Extreme Genetic Differentiation Among the Remnant Populations of Marble Trout (Salmo Marmoratus) in Slovenia

Molecular Ecology (2002) 11, 2711–2716

SHORTBlackwell Science, Ltd COMMUNICATION Extreme genetic differentiation among the remnant populations of marble trout (Salmo marmoratus) in Slovenia

L. FUMAGALLI,* A. SNOJ,† D. JESENSEK,‡ F. BALLOUX,*§ T. JUG,† O. DURON,¶ F. BROSSIER,¶ A. J. CRIVELLI** and P. BERREBI¶ *Institut d’Ecologie, Laboratoire de Biologie de la Conservation, Bâtiment de Biologie, 1015 Lausanne, Switzerland; †Department of Animal Science, University of Ljubljana, Dom9ale, Slovenia; ‡Anglers’ Society of Tolmin, Most na So8i, Slovenia; §I.C.A.P.B., University of Edinburgh, Edinburgh, UK; ¶UMR 5119 ‘Ecosystèmes Lagunaires’, Université Montpellier II, Montpellier, France, **Station Biologique de la Tour du Valat, Arles, France

Abstract Populations of the marble trout (Salmo marmoratus) have declined critically due to introgression by brown trout (Salmo trutta) strains. In order to define strategies for long-term conservation, we examined the genetic structure of the 8 known pure populations using 15 microsatellite loci. The analyses reveal extraordinarily strong genetic differentiation among populations separated by < 15 km, and extremely low levels of intrapopulation genetic variability. As natural recolonization seems highly unlikely, appropriate management and conservation strategies should comprise the reintroduction of pure populations from mixed stocks (translocation) to avoid further loss of genetic diversity. Keywords: conservation genetics, hybridization, introgression, microsatellites, population structure, Salmo marmoratus Received 21 May 2002; revision received 5 September 2002; accepted 5 September 2002

former Yugoslavia and Albania), and is characterized by a Introduction particular marbled colour and a very large size (up to 25 Hybridization and introgression, induced by anthropogenic kg in weight). Molecular studies [mitochondrial DNA activities such as the introduction of nonindigenous taxa (mtDNA) sequence data] have shown that within the Po and habitat modification, have led to the extinction of basin (Italy), all marmoratus populations form a distinct many native populations and species. Subsequent genetic evolutionary lineage among the brown trout (Salmo trutta) mixing can lead to a loss of genotypically distinct popu- population complex. Before massive stockings in recent lations, in particular when hybrids are fertile and mate years, brown and marble trout were parapatric, behaving both among themselves and with parental (nonhybrid) as true species (Giuffra et al. 1994, 1996). Hybridization individuals, resulting in the creation of hybrid swarms (see between marble and brown trout has been so extensive that Leary et al. 1995). The consequences of these phenomena nowadays morphologically typical marmoratus forms are can be particularly harmful for rare, threatened species very rare, and the species is considered to be one of the coming into contact with more abundant ones. In fish, most endangered freshwater fish of the Adriatic basin hybridization is especially common because of the exten- (Pov6 1995; Delling et al. 2000). Within the Slovenian dis- sive introduction of nonnative species for angling or tribution, introduction of nonindigenous brown trout started commercial purposes, and because interspecific hybrids in 1906 and hybrids now dominate most rivers, with the are often fertile (Rhymer & Simberloff 1996; Allendorf exception of secluded headwater streams of the So5a River et al. 2001). basin separated from downstream hybrid populations by The marble trout (Salmo marmoratus) has a restricted impenetrable natural barriers (Pov6 et al. 1996). Recent native range within the Adriatic basin (northern Italy, allozyme and mtDNA investigations revealed within such isolated streams the existence of eight genetically pure Correspondence: Luca Fumagalli. Fax: 41 21 692 4165; E-mail: marmoratus populations, according to diagnostic loci [email protected] (Berrebi et al. 2000; Plesko & Budihna 2000; Snoj et al. 2000).

2712 L. FUMAGALLI ET AL.

Fig. 1 Map showing sampling localities of Salmo marmoratus within the So5a River basin in Slovenia. The So5a River system, representing the single Slovenian drainage inhabited by S. marmoratus, lies in the northwestern part of Slovenia, and belongs to the Adriatic basin. Overall it covers ≈ 2300 km2, but the study area where tributaries of the So5a River have been shown to still host pure S. marmoratus populations (see text for further details) covers ≈ 900 km2. All sampling locations are separated downstream from the hybridization zone by several impassable barriers (waterfalls) preventing upstream immigration, and have presumably never been influenced by any fishing and stocking activities. Note that the natural Predelica population went extinct in 2000 due to an important landslide, however, a hatchery-reared population derived from wild Predelica still exists.

The isolated nature of these populations makes them Polymerase chain reactions (PCR) were performed in 10 µL extremely vulnerable to stochastic factors, and the pro- reaction mixtures containing 25 ng DNA, 0.5 µm of each µ × bability of spontaneous colonization of new habitats seems primer, 1.5–2 mm MgCl2, 200 m each dNTP, 1 reaction extremely reduced. Furthermore, the small size of the buffer and 0.25 units of Taq DNA polymerase (Applied remnant populations could also decrease their genetic Biosystems, Gibco BRL or QIAGEN). The PCR profile diversity, potentially leading to fixation of deleterious consisted of 30 iterations of 95 °C for 30 s, 48 °C (locus alleles and to a lack of evolvability in response to environ- BFRO 003), 50 °C (BS131, MST85), 55 °C (Ssa171, Str58, mental changes (Frankham et al. 2002). As the applicability Str591), 58 °C (Str24, MST15, MST73, MST543ae) or 60 °C of conservation strategies critically depends on prior (T3-13, Str60, BFRO 001, BFRO 002, Ssa197) for 30 s, 72 °C knowledge of both within-population genetic diversities for 30 s. Amplified products were genotyped either with and between-population differentiation, we performed ABI PRISM 377 DNA Sequencer or ABI Prism 310 using a genetic survey of all known pure populations of the genescan analysis 2.1 software (Applied Biosystems). marbled trout using 15 microsatellites. Gene diversities comprising observed (HO), expected within-subpopulation (HS) and expected overall heterozygosities (H ) were estimated following Nei & Chesser Materials and Methods T (1983). Genotypic disequilibrium between loci in each A total of 159 samples of Salmo marmoratus were obtained sample and deviations from Hardy–Weinberg equilibrium from the headwaters of 8 tributaries of the So5a River by (HWE) within samples were tested based on 14 560 means of electrofishing (Fig. 1). DNA was extracted using permutations and 2000 randomizations, respectively. the DNeasy Tissue kit (QIAGEN), following supplier’s Wright’s fixation indices for within-subpopulation devi- instructions. Twelve microsatellite loci isolated and ation from random mating (FIS), as well as pairwise sub- characterized from other salmonid species (Str15, Str60, population differentiation (FST), were estimated following Str73: Estoup et al. 1993; Str24: Poteaux 1995; MST85, Weir & Cockerham (1984). Deviation from random mating

MST591: Presa & Guyomard 1996; Ssa171, Ssa197: O’Reilly within populations (FIS) per locus and sample were com- et al. 1996; BS131, T3-13, MST543ae: Estoup et al. 1998; puted with a bootstrap procedure (2000 randomizations). Strutta-58: Poteaux et al. 1999) and three from S. marm- Statistical support for pairwise population differentiation oratus (BFRO 001: Snoj et al. 1997; BFRO 002: SuSnik et al. was obtained through exact G-tests on allelic frequencies as 1997; BFRO 003: Snoj et al. 2000) were amplified and scored. described by Goudet et al. (1996) with 2000 randomizations.

GENETIC DIFFERENTIATION IN THE MARBLE TROUT 2713

Table 1 Genetic variability as determined Proportion of from 14 microsatellite loci for eight rem- polymorphic Mean no. of nant populations of Salmo marmoratus in N loci alleles/locus HO HS Slovenia

Predelica 22 0.57 2.00 0.233 0.219 Zadlascica 20 0.43 1.57 0.149 0.162 Lipovscek 20 0.36 1.50 0.191 0.156 Huda Grapa 20 0.14 1.14 0.046 0.039 Sevnica 20 0.29 1.29 0.097 0.088 Studenc 20 0.29 1.71 0.136 0.150 Trebuscica 19 0.29 1.43 0.103 0.094 U. Idrijca 18 0.36 1.57 0.119 0.155 Overall 159 0.64 3.50 0.135 0.133

Reported significance levels are after strict Bonferroni populations are clearly discriminated: Huda Grapa and corrections based on the indicative adjusted P-value. All ZadlaS5ica placed at the positive end of the first axis are in summary statistics and tests mentioned above have been fact discriminated by the third axis (not shown, inertia computed using fstat Version 2.9.3.2 (Goudet 1995). We 12.9%), and successively LipovS5ek, Predelica and a group further performed a factorial correspondence analysis of four populations at the negative end of the first axis and (CA) using genetix software (Belkhir et al. 1998). along the second axis. This last group of populations (all located within the Idrijca drainage) is composed of largely overlapping individuals. Results Tests for HWE indicated that locus Str24 was characterized Discussion by a significant heterozygote deficiency (P = 0.0005), suggesting the presence of null alleles, and was removed Our study reveals extraordinarily strong genetic differ- from further analyses. All other loci tested were at HWE entiation among populations on a restricted geographical and in genotypic equilibrium (P < 0.05). For the remaining range. For instance, populations of LipovS5ek and Huda 14 microsatellite loci, the number of alleles per locus Grapa, separated by a waterway of < 15 km, showed an ranged from 1 (5 loci were homozygous and monomorphic average pairwise FST value of 0.79, the extreme level being across the 8 populations) to 12 (average = 3.5), with a total found between TrebuS5ica and Huda Grapa (0.88), of 49 alleles across 14 loci. Expected heterozygosities per separated by 28.5 km (Fig. 1). These extremely high FST locus within samples (HS) ranged from 0 to 0.51, with an estimates exceed by far the highest values reported average of 0.13, whereas expected overall heterozygosity between fish populations at a micro- or macrogeographical

(HT) averaged 0.36 (range per locus: 0–0.85). Observed scale and are to the best of our knowledge the highest heterozygosity (HO) values varied from 0 to 0.56, with an differentiation ever recorded with microsatellites at an average of 0.135. We found 16 population-specific (private) intraspecific level. For example, levels of differentiation alleles (representing 33% of the total number of alleles reported in other salmonids on a larger geographical sampled). Genetic diversities for the eight populations of scale are much lower (average FST value of 0.33 in Taylor Salmo marmoratus are summarized in Table 1. et al. 2001; 0.36 in Koskinen et al. 2001; 0.37 in Angers & There was evidence for significant deviation from Bernatchez 1998). The remnant marble trout populations random mating in none of the eight analysed populations are thus completely isolated from each other and from the − (global FIS = 0.014), suggesting the absence of a within- main river system as immigration from downstream is sample substructure. However, between-population precluded by impassable barriers. Even in the complete genetic subdivision was very strong pointing to an extreme absence of genetic exchanges, FST values are generally value of FST = 0.66 (P < 0.001). Furthermore, all pairwise much lower for microsatellites because of their high population differentiation estimates (pairwise FST) were mutation rates increasing within-population genetic high, ranging from 0.31 to 0.88, with all being significant variability, and thus avoiding within-population fixa- (P < 0.01) after strict Bonferroni correction for multiple tion of alleles (Balloux & Lugon-Moulin 2002). However, tests. in these populations, microsatellite allele numbers and Figure 2 illustrates the position of the eight populations heterozygosity estimates were remarkably low compared on the main factorial plane described by the two main with genetic variation at microsatellite loci commonly axes of the CA analysis. In this diagram, five groups of observed in other freshwater fish species (DeWoody &

Fig. 2 Projection diagram of the first two axes from the factorial analysis based on the genotypes of 14 microsatellite loci from 8 marble trout populations from the So5a River system, Slovenia. The first axis accounts for 15.9% of the total inertia contained in the dataset and the second axis for 14.3% (cumulative inertia is thus 30% with 47 axes). Without structure, these axes should gather only ≈ 4%.

Avise 2000). This implies a strong impact of local genetic Bavec & Tulaczyk 2002). Altogether, these complex and drift and points to low effective population sizes due to the recurrent events of river isolation and interconnection may absence of immigration. also have contributed to the particular situation of local Patterns of population structure reflect both current and genetic pattern, by separating and/or maintaining some of historical levels of gene flow. Despite the extreme genetic the pure marmoratus populations in contact. Further structuration demonstrated by the microsatellite analysis, evidence suggesting distinct drainage patterns stems from the presence of a single marmoratus mtDNA control region the distribution of other species. For instance, the stone haplotype (Ma1) in Northern Italy (Giuffra et al. 1994) and crayfish (Austropotamobius torrentium) inhabits the Danu- in three pure Slovenian populations analysed so far (Snoj bian system but also the upper part of the Idrijca and Baca et al. 2000) indicates relatively recent connectivity prior to drainages, whereas the rest of the Idrijca drainage and the the fragmentation of habitats. Several lines of evidence So5a River are occupied by the white-clawed crayfish support the idea that the pure populations within the (A. pallipes) (Budihna 2001). Idrijca drainage (Sevnica, Studenc, TrebuS5ica and Upper Since the discovery of new pure populations in Slovenia Idrijca; see Fig. 1) belonged to an independent river system. seems unlikely (Crivelli et al. 2000), management efforts They clustered together in the CA analysis (Fig. 2) and should focus on maintaining and expanding these remain- generally exhibit lower reciprocal levels of genetic struc- ing populations. Their geographical isolation protects turing (mean = 0.49). Geological events during the Pliocene them from natural hybridization, hence the presence of and climate fluctuations during the Pleistocene had a hybrids within the main river does not represent a threat substantial impact on the evolution of the Idrijca drainage. per se for their survival. Similarly, excessive fishing pres- Originally, its upper part flowed into the Danubian river sure or stocking with domestic brown trout strains does system from northwest to southeast. Later, by the pro- not seem particularly worrying, as access to most of these cess of retrograde erosion, it became connected with what streams (often located within narrow valleys and canyons) is today the modern Idrijca drainage, which flows in is generally very arduous, and stocking has been banned the opposite direction southeast to northwest (Jane6 et al. legally since 1996. Nevertheless, it is important to give 1997). During the climate fluctuations of Pleistocene, the highest protection priority to these genetically distinct Idrijca drainage has never been covered by ice, in contrast populations (management units within a single evolution- to the upper So5a Valley, and some interconnections with ary significant unit, sensu Moritz 1994), which are among the Danubian river system may have occurred (Melik 1963; the very few nonintrogressed populations documented

© 2002 Blackwell Science Ltd, Molecular Ecology, 11, 2711–2716 GENETIC DIFFERENTIATION IN THE MARBLE TROUT 2715 within the entire species’ range. As the populations are Balloux F, Lugon-Moulin N (2002) The estimation of population extremely isolated and small, and because there is no differentiation with microsatellite markers. Molecular Ecology, potential of spontaneous colonization of new habitats 11, 155–165. Bavec M, Tulaczyk SM (2002) New simple mathematical model to through dispersal, the risk of local extinction due to natural help evaluating the extent of the late-Quarternary valley glacier factors (e.g. diseases, catastrophes) is very high. In par- in the Upper So5a region. Geologija, 45, 255–268. ticular, the So5a River area is tectonically very active and Belkhir K, Borsa P, Goudet J, Chikhi L, Bonhomme F (1998) frequently affected by severe landslides. 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