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Phylogenetic approaches to delimit genetic lineages of the Mytilus complex of South America: how many species are there?

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

The genus Mytilus, as defined by Linnaeus (1758), consists of two types of species: Those that are hard-shelled for which two species, Mytilus californianus and Mytilus coruscus, have been defined; and those that are smooth-shelled for which three species, Mytilus edulis, Mytilus galloprovincialis, and Mytilus trossulus, have been recognized. Despite this, over the past decade or so, there has been an extensive debate about the species definitions of the latter group of Mytilus species. This debate has arisen mainly due to three factors: (1) the great morphological and morphometric similarity of the valves (McDonald et al. 1991) of smooth-shelled species; (2) the ability of these species to interbreed and produce viable hybrid offspring, and (3) the great genetic similarity or unclear genetic patterns seen among the smooth-shelled species.

Furthermore, cross breeding experiments have been conducted and viable hybrids were successfully produced both in controlled (Toro et al. 2012) and natural environments (McDonald & Koehn 1988, Inoue et al. 1995, Wilhelm & Hilbish 1998, Rawson et al. 1999, Brannock et al. 2009, Westfall & Gardner 2010, Larrain et al. 2012, Toro et al. 2012, Lourenco et al. 2015). In addition to this, phenotypic studies have shown that smooth-shelled Mytilus are highly plastic such that the valve morphology is largely determined by the organism's local environment (McDonald et al. 1991).

Because of this lack of clarity in morphological identification, smooth-shelled Mytilus have been grouped into a species complex, or the Mytilus edulis complex. Within the definition of this complex, the species should have a common evolutionary ancestor or an "edulis type." As it is not known when the three identified species of this complex diverged from the common ancestor, this complex could still be undergoing speciation that would make it very difficult to delimit species (Gosling 1992).

The delimitation of species becomes even more complex when analyzed using the existing information for the groups present in the southern hemisphere because different genetics groups has been found. Taxonomic identification is even more difficult when trying to identify Mytilus species present along the coast of Chile. This is the case because sampling along the coast of Chile has been sparse.

The Mytilus mussel in Chile is of high economic importance because it is produced in large volumes in aquaculture. Chile is now one of the largest mussel producers in the world (FAO 2014), and Chilean Mytilus is one of the top five mollusc species produced in world aquaculture with production of 288,000 tons in 2011 (FAO 2013). The aquaculture production of this resource is sustained entirely from natural populations because the seeds for aquaculture are obtained from the natural environment (Uriarte 2008). For the sustainable aquaculture of this species, it is important to know about the species distinctions and population dynamics of this resource.

Along the Chilean coast, natural beds of Mytilus are found from 38[degrees]S to the southern tip of the country at 54[degrees]S (Toro et al. 2006). This species was initially described as Mytilus chilensis (Hupe, 1854), and the name M. chilensis has been used recurrently in the literature in various areas of research. In fact, more than 150 citations can be found in the ISI web of science using this name for the Chilean mussel species (e.g., Enriquez et al. 1992, Jaramillo et al. 1992, Simpfendorfer et al. 1995, Toro 1998, Labarta et al. 2002, Rego et al. 2002, Velasco & Navarro 2002, Velasco & Navarro 2003, Toro et al. 2004, Krapivka et al. 2007, Ouagajjou et al. 2011, Ibarrola et al. 2012, Larrain et al. 2014, Nunez-Acuiia & Gallardo-Escarate 2014, Oyarzun et al. 2014, Vallejos et al. 2014). The Chilean mussel can also be found under the name Mytilus edulis chilensis (Gray et al. 1997, Gray et al. 1999, Mercado et al. 2005, Arenas et al. 2006, Duarte et al. 2011), and recently as Mytilus edulis platensis (Borsa et al. 2012, Diaz & Campos 2014). The name M. edulis platensis was derived for the Mytilus species that inhabits the coast of Argentina.

Many genetic studies of smooth-shelled Mytilus have been conducted, and some of these have used allozymes (McDonald et al. 1991, Carcamo et al. 2005, Toro et al. 2006), nuclear markers (Daguin & Borsa 2000, Wood et al. 2003, Westfall & Gardner 2010), restriction fragment length polymorphism (RFLP) (Westfall et al. 2010), mitochondrial DNA (Hilbish et al. 2000, Gerard et al. 2008), or microsatellites (Ouagajjou et al. 2011). Specifically, the evolutionary and phylogenetic relationships within the Mytilus group have been analyzed using high-resolution markers and DNA sequencing. This work has mainly been done by Hilbish et al. (2000) and Gerard et al. (2008). One study by Hilbish et al. (2000) aimed to determine that Mytilus origin location or migration route could have led to the antitropical distribution exhibited by the genus. In this study, 47 Mytilus samples from both hemispheres were analyzed using RFLP analysis. The results of the study indicate the existence of two migration events from the northern to the southern hemisphere, both via an Atlantic route. Occurring in the Pleistocene, one of these events is considered to be ancient whereas the other migration event is thought to have occurred more recently. Following this, another study, employing a greater sampling effort and using Cytochrome oxidase I (COI) gene sequences, reassessed the time of divergence of Mytilus species (Gerard et al. 2008). This study was conducted to define the times at which the Mytilus genus split and colonized the southern hemisphere. By analyzing 171 COI sequences COI and 224 16S RNA ribosomal subunit (16S) sequences, this study detected genetic population differentiation between the hemispheres. Specifically, populations from southern Chile, Kerguelen, Tasmania, and New Zealand were found to be different and were found to have diverged from the northern clade between 0.5 and 1.3 million years ago. Following these results, the northern clade of Mytilus consists of populations found in the northern hemisphere, Western Australia, South Africa, and northern Chile (Dichato).

More recently, results gathered from RFLP and nuclear gene polymorphism data have indicated the presence of Mytilus trossulus, Mytilus galloprovincialis, and possible hybrids in Chile. Both M. trossulus and M. galloprovincialis were found off the coast of the Bio-Bio Region of Chile, however the Mytilus hybrids were found in regions where Chilean mussels are farmed (Larrain et al. 2012). Further to this, results of a detailed review also indicate that Mytilus is genetically distinct in the two hemispheres (Borsa et al. 2012). Also, this review shows that Chilean Mytilus are different from northern hemisphere Mytilus. Because of this, Chilean Mytilus should be identified as Mytilus edulis platensis following taxonomic priority rules.

Based on the results of previous work, the study here in presented attempts to determine if the phylogenetic patterns found by Hilbish et al. (2000) and Gerard et al. (2008) are present in this system (South America). The study emphasizes the Chilean coast, adding information from the southern cone of South America. Furthermore, here the possible presence of Mytilus trossulus and its hybrids in Chile is assessed. Finally, a taxonomically suitable identification of the Mytilus species present in Chile is proposed.

MATERIALS AND METHODS

Samples of Mytilus were collected from eight locations along the coast of Chile (see Table 1 for details). Of these, samples from Tumbes (TUM) were determined to be Mytilus galloprovincialis (Toro et al. 2006, Astorga 2012, Tarifeno et al. 2012) and samples from the other seven locations were determined to be Mytilus edulis platensis (Table 1). In addition, samples from two other locations in the southern cone of South America were analyzed. These locations included Montevideo, Uruguay and Puerto Deseado, Argentina. All of the samples from the southern cone of Southern America-Chile. Argentina, and Uruguay--excluding TUM, will be called Mytilus from South America.

Deoxyribose nucleic acid was only extracted from the mantle of female mussels using a mollusc extraction kit (E.Z. N.A.) following the manufacturer's instructions. The COI mitochondrial gene was amplified using the HCO and LCO universal primers (Folmer et al. 1994). The 16S RNA ribosomal subunit (16S) was amplified using the 16SAR and 16SBR primers (Palumbi et al. 1991). The sequences were edited and aligned utilizing the BIOEDIT 5.0.9 program (Hall 1999).

In total, 426 COI sequences and 190 16S sequences of different Mytilus species were compared; the sequences came from samples that encompass a worldwide distribution. Of the 426 COI sequences analyzed, 127 were newly generated for this study and 299 were obtained from the National Center for Biotechnology Information database (www.ncbi.nlm.nih.gov). In addition, of the 190 16S sequences analyzed in this study, 93 were originally sequenced, and 97 were mined from National Center for Biotechnology Information (details in Table 1 and Fig. 1).

Standard genetic diversity indices including the number of segregating sites (S), the number of haplotypes (h), haplotype diversity, nucleotide diversity ([pi]), and the mean number of pairwise differences ([pi]) were estimated for each group and gene, using DnaSP v5.00.04 (Librado & Rozas 2009). The software MEGA 6.0 (Tamura et al. 2013) was used to estimate genetic divergence between samples using the number of base substitutions per site and by averaging over all of the sequence pairs between groups. The significance and SE of the values were evaluated by 1000 bootstrap replicates. Moreover, the percentage of different nucleotides was measured using DnaSP v5.00.04 (Librado & Rozas 2009). To perform this analysis, the groups were separated following the criteria of Gerard et al. (2008) where haplotypes in the northern hemisphere and southern hemisphere are defined for galloprovincialis.

Because the 16S gene is commonly used to build marine invertebrate phylogenies and to identify operational taxonomic units and species (for example de Luna Sales et al. 2013, Pulliandre et al. 2014), this gene was used for the phylogenetic analyses. Maximum Likelihood reconstructions were performed using the best substitution model, as is implemented in the software MEGA 6.0 (Tamura et al. 2013). Bayesian inference method (BI) was performed using the software Mr. Bayes (Huelsenbeck & Ronquist 2001). The evolutionary model was chosen according to the BIC Model Selection as implemented in Modeltest 3.7 (Posada & Crandall 1998). Posterior probabilities were estimated over 10,000,000 generations via one run of four simultaneous Markov Chain Monte Carlo chains with every 1,000th tree saved. The first 10% burn-in was discarded following suggestions by Felsenstein (1985). In the phylogenies, 16S sequences of Mytilus californianus and Mytilus coruscus were chosen as out-groups (Gerard et al. 2008).

The COI gene is a commonly used marker for phylogeo-graphic studies. This molecular marker can be used to determine the number of species and the relationships among haplotypes (for example Cardenas et al. 2009, Haye et al. 2014). The relationships among the observed haplotypes in this study were assessed by constructing median joining networks (Bandelt et al. 1999) using the software Network v4.613 (www.fluxus-engineering. com). To determine connections in the network, a star contraction procedure was applied before network calculation was performed (Forster et al. 1996). After calculating the network, maximum parsimony analysis was used to filter out uninformative branches (Polzin & Daneshmand 2003). Two different analyses were performed: first, the number of shared haplotypes and the distribution of haplotypes among species were visualized by building a network with the entire dataset including all of the analyzed species. Here a total of 426 COI sequences each having at least 399 bp were used to build the network. The second analysis was performed exclusively with samples from South America. The aim of this analysis was to identify the spatial pattern of the haplotype distribution. For this second analysis, a database composed of sequences of 127 individuals was used and each sequence used was 667 bp in length.

RESULTS

The genetic diversity indices calculated for the smooth-shelled species of Mytilus showed the same uniform diversity distribution regardless of the gene analyzed (Table 2 for COI and Table 3 for 16S). Between pairs of groups (species), there was low genetic divergence, again regardless of the gene tested. Despite this, slight differentiation was observed between northern hemisphere Mytilus galloprovincialis [with northen haplotype (NH)] and Mytilus edulis using the COI gene. It should be noted that differentiation was observed between the two groups of M. galloprovincialis [NH with southern haplotype (SH)]; and within the same hemisphere the groups were more similar to each than to those from the other hemisphere (M. edulis with M. galloprovincialis from the northern hemisphere, and Mytilus from South America with M. galloprovincialis from southern hemisphere) (Table 4 for COI gene). The genetic divergence based on the 16S data showed a similar pattern to that observed for the COI gene (Table 5 for 16S). In both datasets, the smallest divergence observed was between the samples of Mytilus from South America and the M. galloprovincialis samples that have the SH. The level of genetic differentiation between Mytilus from South America and the M. galloprovincialis-SH was 1.91% based on the COI gene and 0.27% based on the 16S gene. The divergence between Mytilus from South America and the M. galloprovincialis-NH was 1.73% for the COI gene and 1.57% for the 16S gene. For the comparison between Mytilus from South America and M. edulis, the value was 1.86% based on the COI gene and 1.13% based on 16S. The percent divergence between M. galloprovincialis-SH and M. edulis is 2.20% for the COI gene and 2.45% for 16S. Between M. galloprovincialis-NH and M. edulis, 1.05% and 2.67% divergence was found for COI and 16S, respectively. Finally, the percentage of different nucleotides between M. galloprovincialis-SH and -NH was 2.40% based on the COI gene and 2.53% based on 16S.

Phylogenetic reconstruction with the 16S gene showed a similar topology independent of whether the maximum likelihood (ML) or BI analysis method was used (Fig. 2). In this reconstruction, three well-supported clades were detected. The first clade was composed of samples of Mytilus edulis, Mytilus galloprovincialis-NH, one sample of M. galloprovincialis-SH, and samples from TUM (Chile) in South America. The second clade is composed of samples of M. edulis, M. galloprovincialis-NH, and Mytilus trossulus with only one South America sample coming from Uruguay. The third clade is composed of samples of M. galloprovinciales-NH and -SH, M. edulis, and samples from South America collected in TUM (Chile). The sequences of other samples from South America and samples of M. galloprovincialis-SH clustered into a large polytomy indicating considerable uncertainty in the relationship among these taxa.

The network analysis using the whole dataset, i.e., including samples from Mytilus edulis, Mytilus trossulus, Mytilus galloprovincialis-NH, M. galloprovincialis-SH and Mytilus from South America, is shown in Figure 3. The network indicates the existence of two haplotypes of high frequency separated by six mutational steps. One of these haplotypes was observed in a total of 114 individuals including samples from M. edulis, M. galloprovincialis-NH, and in several South America and M. galloprovincialis-SH samples. Several other lower frequency haplotypes are connected to the one large haplotype previously described. These haplotypes are separated by a distance of one mutational step and are mainly from the M. galloprovincialis-NH and M. edulis samples. Other high frequency haplotypes were found in 69 samples. These were mainly found in samples from South America, but there were also some detected in the M. galloprovincialis-SH and -NH samples. In addition, the M. trossulus haplotypes were independent from the main network; these haplotypes were separated from the main network by 34 mutational steps.

When samples of Mytilus from South America were analyzed (Fig. 4), one central high frequency haplotype was observed in a total of 28 individuals from Puerto Saavedra. Queule, Chaihuin, Yaldad, Puerto Balmaceda; Punta Arenas, and Puerto Deseado. In addition, several other haplotypes were detected; these included individuals from several sites with no apparent spatial segregation of the genetic diversity. This suggests that there is no spatial structure to Mytilus populations in South America. However, exceptions to this include three haplotypes from ARG that are nine mutational steps away from the rest of the network.

DISCUSSION

This study supports the hypothesis proposed by Hilbish et al. (2000) and Gerard et al. (2008) as two main groups of Mytilus galloprovincialis were found. In addition, a clear separation of Mytilus trossulus and other Mytilus species was found. Further to this, there is no evidence of the presence of this species or hybrids in Chile. Finally, there is evidence that South America samples are taxonomically independent. This also suggests that there has been a recent introduction of M. galloprovincialis-NH in TUM and ARG.

The results herein presented confirm the low phylogenetic divergence between groups within the Mytilus edulis complex, in comparison, with the other Mytilus species. The low genetic diversity within the M. edulis complex is to be expected, as it is known that these species interbreed (Beaumont et al. 2004, Beaumont et al. 2008, Gosling et al. 2008, Klibansky & McCartney 2014, Lourenco et al. 2015) and have few distinct morphological characteristics (Daguin & Borsa 1999, Oyarzun et al. 2014). Despite this, when the genetic and phylogenetic divergences of this species complex are analyzed in detail, differentiation can be found. Phylogenetic analysis of the COI gene sequence data shows a separation of the northern hemisphere samples from the southern hemisphere samples. This generates a clade of northern haplotypes plus some groups present in the southern hemisphere. This clade includes samples from South Africa, Western Australia, and TUM on the coast of Chile. It is possible that this may have resulted from a more recent introduction (Branch & Stefanni 2004, Braby & Somero 2005, Robinson et al. 2007, Lockwood & Somero 2011). The presence of Mytilus galloprovincialis in TUM has been reported to be a recent introduction of galloprovincialis (Branch & Stefanni 2004, Castilla & Neill 2009). Therefore, this recent introduction may also have resulted from the second trans-equatorial migration from the northern hemisphere as proposed by Hilbish et al. (2000).

This separation of haplotypes into clusters associated with the hemispheres has already been detected by Gerard et al. (2008). This study demonstrates that there are great differences between southern hemisphere Mytilus samples. This is confirmed in the present work by the high genetic variability seen in the galloprovincialis-SH samples. The greatest number of different nucleotides was found within this group (Table 2).

The results herein presented indicate that there is little genetic differentiation between species of the Mytilus edulis complex. In general, a greater similarity was observed between M. edulis and Mytilus galloprovincialis from the northern hemisphere than between Mytilus from South America and edulis.

Using both markers, phylogenetic analyses of the South American samples show a separation of the data into a single clade with low genetic differentiation within this clade. These samples also form a group with the Kerguelen samples, as was also detected by Gerard et al. (2008). It is possible that South American Mytilus are more genetically distinct from the Mytilus edulis group (1.5%) and from the northern hemisphere galloprovincialis (1.7%) than they are from the southern hemisphere galloprovincialis (1.0%). The divergence between Mytilus of the northern and southern hemispheres has been estimated as 1.4% (Hilbish et al. 2000). Interestingly, this is the same as that observed in this study if the average of the divergence rate of galloprovincialis from the southern hemisphere and that of Mytilus from South America (1.4%) is considered; The other study measuring divergence rates also considered these together (Hilbish et al. 2000). It is possible that due to Atlantic transequatorial colonization, these new southern hemisphere groups have undergone local differentiation. This differentiation would have created at least two groups including one in South America and another in the southwest Pacific encompassing New Zealand, Tasmania, and eastern Australia. This group in the south hemisphere may in turn be structured, as shown by Gerard et al. (2008, 2015). Finally, as a result of more recent migration (Hilbish et al. 2000), there is likely a group consisting of northern haplotypes. This would include the groups of South Africa and Western Australia. The divergence results would suggest an absence of gene flow between the hemispheres and potential differentiation through local adaptation. This is in contrast with the situation between Mytilus galloprovincialis and M. edulis observed in the northern hemisphere where introgression and some degree of gene flow have been reported (Quesada et al. 1998. Bierne et al. 2002). Based on the analysis of the sperm ultrastructure of the South American samples (i.e. Mytilus chilensis) and M. galloprovincialis samples from the Chilean coast, differences have been found which corroborate the current differentiation between these groups. From this, it is thought that sperm ultrastructures could be used as characteristics for taxonomic identification (Oyarzun et al. 2014). This being said, spermatozoa differentiation has not prevented reciprocal reproduction between samples of South America and M. galloprovincialis (Toro et al. 2012). These results indicate that there is greater differentiation of Mytilus between the hemispheres than between the classic species. Furthermore, Mytilus from South America are consistently different from edulis and galloprovincialis.

Thus, to return to the three initial questions, the following conclusions are made. First, the hypotheses of Hilbish et al. (2000) and Gerard et al. (2008) could be corroborated with exhaustive sampling of the coast of the southern cone of South America. Second, no trossulus haplotypes were detected among the South American samples. Because the level of divergence within this group has been shown to be very high, one would have expected to detect this species rapidly. Therefore, the presence of trossulus and its hybrids along the Chilean coast is discounted in contrast to that reported by Larrain et al. (2012). Finally, the taxonomic status of the group present along the coast of South America cannot be easily defined.

Based on the results presented, Mytilus from South America are genetically distinct from edulis. It is possible then that South American Mytilus should not be considered as a subspecies of Mytilus edulis as it has recently been called (M. edulis platensis). Instead, this group should be treated as a differentiated cluster based on its high divergence from the edulis group. If however, the proposal of the edulis complex is put forth, all the species identified to date would be subspecies of this group including M. edulis edulis; M. edulis galloprovincialis, M. edulis planulatus and M. edulis platensis. This is not feasible. Considering the great divergence between the two haplotypes of galloprovincialis, which is greater than their divergence from edulis, a different identification scheme is proposed. The following is suggested: M. edulis should be the name for species from the northern hemisphere; Mytilus galloprovincialis should be the name used for species from the northern hemisphere and for the exceptions present in the southern hemisphere. In the southern hemisphere, Mytilus planulatus should be the named used for specimens from South America, Kerguelen, and the southwest Pacific (eastern Australia, Tasmania and New Zealand); or perhaps two differentiated groups should exist for the southern hemisphere, namely planulatus and plalensis. Borsa et al. (2012) proposed this later identification schemed based on the analysis of the literature existing at the date of their work. Following the rules of taxonomic priority, the name frequently used in the literature for samples from the Chilean coast (Mytilus chilensis) would cease to be used.

ACKNOWLEDGMENTS

We thank Project Fondecyt 1130716, to the Millennium Nucleus Center for the Study of Multiple drivers on Marine Socio-Ecological Systems (MUSELS) by MINECON Project NC120086. We also thank Dr. Jorge Toro, who collected some of the samples for this study.

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DOI: 10.2983/035.034.0322

MARCELA P. ASTORGA, (1) * LEYLA CARDENAS (2) AND JAIME VARGAS (1)

(1) Instituto de Acuicultura, Universidad Austral de Chile, P.O. 1327, Puerto Mont, Chile; (2) Instituto de Ciencias Ambientales y Evolutivas, Universidad Austral de Chile, Valdivia, Chile

* Corresponding author. E-mail: marcelaastorga@uach.cl

TABLE 1.
Samples of Mytilus species from around the world
were used in this work.

Samples from South America

Locality            Abbreviation                Lat/Long

Tumbes                  TUM        36[degrees] 36' S/73[degrees] 04' O
Puerto Saavedra         PSA        38[degrees] 47' S/73[degrees] 23' O
Queule                  QUE        39[degrees] 23' S/73[degrees] 13' O
Chaihuin                CHA        39[degrees] 56' S/73[degrees] 35' O
Huinay                  HUI        42[degrees] 22' S/72[degrees] 24' O
Yaldad                  YAL        43[degrees] 06' S/73[degrees] 42' O
Puerto Balmaceda        PRM        43[degrees] 45' S/72[degrees] 58' O
Punta Arenas            PTA        53[degrees] 25' S/69[degrees] 23' O
Puerto Deseado          ARG        47[degrees] 45' S/65[degrees] 53' O
Montevideo              URU        34[degrees] 50' S/56[degrees] 10' O
Total

Samples from South America

Locality            N 16S   N COI         Species

Tumbes               10      12     M. galloprovincialis
Puerto Saavedra      12       9     Mytilus
Queule               15      12     Mvtilus
Chaihuin              9      10     Mytilus
Huinay                8      13     Mvtilus
Yaldad               11      16     Mytilus
Puerto Balmaceda      8      11     Mvtilus
Punta Arenas         12      29     Mvtilus
Puerto Deseado        0       7     Mvtilus
Montevideo            7       8     Mvtilus
Total                92      127

Samples from South America

Locality                         GenBank 16 /COI

Tumbes              KP052886-905 / KP052906-927
Puerto Saavedra     KM452802-06; KR153801-07 / KR066669-77
Queule              KR 153811-20; KM452797-801 / KR066657-68
Chaihuin            KR 153794-97; KM452807-11 / KR066678-87
Huinay              KR153790-93;834;KM452814-16 / KR066688-700
Yaldad              KR3821-26; KM452817-21 / KR066701-16
Puerto Balmaceda    KR153198-800; KM452822-26 / KR066717-27
Punta Arenas        KP052859-70 / KR066728-56
Puerto Deseado      KR066757-63
Montevideo          KR 153827-33 / KR066764-71
Total

Samples from Other Coasts for COI

Locality, Country                 N          Species

Dichato, Chile                   6     Mytilus sp.
Patagonia, Chile                 1     Mytilus sp.
Maullin, Chile                   1     Mytilus sp.
Samos, Grecia                    7     M. galloprovincialis
Morgat, Francia                  1     M. galloprovincialis
Grecia                           1     M. galloprovincialis
Auckland, New Zealand            10    M. galloprovincialis
Lyttelton, New Zealand           8     M. galloprovincialis
Capetown, SouthAfrica            14    M. galloprovincialis
Sydney, Australia                15    M. galloprovincialis
Campbell, Canada                 1     M. galloprovincialis
Hobart, Australia                9     M. galloprovincialis
Chioggia, Italia                 1     M. galloprovincialis
Nedlands, Australia Oeste        1     M. galloprovincialis
Kerguelen, French                1     M. galloprovincialis
New Zealand                      3     M. galloprovincialis
Tasmania, Australia              2     M. galloprovincialis
Kerguelen, French                2     M. galloprovincialis
Ciudad del Cabo, South Africa    21    M. galloprovincialis
South Africa                     1     M. galloprovincialis
Atlantico Norte                  39    M. galloprovincialis
Kanagawa, Japan                  1     M. galloprovincialis
Roscoff, France                  1     M. edulis
Greenland                        3     M. edulis
Atlantico Norte                  3     M. edulis
Kieler Bucht. Germany            134   M. edulis
Mahone Bay, Canada               7     M. trossulus

Locality, Country                       GenBank          Haplotype

Dichato, Chile                   AM905174-79                 S
Patagonia, Chile                 AM905195                    S
Maullin, Chile                   AM905186                    5
Samos, Grecia                    AY 130054-60                N
Morgat, Francia                  AY497292                    N
Grecia                           DQ403169                    N
Auckland, New Zealand            DQ864378-87                 S
Lyttelton, New Zealand           DQ864426-33                 S
Capetown, SouthAfrica            DQ864397-410                N
Sydney, Australia                DQ86411-25                  S
Campbell, Canada                 DQ864416                    N
Hobart, Australia                DQ864388-96                 S
Chioggia, Italia                 AM905225                    N
Nedlands, Australia Oeste        AM905215                    N
Kerguelen, French                AM905211                    S
New Zealand                      AM905146; 49; 55            S
Tasmania, Australia              AM905161; 62; 65; 68        s
Kerguelen, French                AM905196-97                 N
Ciudad del Cabo, South Africa    DQ351477-97                 N
South Africa                     DQ917605                    N
Atlantico Norte                  AF241997-20031; 33-35       N
Kanagawa, Japan                  AB076943                    N
Roscoff, France                  AY130053
Greenland                        EU915572-4
Atlantico Norte                  AF241969-71
Kieler Bucht. Germany            JF825556-689
Mahone Bay, Canada               AY130061-67

Locality, Country                         Reference

Dichato, Chile                   Gerard et al. (2008)
Patagonia, Chile                 Gerard et al. (2008)
Maullin, Chile                   Gerard et al. (2008)
Samos, Grecia                    Riginos et al. (2004)
Morgat, Francia                  Cao et al. (2004)
Grecia                           Venetis et al. (2006)
Auckland, New Zealand            Unpublished
Lyttelton, New Zealand           Unpublished
Capetown, SouthAfrica            Unpublished
Sydney, Australia                Unpublished
Campbell, Canada                 Unpublished
Hobart, Australia                Unpublished
Chioggia, Italia                 Gerard et al. (2008)
Nedlands, Australia Oeste        Gerard et al. (2008)
Kerguelen, French                Gerard et al. (2008)
New Zealand                      Gerard et al. (2008)
Tasmania, Australia              Gerard et al. (2008)
Kerguelen, French                Gerard et al. (2008)
Ciudad del Cabo, South Africa    Zardi et al. (2007)
South Africa                     Wood et al. (2007)
Atlantico Norte                  Wares and Cumminghan (2001)
Kanagawa, Japan                  Matsumoto (2003)
Roscoff, France                  Riginos et al. (2004)
Greenland                        Riginos and Henzler (2008)
Atlantico Norte                  Wares and Cumminghan (2001)
Kieler Bucht. Germany            Steinert et al. (2012)
Mahone Bay, Canada               Riginos et al. (2004)

Samples from Other Coasts for 16S

Locality             N            Species               GenBank

Castro, Chile        1     Mytilus sp.             AF179447
Mediterranean Sea    1     M. galloprovincialis    DQ836018
South Hemisphere     2     M. galloprovincialis    GQ4553981; 88
San Diego, CA        1     M. galloprovincialis    MGU22885
Japan                15    M. galloprovincialis    KC835224-6; 8;
                                                     30-2; 42-6-51
New Zealand          4     M. galloprovincialis    AM904568-70; 72;
Tasmania             2     M. galloprovincialis    AM904579; 80;
Kerguelen            2     M. galloprovincialis    AM904589; 90;
Australia            3     M. galloprovincialis    AM904592-94
Australia            1     M. galloprovincialis    AF179448
Kerguelen            1     M. galloprovincialis    AF179449
New Zealand          2     M. galloprovincialis    AF179452-53
Falkland             1     M. galloprovincialis    AF179457
New Zealand          1     M. galloprovincialis    AF179459
Australia            3     M. galloprovincialis    AF179460-62
                     8     M. galloprovincialis    GQ472141-45; 51-53
                     1     M. galloprovincialis    AF317056
Marruecos,           2     M. galloprovincialis    KT021638-39
  Africa (AFR)
Ria Arousa,          3     M. galloprovincialis    KT021640-42
  Spain (RI)
Puerto Balleira,     4     M. galloprovincialis    KT021643-46
  Spain (PB)
Bergen, Norway       2     M. edulis               HQ832566; 71
                     1     M. edulis               GQ455405
                     2     M. edulis               AF317054-55
                     1     M. edulis               U22866
                     3     M. edulis               MEU22866-68
Lewes, DE            6     M. edulis               AF023546-51
                     2     M. edulis               AJ293730; 38
                     1     M. edulis               KC429249
Kola Bay;            5     M. trossulus            HQ832566-70
  Gremikha,
  Russia
                     1     M. trossulus            GQ455404
Japan                5     M. trossulus            KC835211; 13;
                                                     15; 35; 41
California,          1     M. californianus        NC015993
  Santa Cruz
                     1     M. californianus        AF317544
                     1     M. coruscus             AF317545
                     5     M. coruscus             GQ472146-50
United Kingdom       1     Mytilus sp.             AF023590
                             Hybrid gallojedulis

Locality             N     Haplotype            Reference

Castro, Chile        1         S       Hilbish et al. (2000)
Mediterranean Sea    1         N       Terranova et al. (2007)
South Hemisphere     2         S       Westfall et al. (2010)
San Diego, CA        1         N       Rawson and Hilbish (1995)
Japan                15        N       Brannock et al. (2013)

New Zealand          4         S       Gerard et al. (2008)
Tasmania             2         S       Gerard et al. (2008)
Kerguelen            2         s       Gerard et al. (2008)
Australia            3         s       Gerard et al. (2008)
Australia            1         s       Hilbish et al. (2000)
Kerguelen            1         s       Hilbish et al. (2000)
New Zealand          2         s       Hilbish et al. (2000)
Falkland             1         s       Hilbish et al. (2000)
New Zealand          1         S       Hilbish et al. (2000)
Australia            3         S       Hilbish et al. (2000)
                     8       n.i.      Liu et al. (2011)
                     1       n.i.      Unpublished
Marruecos,           2         N       Present work
  Africa (AFR)
Ria Arousa,          3         N       Present work
  Spain (RI)
Puerto Balleira,     4         N       Present work
  Spain (PB)
Bergen, Norway       2                 Vainola and Strelkov (2011)
                     1                 Westfall et al. (2010)
                     2                 Unpublished
                     1                 Rawson and Hilbish (1995)
                     3                 Rawson and Hilbish (1995)
Lewes, DE            6                 Rawson and Hilbish (1995)
                     2                 Unpublished
                     1                 Sharma et al. (2013)
Kola Bay;            5                 Vainola and Strelkov (2011)
  Gremikha,
  Russia
                     1                 Westfall et al. (2010)
Japan                5                 Brannock et al. (2013)

California,          1                 Cao et al. (2009)
  Santa Cruz
                     1                 Unpublished
                     1                 Unpublished
                     5                 Liu et al. (2011)
United Kingdom       1                 Rawson and Hilbish (1995)

n.i., not informated.

The GenBank code and the author of the sequences are shown.
All of the sequences of Mytilus from South America are
indicated in A and were generated in this work. For Mytilus
galloprovincialis the haplotype is indicated as either
northern (N) or southern (S).

TABLE 2.
Indices of genetic variability based on mtDNA (COI)
sequences for the Mytilus species.

                                    Mytilus
                                 South America

No sequences                          117
S (polymorphic sites)                  81
Nh                                     53
Hd                            0.927 [+ or -] 0.017
Nucleotide diversity P        0.008 [+ or -] 0.001
No different nucleotides K    4.252 [+ or -] 1.442

                                    Mytilus
                               galloprovincialis-
                                       NH

No sequences                           99
S (polymorphic sites)                  31
Nh                                     28
Hd                            0.808 [+ or -] 0.035
Nucleotide diversity P        0.013 [+ or -] 0.002
No different nucleotides K    3.187 [+ or -] 1.084

                                       M.
                              galloprovincialis-
                                       SH

No sequences                           54
S (polymorphic sites)                  63
Nh                                     28
Hd                            0.932 [+ or -] 0.021
Nucleotide diversity P        0.038 [+ or -] 0.004
No different nucleotides K   14.185 [+ or -] 4.907

                                     Mytilus
                                     edulis

No sequences                          141
S (polymorphic sites)                  48
Nh                                     41
Hd                            0.901 [+ or -] 0.02
Nucleotide diversity P        0.009 [+ or -] 0.0007
No different nucleotides K    3.259 [+ or -] 1.102

                                    Mytilus
                                   trossulus

No sequences                          15
S (polymorphic sites)                 16
Nh                                    12
Hd                           0.962 [+ or -] 0.04
Nucleotide diversity P        0.01 [+ or -] 0.001
No different nucleotides K    3.81 [+ or -] 1.451

Nh, number of haplotypes; Hd, haplotypes diversity.
Hd, P, and K. with SD.

TABLE 3.
Indices of genetic variability based on mtDNA (16S)
sequences of the Mytilus species.

                                                 Mytilus
                          Mytilus               gullopro
                       South America           vincialis-NH

No sequences                84                      61
S (polymorphic              22                      37
  sites)
Nh                          15                      17
Hd                 0.519 [+ or -] 0.066    0.826 [+ or -] 0.037
Nucleotide         0.002 [+ or -] 0.0005   0.027 [+ or -] 0.004
  diversity P
No different       0.843 [+ or -] 0.288    9.806 [+ or -] 3.378
  nucleotides K

                            M.
                    galloprovincialis-
                            SH               Mytilus edulis

No sequences                14                     25
S (polymorphic              13                     33
  sites)
Nh                          10                     14
Hd                 0.89 [+ or -] 0.081    0.890 [+ or -] 0.052
Nucleotide         0.006 [+ or -] 0.002   0.029 [+ or -] 0.004
  diversity P
No different       2.198 [+ or -] 0.845   11.053 [+ or -] 3.99
  nucleotides K

                    Mytilus trossulus

No sequences                18
S (polymorphic              36
  sites)
Nh                          6
Hd                 0.627 [+ or -] 0.124
Nucleotide         0.012 [+ or -] 0.009
  diversity P
No different       4.464 [+ or -] 1.663
  nucleotides K

Nh, number of haplotypes; Hd, haplotypes diversity.
Hd, P, and K with SD.

TABLE 4.

Estimates of evolutionary divergence between groups
using COI sequences.

                        Mytilus      Mytilus               M.
                         South       gullopro      galloprovincialis-
                        America    vincialis-NH            SH

Mytilus South           0.007#    [+ or -] 0.014     [+ or -] 0.011
  America
M.                       0.035             0.017#    [+ or -] 0.016
  galloprovincialis-
  NH
M.                       0.033             0.041              0.039#
  galloprovincialis-
  SH
M. edulis                0.030             0.015              0.039
M. trossulus             0.438             0.472              0.490

                           Mytilus          Mytilus
                            edulis         trossulus

Mytilus South           [+ or -] 0.013   [+ or -] 0.199
  America
M.                      [+ or -] 0.006   [+ or -] 0.222
  galloprovincialis-
  NH
M.                      [+ or -] 0.015   [+ or -] 0.242
  galloprovincialis-
  SH
M. edulis                        0.011#  [+ or -] 0.202
M. trossulus                     0.448            0.011#

The number of base substitutions per site averaged over all
sequence pairs between groups are shown. SE estimate(s) are
shown above the diagonal and were obtained by bootstrapping
(1,000 replicates). The number of base substitutions per
site averaged over all sequence pairs within each group are
shown bold on the diagonal. Analyses were conducted using
the Tamura-Nei 93 model + G. The rate variation among sites
was modeled with a gamma distribution
(shape parameter = 0.241)

Note: bold indicated with #.

TABLE 5.
Estimates of evolutionary divergence between groups
using 16S sequences.

                         Mytilus      Mytilus               M.
                          South       gallopro      galloprovincialis-
                         America    vincialis-NH            SH

Mytilus South America     0.002#    [+ or -] 0.005     [+ or -] 0.001
M. galloprovincialis-     0.019              0.026#    [+ or -] 0.005
  NH
M. galloprovincialis-     0.005              0.021              0.008#
  SH
M. edulis                 0.034              0.038               0.036
M. trossulus              0.098              0.103               0.107

                            Mytilus           Mytilus
                             edulis          trossulus

Mytilus South America    [+ or -] 0.008    [+ or -] 0.024
M. galloprovincialis-    [+ or -] 0.009    [+ or -] 0.024
  NH
M. galloprovincialis-    [+ or -] 0.008    [+ or -] 0.024
  SH
M. edulis                         0.042#   [+ or -] 0.022
M. trossulus                      0.100             0.031#

The number of base substitutions per site averaged over all
sequence pairs between groups are shown. SE estimate(s) are
shown above the diagonal. The number of base substitutions
per site averaged over all sequence pairs within each group
are shown bold on the diagonal. Analyses were conducted
using the Tamura-3-parameters model. The rate variation
among sites was modeled with a gamma distribution (shape
parameter = 0.319)

Note: bold indicated with #.
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Article Details
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Author:Astorga, Marcela P.; Cardenas, Leyla; Vargas, Jaime
Publication:Journal of Shellfish Research
Article Type:Report
Geographic Code:3CHIL
Date:Dec 1, 2015
Words:8798
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