (Affymetrix) following manufacturer’s instructions. DNA sequencing reactions were carried out in the direct and reverse senses using the M13F (-21) and M13R (-27) primers. The resulting products were resolved in an ABI 3130 Genetic Analyzer (Applied Biosystems). 2.3. DNA analysis Each consensus sequence was obtained after assembling the direct and reverse traces with SEQSCAPE v2.5 (Applied Biosystems, Foster City, CA), and aligned to each other employing MEGA 5.0 (Tamura et al., 2011). The list of haplotypes and their frequencies were calculated using DNAspV5 (Librado and Rozas, 2009). Pairwise distances among haplotypes were estimated using ARLEQUIN 3.5 (Excoffier and Lischer, 2010). These distances were employed in the software HapStar 0.5 (Teacher and Griffiths, 2011) with the “Minimum Spanning Network” option for the graphic representation of the haplotype network. 3. Results A total of 73 DNA sequences were used to produce a COI alignment 656 bp in length, including 32 polymorphic sites from which eight were parsimony informative. A total of 29 different haplotypes were found and 23 of them were detected only once. Three of the haplotypes were observed in 10 or more individuals and comprised 58% of all the sequences present in the dataset. A COI haplotype network (Fig. 1) showed a star-like pattern with a central haplotype representing 14% of the sampled individuals from the three areas (Mediterranean Sea, North East Atlantic and North West Atlantic). This haplotype was connected with the other two most common, only one mutation away. All other haplotypes were separated from these main three, most of them only one or two mutations apart. There was no clear geographic structure in the network because these common COI haplotypes were shared among all the represented areas. Furthermore, four of the six haplotypes represented by more than one individual were shared between at least two different areas. 4. Discussion The North Atlantic codling Lepidion eques is considered a valid species with a North Atlantic distribution in two of the most consulted fish faunas (Cohen, 1986; Cohen et al., 1990). The same result is obtained when the main two electronic repositories, The Catalog of Fishes (Eschmeyer et al., 2016) and FishBase (Froese and Pauly, 2016), are searched for using this species name as query. The latter considers the Mediterranean codling Lepidion lepidion a separated species with a Mediterranean distribution while the former also gives it an eastern Atlantic distribution.
8 D. Barros-García et al. / Biochemical Systematics and Ecology 68 (2016) 6e10 Fig. 1. COI haplotype network of Lepidion lepidion and L. eques populations. Each circle represents a unique haplotype, with the area being proportional to its frequency in three regions: red (Mediterranean Sea), green (North East Atlantic) and blue (North West Atlantic). The size of the circles is proportional to the number of individuals sampled. Capture locations of individuals are shown with triangles. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
D. Barros-García et al. / Biochemical Systematics and Ecology 68 (2016) 6e10
9
A previous investigation comparing 26 specimens of Lepidion from Mediterranean and Atlantic Spanish waters showed no morphological or DNA barcoding differences between sampling sites, suggesting that all the specimens were conspecific ~o n et al., 2013). An increase in both the number of specimens and sampling sites in the North Atlantic is now offering (Ban similar molecular results, with a variety of COI haplotypes shared among the distribution areas (Mediterranean Sea, North East and North West Atlantic). This result rejects the possibility of a limited distribution of the taxa due to biogeographical barriers and, therefore, the presence of different species. Furthermore, the high frequency of common haplotypes between species and distribution areas makes a possible hybridization hypothesis unlikely, and supports a scenario where L. eques should be considered a junior synonym of L. lepidion. As a consequence of this erroneous classification, L. lepidion has been traditionally considered an endemism of the Mediterranean Sea. However, the Gibraltar sill is not an impenetrable barrier for such deep-water fishes and a certain number of so-called Mediterranean Sea endemic species have been later captured in the Atlantic Ocean, or made synonyms of Atlantic species, as occurs in this case. Eggs and early stages of morid fishes are pelagic (Cohen, 1986), and fry of L. eques has been captured in surface waters in several locations off Iceland and western Scotland, for example (reviewed in Templeman, 1970); more recently, a pelagic juvenile of Lepidion inosimae has been collected off northeastern Japan (Okamoto et al., 2009). The presence of these pelagic forms would allow the colonization of the Mediterranean Sea via the inflowing surface current across the Gibraltar sill. This passage, 12.9 km wide and with a depth of 286e900 m of depth, is the single connection between the Atlantic Ocean and the Mediterranean Sea. The reopening of the Strait of Gibraltar 5 million years ago, led to the restocking of the Mediterranean Sea with fauna and flora from the Atlantic Ocean (Coll et al., 2010) and, nowadays, it is estimated that almost 59% of the Mediterranean fishes have an Atlantic origin (Psomadakis et al., 2012). Regardless of their morphological differences, the Lepidion individuals collected from across the western Mediterranean and northern Atlantic appear to belong to a single demographic unit. The facts mentioned above would support that Lepidion lepidion is not endemic in the Mediterranean, showing instead an Atlantic-Mediterranean distribution, as is also observed in ~o n et al., 2010). its congener Lepidion guentheri (Ban Acknowledgements ~ ol de OceanThe authors would like to thank the staff involved in the following research surveys of the “Instituto Espan ografía”: INDEMARES-BANGAL 0811, INDEMARES-AVILES 0412, FLEMISH CAP 2015, PLATUXA 2015, 3FNL 2015 and PORCUPINE 2015. We are deeply grateful to J. Cartes (ICM-CSIC, Barcelona, Spain), for providing specimens of L. lepidion from the Mediterranean Sea, in the framework of the project ANTROMARE (CTM2009-12214-C02-01-MAR), granted by the Spanish Ministry of Science and Technology. We also extend our thanks to the crews of the Research Vessels “Miguel Oliver” and “Vizconde de Eza” (Ministry of Agriculture, Food and Environment, Spain). This investigation was partially funded through the research project ECOMARG 3 (Ministry of Agriculture, Food and Environment, Spain) and the EC LIFEþ “Nature Biodiversity” INDEMARES project (07/NAT/E/000732). This results partially fulfil the PhD degree requirements of DBG at the University of Vigo. References ~o n, R., Arronte, J.C., Heredia, J., Pis-Milla n, J.A., 2010. First record of a specimen of Lepidion guentheri (Giglioli, 1880) (Gadiformes: Moridae) with Ban melanistic coloration. J. Appl. Ichth 26, 602e605. http://dx.doi.org/10.1111/j.1439-0426.2010.01472.x. ~o n, R., Arronte, J.C., V Ban azquez-Dorado, S., del Río, J.L., de Carlos, A., 2013. DNA barcoding of the genus Lepidion (Gadiformes: Moridae) with recognition of Lepidion eques as a junior synonym of Lepidion lepidion. Mol. Ecol. Resour. 13, 189e199. http://dx.doi.org/10.1111/1755-0998.12045. Cohen, D.M., 1986. Moridae. In: Whitehead, P.J.P., Bauchot, M.-L., Hureau, J.-C., Nielsen, J., Tortonese, E. (Eds.), Fishes of the Northeastern Atlantic and the Mediterranean, vol. 2. UNESCO, Paris, pp. 713e723. Cohen, D.M., Inada, T., Iwamoto, T., Scialabba, N., 1990. FAO Species Catalogue. In: Gadiform Fishes of the World (Order Gadiformes). An Annotated and Illustrated Catalogue of Cods, Hakes, Grenadiers and Other Gadiform Fishes Known to Date, vol. 10. FAO, Rome. Coll, M., Piroddi, C., Steenbeek, J., Kaschner, K., Ben Rais Lasram, F., Aguzzi, J., et al., 2010. The biodiversity of the Mediterranean sea: estimates, patterns, and threats. PLoS ONE 5, e11842. http://dx.doi.org/10.1371/journal.pone.0011842. Collett, R., 1905. Fiske indsamlede under “Michael Sars”s togter i Nordhavet 1900-1902. Rep. Norw. Fish. Mar. Invest. 2, 1e151. Eschmeyer, W.N., Fricke, R., van der Laan, R. (eds). Catalog of Fishes: Genera, Species, References. (http://researcharchive.calacademy.org/research/ ichthyology/catalog/fishcatmain.asp (accessed 01.06.2016). Excoffier, L., Lischer, H.E., 2010. Arlequin suite ver 3.5: a new series of programs to perform population genetics analyses under Linux and Windows. Mol. Ecol. Resour. 10, 564e567. http://dx.doi.org/10.1111/j.1755-0998.2010.02847.x. Froese, R., Pauly, D. (Eds.), 2016. FishBase. World Wide Web Electronic Publication. www.fishbase.org. version 01/2016 (accessed 01.06.2016). Grey, M., 1956. The Distribution of Fishes Found below a Depth of 2000 Meters. Chicago Natural History Museum, Chicago. Günther, A., 1887. Report on the deep-sea fishes collected by H.M.S. Challenger during the years 1873-76. In: Thomson, C.W., Murray, J. (Eds.), Report of the Scientific Results of the Voyage of the H.M.S. Challenger during the Years, Zoology Vol. XXII. Eyre & Spottiswoode, London, pp. 1873e1876. Ivanova, N., Zemlak, T.S., Hanner, R.H., Hebert, P.D.N., 2007. Universal primer cocktails for fish DNA barcoding. Mol. Ecol. Notes 7, 544e548. Librado, P., Rozas, J., 2009. DNAspV5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics 25, 1451e1452. http://dx.doi.org/10. 1093/bioinformatics/btp187. Norman, J.R., 1935. Coast fishes. Part I. The south Atlantic. “Discovery” Rep. 12, 1e58. Okamoto, M., Watanabe, Y., Asahida, T., 2009. Descriptive morphology of pelagic juvenile of Lepidion inosimae (Gadiformes: Moridae) collected from off northeastern Japan. Ichthiol. Res. 56, 76e81. http://dx.doi.org/10.1007/s10228-008-0052-3. Psomadakis, P.N., Giustino, S., Vacchi, M., 2012. Mediterranean fish biodiversity: an updated inventory with focus on the Ligurian and Tyrrhenian seas. Zootaxa 3263, 1e46. ces ichthyologiques capture es au cours des campagnes de l’Institut des Pe ^ches en Me diterrane e (1957-61). Raimbault, R., 1963. Notes sur certaines espe ^ches Marit. 27, 161e176. Revue des Travaux de l’Inst. Sci. Tech. des Pe partement des Alpes maritimes. F. Schoell, Paris. Risso, A., 1810. Ichtyologie de Nice ou histoire naturelle des poisons du de
10
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sultats des Campagnes scientifiques du Prince Albert Ier de Monaco. Poissons provenant des campagnes du yacht ’Princesse Alice’ (1891Roule, L., 1919. Re 1913) et du yacht ’Hirondelle II’ (1914), 52, pp. 1e191. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., Kumar, S., 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28, 2731e2739. http://dx.doi.org/10.1093/molbev/msr121. Teacher, A.G., Griffiths, D.J., 2011. HapStar: automated haplotype network layout and visualization. Mol. Ecol. Resour. 11, 151e153. http://dx.doi.org/10.1111/j. 1755-0998.2010.02890.x. Templeman, W., 1970. A review of the morid fish genus Lepidion of the North Atlantic with first records of Lepidion eques from the western North Atlantic. J. Fish. Res. Board Can. 27, 457e498.
Contents lists available at ScienceDirect
Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco
New data reinforcing the taxonomic status of Lepidion eques as synonym of Lepidion lepidion (Teleostei, Gadiformes) ~o n b, Juan-Carlos Arronte c, Alejandro de David Barros-García a, *, Rafael Ban a Carlos a
Department of Biochemistry, Genetics and Immunology, University of Vigo, C/ Fonte das Abelleiras s/n, 36310 Vigo, Spain Instituto de Investigaciones Marinas, Consejo Superior de Investigaciones Científicas (IIM-CSIC), C/ Eduardo Cabello, 6, 36208 Vigo, Spain c tico Rodrigo Uría s/n, 33071 Oviedo, Spain Department of Biology of Organisms and Systems, University of Oviedo, C/ Catedra b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 February 2016 Received in revised form 10 June 2016 Accepted 26 June 2016
The Mediterranean morid codling Lepidion lepidion is thought to be endemic, yet its taxonomic distinctiveness from the morphologically similar and more wide-ranging Atlantic Lepidion eques is unresolved, and has been controversial since the beginning of the twentieth century. Despite the abundant taxonomic literature questioning the interspecific relationship between these taxa, their current status remains unchanged. To elucidate the differentiation of the specimens identified as L. lepidion and L. eques collected across much of their geographic ranges, the sequence divergence of the cytochrome oxidase I “DNA barcode” gene of the mitochondrial genome was evaluated. A network analysis indicates that the most observed haplotypes are common to both species throughout their Mediterranean and North Atlantic distribution areas. This molecular evidence suggests the absence of biogeographical barriers and is insufficient to support the different species designations, giving L. eques the taxonomic status of junior synonym of L. lepidion. © 2016 Elsevier Ltd. All rights reserved.
Keywords: COI haplotype Morid codling Mitochondrial DNA Systematics
1. Introduction The Mediterranean Lepidion lepidion (Risso, 1810) and the Atlantic Lepidion eques (Günther, 1887) are two codling fish species included in the family Moridae. Their taxonomic relationship has been controversial since the beginning of the twentieth century. The first known species was the former, described in one specimen from Nice, France, in the northwestern Mediterranean Sea (Risso, 1810). Afterwards, Lepidion eques was described in the northeastern Atlantic, following the examination of specimens captured in the Faroe Channel; a larger eye and shorter head appearing to be the key distinctive traits (Günther, 1887). However, the taxonomic similarity between these two species was soon pointed out by several authors (Collett, 1905; Roule, 1919; Norman, 1935; Grey, 1956; Raimbault, 1963). A more recent and comprehensive revision of the morphology of these two Lepidion species concluded that both could be considered to have a sub specific rather than a specific
Abbreviations: COI, cytochrome c oxidase I. * Corresponding author. ~o n), [email protected] (J.-C. Arronte), [email protected] E-mail addresses: [email protected] (D. Barros-García), [email protected] (R. Ban (A. de Carlos). http://dx.doi.org/10.1016/j.bse.2016.06.017 0305-1978/© 2016 Elsevier Ltd. All rights reserved.
D. Barros-García et al. / Biochemical Systematics and Ecology 68 (2016) 6e10
7
relationship, although it would be unwise to make L. eques a synonym of L. lepidion (Templeman, 1970). This criterion remained unchanged until recently, when COI DNA sequencing along with morphological and meristic analysis of a number of Lepidion specimens from the eastern and western coasts of the Iberian Peninsula found no specific differences between the Mediterranean Lepidion lepidion and the Atlantic Lepidion eques, suggesting that the latter is a junior synonym of the former ~o n et al., 2013). The aim of this investigation was to carry on a molecular analysis on specimens captured in their dis(Ban tribution areas in order to confirm the synonymy status of these two Lepidion species. 2. Materials and methods 2.1. Sample collection and specimen identification Between 2007 and 2015, 72 specimens, 20 identified as L. lepidion and 52 as L. eques, were collected by commercial and research vessels at diverse locations (Fig. 1). The specimens of L. lepidion have a western Mediterranean origin, at the Balearic basin, off Barcelona. The L. eques specimens were captured in several locations in the eastern Atlantic, such as the Galicia Bank s Canyon, in the Bay of Biscay, (10 samples) and Porcupine seamount, in the northwestern Spanish waters, (26 samples), Avile Sea Bight, in the southwest of Ireland, (13 samples). Three specimens have a northwestern Atlantic origin and were captured in Canadian waters. The COI barcode of one sample from Greenland was mined from BOLD (GLF150-14). The specimens were immediately frozen on board and, once in the laboratory, muscle samples were removed from thawed individuals and stored in 95% ethanol. The DNA barcodes included in this study are deposited in GenBank under accession numbers JX437971eJX437986, JX437989eJX437998, KT351924eKT351953 and KT989513eKT989528. 2.2. DNA extraction, PCR amplification and sequencing DNA was extracted by means of the E.Z.N.A Tissue DNA Kit from OMEGA bio-tek. The standard 50 barcoding region of COI (ca. 650 bp) was amplified using the primers cocktail COI-3 (Ivanova et al., 2007), with Thermo Scientific Phire Green Hot Start II PCR Master Mix and reaction conditions as follows: 98 C for 30 s followed by 35 cycles of 98 C for 5 s, annealing at 52 C for 5 s and 72 C for 10 s, with a final extension at 72 C for 1 min. COI amplicon bands were visualised on 1.2% agarose gels (Seakem LEAgarose), stained with ethidium bromide and purified with ExoSAP-IT (Affymetrix) following manufacturer’s instructions. DNA sequencing reactions were carried out in the direct and reverse senses using the M13F (-21) and M13R (-27) primers. The resulting products were resolved in an ABI 3130 Genetic Analyzer (Applied Biosystems). 2.3. DNA analysis Each consensus sequence was obtained after assembling the direct and reverse traces with SEQSCAPE v2.5 (Applied Biosystems, Foster City, CA), and aligned to each other employing MEGA 5.0 (Tamura et al., 2011). The list of haplotypes and their frequencies were calculated using DNAspV5 (Librado and Rozas, 2009). Pairwise distances among haplotypes were estimated using ARLEQUIN 3.5 (Excoffier and Lischer, 2010). These distances were employed in the software HapStar 0.5 (Teacher and Griffiths, 2011) with the “Minimum Spanning Network” option for the graphic representation of the haplotype network. 3. Results A total of 73 DNA sequences were used to produce a COI alignment 656 bp in length, including 32 polymorphic sites from which eight were parsimony informative. A total of 29 different haplotypes were found and 23 of them were detected only once. Three of the haplotypes were observed in 10 or more individuals and comprised 58% of all the sequences present in the dataset. A COI haplotype network (Fig. 1) showed a star-like pattern with a central haplotype representing 14% of the sampled individuals from the three areas (Mediterranean Sea, North East Atlantic and North West Atlantic). This haplotype was connected with the other two most common, only one mutation away. All other haplotypes were separated from these main three, most of them only one or two mutations apart. There was no clear geographic structure in the network because these common COI haplotypes were shared among all the represented areas. Furthermore, four of the six haplotypes represented by more than one individual were shared between at least two different areas. 4. Discussion The North Atlantic codling Lepidion eques is considered a valid species with a North Atlantic distribution in two of the most consulted fish faunas (Cohen, 1986; Cohen et al., 1990). The same result is obtained when the main two electronic repositories, The Catalog of Fishes (Eschmeyer et al., 2016) and FishBase (Froese and Pauly, 2016), are searched for using this species name as query. The latter considers the Mediterranean codling Lepidion lepidion a separated species with a Mediterranean distribution while the former also gives it an eastern Atlantic distribution.
8 D. Barros-García et al. / Biochemical Systematics and Ecology 68 (2016) 6e10 Fig. 1. COI haplotype network of Lepidion lepidion and L. eques populations. Each circle represents a unique haplotype, with the area being proportional to its frequency in three regions: red (Mediterranean Sea), green (North East Atlantic) and blue (North West Atlantic). The size of the circles is proportional to the number of individuals sampled. Capture locations of individuals are shown with triangles. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
D. Barros-García et al. / Biochemical Systematics and Ecology 68 (2016) 6e10
9
A previous investigation comparing 26 specimens of Lepidion from Mediterranean and Atlantic Spanish waters showed no morphological or DNA barcoding differences between sampling sites, suggesting that all the specimens were conspecific ~o n et al., 2013). An increase in both the number of specimens and sampling sites in the North Atlantic is now offering (Ban similar molecular results, with a variety of COI haplotypes shared among the distribution areas (Mediterranean Sea, North East and North West Atlantic). This result rejects the possibility of a limited distribution of the taxa due to biogeographical barriers and, therefore, the presence of different species. Furthermore, the high frequency of common haplotypes between species and distribution areas makes a possible hybridization hypothesis unlikely, and supports a scenario where L. eques should be considered a junior synonym of L. lepidion. As a consequence of this erroneous classification, L. lepidion has been traditionally considered an endemism of the Mediterranean Sea. However, the Gibraltar sill is not an impenetrable barrier for such deep-water fishes and a certain number of so-called Mediterranean Sea endemic species have been later captured in the Atlantic Ocean, or made synonyms of Atlantic species, as occurs in this case. Eggs and early stages of morid fishes are pelagic (Cohen, 1986), and fry of L. eques has been captured in surface waters in several locations off Iceland and western Scotland, for example (reviewed in Templeman, 1970); more recently, a pelagic juvenile of Lepidion inosimae has been collected off northeastern Japan (Okamoto et al., 2009). The presence of these pelagic forms would allow the colonization of the Mediterranean Sea via the inflowing surface current across the Gibraltar sill. This passage, 12.9 km wide and with a depth of 286e900 m of depth, is the single connection between the Atlantic Ocean and the Mediterranean Sea. The reopening of the Strait of Gibraltar 5 million years ago, led to the restocking of the Mediterranean Sea with fauna and flora from the Atlantic Ocean (Coll et al., 2010) and, nowadays, it is estimated that almost 59% of the Mediterranean fishes have an Atlantic origin (Psomadakis et al., 2012). Regardless of their morphological differences, the Lepidion individuals collected from across the western Mediterranean and northern Atlantic appear to belong to a single demographic unit. The facts mentioned above would support that Lepidion lepidion is not endemic in the Mediterranean, showing instead an Atlantic-Mediterranean distribution, as is also observed in ~o n et al., 2010). its congener Lepidion guentheri (Ban Acknowledgements ~ ol de OceanThe authors would like to thank the staff involved in the following research surveys of the “Instituto Espan ografía”: INDEMARES-BANGAL 0811, INDEMARES-AVILES 0412, FLEMISH CAP 2015, PLATUXA 2015, 3FNL 2015 and PORCUPINE 2015. We are deeply grateful to J. Cartes (ICM-CSIC, Barcelona, Spain), for providing specimens of L. lepidion from the Mediterranean Sea, in the framework of the project ANTROMARE (CTM2009-12214-C02-01-MAR), granted by the Spanish Ministry of Science and Technology. We also extend our thanks to the crews of the Research Vessels “Miguel Oliver” and “Vizconde de Eza” (Ministry of Agriculture, Food and Environment, Spain). This investigation was partially funded through the research project ECOMARG 3 (Ministry of Agriculture, Food and Environment, Spain) and the EC LIFEþ “Nature Biodiversity” INDEMARES project (07/NAT/E/000732). This results partially fulfil the PhD degree requirements of DBG at the University of Vigo. References ~o n, R., Arronte, J.C., Heredia, J., Pis-Milla n, J.A., 2010. First record of a specimen of Lepidion guentheri (Giglioli, 1880) (Gadiformes: Moridae) with Ban melanistic coloration. J. Appl. Ichth 26, 602e605. http://dx.doi.org/10.1111/j.1439-0426.2010.01472.x. ~o n, R., Arronte, J.C., V Ban azquez-Dorado, S., del Río, J.L., de Carlos, A., 2013. DNA barcoding of the genus Lepidion (Gadiformes: Moridae) with recognition of Lepidion eques as a junior synonym of Lepidion lepidion. Mol. Ecol. Resour. 13, 189e199. http://dx.doi.org/10.1111/1755-0998.12045. Cohen, D.M., 1986. Moridae. In: Whitehead, P.J.P., Bauchot, M.-L., Hureau, J.-C., Nielsen, J., Tortonese, E. (Eds.), Fishes of the Northeastern Atlantic and the Mediterranean, vol. 2. UNESCO, Paris, pp. 713e723. Cohen, D.M., Inada, T., Iwamoto, T., Scialabba, N., 1990. FAO Species Catalogue. In: Gadiform Fishes of the World (Order Gadiformes). An Annotated and Illustrated Catalogue of Cods, Hakes, Grenadiers and Other Gadiform Fishes Known to Date, vol. 10. FAO, Rome. Coll, M., Piroddi, C., Steenbeek, J., Kaschner, K., Ben Rais Lasram, F., Aguzzi, J., et al., 2010. The biodiversity of the Mediterranean sea: estimates, patterns, and threats. PLoS ONE 5, e11842. http://dx.doi.org/10.1371/journal.pone.0011842. Collett, R., 1905. Fiske indsamlede under “Michael Sars”s togter i Nordhavet 1900-1902. Rep. Norw. Fish. Mar. Invest. 2, 1e151. Eschmeyer, W.N., Fricke, R., van der Laan, R. (eds). Catalog of Fishes: Genera, Species, References. (http://researcharchive.calacademy.org/research/ ichthyology/catalog/fishcatmain.asp (accessed 01.06.2016). Excoffier, L., Lischer, H.E., 2010. 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