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, Larra ın 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 organism s 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, Larra ın identified species of this complex diverged from the common et al. 2014, Nu nez-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, D ıaz & 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, G erard 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 G erard 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 (G erard 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. Following these results, the northern clade diversity, nucleotide diversity (p1), and the mean number of of Mytilus consists of populations found in the northern pairwise differences (p1) were estimated for each group and hemisphere, Western Australia, South Africa, and northern gene, using DnaSP v5.00.04 (Librado & Rozas 2009). The Chile (Dichato). software MEGA 6.0 (Tamura et al. 2013) was used to More recently, results gathered from RFLP and nuclear estimate genetic divergence between samples using the num- gene polymorphism data have indicated the presence of Mytilus ber of base substitutions per site and by averaging over all of trossulus, Mytilus galloprovincialis, and possible hybrids in the sequence pairs between groups. The significance and SE Chile. Both M. trossulus and M. galloprovincialis were found of the values were evaluated by 1000 bootstrap replicates. off the coast of the Bio-Bio Region of Chile, however the Moreover, the percentage of different nucleotides was mea- Mytilus hybrids were found in regions where Chilean mussels sured using DnaSP v5.00.04 (Librado & Rozas 2009). To are farmed (Larra ın et al. 2012). Further to this, results of perform this analysis, the groups were separated following a detailed review also indicate that Mytilus is genetically distinct the criteria of G erard et al. (2008) where haplotypes in the in the two hemispheres (Borsa et al. 2012). Also, this review northern hemisphere and southern hemisphere are defined shows that Chilean Mytilus are different from northern hemi- for galloprovincialis. sphere Mytilus. Because of this, Chilean Mytilus should be Because the 16S gene is commonly used to build marine identified as Mytilus edulis platensis following taxonomic invertebrate phylogenies and to identify operational taxo- priority rules. nomic units and species (for example de Luna Sales et al. Based on the results of previous work, the study here in 2013, Pulliandre et al. 2014), this gene was used for the presented attempts to determine if the phylogenetic patterns phylogenetic analyses. Maximum Likelihood reconstructions found by Hilbish et al. (2000) and G erard et al. (2008) are were performed using the best substitution model, as is present in this system (South America). The study emphasizes implemented in the software MEGA 6.0 (Tamura et al. the Chilean coast, adding information from the southern cone 2013). Bayesian inference method (BI) was performed using of South America. Furthermore, here the possible presence of the software Mr. Bayes (Huelsenbeck & Ronquist 2001). The Mytilus trossulus and its hybrids in Chile is assessed. Finally, evolutionary model was chosen according to the BIC Model a taxonomically suitable identification of the Mytilus species Selection as implemented in Modeltest 3.7 (Posada & Crandall present in Chile is proposed. 1998). Posterior probabilities were estimated over 10,000,000 PHYLOGENETIC ANALYSIS OF MYTILUS FROM SOUTH AMERICA 3
TABLE 1. Samples of Mytilus species from around the world were used in this work.
Samples from South America Locality Abbreviation Lat/Long N 16S N COI Species GenBank 16 /COI Tumbes TUM 36° 36# S/73° 04# O1012M. galloprovincialis KP052886–905 / KP052906–927 Puerto Saavedra PSA 38° 47# S/73° 23# O12 9Mytilus KM452802–06; KR153801–07 / KR066669–77 Queule QUE 39° 23# S/73° 13# O1512Mytilus KR153811–20; KM452797–801 / KR066657–68 Chaihu ın CHA 39° 56# S/73° 35# O9 10Mytilus KR153794–97; KM452807–11 / KR066678–87 Huinay HUI 42° 22# S/72° 24# O8 13Mytilus KR153790–93;834;KM452814–16 / KR066688–700 Yaldad YAL 43° 06# S/73° 42# O1116Mytilus KR3821–26; KM452817–21 / KR066701–16 Puerto Balmaceda PRM 43° 45# S/72° 58# O8 11Mytilus KR153198–800; KM452822–26 / KR066717–27 Punta Arenas PTA 53° 25# S/69° 23# O1229Mytilus KP052859–70 / KR066728–56 Puerto Deseado ARG 47° 45# S/65° 53# O0 7Mytilus KR066757–63 Montevideo URU 34° 50# S/56° 10# O7 8Mytilus KR153827–33 / KR066764–71 Total 92 127 Samples from Other Coasts for COI Locality, Country N Species GenBank Haplotype Reference Dichato, Chile 6 Mytilus sp. AM905174–79 S G erard et al. (2008) Patagonia, Chile 1 Mytilus sp. AM905195 S G erard et al. (2008) Maull ın, Chile 1 Mytilus sp. AM905186 S G erard et al. (2008) Samos, Grecia 7 Mytilus galloprovincialis AY130054–60 N Riginos et al. (2004) Morgat, Francia 1 M. galloprovincialis AY497292 N Cao et al. (2004) Grecia 1 M. galloprovincialis DQ403169 N Venetis et al. (2006) Auckland, New Zealand 10 M. galloprovincialis DQ864378–87 S Unpublished Lyttelton, New Zealand 8 M. galloprovincialis DQ864426–33 S Unpublished Capetown, SouthAfrica 14 M. galloprovincialis DQ864397–410 N Unpublished Sydney, Australia 15 M. galloprovincialis DQ86411–25 S Unpublished Campbell, Canada 1 M. galloprovincialis DQ864416 N Unpublished Hobart, Australia 9 M. galloprovincialis DQ864388–96 S Unpublished Chioggia, Italia 1 M. galloprovincialis AM905225 N G erard et al. (2008) Nedlands, Australia Oeste 1 M. galloprovincialis AM905215 N G erard et al. (2008) Kerguelen, French 1 M. galloprovincialis AM905211 S G erard et al. (2008) New Zealand 3 M. galloprovincialis AM905146; 49; 55 S G erard et al. (2008) Tasmania, Australia 2 M. galloprovincialis AM905161; 62; 65; 68 S G erard et al. (2008) Kerguelen, French 2 M. galloprovincialis AM905196–97 N Gerard et al. (2008) Ciudad del Cabo, South Africa 21 M. galloprovincialis DQ351477–97 N Zardi et al. (2007) South Africa 1 M. galloprovincialis DQ917605 N Wood et al. (2007) Atlantico Norte 39 M. galloprovincialis AF241997–20031; 33–35 N Wares and Cumminghan (2001) Kanagawa, Japan 1 M. galloprovincialis AB076943 N Matsumoto (2003) Roscoff, France 1 Mytilus edulis AY130053 Riginos et al. (2004) Greenland 3 M. edulis EU915572–4 Riginos and Henzler (2008) Atlantico Norte 3 M. edulis AF241969–71 Wares and Cumminghan (2001) Kieler Bucht, Germany 134 M. edulis JF825556–689 Steinert et al. (2012) Mahone Bay, Canada 7 Mytilus trossulus AY130061–67 Riginos et al. (2004) Samples from Other Coasts for 16S Locality N Species GenBank Haplotype Reference Castro, Chile 1 Mytilus sp. AF179447 S Mediterranean Sea 1 M. galloprovincialis DQ836018 N Terranova et al. (2007) South Hemisphere 2 M. galloprovincialis GQ4553981; 88 S Westfall et al. (2010) San Diego, CA 1 M. galloprovincialis MGU22885 N Rawson and Hilbish (1995) Japan 15 M. galloprovincialis KC835224–6; 8; 30–2; 42–6–51 N Brannock et al. (2013) New Zealand 4 M. galloprovincialis AM904568–70; 72; S G erard et al. (2008) Tasmania 2 M. galloprovincialis AM904579; 80; S G erard et al. (2008) Kerguelen 2 M. galloprovincialis AM904589; 90; S G erard et al. (2008) Australia 3 M. galloprovincialis AM904592–94 S G erard et al. (2008) Australia 1 M. galloprovincialis AF179448 S Hilbish et al. (2000) Kerguelen 1 M. galloprovincialis AF179449 S Hilbish et al. (2000) New Zealand 2 M. galloprovincialis AF179452–53 S Hilbish et al. (2000) Falkland 1 M. galloprovincialis AF179457 S Hilbish et al. (2000)
continued on next page 4 ASTORGA ET AL.
TABLE 1. continued
Samples from Other Coasts for 16S Locality N Species GenBank Haplotype Reference New Zealand 1 M. galloprovincialis AF179459 S Hilbish et al. (2000) Australia 3 M. galloprovincialis AF179460–62 S Hilbish et al. (2000) 8 M. galloprovincialis GQ472141–45; 51–53 n.i. Liu et al. (2011) 1 M. galloprovincialis AF317056 n.i. Unpublished Marruecos, Africa (AFR) 2 M. galloprovincialis KT021638–39 N Present work Ria Arousa, Spain (RI) 3 M. galloprovincialis KT021640–42 N Present work Puerto Balleira, Spain (PB) 4 M. galloprovincialis KT021643–46 N Present work Bergen, Norway 2 M. edulis HQ832566; 71 Vain€ ol€ a€ and Strelkov (2011) 1 M. edulis GQ455405 Westfall et al. (2010) 2 M. edulis AF317054–55 Unpublished 1 M. edulis U22866 Rawson and Hilbish (1995) 3 M. edulis MEU22866–68 Rawson and Hilbish (1995) Lewes, DE 6 M. edulis AF023546–51 Rawson and Hilbish (1995) 2 M. edulis AJ293730; 38 Unpublished 1 M. edulis KC429249 Sharma et al. (2013) Kola Bay; Gremikha, Russia 5 M. trossulus HQ832566–70 Vain€ ol€ a€ and Strelkov (2011) 1 M. trossulus GQ455404 Westfall et al. (2010) Japan 5 M. trossulus KC835211; 13; 15; 35; 41 Brannock et al. (2013) California, Santa Cruz 1 Mytilus californianus NC015993 Cao et al. (2009) 1 M. californianus AF317544 Unpublished 1 M. coruscus AF317545 Unpublished 5 M. coruscus GQ472146–50 Liu et al. (2011) United Kingdom 1 Mytilus sp. Hybrid gallo/edulis AF023590 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).
generations via one run of four simultaneous Markov individuals was used and each sequence used was 667 bp in Chain Monte Carlo chains with every 1,000th tree saved. length. The first 10% burn-in was discarded following suggestions by Felsenstein (1985). In the phylogenies, 16S sequences of RESULTS Mytilus californianus and Mytilus coruscus were chosen as out- groups (G erard et al. 2008). The genetic diversity indices calculated for the smooth- The COI gene is a commonly used marker for phylogeo- shelled species of Mytilus showed the same uniform diversity graphic studies. This molecular marker can be used to distribution regardless of the gene analyzed (Table 2 for COI T2 determine the number of species and the relationships among and Table 3 for 16S). Between pairs of groups (species), there T3 haplotypes (for example Cardenas et al. 2009, Haye et al. was low genetic divergence, again regardless of the gene tested. 2014). The relationships among the observed haplotypes in Despite this, slight differentiation was observed between north- this study were assessed by constructing median joining ern hemisphere Mytilus galloprovincialis [with northen haplo- networks (Bandelt et al. 1999) using the software Network type (NH)] and Mytilus edulis using the COI gene. It should be v4.613 (www.fluxus-engineering.com). To determine con- noted that differentiation was observed between the two groups nections in the network, a star contraction procedure was of M. galloprovincialis [NH with southern haplotype (SH)]; and applied before network calculation was performed (Forster within the same hemisphere the groups were more similar to et al. 1996). After calculating the network, maximum parsi- each than to those from the other hemisphere (M. edulis with mony analysis was used to filter out uninformative branches M. galloprovincialis from the northern hemisphere, and Mytilus (Polzin & Daneshmand 2003). Two different analyses were from South America with M. galloprovincialis from southern performed: first, the number of shared haplotypes and the hemisphere) (Table 4 for COI gene). The genetic divergence T4 distribution of haplotypes among species were visualized by based on the 16S data showed a similar pattern to that observed building a network with the entire dataset including all of the for the COI gene (Table 5 for 16S). In both datasets, the smallest T5 analyzed species. Here a total of 426 CO1 sequences each divergence observed was between the samples of Mytilus from having at least 399 bp were used to build the network. The South America and the M. galloprovincialis samples that have second analysis was performed exclusively with samples the SH. The level of genetic differentiation between Mytilus from South America. The aim of this analysis was to identify from South America and the M. galloprovincialis-SH was the spatial pattern of the haplotype distribution. For this 1.91% based on the COI gene and 0.27% based on the 16S second analysis, a database composed of sequences of 127 gene. The divergence between Mytilus from South America and PHYLOGENETIC ANALYSIS OF MYTILUS FROM SOUTH AMERICA 5
Figure 1. Map of geographic distribution of the samples analyzed of Mytilus species.
the M. galloprovincialis-NH was 1.73% for the COI gene and composed of samples of M. edulis, M. galloprovincialis-NH, and 1.57% for the 16S gene. For the comparison between Mytilus Mytilus trossulus with only one South America sample coming from South America and M. edulis, the value was 1.86% based from Uruguay. The third clade is composed of samples of on the COI gene and 1.13% based on 16S. The percent M. galloprovinciales-NH and -SH, M. edulis, and samples from divergence between M. galloprovincialis-SH and M. edulis is South America collected in TUM (Chile). The sequences of other 2.20% for the COI gene and 2.45% for 16S. Between samples from South America and samples of M. galloprovincialis- M. galloprovincialis-NH and M. edulis, 1.05% and 2.67% divergence SH clustered into a large polytomy indicating considerable was found for COI and 16S, respectively. Finally, the percentage uncertainty in the relationship among these taxa. of different nucleotides between M. galloprovincialis-SH and The network analysis using the whole dataset, i.e., including -NH was 2.40% based on the COI gene and 2.53% based on 16S. samples from Mytilus edulis, Mytilus trossulus, Mytilus gallo- Phylogenetic reconstruction with the 16S gene showed a sim- provincialis-NH, M. galloprovincialis-SH and Mytilus from ilar topology independent of whether the maximum likelihood South America, is shown in Figure 3. The network indicates F3 F2 (ML) or BI analysis method was used (Fig. 2). In this reconstruc- the existence of two haplotypes of high frequency separated by tion, three well-supported clades were detected. The first clade six mutational steps. One of these haplotypes was observed was composed of samples of Mytilus edulis, Mytilus galloprovin- in a total of 114 individuals including samples from M. edulis, cialis-HN, one sample of M. galloprovincialis-SH, and samples M. galloprovincialis-NH, and in several South America and from TUM (Chile) in South America. The second clade is M. galloprovincialis-SH samples. Several other lower frequency
TABLE 2. Indices of genetic variability based on mtDNA (COI) sequences for the Mytilus species.
Mytilus Mytilus M. South galloprovincialis- galloprovincialis- Mytilus Mytilus America NH SH edulis trossulus N° sequences 117 99 54 141 15 S (polymorphic sites) 81 31 63 48 16 Nh 53 28 28 41 12 Hd 0.927 ± 0.017 0.808 ± 0.035 0.932 ± 0.021 0.901 ± 0.02 0.962 ± 0.04 Nucleotide diversity P 0.008 ± 0.001 0.013 ± 0.002 0.038 ± 0.004 0.009 ± 0.0007 0.01 ± 0.001 N° different nucleotides K 4.252 ± 1.442 3.187 ± 1.084 14.185 ± 4.907 3.259 ± 1.102 3.81 ± 1.451
Nh, number of haplotypes; Hd, haplotypes diversity. Hd, P, and K with SD. 6 ASTORGA ET AL.
TABLE 3. Indices of genetic variability based on mtDNA (16S) sequences of the Mytilus species.
Mytilus M. Mytilus galloprovincialis- galloprovincialis- Mytilus Mytilus South America NH SH edulis trossulus N° sequences 84 61 14 25 18 S (polymorphic sites) 22 37 13 33 36 Nh 15 17 10 14 6 Hd 0.519 ± 0.066 0.826 ± 0.037 0.89 ± 0.081 0.890 ± 0.052 0.627 ± 0.124 Nucleotide diversity P 0.002 ± 0.0005 0.027 ± 0.004 0.006 ± 0.002 0.029 ± 0.004 0.012 ± 0.009 N° different nucleotides K 0.843 ± 0.288 9.806 ± 3.378 2.198 ± 0.845 11.053 ± 3.99 4.464 ± 1.663
Nh, number of haplotypes; Hd, haplotypes diversity. Hd, P, and K with SD.
haplotypes are connected to the one large haplotype previously there has been a recent introduction of M. galloprovincialis-NH described. These haplotypes are separated by a distance of one in TUM and ARG. mutational step and are mainly from the M. galloprovincialis-NH The results herein presented confirm the low phylogenetic and M. edulis samples. Other high frequency haplotypes were found divergence between groups within the Mytilus edulis complex, in in 69 samples. These were mainly found in samples from South comparison, with the other Mytilus species. The low genetic America, but there were also some detected in the M. galloprovin- diversity within the M. edulis complex is to be expected, as it is cialis-SH and -NH samples. In addition, the M. trossulus haplo- known that these species interbreed (Beaumont et al. 2004, types were independent from the main network; these haplotypes Beaumont et al. 2008, Gosling et al. 2008, Klibansky & were separated from the main network by 34 mutational steps. McCartney 2014, Lourenco et al. 2015) and have few distinct When samples of Mytilus from South America were analyzed morphological characteristics (Daguin & Borsa 1999, Oyarzun F4 (Fig. 4), one central high frequency haplotype was observed in et al. 2014). Despite this, when the genetic and phylogenetic a total of 28 individuals from Puerto Saavedra, Queule, Chaihu ın, divergences of this species complex are analyzed in detail, Yaldad, Puerto Balmaceda; Punta Arenas, and Puerto Deseado. differentiation can be found. Phylogenetic analysis of the COI In addition, several other haplotypes were detected; these included gene sequence data shows a separation of the northern hemi- individuals from several sites with no apparent spatial segregation sphere samples from the southern hemisphere samples. This of the genetic diversity. This suggests that there is no spatial generates a clade of northern haplotypes plus some groups structure to Mytilus populations in South America. However, present in the southern hemisphere. This clade includes samples exceptions to this include three haplotypes from ARG that are from South Africa, Western Australia, and TUM on the coast nine mutational steps away from the rest of the network. of Chile. It is possible that this may have resulted from a more recent introduction (Branch & Stefanni 2004, Braby & Somero DISCUSSION 2005, Robinson et al. 2007, Lockwood & Somero 2011). The presence of Mytilus galloprovincialis in TUM has been reported This study supports the hypothesis proposed by Hilbish et al. to be a recent introduction of galloprovincialis (Branch & (2000) and G erard et al. (2008) as two main groups of Mytilus Stefanni 2004, Castilla & Neill 2009). Therefore, this recent galloprovincialis were found. In addition, a clear separation of introduction may also have resulted from the second trans- Mytilus trossulus and other Mytilus species was found. Further equatorial migration from the northern hemisphere as proposed to this, there is no evidence of the presence of this species or by Hilbish et al. (2000). hybrids in Chile. Finally, there is evidence that South America This separation of haplotypes into clusters associated samples are taxonomically independent. This also suggests that with the hemispheres has already been detected by G erard
TABLE 4. Estimates of evolutionary divergence between groups using COI sequences.
Mytilus M. Mytilus galloprovincialis- galloprovincialis- Mytilus Mytilus South America NH SH edulis trossulus Mytilus South America 0.007 ±0.014 ±0.011 ±0.013 ±0.199 M. galloprovincialis-NH 0.035 0.017 ±0.016 ±0.006 ±0.222 M. galloprovincialis-SH 0.033 0.041 0.039 ±0.015 ±0.242 M. edulis 0.030 0.015 0.039 0.011 ±0.202 M. trossulus 0.438 0.472 0.490 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 ¼ 0.241) PHYLOGENETIC ANALYSIS OF MYTILUS FROM SOUTH AMERICA 7
TABLE 5. Estimates of evolutionary divergence between groups using 16S sequences.
Mytilus M. Mytilus galloprovincialis- galloprovincialis- Mytilus Mytilus South America NH SH edulis trossulus Mytilus South America 0.002 ±0.005 ±0.001 ±0.008 ±0.024 M. galloprovincialis-NH 0.019 0.026 ±0.005 ±0.009 ±0.024 M. galloprovincialis-SH 0.005 0.021 0.008 ±0.008 ±0.024 M. edulis 0.034 0.038 0.036 0.042 ±0.022 M. trossulus 0.098 0.103 0.107 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 ¼ 0.319) et al. (2008). This study demonstrates that there are great variability seen in the galloprovincialis-SH samples. The differences between southern hemisphere Mytilus samples. greatest number of different nucleotides was found within This is confirmed in the present work by the high genetic this group (Table 2).
Figure 2. Phylogenetic relationship of Mytilus sp. using the 16S gene. Maximum likelihood (ML) and Bayesian inference (BI) analysis showed the same topology. The value above each branch indicates the bootstrap percentages for ML (left) and posterior probabilities in percentages for BI (right) analysis. 8 ASTORGA ET AL.
Figure 3. Minimum spanning network of haplotypes of COI gene of Mytilus species. The size of each circle is proportional to the absolute haplotype frequency, the total number of individuals in each haplotype is indicated inside the circle. The branch lengths are proportional to the mutational steps unless otherwise indicated by black lines or numerals.
The results herein presented indicate that there is little genetic considered; The other study measuring divergence rates also differentiation between species of the Mytilus edulis complex. In considered these together (Hilbish et al. 2000). It is possible general, a greater similarity was observed between M. edulis and that due to Atlantic transequatorial colonization, these new Mytilus galloprovincialis from the northern hemisphere than southern hemisphere groups have undergone local differenti- between Mytilus from South America and edulis. ation. This differentiation would have created at least two Using both markers, phylogenetic analyses of the South groups including one in South America and another in the American samples show a separation of the data into a single southwest Pacific encompassing New Zealand, Tasmania, and clade with low genetic differentiation within this clade. These eastern Australia. This group in the south hemisphere may in samples also form a group with the Kerguelen samples, as was turn be structured, as shown by G erard et al. (2008, 2015). also detected by G erard et al. (2008). It is possible that South Finally, as a result of more recent migration (Hilbish et al. American Mytilus are more genetically distinct from the 2000), there is likely a group consisting of northern haplo- Mytilus edulis group (1.5%) and from the northern hemisphere types. This would include the groups of South Africa and galloprovincialis (1.7%) than they are from the southern Western Australia. The divergence results would suggest an hemisphere galloprovincialis (1.0%). The divergence between absence of gene flow between the hemispheres and potential Mytilus of the northern and southern hemispheres has been differentiation through local adaptation. This is in contrast estimated as 1.4% (Hilbish et al. 2000). Interestingly, this is the with the situation between Mytilus galloprovincialis and same as that observed in this study if the average of the M. edulis observed in the northern hemisphere where introgres- divergence rate of galloprovincialis from the southern hemi- sion and some degree of gene flow have been reported sphere and that of Mytilus from South America (1.4%) is (Quesada et al. 1998, Bierne et al. 2002). Based on the analysis PHYLOGENETIC ANALYSIS OF MYTILUS FROM SOUTH AMERICA 9
Figure 4. Minimum spanning network of haplotypes of COI gen of Mytilus from South America. The size of each circle is proportional to absolute haplotype frequency, circles are separated by one mutational step, unless otherwise indicated by black lines or numerals. of the sperm ultrastructure of the South American samples Finally, the taxonomic status of the group present along the coast (i.e., Mytilus chilensis)andM. galloprovincialis samples from of South America cannot be easily defined. the Chilean coast, differences have been found which corrob- Based on the results presented, Mytilus from South Amer- orate the current differentiation between these groups. From ica are genetically distinct from edulis. It is possible then that this, it is thought that sperm ultrastructures could be used as South American Mytilus should not be considered as a sub- characteristics for taxonomic identification (Oyarzun et al. species of Mytilus edulis as it has recently been called (M. edulis 2014). This being said, spermatozoa differentiation has not platensis). Instead, this group should be treated as a differen- prevented reciprocal reproduction between samples of South tiated cluster based on its high divergence from the edulis America and M. galloprovincialis (Toro et al. 2012). These group. If however, the proposal of the edulis complex is put results indicate that there is greater differentiation of Mytilus forth, all the species identified to date would be subspecies of between the hemispheres than between the classic species. this group including M. edulis edulis; M. edulis galloprovincia- Furthermore, Mytilus from South America are consistently lis, M. edulis planulatus and M. edulis platensis. This is not different from edulis and galloprovincialis. feasible. Considering the great divergence between the two Thus, to return to the three initial questions, the following haplotypes of galloprovincialis, which is greater than their conclusions are made. First, the hypotheses of Hilbish et al. divergence from edulis, a different identification scheme is (2000) and G erard et al. (2008) could be corroborated with proposed. The following is suggested: M. edulis should be the exhaustive sampling of the coast of the southern cone of South name for species from the northern hemisphere; Mytilus America. Second, no trossulus haplotypes were detected galloprovincialis should be the name used for species from among the South American samples. Because the level of the northern hemisphere and for the exceptions present in the divergence within this group has been shown to be very high, southern hemisphere. In the southern hemisphere, Mytilus one would have expected to detect this species rapidly. Therefore, planulatus should be the named used for specimens from the presence of trossulus and its hybrids along the Chilean coast is South America, Kerguelen, and the southwest Pacific (eastern discounted in contrast to that reported by Larra ın et al. (2012). Australia, Tasmania and New Zealand); or perhaps two 10 ASTORGA ET AL. differentiated groups should exist for the southern hemi- ACKNOWLEDGMENTS sphere, namely planulatus and platensis.Borsaetal.(2012) proposed this later identification schemed based on the anal- We thank Project Fondecyt 1130716, to the Millennium ysis of the literature existing at the date of their work. Nucleus Center for the Study of Multiple drivers on Marine Following the rules of taxonomic priority, the name frequently Socio-Ecological Systems (MUSELS) by MINECON Project used in the literature for samples from the Chilean coast NC120086. We also thank Dr. Jorge Toro, who collected some (Mytilus chilensis) would cease to be used. of the samples for this study.
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