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Evolutionary relationship between marble trout of the northern and the southern Adriatic basin
Molecular Phylogenetics and Evolution 59 (2011) 761–766
Contents lists available at ScienceDirect
Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev
Short Communication
Evolutionary relationship between marble trout of the northern and the southern Adriatic basin G. Pustovrh, S. Sušnik Bajec, A. Snoj ⇑ Department of Animal Science, Biotechnical Faculty, University of Ljubljana, Groblje 3, SI-1230 Domzˇale, Slovenia
a r t i c l e
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Article history: Received 30 November 2010 Revised 15 March 2011 Accepted 20 March 2011 Available online 1 April 2011 Keywords: S. marmoratus Salmo trutta Multilocus phylogeny Nuclear DNA Mitochondrial DNA
a b s t r a c t Marble trout (Salmo marmoratus) populate two geographically separated areas in the northern and southern parts of the Adriatic Sea drainage. Although morphologically similar, each population is distinguished by a different set of unrelated mitochondrial haplotypes, suggesting that they have evolved from different ancestors. Due to a possible discordance between mitochondrial and species phylogeny, we performed phylogenetic analysis based on 22 nuclear loci. The results inferred from Maximum-likelihood and Bayesian Inference analysis revealed that northern and southern populations are closely related, forming a monophyletic group. This observation is concordant with the present marble trout classification, which considers both populations as conspecific. On the other hand, our findings are in marked contrast to those of previous mtDNA-based studies and highlight potential dangers of making phylogenetic inferences from mtDNA alone. Reasons for discordance between mtDNA and nDNA phylogeny are discussed with incomplete lineage sorting proposed as the most parsimonious explanation for mtDNA divergence in marble trout. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Marble trout (Salmo marmoratus), characterized by its marbledcolor pattern, inhabit both the northern part of the Adriatic Sea drainage in Italy and Slovenia and the southern part, including rivers in Bosnia–Herzegovina (River Neretva system) and Montenegro (River Skadar system). Studies on northern populations are numerous and include morphological (Delling, 2002), allozyme (Berrebi et al., 2000; Giuffra et al., 1994) and DNA analysis (control region (CR) mtDNA (Bernatchez, 2001; Giuffra et al., 1994; Snoj et al., 2000), RAPDs (Jug et al., 2004), microsatellite DNA (Fumagalli et al., 2002; Meraner et al., 2010) and nuclear SNPs (Sušnik et al., 2008)) of several marble trout stocks. In contrast southern populations have been considerably less studied. Initial work consisted of osteological analysis of River Neretva marble trout (Dorofeeva et al., 1983), while at the molecular level, southern marble trout were included in a recent survey that focused primarily on the taxonomic status of Salmo dentex (Snoj et al., 2010). With the exception of CR mtDNA, different sets of molecular markers have been used in the studies on northern and southern populations, limiting comparison of the data. All of the northern populations were found to be fixed for a group of closely related mtDNA (MA) haplotypes, whereby a dis⇑ Corresponding author. Fax: +386 1 3203 888. E-mail addresses: [email protected] (G. Pustovrh), [email protected] (S. Sušnik Bajec), [email protected] (A. Snoj). 1055-7903/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2011.03.024
tinct mtDNA phylogenetic lineage, i.e. marmoratus lineage (Bernatchez et al., 1992), was recognized. Recent analysis of Balkan trouts based on CR mtDNA sequencing revealed that southern populations of marble trout do not exhibit MA haplotypes but rather so-called Adriatic (AD) haplotypes (Razpet et al., 2007; Snoj et al., 2010). This group of haplotypes was previously reported to be associated with brown trout (Salmo trutta) of the Mediterranean basin (Cortey et al., 2004), including the Adriatic drainage (Snoj et al., 2010; Sušnik et al., 2007), where it had been first observed (Bernatchez et al., 1992). On the other hand, marmoratus haplotypes have been detected in Mediterranean brown trout, including rivers in Greece (Apostolidis et al., 1997), Dalmatia (Bernatchez, 2001), central Italy (Splendiani et al., 2006), Albania (Snoj et al., 2009) and Corsica (authors’ unpublished data). These observations indicate that the specific color pattern of marble trout is not strictly related with MA haplotypes and suggest that marble trout from the northern Adriatic basin and those from the southern part may represent divergent evolutionary lineages and that their similar color pattern has been acquired independently. Given that the marble-color pattern was found also in unrelated S. trutta from the Ottra River in Norway (Skaala and Solberg, 1997), convergent evolution of this trait seems plausible also in the case of marble trout. Nevertheless, due to possible incongruence between phylogenetic reconstructions derived from mtDNA and from nDNA, as shown previously in several studies (e.g., Renoult et al., 2009; Sušnik et al., 2007; Wiens et al., 2010), the phylogenetic relationship between northern and southern populations will remain
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questionable until both populations are compared using also nDNA markers. The present study aimed to genotype marble trout from both the northern and southern parts of the Adriatic drainage along with other, native trout inhabiting Adriatic rivers using a novel set of nuclear loci (Pustovrh et al., 2010) designed for identifying trouts and their hybrids in the genus Salmo. 2. Material and methods 2.1. Samples and DNA isolation Fin clips of 36 individuals were analyzed (Table S1: Supplementary data). Eighteen individuals were marble trout (S. marmoratus) and twelve brown trout (S. trutta) of two phylogenetic lineages (Adriatic and Mediterranean). In addition, so-called dentex trout (S. dentex) from two locations in the Adriatic basin and Salmo salar as outgroup were included in the analysis. Genetic purity and phylogenetic origin of marble and brown trout specimens were previously determined with mtDNA and microsatellite DNA analysis (Fumagalli et al., 2002; Jug et al., 2005; Razpet et al., 2007; Sušnik et al., 2007). Total DNA was isolated from fin tissue preserved in 96% ethanol following the high-salt extraction protocol described by Miller et al. (1988).
performed with the settings advised by the author (Zwickl, 2006), where run was set for unlimited number of generations and automatic termination following 20,000 generations without a meaningful (ln L increase of 0.01) change in score. In addition, Bayesian Inference (BI) analysis was performed using MrBayes 3.1.2 (Ronquist and Huelsenbeck, 2003). Prior model selection for each partition (locus) was determined using the Akaike Information Criterion (AIC) calculated in MrModeltest 2.3 (Nylander et al., 2004) in conjunction with PAUP. Random starting trees were used and four Markov chains were run for one million generations, nucmodel = 4by4, nruns = 2, tree-sampling frequencies of 1 in 100 (10,000 trees saved). Convergence was assessed by inspecting the cumulative posterior probabilities of clades using the online program Are We There Yet? (AWTY; Nylander et al., 2008). Four nuclear loci (TFG-beta, tnfa, TF, RH) that were assigned to protein-coding regions were tested for positive selection (HA: dN > dS) by the Nei–Gojobori method (Nei and Gojobori, 1986) using MEGA version 4 (Tamura et al., 2007). Although none of the loci containing coding regions proved to be under strong selective pressure, their potential role in selection was considered. Phylogenetic analyses were therefore also performed on nuclear loci that either do not contain coding regions or have no annotated hits among the blast results (18 loci altogether). All analyses were performed under the same settings as described above to enable direct comparison of both phylogenetic resolutions.
2.2. Description of nuclear loci 3. Results Twenty-two nuclear loci were used in phylogenetic analysis. Description of 18 loci, PCR primers and conditions are given in Pustovrh et al. (2010); see also Table 1. Four loci (rhodopsin, somatolactin, SILVA and transferrin) characterized to have an allele diagnostic for marble trout in the northern Adriatic basin (Sušnik et al., 2008) were added to the data set. PCR primers and PCR conditions for additional four loci are described in Sušnik et al. (2008); see also Table 1. Amplified DNA fragments were run on a 1.5% agarose gel and purified using the QIAEX II Gel Extraction Kit (QIAGEN). Approximately 100 ng of purified PCR product was used in cycle sequencing reactions following BigDyeÒ Terminator v3.1 Cycle Sequencing protocols (Applied Biosystems), applying forward primers. The amplified, fluorescently labeled and terminated DNA was salt-precipitated and analyzed with an ABI 3130 XL Genetic Analyser. 2.3. Alignment, data partitioning and phylogenetic analysis Sequences of all 22 loci amplified for each individual sample were combined in the same order as reported in Table 1 and aligned using the default parameters in CLUSTAL_W (Thompson et al., 1994). The final alignment was archived in TreeBase under submission number 11,254 (http://purl.org/phylo/treebase/phylows/study/TB2:S11264?x-access-code=42c459a14ed3e5770299a 33fab0b3f90&format=html). Phylogenies were estimated by maximum-likelihood (ML) analysis as implemented in program GARLI Version 0.96b8 (Zwickl, 2006). This program performs implementation of the ML method equivalent to PAUP version 4.0b10 (Swofford, 2002), and therefore the likelihood scores obtained by each program are directly comparable. To avoid over partitioning and yet still effectively deal with heterogeneity each locus was used as a criterion to define a partition. Prior model selection for each partition (locus) was determined using the Bayesian information criterion (BIC) calculated in MODELTEST v 3.06 (Posada, 2008) in conjunction with PAUP. For ML analysis, 2000 bootstrap replicates were carried out to identify the best partitioning scheme. Analysis was
Twenty-two nuclear loci were successfully amplified in all samples, except in outgroup species S. salar, for which only seven loci could be amplified and sequenced. Sequences of eleven loci from S. salar were obtained from Genbank database (see Table S1: Supplementary data), though four loci were still missing in the final alignment. All new sequences were deposited in GenBank (Accession Numbers in Table 1). In the final alignment of joint sequences consisting of ca. 7940 bp, 84 variable sites, 70 of which were parsimony informative, and six indels, were detected. The most appropriate models of evolution for ML and BI phylogenetic analyses and for each of 22 partitions are reported in Table 1. Both phylogenetic analyses recovered, with strong support, monophyly of northern and southern marble trout populations (Fig. 1). However, resolution within the marble trout clade was less pronounced. Both BI and ML analyses based on all 22 loci, supported monophyly of northern populations, but not of two southern populations. On the other hand, the results of ML analyses, without four coding regions, supported monophyly of southern and northern populations, while BI analysis, without four coding regions, supported monophyly of southern populations but not of northern populations. Northern populations of marble trout were divided into three groups corresponding to their geographical distribution. Samples of S. dentex from River Neretva clustered together with samples from southern marble trout populations. Other samples of brown trout from the Adriatic and Mediterranean drainage, including S. dentex from Montenegro, were placed in a moderately supported sister clade to marble trout. 4. Discussion Multilocus phylogeny inferred from nuclear genes revealed that northern and southern population of marble trout are closely related and that they—in contrast to other trout inhabiting the
Table 1 Locus name, primer sequences, annealing temperature, GeneBank accession numbers of amplified sequences and nucleotide substitution model for each partition (locus). Locus
GP1
Primer sequence
a
GP4 GP5 GP14 GP16
b
GP31
GP37 GP38 GP42 GP73 GP81 GP85 GP94 HMG1 SS2 TFGB-beta tnfa RH SILVA SL TF a b c
c
Source
Genbank accession number
Nucleotide substitution model ML-Model test
BI-MrModel test
56
Pustovrh et al. (2010)
HM066793, HM066804-HM066818, HM635370-HM635372
TrNef
GTR
60
Pustovrh et al. (2010)
HM066821, HM066825, HM635375-HM635376
F81
F81
52
Pustovrh et al. (2010)
HM066826-HM066827, HM066830-HM066832, HM635378-HM635379
JC
HKY
61
Pustovrh et al. (2010)
HM635380-HM635384
JC
F81
60
Pustovrh et al. (2010)
HM066835, HM066841-HM066842, HM635386
F81
F81
52
Pustovrh et al. (2010)
HM066845-HM066846, HM635387
JC
JC+I
61
Pustovrh et al. (2010)
HM066852-HM066861, HM635389-HM635391
K80
HKY
52
Pustovrh et al. (2010)
HM066863, HM066869-HM066873
TPM2uf
HKY
56
Pustovrh et al. (2010)
HM066880, HM066882, HM635392-HM635393
JC
JC+I
61
Pustovrh et al. (2010)
HM066884-HM066885, HM066888-HM066890, HM635395
F81
F81
61
Pustovrh et al. (2010)
HM066893, HM066898-HM066901, HM635396
F81
HKY
61
Pustovrh et al. (2010)
HM635402, HM635405-HM635409, HM635411-HM635412
F81
HKY
61
Pustovrh et al. (2010)
HM066916-HM066925, HM635415-HM635418
TPM2uf+G
GTR+G
52
Pustovrh et al. (2010)
HM066928-HM066934, HM635420
F81
HKY+I
61
Ryynanen and Primmer (2006)
HM066734, HM066737, HM635469
F81
F81+I
61
Ryynanen and Primmer (2006)
HM066740-HM066745, HM635458-HM635460
F81
HKY+I
61
Ryynanen and Primmer (2006)
HM066759, HM066761-HM066765, HM635472-HM635473
JC
JC+I
56
Ryynanen and Primmer (2006)
HM066769, HM066781-HM066784, HM635474-HM635476
JC+G
GTR+G
60
Sušnik et al. (2008)
HM635423, HM635425-HM635430, HM635432-HM635433
F81
F81
60
Sušnik et al. (2008)
HM635434-HM635435, HM635437, HM635440-HM635444, HM635446
K80
K80+I
52
Sušnik et al. (2008)
HM635451, HM635455-HM635457
F81
HKY
60
Sušnik et al. (2008)
HM635463-HM635467, HM635469-HM635470
JC
HKY
G. Pustovrh et al. / Molecular Phylogenetics and Evolution 59 (2011) 761–766
GP34
F: 5’-AGGTGGTAGAATGGACAAGTCA-3’ R: 5’-AGGGAGGGAGGGATAAGAG-3’ F: 5’-GACTGGGATTACTAGATATGGG-3’ R: 5’-GAAACCGGAATGGATAG-3’ F: 5’-GATTCCTTGGTTGGACTTGATTGTT-3’ R: 5’-AAGAGCTCCAGTGGTCCGATA-3’ F: 5’-CGGAGCAGAGGGAGTTGAGT-3’ R: 5’-ACCTGCACACTAATAAAACAACAAA-3’ F: 5’-AAGGGCACATATAAACGAACA-3’ R: 5’-ATGGTGATGAAGAAGGTGGTTATGG-3’ F: 5’-AGACCAGGAGGGTATTACTAACACA-3’ R: 5’-AAGTTTCAGTACTTGGCATTGAT-3’ F: 5’-CTTTGAACTGTTTGGCATGTAGG-3’ R: 5’-AACACCACAGGCCACTATTT-3’ F: 5’-GTATGTGCCATATTTCTATGCTT-3’ R: 5’-CAGAATGTCAGCCAAACTCC-3’ F: 5’-CAAAGAAATATGGCAAAC-3’ R: 5’-AGCCATTCATTGTTGATAAT-3’ F: 5’-TATCAGGTCATCCCAATGTCAAG-3’ R: 5’-ATAATCAATGTACATGCGAAAA-3’ F: 5’-GGCACTCCCTGTATATAGCTTC-3’ R: 5’-AGAGCTCACGGTTTTACCA-3’ F: 5’-TGGGTACTAGCTAGCTATATATGA-3’ R: 5’-CCAAGTGATTGTGTTGATATGGT-3’ F: 5’-CAGCATCCAGCCCAATATCAT-3’ R: 5’-CACTGCCCACTTGTTTGTGTTAGA-3’ F: 5’-TTCTATATTGTGCAGTTAGCTGT-3’ R: 5’-TACCTTTACCTTATGGGA-3’ F: 5’-TCAATCCCTTGATAGTTGTCTTTG-3’ R: 5’-GGGACACCCTGATTTTAATTGTAG-3’ F: 5’-CACTGCAGTAAGGATTATCTGCTT-3’ R: 5’-GAGGAAGAGCGAGGAAGATATAAAG-3’ F: 5’-GTGAGACTATCCTTTATTCCAACG-3’ R: 5’-TGTGGTTGGGTAAACAACAGTAGA-3’ F: 5’-CTTTTCATTGAATTGCTTCTCACT-3’ R: 5’-GCAGTAGAAGGAAGAAAGGTTCAT-3’ F: 5’-CXTATGAATAYCCTAGTACTACC-3’ R: 5’-CCRCAGCACARCGTGGTGATCATG-3’ F: 5’-CATACAACTGGGACTTTGGTG-3’ R: 5’-TTACTGTAGCTCCCTGTGTGG-3’ F: 5’-TGGCCCGTTGAATCCATATAAAG-3’ R: 5’-ACTGTGAAACACTAAGCTCTCCA-3’ F: 5’-CCAGTCTCCTTTTACCCCTACT-3’ R: 5’-CTTGACGGCCACCAGTTT-3’
Ta (°C)
98%(342/347) homology to S. salar interferon-inducible GTPase b and interferon-inducible GTPase_a genes [GenBank:EU221179.1]. 86%(274/315) homology to S. salar microsatellite Hae018 sequence [GenBank:AF271480.1]. 88%(174/197) homology to O. mykiss conserved noncoding element 1117-1131-i genomic sequences [GenBank:FJ356120.1].
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Fig. 1. Phylogenetic tree based upon Bayesian inference analysis of combined nuclear DNA data from Salmo trutta spp. samples inhabiting the Adriatic drainage. Support values refer to Bayesian inference (top) and maximum-likelihood (bottom) methods. Values < 50 are marked with ‘‘/’’. (A) Phylogenetic tree based on all 22 nuclear loci; (B) phylogenetic tree based on 18 loci; four coding loci (TFG-beta, tnfa, TF, RH), potentially under selection pressure, were removed from the analysis.
Mediterranean river basin—form a monophyletic group. This observation is concordant with the current marble trout classification, which considers both populations as conspecific. On the other hand, our results are in marked contrast to the notion of polyphyletic origin of marble trout, as inferred from the occurrence of unrelated CR mtDNA haplotypes (Snoj et al., 2010). Interestingly, mitochondrial and nDNA results were congruent in S. dentex, whose embedment either in S. marmoratus or in S. trutta (Fig. 1) clearly indicated its polyphyletic origin as proposed previously by Snoj et al. (2010). Discordance between mtDNA trees and nDNA phylogeny is a common phenomenon and generally explained by incomplete lineage sorting (incomplete and stochastic sorting of ancestral polymorphisms) or introgression between related species or differentiated lineages (introgressive hybridization; reviewed in Maddison, 1997).
Incomplete lineage sorting may represent a problem for organismal phylogeny if the time needed for haplotypes within a lineage to coalesce is greater than the time between successive speciation events (Page and Holmes, 2000). This phenomenon may be well applied to trouts of the genus Salmo, including S. trutta and S. marmoratus, which are known to have diverged very rapidly in the Pleistocene (Bernatchez, 2001). Moreover, both population groups of marble trout appear to be fixed, either for a single haplotype (as in the Neretva and Skadar basins; Snoj et al., 2010) or for a group of highly related haplotypes (MA haplotypes that probably emerged from a single plesiomorphic haplotype during marble trout evolution), which in brown trout of the Adriatic drainage represent only part of a much larger genetic polymorphism. Limited haplotype polymorphism found in marble trout populations is congruent with evidence of severe Pleistocene bottlenecks of marble trout populations and restricted habitat availability (Bernatchez, 2001;
G. Pustovrh et al. / Molecular Phylogenetics and Evolution 59 (2011) 761–766
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Fig. 1 (continued)
Snoj et al., 2010). Introgression, as the other option of mitochondrial–nuclear discordance, could have hypothetically also played a role in this case. Infiltration of mitochondrial genomes of one species into the original mitochondrial genome pool of another has been identified in several organisms (see Renoult et al., 2009, for examples). The most extreme, and very rarely detected, case of introgression is replacement of complete original mtDNA of an introgressed species with that of another, termed mtDNA capture. For example, a subspecies of softmouth trout (Salmo obtusirostrris salonitana) from the River Jadro (Croatia) has a mtDNA haplotype that is indicative of brown trout. However, all nuclear genes that have been examined appear to be characteristic of softmouth trout (Sušnik et al., 2007). Mitochondrial DNA capture could theoretically be attributed also to marble trout. In such a scenario, mtDNA capture of the incipient marble trout population (i.e., when northern and southern populations were last in contact) and subsequent divergence of this population into two is unlikely considering the substantial genetic distance between them (1%; Snoj et al.,
2009); thus extinction of the original mitochondrial genome of marble trout and its replacement with that of brown trout must have occurred twice in two geographically separated populations of the same species. Hypothetically, the effects of genetic drift and other factors, responsible for reduced fitness of marble trout females compared to introgressing brown trout females, may explain this scenario mtDNA capture. However, given the relatively non-parsimonious explanation of this scenario it seems more realistic that the mitochondrial–nuclear discordance found between northern and southern populations of marble trout results from incomplete lineage sorting. Although nuclear DNA data reveal monophylogeny of northern and southern populations of marble trout and indicate conspecificity, we have here shown that certain genetic differences do exist between them. As these populations have most probably been separated for a long geological time (at least since Würm glaciation), there is a possibility that they have developed locally adapted phenotypic traits as a result of selective differences. However, since no
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morphological and ecological analyses have been undertaken on both populations, this suggestion is impossible to prove. Acknowledgment We would like to that D. Jesenšek, I. Bogut, B. Glamuzina, N. Pojskic´, D. Mrdak, J. Schöffmann and R. Šanda for providing samples. Special thanks go to I. Wilson for editing and correcting the manuscript.
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Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev
Short Communication
Evolutionary relationship between marble trout of the northern and the southern Adriatic basin G. Pustovrh, S. Sušnik Bajec, A. Snoj ⇑ Department of Animal Science, Biotechnical Faculty, University of Ljubljana, Groblje 3, SI-1230 Domzˇale, Slovenia
a r t i c l e
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Article history: Received 30 November 2010 Revised 15 March 2011 Accepted 20 March 2011 Available online 1 April 2011 Keywords: S. marmoratus Salmo trutta Multilocus phylogeny Nuclear DNA Mitochondrial DNA
a b s t r a c t Marble trout (Salmo marmoratus) populate two geographically separated areas in the northern and southern parts of the Adriatic Sea drainage. Although morphologically similar, each population is distinguished by a different set of unrelated mitochondrial haplotypes, suggesting that they have evolved from different ancestors. Due to a possible discordance between mitochondrial and species phylogeny, we performed phylogenetic analysis based on 22 nuclear loci. The results inferred from Maximum-likelihood and Bayesian Inference analysis revealed that northern and southern populations are closely related, forming a monophyletic group. This observation is concordant with the present marble trout classification, which considers both populations as conspecific. On the other hand, our findings are in marked contrast to those of previous mtDNA-based studies and highlight potential dangers of making phylogenetic inferences from mtDNA alone. Reasons for discordance between mtDNA and nDNA phylogeny are discussed with incomplete lineage sorting proposed as the most parsimonious explanation for mtDNA divergence in marble trout. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Marble trout (Salmo marmoratus), characterized by its marbledcolor pattern, inhabit both the northern part of the Adriatic Sea drainage in Italy and Slovenia and the southern part, including rivers in Bosnia–Herzegovina (River Neretva system) and Montenegro (River Skadar system). Studies on northern populations are numerous and include morphological (Delling, 2002), allozyme (Berrebi et al., 2000; Giuffra et al., 1994) and DNA analysis (control region (CR) mtDNA (Bernatchez, 2001; Giuffra et al., 1994; Snoj et al., 2000), RAPDs (Jug et al., 2004), microsatellite DNA (Fumagalli et al., 2002; Meraner et al., 2010) and nuclear SNPs (Sušnik et al., 2008)) of several marble trout stocks. In contrast southern populations have been considerably less studied. Initial work consisted of osteological analysis of River Neretva marble trout (Dorofeeva et al., 1983), while at the molecular level, southern marble trout were included in a recent survey that focused primarily on the taxonomic status of Salmo dentex (Snoj et al., 2010). With the exception of CR mtDNA, different sets of molecular markers have been used in the studies on northern and southern populations, limiting comparison of the data. All of the northern populations were found to be fixed for a group of closely related mtDNA (MA) haplotypes, whereby a dis⇑ Corresponding author. Fax: +386 1 3203 888. E-mail addresses: [email protected] (G. Pustovrh), [email protected] (S. Sušnik Bajec), [email protected] (A. Snoj). 1055-7903/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2011.03.024
tinct mtDNA phylogenetic lineage, i.e. marmoratus lineage (Bernatchez et al., 1992), was recognized. Recent analysis of Balkan trouts based on CR mtDNA sequencing revealed that southern populations of marble trout do not exhibit MA haplotypes but rather so-called Adriatic (AD) haplotypes (Razpet et al., 2007; Snoj et al., 2010). This group of haplotypes was previously reported to be associated with brown trout (Salmo trutta) of the Mediterranean basin (Cortey et al., 2004), including the Adriatic drainage (Snoj et al., 2010; Sušnik et al., 2007), where it had been first observed (Bernatchez et al., 1992). On the other hand, marmoratus haplotypes have been detected in Mediterranean brown trout, including rivers in Greece (Apostolidis et al., 1997), Dalmatia (Bernatchez, 2001), central Italy (Splendiani et al., 2006), Albania (Snoj et al., 2009) and Corsica (authors’ unpublished data). These observations indicate that the specific color pattern of marble trout is not strictly related with MA haplotypes and suggest that marble trout from the northern Adriatic basin and those from the southern part may represent divergent evolutionary lineages and that their similar color pattern has been acquired independently. Given that the marble-color pattern was found also in unrelated S. trutta from the Ottra River in Norway (Skaala and Solberg, 1997), convergent evolution of this trait seems plausible also in the case of marble trout. Nevertheless, due to possible incongruence between phylogenetic reconstructions derived from mtDNA and from nDNA, as shown previously in several studies (e.g., Renoult et al., 2009; Sušnik et al., 2007; Wiens et al., 2010), the phylogenetic relationship between northern and southern populations will remain
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questionable until both populations are compared using also nDNA markers. The present study aimed to genotype marble trout from both the northern and southern parts of the Adriatic drainage along with other, native trout inhabiting Adriatic rivers using a novel set of nuclear loci (Pustovrh et al., 2010) designed for identifying trouts and their hybrids in the genus Salmo. 2. Material and methods 2.1. Samples and DNA isolation Fin clips of 36 individuals were analyzed (Table S1: Supplementary data). Eighteen individuals were marble trout (S. marmoratus) and twelve brown trout (S. trutta) of two phylogenetic lineages (Adriatic and Mediterranean). In addition, so-called dentex trout (S. dentex) from two locations in the Adriatic basin and Salmo salar as outgroup were included in the analysis. Genetic purity and phylogenetic origin of marble and brown trout specimens were previously determined with mtDNA and microsatellite DNA analysis (Fumagalli et al., 2002; Jug et al., 2005; Razpet et al., 2007; Sušnik et al., 2007). Total DNA was isolated from fin tissue preserved in 96% ethanol following the high-salt extraction protocol described by Miller et al. (1988).
performed with the settings advised by the author (Zwickl, 2006), where run was set for unlimited number of generations and automatic termination following 20,000 generations without a meaningful (ln L increase of 0.01) change in score. In addition, Bayesian Inference (BI) analysis was performed using MrBayes 3.1.2 (Ronquist and Huelsenbeck, 2003). Prior model selection for each partition (locus) was determined using the Akaike Information Criterion (AIC) calculated in MrModeltest 2.3 (Nylander et al., 2004) in conjunction with PAUP. Random starting trees were used and four Markov chains were run for one million generations, nucmodel = 4by4, nruns = 2, tree-sampling frequencies of 1 in 100 (10,000 trees saved). Convergence was assessed by inspecting the cumulative posterior probabilities of clades using the online program Are We There Yet? (AWTY; Nylander et al., 2008). Four nuclear loci (TFG-beta, tnfa, TF, RH) that were assigned to protein-coding regions were tested for positive selection (HA: dN > dS) by the Nei–Gojobori method (Nei and Gojobori, 1986) using MEGA version 4 (Tamura et al., 2007). Although none of the loci containing coding regions proved to be under strong selective pressure, their potential role in selection was considered. Phylogenetic analyses were therefore also performed on nuclear loci that either do not contain coding regions or have no annotated hits among the blast results (18 loci altogether). All analyses were performed under the same settings as described above to enable direct comparison of both phylogenetic resolutions.
2.2. Description of nuclear loci 3. Results Twenty-two nuclear loci were used in phylogenetic analysis. Description of 18 loci, PCR primers and conditions are given in Pustovrh et al. (2010); see also Table 1. Four loci (rhodopsin, somatolactin, SILVA and transferrin) characterized to have an allele diagnostic for marble trout in the northern Adriatic basin (Sušnik et al., 2008) were added to the data set. PCR primers and PCR conditions for additional four loci are described in Sušnik et al. (2008); see also Table 1. Amplified DNA fragments were run on a 1.5% agarose gel and purified using the QIAEX II Gel Extraction Kit (QIAGEN). Approximately 100 ng of purified PCR product was used in cycle sequencing reactions following BigDyeÒ Terminator v3.1 Cycle Sequencing protocols (Applied Biosystems), applying forward primers. The amplified, fluorescently labeled and terminated DNA was salt-precipitated and analyzed with an ABI 3130 XL Genetic Analyser. 2.3. Alignment, data partitioning and phylogenetic analysis Sequences of all 22 loci amplified for each individual sample were combined in the same order as reported in Table 1 and aligned using the default parameters in CLUSTAL_W (Thompson et al., 1994). The final alignment was archived in TreeBase under submission number 11,254 (http://purl.org/phylo/treebase/phylows/study/TB2:S11264?x-access-code=42c459a14ed3e5770299a 33fab0b3f90&format=html). Phylogenies were estimated by maximum-likelihood (ML) analysis as implemented in program GARLI Version 0.96b8 (Zwickl, 2006). This program performs implementation of the ML method equivalent to PAUP version 4.0b10 (Swofford, 2002), and therefore the likelihood scores obtained by each program are directly comparable. To avoid over partitioning and yet still effectively deal with heterogeneity each locus was used as a criterion to define a partition. Prior model selection for each partition (locus) was determined using the Bayesian information criterion (BIC) calculated in MODELTEST v 3.06 (Posada, 2008) in conjunction with PAUP. For ML analysis, 2000 bootstrap replicates were carried out to identify the best partitioning scheme. Analysis was
Twenty-two nuclear loci were successfully amplified in all samples, except in outgroup species S. salar, for which only seven loci could be amplified and sequenced. Sequences of eleven loci from S. salar were obtained from Genbank database (see Table S1: Supplementary data), though four loci were still missing in the final alignment. All new sequences were deposited in GenBank (Accession Numbers in Table 1). In the final alignment of joint sequences consisting of ca. 7940 bp, 84 variable sites, 70 of which were parsimony informative, and six indels, were detected. The most appropriate models of evolution for ML and BI phylogenetic analyses and for each of 22 partitions are reported in Table 1. Both phylogenetic analyses recovered, with strong support, monophyly of northern and southern marble trout populations (Fig. 1). However, resolution within the marble trout clade was less pronounced. Both BI and ML analyses based on all 22 loci, supported monophyly of northern populations, but not of two southern populations. On the other hand, the results of ML analyses, without four coding regions, supported monophyly of southern and northern populations, while BI analysis, without four coding regions, supported monophyly of southern populations but not of northern populations. Northern populations of marble trout were divided into three groups corresponding to their geographical distribution. Samples of S. dentex from River Neretva clustered together with samples from southern marble trout populations. Other samples of brown trout from the Adriatic and Mediterranean drainage, including S. dentex from Montenegro, were placed in a moderately supported sister clade to marble trout. 4. Discussion Multilocus phylogeny inferred from nuclear genes revealed that northern and southern population of marble trout are closely related and that they—in contrast to other trout inhabiting the
Table 1 Locus name, primer sequences, annealing temperature, GeneBank accession numbers of amplified sequences and nucleotide substitution model for each partition (locus). Locus
GP1
Primer sequence
a
GP4 GP5 GP14 GP16
b
GP31
GP37 GP38 GP42 GP73 GP81 GP85 GP94 HMG1 SS2 TFGB-beta tnfa RH SILVA SL TF a b c
c
Source
Genbank accession number
Nucleotide substitution model ML-Model test
BI-MrModel test
56
Pustovrh et al. (2010)
HM066793, HM066804-HM066818, HM635370-HM635372
TrNef
GTR
60
Pustovrh et al. (2010)
HM066821, HM066825, HM635375-HM635376
F81
F81
52
Pustovrh et al. (2010)
HM066826-HM066827, HM066830-HM066832, HM635378-HM635379
JC
HKY
61
Pustovrh et al. (2010)
HM635380-HM635384
JC
F81
60
Pustovrh et al. (2010)
HM066835, HM066841-HM066842, HM635386
F81
F81
52
Pustovrh et al. (2010)
HM066845-HM066846, HM635387
JC
JC+I
61
Pustovrh et al. (2010)
HM066852-HM066861, HM635389-HM635391
K80
HKY
52
Pustovrh et al. (2010)
HM066863, HM066869-HM066873
TPM2uf
HKY
56
Pustovrh et al. (2010)
HM066880, HM066882, HM635392-HM635393
JC
JC+I
61
Pustovrh et al. (2010)
HM066884-HM066885, HM066888-HM066890, HM635395
F81
F81
61
Pustovrh et al. (2010)
HM066893, HM066898-HM066901, HM635396
F81
HKY
61
Pustovrh et al. (2010)
HM635402, HM635405-HM635409, HM635411-HM635412
F81
HKY
61
Pustovrh et al. (2010)
HM066916-HM066925, HM635415-HM635418
TPM2uf+G
GTR+G
52
Pustovrh et al. (2010)
HM066928-HM066934, HM635420
F81
HKY+I
61
Ryynanen and Primmer (2006)
HM066734, HM066737, HM635469
F81
F81+I
61
Ryynanen and Primmer (2006)
HM066740-HM066745, HM635458-HM635460
F81
HKY+I
61
Ryynanen and Primmer (2006)
HM066759, HM066761-HM066765, HM635472-HM635473
JC
JC+I
56
Ryynanen and Primmer (2006)
HM066769, HM066781-HM066784, HM635474-HM635476
JC+G
GTR+G
60
Sušnik et al. (2008)
HM635423, HM635425-HM635430, HM635432-HM635433
F81
F81
60
Sušnik et al. (2008)
HM635434-HM635435, HM635437, HM635440-HM635444, HM635446
K80
K80+I
52
Sušnik et al. (2008)
HM635451, HM635455-HM635457
F81
HKY
60
Sušnik et al. (2008)
HM635463-HM635467, HM635469-HM635470
JC
HKY
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GP34
F: 5’-AGGTGGTAGAATGGACAAGTCA-3’ R: 5’-AGGGAGGGAGGGATAAGAG-3’ F: 5’-GACTGGGATTACTAGATATGGG-3’ R: 5’-GAAACCGGAATGGATAG-3’ F: 5’-GATTCCTTGGTTGGACTTGATTGTT-3’ R: 5’-AAGAGCTCCAGTGGTCCGATA-3’ F: 5’-CGGAGCAGAGGGAGTTGAGT-3’ R: 5’-ACCTGCACACTAATAAAACAACAAA-3’ F: 5’-AAGGGCACATATAAACGAACA-3’ R: 5’-ATGGTGATGAAGAAGGTGGTTATGG-3’ F: 5’-AGACCAGGAGGGTATTACTAACACA-3’ R: 5’-AAGTTTCAGTACTTGGCATTGAT-3’ F: 5’-CTTTGAACTGTTTGGCATGTAGG-3’ R: 5’-AACACCACAGGCCACTATTT-3’ F: 5’-GTATGTGCCATATTTCTATGCTT-3’ R: 5’-CAGAATGTCAGCCAAACTCC-3’ F: 5’-CAAAGAAATATGGCAAAC-3’ R: 5’-AGCCATTCATTGTTGATAAT-3’ F: 5’-TATCAGGTCATCCCAATGTCAAG-3’ R: 5’-ATAATCAATGTACATGCGAAAA-3’ F: 5’-GGCACTCCCTGTATATAGCTTC-3’ R: 5’-AGAGCTCACGGTTTTACCA-3’ F: 5’-TGGGTACTAGCTAGCTATATATGA-3’ R: 5’-CCAAGTGATTGTGTTGATATGGT-3’ F: 5’-CAGCATCCAGCCCAATATCAT-3’ R: 5’-CACTGCCCACTTGTTTGTGTTAGA-3’ F: 5’-TTCTATATTGTGCAGTTAGCTGT-3’ R: 5’-TACCTTTACCTTATGGGA-3’ F: 5’-TCAATCCCTTGATAGTTGTCTTTG-3’ R: 5’-GGGACACCCTGATTTTAATTGTAG-3’ F: 5’-CACTGCAGTAAGGATTATCTGCTT-3’ R: 5’-GAGGAAGAGCGAGGAAGATATAAAG-3’ F: 5’-GTGAGACTATCCTTTATTCCAACG-3’ R: 5’-TGTGGTTGGGTAAACAACAGTAGA-3’ F: 5’-CTTTTCATTGAATTGCTTCTCACT-3’ R: 5’-GCAGTAGAAGGAAGAAAGGTTCAT-3’ F: 5’-CXTATGAATAYCCTAGTACTACC-3’ R: 5’-CCRCAGCACARCGTGGTGATCATG-3’ F: 5’-CATACAACTGGGACTTTGGTG-3’ R: 5’-TTACTGTAGCTCCCTGTGTGG-3’ F: 5’-TGGCCCGTTGAATCCATATAAAG-3’ R: 5’-ACTGTGAAACACTAAGCTCTCCA-3’ F: 5’-CCAGTCTCCTTTTACCCCTACT-3’ R: 5’-CTTGACGGCCACCAGTTT-3’
Ta (°C)
98%(342/347) homology to S. salar interferon-inducible GTPase b and interferon-inducible GTPase_a genes [GenBank:EU221179.1]. 86%(274/315) homology to S. salar microsatellite Hae018 sequence [GenBank:AF271480.1]. 88%(174/197) homology to O. mykiss conserved noncoding element 1117-1131-i genomic sequences [GenBank:FJ356120.1].
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Fig. 1. Phylogenetic tree based upon Bayesian inference analysis of combined nuclear DNA data from Salmo trutta spp. samples inhabiting the Adriatic drainage. Support values refer to Bayesian inference (top) and maximum-likelihood (bottom) methods. Values < 50 are marked with ‘‘/’’. (A) Phylogenetic tree based on all 22 nuclear loci; (B) phylogenetic tree based on 18 loci; four coding loci (TFG-beta, tnfa, TF, RH), potentially under selection pressure, were removed from the analysis.
Mediterranean river basin—form a monophyletic group. This observation is concordant with the current marble trout classification, which considers both populations as conspecific. On the other hand, our results are in marked contrast to the notion of polyphyletic origin of marble trout, as inferred from the occurrence of unrelated CR mtDNA haplotypes (Snoj et al., 2010). Interestingly, mitochondrial and nDNA results were congruent in S. dentex, whose embedment either in S. marmoratus or in S. trutta (Fig. 1) clearly indicated its polyphyletic origin as proposed previously by Snoj et al. (2010). Discordance between mtDNA trees and nDNA phylogeny is a common phenomenon and generally explained by incomplete lineage sorting (incomplete and stochastic sorting of ancestral polymorphisms) or introgression between related species or differentiated lineages (introgressive hybridization; reviewed in Maddison, 1997).
Incomplete lineage sorting may represent a problem for organismal phylogeny if the time needed for haplotypes within a lineage to coalesce is greater than the time between successive speciation events (Page and Holmes, 2000). This phenomenon may be well applied to trouts of the genus Salmo, including S. trutta and S. marmoratus, which are known to have diverged very rapidly in the Pleistocene (Bernatchez, 2001). Moreover, both population groups of marble trout appear to be fixed, either for a single haplotype (as in the Neretva and Skadar basins; Snoj et al., 2010) or for a group of highly related haplotypes (MA haplotypes that probably emerged from a single plesiomorphic haplotype during marble trout evolution), which in brown trout of the Adriatic drainage represent only part of a much larger genetic polymorphism. Limited haplotype polymorphism found in marble trout populations is congruent with evidence of severe Pleistocene bottlenecks of marble trout populations and restricted habitat availability (Bernatchez, 2001;
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Fig. 1 (continued)
Snoj et al., 2010). Introgression, as the other option of mitochondrial–nuclear discordance, could have hypothetically also played a role in this case. Infiltration of mitochondrial genomes of one species into the original mitochondrial genome pool of another has been identified in several organisms (see Renoult et al., 2009, for examples). The most extreme, and very rarely detected, case of introgression is replacement of complete original mtDNA of an introgressed species with that of another, termed mtDNA capture. For example, a subspecies of softmouth trout (Salmo obtusirostrris salonitana) from the River Jadro (Croatia) has a mtDNA haplotype that is indicative of brown trout. However, all nuclear genes that have been examined appear to be characteristic of softmouth trout (Sušnik et al., 2007). Mitochondrial DNA capture could theoretically be attributed also to marble trout. In such a scenario, mtDNA capture of the incipient marble trout population (i.e., when northern and southern populations were last in contact) and subsequent divergence of this population into two is unlikely considering the substantial genetic distance between them (1%; Snoj et al.,
2009); thus extinction of the original mitochondrial genome of marble trout and its replacement with that of brown trout must have occurred twice in two geographically separated populations of the same species. Hypothetically, the effects of genetic drift and other factors, responsible for reduced fitness of marble trout females compared to introgressing brown trout females, may explain this scenario mtDNA capture. However, given the relatively non-parsimonious explanation of this scenario it seems more realistic that the mitochondrial–nuclear discordance found between northern and southern populations of marble trout results from incomplete lineage sorting. Although nuclear DNA data reveal monophylogeny of northern and southern populations of marble trout and indicate conspecificity, we have here shown that certain genetic differences do exist between them. As these populations have most probably been separated for a long geological time (at least since Würm glaciation), there is a possibility that they have developed locally adapted phenotypic traits as a result of selective differences. However, since no
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morphological and ecological analyses have been undertaken on both populations, this suggestion is impossible to prove. Acknowledgment We would like to that D. Jesenšek, I. Bogut, B. Glamuzina, N. Pojskic´, D. Mrdak, J. Schöffmann and R. Šanda for providing samples. Special thanks go to I. Wilson for editing and correcting the manuscript.
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