Journal of Shellfish Research, Vol. 34, No. 3, 1–12, 2015. PHYLOGENETIC APPROACHES TO DELIMIT GENETIC LINEAGES OF THE MYTILUS COMPLEX OF SOUTH AMERICA: HOW MANY SPECIES ARE THERE? MARCELA P. ASTORGA,1* LEYLA CARDENAS2 AND JAIME VARGAS1 1Instituto de Acuicultura, Universidad Austral de Chile, P.O. 1327, Puerto Mont, Chile; 2Instituto de Ciencias Ambientales y Evolutivas, Universidad Austral de Chile, Valdivia, Chile ABSTRACT The aim of the present work is to increase the general knowledge about an economically important mussel species in Chile. Species of Mytilus are present in the southern cone of South America; however, there is still some controversy about species identification of samples from this area. The study herein presented attempts to: (1) corroborate the phylogenetic hypothesis defined for the Mytilus edulis species complex including taxa from a worldwide distribution; (2) evaluate the possible presence of the species Mytilus trossulus along the Chilean coast and determine if M. trossulus hybridizes with the local species; and (3) provide detailed data collected along the Chilean coast to help define the taxonomic status of Mytilus in South America. To this end, exhaustive sampling was conducted; Mytilus was collected from the Chilean coast and from the coasts of Argentina and Uruguay. Phylogenetic analysis and genetic divergence estimators were used to compare 426 Cytochrome oxidase I mitochondrial gene sequences and 190 16S RNA sequences of Mytilus species sampled from around the world. Following this, the time of divergence between northern hemisphere and southern hemisphere clades of Mytilus species was estimated. In addition, neither M. trossulus nor any associated hybrids were found along the Chilean coast. Finally, the identification of samples from the southern cone of South America is discussed including whether the samples should be identified as Mytilus planulatus or Mytilus platensis. In accordance with the taxonomic priority rules and the results presented here, the species identification frequently used in the literature for samples from the Chilean coast (Mytilus chilensis) may cease to be used. KEY WORDS: mussel, Mytilus, Mytilus edulis complex, South America, COI, 16S INTRODUCTION ancestor, this complex could still be undergoing speciation that would make it very difficult to delimit species (Gosling 1992). The genus Mytilus, as defined by Linnaeus (1758), consists of The delimitation of species becomes even more complex two types of species: Those that are hard-shelled for which two when analyzed using the existing information for the groups species, Mytilus californianus and Mytilus coruscus, have been present in the southern hemisphere because different genetics defined; and those that are smooth-shelled for which three groups has been found. Taxonomic identification is even more species, Mytilus edulis, Mytilus galloprovincialis, and Mytilus difficult when trying to identify Mytilus species present along trossulus, have been recognized. Despite this, over the past the coast of Chile. This is the case because sampling along the decade or so, there has been an extensive debate about the coast of Chile has been sparse. species definitions of the latter group of Mytilus species. This The Mytilus mussel in Chile is of high economic importance debate has arisen mainly due to three factors: (1) the great because it is produced in large volumes in aquaculture. Chile is morphological and morphometric similarity of the valves now one of the largest mussel producers in the world (FAO (McDonald et al. 1991) of smooth-shelled species; (2) the ability 2014), and Chilean Mytilus is one of the top five mollusc species of these species to interbreed and produce viable hybrid off- produced in world aquaculture with production of 288,000 tons spring, and (3) the great genetic similarity or unclear genetic in 2011 (FAO 2013). The aquaculture production of this patterns seen among the smooth-shelled species. resource is sustained entirely from natural populations because Furthermore, cross breeding experiments have been con- the seeds for aquaculture are obtained from the natural ducted and viable hybrids were successfully produced both in environment (Uriarte 2008). For the sustainable aquaculture controlled (Toro et al. 2012) and natural environments of this species, it is important to know about the species (McDonald & Koehn 1988, Inoue et al. 1995, Wilhelm & Hilbish distinctions and population dynamics of this resource. 1998, Rawson et al. 1999, Brannock et al. 2009, Westfall & Along the Chilean coast, natural beds of Mytilus are found Gardner 2010, Larraın et al. 2012, Toro et al. 2012, Lourenco from 38° S to the southern tip of the country at 54° S (Toro et al. et al. 2015). In addition to this, phenotypic studies have shown 2006). This species was initially described as Mytilus chilensis that smooth-shelled Mytilus are highly plastic such that the valve (Hupe, 1854), and the name M. chilensis has been used re- morphology is largely determined by the organismÕs local currently in the literature in various areas of research. In fact, environment (McDonald et al. 1991). more than 150 citations can be found in the ISI web of science Because of this lack of clarity in morphological identifica- using this name for the Chilean mussel species (e.g., Enriquez tion, smooth-shelled Mytilus have been grouped into a species et al. 1992, Jaramillo et al. 1992, Simpfendorfer€ et al. 1995, Toro complex, or the Mytilus edulis complex. Within the definition of 1998, Labarta et al. 2002, Rego et al. 2002, Velasco & Navarro this complex, the species should have a common evolutionary 2002, Velasco & Navarro 2003, Toro et al. 2004, Krapivka et al. ancestor or an ‘‘edulis type.’’ As it is not known when the three 2007, Ouagajjou et al. 2011, Ibarrola et al. 2012, Larraın identified species of this complex diverged from the common et al. 2014, Nunez-Acu~ na~ & Gallardo-Escarate 2014, Oyarzun et al. 2014, Vallejos et al. 2014). The Chilean mussel can also be *Corresponding author. E-mail: [email protected] found under the name Mytilus edulis chilensis (Gray et al. 1997, DOI: 10.2983/035.034.0300 Gray et al. 1999, Mercado et al. 2005, Arenas et al. 2006, Duarte 1 2 ASTORGA ET AL. et al. 2011), and recently as Mytilus edulis platensis (Borsa et al. MATERIALS AND METHODS 2012, Dıaz & Campos 2014). The name M. edulis platensis was Samples of Mytilus were collected from eight locations along derived for the Mytilus species that inhabits the coast of Argentina. the coast of Chile (see Table 1 for details). Of these, samples T1 Many genetic studies of smooth-shelled Mytilus have been from Tumbes (TUM) were determined to be Mytilus gallopro- conducted, and some of these have used allozymes (McDonald ~ et al. 1991, Carcamo et al. 2005, Toro et al. 2006), nuclear vincialis (Toro et al. 2006, Astorga 2012, Tarifeno et al. 2012) markers (Daguin & Borsa 2000, Wood et al. 2003, Westfall & and samples from the other seven locations were determined to Gardner 2010), restriction fragment length polymorphism be Mytilus edulis platensis (Table 1). In addition, samples from (RFLP) (Westfall et al. 2010), mitochondrial DNA (Hilbish two other locations in the southern cone of South America were et al. 2000, Gerard et al. 2008), or microsatellites (Ouagajjou analyzed. These locations included Montevideo, Uruguay and et al. 2011). Specifically, the evolutionary and phylogenetic Puerto Deseado, Argentina. All of the samples from the relationships within the Mytilus group have been analyzed southern cone of Southern America–Chile, Argentina, and using high-resolution markers and DNA sequencing. This Uruguay—excluding TUM, will be called Mytilus from South work has mainly been done by Hilbish et al. (2000) and Gerard America. et al. (2008). One study by Hilbish et al. (2000) aimed to Deoxyribose nucleic acid was only extracted from the determine that Mytilus origin location or migration route mantle of female mussels using a mollusc extraction kit (E.Z. could have led to the antitropical distribution exhibited by the N.A.) following the manufacturer’s instructions. The COI genus. In this study, 47 Mytilus samples from both hemi- mitochondrial gene was amplified using the HCO and LCO spheres were analyzed using RFLP analysis. The results of the universal primers (Folmer et al. 1994). The 16S RNA ribo- study indicate the existence of two migration events from the somal subunit (16S) was amplified using the 16SAR and northern to the southern hemisphere, both via an Atlantic 16SBR primers (Palumbi et al. 1991). The sequences were route. Occurring in the Pleistocene, one of these events is edited and aligned utilizing the BIOEDIT 5.0.9 program (Hall considered to be ancient whereas the other migration event is 1999). thought to have occurred more recently. Following this, In total, 426 COI sequences and 190 16S sequences of another study, employing a greater sampling effort and using different Mytilus species were compared; the sequences came Cytochrome oxidase I (COI) gene sequences, reassessed the from samples that encompass a worldwide distribution. Of the time of divergence of Mytilus species (Gerard et al. 2008). This 426 COI sequences analyzed, 127 were newly generated for this study was conducted to define the times at which the Mytilus study and 299 were obtained from the National Center for genus split and colonized the southern hemisphere. By ana- Biotechnology Information database (www.ncbi.nlm.nih.gov). lyzing 171 COI sequences COI and 224 16S RNA ribosomal In addition, of the 190 16S sequences analyzed in this study, 93 subunit (16S) sequences, this study detected genetic popula- were originally sequenced, and 97 were mined from National tion differentiation between the hemispheres. Specifically, Center for Biotechnology Information (details in Table 1 and populations from southern Chile, Kerguelen, Tasmania, and Fig. 1). F1 New Zealand were found to be different and were found to Standard genetic diversity indices including the number of have diverged from the northern clade between 0.5 and 1.3 segregating sites (S), the numberofhaplotypes(h),haplotype million years ago.