Historical demography of brown trout (Salmo trutta) in the Adriatic drainage including the putative S. letnica endemic to Lake Ohrid

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Abstract

We explore the historical demography of the Adriatic lineage of brown trout and more explicitly the colonization and phylogenetic placement of Ohrid trout, based on variation at 12 microsatellite loci and the mtDNA control region. All Adriatic basin haplotypes reside in derived positions in a network that represents the entire lineage. The central presumably most ancestral haplotype in this network is restricted to the Iberian Peninsula, where it is very common, supporting a Western Mediterranean origin for the lineage. The expansion statistic R2, Bayesian based estimates of demographic parameters, and star-like genealogies support expansions on several geographic scales, whereas application of pairwise mismatch analysis was somewhat ambiguous. The estimated time since expansion (155,000 years ago) for the Adriatic lineage was supported by a narrow confidence interval compared to previous studies. Based on microsatellite and mtDNA sequence variation, the endemic Ohrid trout represents a monophyletic lineage isolated from other Adriatic basin populations, but nonetheless most likely evolving from within the Adriatic lineage of brown trout. Our results do not support the existence of population structuring within Lake Ohrid, even though samples included two putative intra-lacustrine forms. In the interests of protecting the unique biodiversity of this ancient ecosystem, we recommend retaining the taxonomic epithet Salmo letnica for the endemic Ohrid trout.

Introduction

Drawing inferences on the historical demography of an organism based on its extant genetic architecture has become a major component of evolutionary research. Historical demography has been increasingly integrated into our conception of the mechanisms of speciation, estimates of divergence times between populations, and assessment of the differing contributions that gene flow, genetic drift, natural selection, and mutation have on the viability of organismal lineages in space and time. A range of analytical techniques is now available aimed at drawing inferences on historical demography from the genetic screening of mitochondrial (Rogers and Harpending, 1992), microsatellite (Kimmel et al., 1998, Reich and Goldstein, 1998) and single nucleotide polymorphism (SNP; Brumfield et al., 2003) loci, as well as combinations of different markers (Hey and Nielsen, 2004, Won and Hey, 2005). Phylogeographic researchers have been particularly active in drawing links between historical demography and genealogy (Avise, 2000, Avise, 2004), as well as demonstrating the effects of non-biological factors, such as climatic oscillations, on extant genetic structure. Indeed, phylogeography has been dominated by studies of temperate organisms, where inferences involving present spatial genetic structure are drawn together with knowledge or speculation on the effects of Pleistocene climatic oscillations (e.g., Hewitt, 2000, Randi, 2006).

In particular, studies of freshwater fishes are obliged to include consideration of the paleo-hydrological dynamics caused by glacial advance and retreat, which have provided intermittent or long-term refugia, trans-basin migratory corridors and isolating barriers. Bernatchez et al. (1992) initiated a flurry of such studies in brown trout, Salmo trutta, through the description of five mitochondrial lineages, four of which were presumed to have originated in allopatry from single major basins (Atlantic, Mediterranean, Adriatic, and Danube), and a fifth prescribed to a unique divergent phenotype (marble trout) within the Adriatic basin. Subsequent studies on brown trout have increased both the genetic and geographic resolution of this basic phylogeographic model, demonstrating both natural (e.g., Estoup et al., 2000, Weiss et al., 2001) and anthropogenic (e.g., Aurelle et al., 2002, Hansen, 2002, Jug et al., 2005) admixture of these five lineages in particular regions.

Bernatchez (2001) provided a summary analysis of brown trout phylogeography, which included assessment of historical demography and dating of the expansion times for each of the five major mtDNA lineages. Despite the work’s comprehensiveness, several studies (e.g., Suárez et al., 2001, Cortey et al., 2004) have brought further complexity to the model and offer new hypotheses concerning the origins of the major mitochondrial lineages. It is especially within so-called southern refugial regions that researchers continue to uncover previously underappreciated structural complexity of brown trout lineages, similar to the patterns found across a broad array of European taxa (Weiss and Ferrand, 2006). For example, the so-called Adriatic lineage is now known to occur across a large area, from Iberia in the west, across the Mediterranean, to as far east as Eastern Turkey, including the Persian Gulf basin (Apostolidis et al., 1997, Bernatchez, 2001, Cortey et al., 2004, Bardakci et al., 2006, Marić et al., 2006). Considering the high frequency, distribution and diversity of Adriatic haplotypes in Iberia, the hypothesis that this lineage arose in the eastern Mediterranean region (Bernatchez, 2001) is unlikely, and thus a western European origin has been proposed (Cortey et al., 2004). Ironically, only a few samples from the Adriatic basin, the namesake of the lineage, were included in these analyses.

The Adriatic drainage may in fact have served as a unique, or even primary, centre for radiation of the genus Salmo as it harbours not only a number of different morphological forms (e.g., Heckel, 1851, Karaman, 1926) but also two endemic species, S. obtusirostris and S. ohridanus, which are phylogenetically placed between S. trutta and S. salar (Snoj et al., 2002, Sušnik et al., 2006). While the Adriatic drainage was not directly affected by Pleistocene glaciers, it was clearly influenced by the large drops in sea-level (100–120 m; Fairbanks, 1989) that took place during glacial maxima as well as other alterations in the paleo-hydrological landscape, at times providing communication between now isolated habitats. The region is also remarkable for the existence of Europe’s oldest permanent body of freshwater, Lake Ohrid (shared between the former Yugoslav Republic of Macedonia and Albania), which originated from the draining of the Paratethys Sea (Hsü, 1978) during the Tertiary period some four to ten million years ago as a result of tectonic shifts (Banarescu, 1991). The level of endemism in Lake Ohrid is exceptionally high. Ten of the 17 fish taxa and 34 of the 38 mollusca taxa inhabiting the lake are thought to be endemic (Stanković, 1960). Putative young pairs of sympatric taxa have been observed among salmonids in many lacustrine systems (e.g., Ferguson and Mason, 1981, Hynes et al., 1996). Lake Ohrid appears to be no exception as within the genus Salmo at least five endemic taxa have been described for the lake: four distinct forms of the so-called Salmo letnica (typicus and balcanicus, both winter spawners; aestivalis, a summer spawner; and lumi, a river spawner) and the highly distinct S. ohridanus, first confirmed as a member of the genus Salmo by Phillips et al. (2000). The taxonomic status of S. letnica, however, is not widely accepted. Both Bernatchez et al. (1992) and Apostolidis et al. (1997) refer to samples from Lake Ohrid as S. trutta, and both these studies as well as the recent work of Sell and Spirkovski (2004) and Sušnik et al. (2006) show that trout from Lake Ohrid are clearly assigned to the Adriatic clade of brown trout, based on mtDNA sequences. Using a direct English translation of the Slavic local common name of trout in Lake Ohrid, we refer to all forms of S. letnica as “Ohrid trout” in this manuscript, noting that “Ohrid trout” is a synonym of the “Lake Ohrid brown trout” described in Sušnik et al. (2006).

Considering the Adriatic basin’s high level of diversity and endemism, inferences concerning the biogeography and historical demography of the Adriatic lineage of brown trout appear incomplete without sampling an adequate number of populations in the basin. Therefore, the following work attempts to: (1) explore the historical demography of the Adriatic lineage, including for the first time comprehensive coverage of the Adriatic basin; (2) explicitly evaluate the likely source, time since colonization and level of isolation of Ohrid trout; (3) assess the diversity and potential differentiation of winter and summer forms of Ohrid trout, especially in consideration of the mtDNA data reported by Sell and Spirkovski (2004). To accomplish these goals, for each sample we screened allelic diversity at 12 microsatellite loci as well as sequence variation of both the 5′ and 3′ end of the mtDNA control region (mtDNA CR). Data were subject to demographic and phylogenetic analysis and, for the mtDNA control region, integrated with existing data on the Adriatic lineage of brown trout.

Section snippets

Samples and DNA extraction

Two hundred and sixty-four trout samples from the Adriatic drainage were included in the analyses. Ohrid trout (N = 144) were purchased from local fishermen and stemmed from seven different locations around the lake, including two putative taxa (reviewed in Stanković, 1960), the winter (S. letnica typicus, N = 70) and summer (S. letnica aestivalis, N = 74) spawning forms. The remaining samples (N = 120) originating from the rivers Neretva, Zeta, Morača, and Cijevna (Table 1; Fig. 1) were caught by

Microsatellites

All microsatellite loci were polymorphic in at least one population. Uncorrected mean number of alleles per locus ranged from four (Str60) to 82 (Ssa-D71), and per population from 5.7 (River Cijevna) to 11.2 (Ohrid trout; Table 2). Mean allelic diversity adjusted for sample size (allelic richness) ranged from 5.4 (River Cijevna) to 8.8 (Ohrid trout; Table 2). Ohrid trout samples were the most polymorphic among the populations analyzed, with an average of 11.5 alleles per locus across all lake

Historical population demography of the Adriatic clade of brown trout

Our analysis of the Adriatic lineage of brown trout includes for the first time comprehensive coverage of the Adriatic basin, including Ohrid trout, which has been considered a distinct species (Salmo letnica; Karaman, 1924, Karaman, 1926, Stanković, 1960). This effort demonstrates that all haplotypes found in the Adriatic basin, including those characterizing Ohrid trout, appear in derived positions with respect to the central and presumably ancestral haplotype (ADcs1) in the lineage (Fig. 3).

Acknowledgments

Research was supported by a Marie Curie postdoctoral fellowship (MEIF-CT-2003-501446) to the first author, as well as the Slovenian Ministry of Education, Science and Sport (Grant No. BI-MK/04-05-006). We would like to thank Prof. Nikola Hristovski from Sveti Kiril i Metodij University, Bitola, and Stojmir Stojanovski from the Hydrobiological Institute, Ohrid, for organizing sampling campaigns. Albanian samples were provided by Mihallaq and Çelnika Shegani and Defrim Lloga with support from the

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