Is genetic variability so important? Non-native salmonids in South America

Journal of Fish Biology (2007) 71 (Supplement D), 136 147 doi:10.1111/j.1095-8649.2007.01674.x, available online at http://www.blackwell-synergy.com Is genetic variability so important? Non-native salmonids in South America A. G. VALIENTE*, F. JUANES, P. NUN EZ AND E. GARCIA-VAZQUEZ* *Departamento Biologia Funcional, Universidad de Oviedo, C/ Julian Claveria s/n, 33006-Oviedo, Spain, Department of Natural Resources Conservation, University of Massachusetts, Amherst, MA 01003, U.S.A. and Centro de Biologia Aplicada del Neuquen, 8371 Junin de los Andes, Neuquen, Argentina Three salmonid species introduced in Patagonian national parks in Argentine have experienced different degrees of expansion. Atlantic salmon Salmo salar is restricted to a few river-lake systems and its populations have been declining over recent years. Both rainbow Oncorhynchus mykiss and brown trout Salmo trutta populations have expanded from their introduction sites and now occupy a wide range of freshwater ecosystems. Genetic variation at the same neutral markers (microsatellite loci) was examined for different populations of the three species acclimatized to the same areas, and compared with that of native populations. Founder effects denoted as reduced variability and great differentiation with respect to the native populations were detected. Significant reduction in variability has not been an obstacle for successful adaptation of rainbow and brown trout, indicating that genetic variability per se cannot be claimed as the reason for their different outcomes in the new habitats. Journal compilation # 2007 The Fisheries Society of the British Isles Key words: exotic species; founder effect; genetic variation; Patagonia; salmonids. INTRODUCTION Loss of genetic diversity increases extinction risk in populations of naturally outbreeding species (Frankham, 2005). Thus, if hybridization with local species is excluded, accidental or deliberate introduction of a few exotic individuals in a foreign ecosystem might not be an important concern for conservation, because their chance of successfully establishing new stable populations would be very low due to low genetic variation. This is not, however, always true. It has been suggested that demography (life-history traits and social structure) is more important than genetics in determining successful adaptation of populations (Lande, 1988). For example, some invaders exhibit low genetic variability (Tsutsui et al., 2000; Sax et al., 2005). The action of a few genes and epistatic interactions could facilitate invasion success (Lee, 2002), rather than general variability or heterozygosity of the genome. Fish invasions (foreign species Author to whom correspondence should be addressed. Tel.: þ34 985102726, þ34 985103076; fax: þ34 985103534; email: egv@fq.uniovi.es 136 Journal compilation # 2007 The Fisheries Society of the British Isles

ADAPTATION OF NON-NATIVE SALMONIDS IN PATAGONIA 137 adapting to new environments) are being increasingly documented in different parts of the world, from New Zealand (Townsend, 2003) to North America (Gido et al., 2004). Invasions may represent good case studies for assessing the relative weight of genetic variability in population adaptation, and thereby aiding in their conservation. Salmonids adapted to South American ecosystems may represent a valuable model for identification of processes leading to adaptation (Riva Rossi et al., 2004). They also represent a concern from the point of view of conservation of native biodiversity, as some species have undergone an expansive growth and have colonized ecosystems, where they have been reported to endanger native species (Baig un & Ferriz, 2003). For example, brown trout Salmo trutta L. is considered a potential invasive species (Townsend, 2003). One century ago, salmonids were first introduced to Argentina. They rapidly acclimatized and adapted to new ecosystems, developing self-sustaining populations of local economic importance. Brown trout embryos were transplanted from Chile to Patagonian national parks between 1915 and 1936 (Del Valle & Nun ez, 1990). The stock initially introduced to Chile in 1905 was imported from Hamburg, Germany (Del Valle & Nun ez, 1990). Atlantic salmon Salmo salar L. embryos were first introduced to Argentina in 1904. They originated from landlocked Atlantic salmon from Sebago Lake (U.S.A.; Tulian, 1908). Argentinean Atlantic salmon populations are restricted to river and lake systems near the Andes Mountain range; migration to the sea is impossible due to impassable dams and reservoirs constructed in all basins during the past century. Finally, rainbow trout Oncorhynchus mykiss (Walbaum) have adapted to different river and lake systems throughout Patagonia since their introduction at the beginning of the 20th century, becoming one of the most conspicuous freshwater fish species in the region (Pascual et al., 2002). Based on genetic analysis, their origin was traced to the McCloud River in California, U.S.A. (Riva Rossi et al., 2004). The Patagonian region is experiencing fast development that endangers pristine environments due to mining (Arribere et al., 2003) and habitat fragmentation by hydroelectric plants and damming (Mugetti et al., 2004). A series of reservoirs was constructed in Neuquen between the 1970s and late 1990s (Fig. 1). Although salmonids are extremely vulnerable to habitat fragmentation due to highly structured populations (Neraas & Spruell, 2001; Morita & Yokota, 2002), both brown and rainbow trout populations persist without any apparent decline in this intensely dammed Patagonian region, where they are also subjected to sport angling. Although Argentine fishing regulations allow a catch of one salmonid per angler per day, Atlantic salmon is a protected species in the Argentinean Patagonia (Official Normative of Sport Fisheries, Reglamento General de Pesca). Only catch-and-release is allowed for Atlantic salmon, and all releases must be in the same area of capture. This regulation was implemented based on scarcity or decline of the species in Patagonia, in contrast to other salmonids. In the Traful Lake (Nahuel Huapi National Park, Neuquen, Argentina), the three species have coexisted for decades but Atlantic salmon have declined since 1983 after the construction of the Alicurá Reservoir (Embalse Alicurá; see Fig. 1). Both rainbow and brown trout have not exhibited signs of depletion and angling fisheries continue for these species as in the rest of the region.

138 A. G. VALIENTE ET AL. FIG. 1. Map showing the location of the Patagonian systems studied.

ADAPTATION OF NON-NATIVE SALMONIDS IN PATAGONIA 139 The main objective of this study was to examine if genetic variability plays a role in the different adaptation of these three salmonids in Patagonia. Variation at eight neutral non-coding hypervariable microsatellite loci was estimated for Atlantic salmon, rainbow trout and brown trout populations inhabiting the Nahuel Huapi National Park (Argentina), and for their donor populations: Sebago Lake (Maine, U.S.A.), McCloud River (California, U.S.A.) and Elbe River (Hamburg, Germany), respectively. POPULATIONS MATERIALS AND METHODS SAMPLED In 2004, scale samples from 50 adults of each of the three salmonid species were obtained from angling catches at the Currhue Grande River (39 529 S; 71 259 W; Neuquen, Andean Patagonia, Argentina; Fig. 1). Another 50 adult samples (scales donated by anglers) were obtained for Atlantic salmon at the Traful Lake and for brown trout from the River Limay in the same region (Fig. 1). Scales (two to five per specimen) were dried and preserved in paper envelopes for DNA extraction. Tissue samples (adipose fin biopsy) of wild native brown trout adults were obtained from two areas of the Elbe River (Germany). Scales were collected from native landlocked Atlantic salmon inhabiting two areas of Maine lakes, Sebago Lake and Grand Lake. Finally, scales samples from rainbow trout were obtained from the McCloud River (U.S.A.) where the fish is native. STUDY AREA AND THE POPULATIONS SAMPLED The Patagonian ecosystem where the three species were collected is at an elevation of 1000 m a.s.l. The ecosystem type is a river- glacial lake system. Other species were also found in the system: the naturalized brook trout Salvelinus fontinalis (Mitchill), and the native Hatcheria macraei (Girard), Galaxias platei (Steindachner) and Galaxias maculatus (Jenyns). The proportion of Atlantic salmon catches over the total number of salmonids angled is shown in Table I as a proxy of S. salar abundance in the last few years (2000 2005) and compared to 20 years ago (1980 1985). These data were obtained from catches reported by anglers in the periods considered. The exact number of catches was not recorded for each of the other salmonids, but their relative mass in the total number of sport catches was roughly estimated as a proxy of their relative abundance. GENETIC ANALYSIS DNA was extracted based on Chelex methodology (Estoup et al., 1996). Eight noncoding hypervariable microsatellite loci were analysed. Microsatellite typing at loci TABLE I. Salmonid catches in the studied system (Currhue Grande River), as a proxy of their relative abundance. Atlantic salmon catches: per cent of Atlantic salmon angled over total catches in the system. Other species: relative abundance of catches (approximate estimate) of the other salmonids studied in the system Atlantic salmon catches (%) Other species 1980 1985 100 None 2000 2005 20 Oncorhynchus mykiss (50%) > Salmo trutta (30%)

140 A. G. VALIENTE ET AL. SSA197, SSA85 and SSA171 (O Reilly et al., 1996), SSOSL85, SSOSL311 and SSOSL417 (Slettan et al., 1995), SSSP (Patterson et al., 2004) and SS4 (Martinez et al., 1999), was based on standard PCR-fragment size determination methodology by capillary electrophoresis in an ABI Prism 3100 DNA Sequencer and the GENSCAN V. 3.7 software at the DNA Sequencing Unit of the University of Oviedo. Annealing temperatures in PCR cycles were the following: loci SS4, SSA85 and SSA171, 58 C; SSOSL85 and SSOSL311, 55 C; SSA197, 64 C; SSSP, 60 C; SSOSL417, 52 C. Reaction mixtures and cycle conditions followed standard protocols (Ayllon et al., 2006). STATISTICAL ANALYSIS Scoring errors, large allele dropout and null alleles at microsatellite loci were checked employing the programme MICROCHECKER (Van Oosterhout et al., 2004). Allele frequencies and number of alleles per locus were determined employing the GENETIX (2000) computer package. Conformity to Hardy Weinberg equilibrium (HWE) was tested employing the programme GENEPOP (Raymond & Rousset, 1995), by the Markov chain method (10 000 dememorization steps, 1000 batches and 10 000 iterations per batch). The same programme was employed for estimating F ST values between pairs of populations as a measure of the genetic differentiation between them. The method for estimating F ST values was a distance method based on the number of different alleles, with 1640 permutations (significance level 005). Bonferroni correction (Rice, 1989) for determining the level of significance in multiple tests was applied when pertinent. Differences between groups of samples, for example between populations acclimated to Patagonia and their sources of origin, for different genetic variables (mean heterozygosity observed and expected under HWE, allelic richness, relatedness) were tested employing the programme FSTAT (Goudet, 1995), version V2.9.3 (2002), with 10 000 iterations. RESULTS The total proportion of Atlantic salmon angling catches relative to other species has declined drastically in the last 20 years (Table I). Although the other two species were already present in the system in the early 1980s, their populations were not large enough to provide substantial sport catches, which were only sporadic in the area. In the last 5 years, however, catches have been principally of rainbow trout, followed by brown trout. These observations indicate a relative decline of the Atlantic salmon population in the area, also reflected in the special protection measures for this species. With respect to their genetic variation at the eight microsatellite loci considered, allele sizes differed among species at many loci, as expected (Table II). After Bonferroni correction, the populations of each species inhabiting the Nahuel Huapi National Park were in HWE at most loci, with some exceptions (brown trout and Atlantic salmon at the SSOSL417, brown trout at the SS4 and rainbow trout at the SSP1605 in the Currhue Grande River). Significant linkage disequilibrium between paired loci was only found for 12 out of 196 comparisons, distributed across the five South American populations and involving different loci. These results indicate that the populations of the three species are in equilibrium in these South American habitats. The donor populations from which South American salmonids were imported still exist in North America (Atlantic salmon and rainbow trout) and Europe (brown trout). They were in HWE at most of the eight loci considered (Table III). F ST between

ADAPTATION OF NON-NATIVE SALMONIDS IN PATAGONIA 141 TABLE II. Microsatellite loci variation in the Patagonian populations (Currhue Grande River) of Oncorhynchus mykiss, Salmo trutta and Salmo salar studied and additional brown trout and Atlantic salmon populations studied, sampled in the Limay River and in the Traful Lake respectively Location Currhue Grande River Limay River Traful Lake Species O. mykiss S. trutta S. salar S. trutta S. salar Locus R Na R Na R Na R Na R Na SSOSL311 124 128 3 128 160 6 114 114 1 128 160 8 114 114 1 SSOSL85 174 176 2 180 188 5 186 204 5 182 186 3 186 204 5 SSOSL417 165 165 1 173 185 6 163 203 5 169 187 7 163 173 5 SSA197 107 111 2 127 145 5 171 221 11 127 139 3 173 221 6 SS4 154 224 3 178 196 9 154 242 11 184 192 4 194 244 11 SSA171 87 99 2 227 255 8 231 249 5 227 249 6 231 249 5 SSA85 102 154 6 112 116 3 106 122 3 106 116 4 106 122 4 SSSP1605 228 348 3 312 438 8 228 264 6 308 438 6 232 306 4 Mean S.D. Na 275 149 625 198 588 352 513 189 513 280 Mean S.D. H e 0354 0256 0606 0135 0499 0239 0590 0188 0473 0305 Mean S.D. H o 0392 0302 0556 0141 0451 0252 0601 0181 0429 0293 R, range of allele sizes; Na, number of alleles per locus; H e, heterozygosity expected under the Hardy Weinberg equilibrium; H o, observed heterozygosity.

142 A. G. VALIENTE ET AL. TABLE III. Microsatellite loci variation in the original populations of Salmo trutta, Salmo salar and Oncorhynchus mykiss from which the Patagonian populations were derived Species S. trutta S. salar O. mykiss Location Gol (Elbe River) Lamitz (Elbe River) Sebago Lake Grand Lake McCloud River Locus R Na R Na R Na R Na R Na SSOSL311 120 150 8 120 164 18 114 116 2 114 116 2 144 146 2 SSOSL85 180 202 3 168 210 8 182 196 6 182 198 7 180 196 3 SSOSL417 169 187 5 157 189 9 165 209 6 165 209 6 165 165 1 SSA197 127 157 6 127 157 9 165 201 9 165 211 11 107 111 2 SS4 154 188 6 154 240 13 186 256 17 186 246 17 154 188 2 SSA171 223 255 10 223 251 9 231 259 11 223 256 10 89 99 2 SSA85 106 116 5 104 116 6 106 232 10 106 146 8 102 128 5 SSSP1605 228 434 7 228 442 10 232 260 7 228 268 7 228 250 2 Mean S.D. Na 63 21 103 37 85 44 85 44 23 12 Mean S.D. H e 0619 0252 0766 0126 0683 0225 0715 0237 0324 0197 Mean S.D. H o 0571 0260 0655 0146 0639 0207 0727 0250 0426 0363 R, range of allele sizes; Na, number of alleles per locus; H e, heterozygosity expected under the Hardy Weinberg equilibrium; H o, observed heterozygosity.

ADAPTATION OF NON-NATIVE SALMONIDS IN PATAGONIA 143 paired donor and derivative populations (Table IV) were significant in all cases, demonstrating that genetic differentiation occurred between the populations native to the northern hemisphere and their descendants adapted to South America. On the other hand, the two brown trout samples obtained from two tributaries of the Elbe River significantly differed from each other, while the two samples of Atlantic salmon obtained from Maine lakes were not statistically significantly different. Rapid population differentiation, characteristic of S. trutta (Ayllon et al., 2006), can explain this result. Significant reduction of genetic variability, particularly marked at allelic richness, has been produced in South American populations of the genus Salmo (Table V) with respect to their donor ancestors for allelic richness, expected heterozygosity, observed heterozygosity and relatedness (one-tailed tests, P < 005 in all cases). This phenomenon was similarly intense for the two species of the genus Salmo, which did not differ from each other in genetic variation. For the rainbow trout, the donor and the derivative populations exhibited similar very low levels of genetic variation at the loci considered. DISCUSSION The South American salmonids considered in this study represent a good example of founder effects. They exhibit only a fraction of the original genetic variability of their donor populations. Reduced genetic variability and genetic differences with the donor are the two key descriptors of founder effects. A newly established population is likely to be much less genetically diverse than the population from which it is derived (Allendorf & Lundquist, 2003); however, they can adapt and colonize new ecosystems even if they are already occupied by natives, a genetic paradox if local adaptation is common and important. The explanation probably lies on a few genes crucial for adaptation and population expansion, as suggested by Lee (2002). In this study, low variability at the eight studied microsatellite loci has not been a handicap for successful adaptation of Atlantic salmon and brown and rainbow trout in the Patagonian lakes. Even populations with extremely reduced variation, like rainbow trout adapted to Currhue Grande Lake, exhibit an expansive trend in the last decades (Table I). Genetic variability, per se, cannot be considered the main factor for adaptation and expansion of a species in a new environment. Low genetic variation was not an obstacle for successful adaptation and competition of rainbow trout, as for other animals (Tsutsui et al., 2000). Although there is no doubt that genetic variation is important to avoid population extinction (Frankham, 2005), it does not seem crucial for adaptation in successful invasive species like brown trout (Townsend, 2003). Even with reduced genetic variation, introduced species can be successful at outcompeting and replacing native species (Allendorf & Lundquist, 2003). In the present study, secondary contact between salmonids also illustrates competition between exotics. It is known that introduced species not only modify native populations but also interact with other exotics, being able to modify their genetic patterns (Gaskin & Schaal, 2002; Vellend et al., 2007). Atlantic salmon populations seem to be declining in Patagonia after an initial period of successful colonization. The present results suggest that differences in genetic

144 A. G. VALIENTE ET AL. TABLE IV. The F ST values between paired populations, below diagonal, and their statistical significance (þ, significant;, non-significant) above diagonal (P ¼ 005). Oncorhynchus mykiss: McCloud and Currhue, donor and derivative populations respectively; Salmo trutta: Gol and Lamitz, samples from the donor Elbe River and Currhue and Limay, South American populations; Salmo salar: Grand Lake and Sebago, donor U.S. populations and Currhue and Traful, South American populations Rainbow trout Brown trout Atlantic salmon Currhue McCloud Currhue Limay Gol Lamitz Traful Currhue Sebago Grand Lake Currhue þ Currhue þ þ þ Traful þ þ þ McCloud 02889 Limay 00722 þ þ Currhue 00384 þ þ Gol 01746 01369 þ Sebago 02409 02275 Lamitz 01473 01346 00779 Grand Lake 02193 02101 00107

ADAPTATION OF NON-NATIVE SALMONIDS IN PATAGONIA 145 TABLE V. Mean genetic variability for Oncorhynchus mykiss, Salmo trutta and Salmo salar from the donor and South American derivative populations O. mykiss S. trutta S. salar Donor Derivative Donor Derivative Donor Derivative Allelic richness 2247 2554 7185 4756 7976 4422 H o 0518 0472 0614 0583 0674 0437 H e 0375 0390 0711 0605 0711 0494 Rel 0024 0069 0141 0152 0016 0090 H e, expected heterozygosity; H o, observed heterozygosity; Rel, related mass. variability were not the reason for the relative success of brown and rainbow trout and Atlantic salmon, as Patagonian Atlantic salmon was not less variable than the trout species at the loci considered. Competition with other species may instead explain the decline in Atlantic salmon. Secondary contact of Atlantic salmon and brown trout in non-native areas has produced a decline, and even extinction in the former, in other regions (Ayllon et al., 2004). The invasive capacity of S. trutta has been repeatedly reported (Townsend, 2003), as well as intense competition between this species and S. salar (Hearn, 1987; Harwood et al., 2001). Additional competition with O. mykiss, the most abundant species in the system from indirect evidences of catches (Table I), may also contribute to Atlantic salmon declines. Juvenile O. mykiss and S. salar are competitors for habitat resources in other locations; steelhead trout (O. mykiss) are usually the superior competitors (Volpe et al., 2001). In conclusion, although founder effects and gene drift occurred in the salmonids introduced in Patagonia one century ago, reduced genetic variability at neutral loci has not been an obstacle for adaptation of S. trutta and O. mykiss to the new ecosystems. Interspecific competition with other salmonids may explain population declines in Patagonian naturalized S. salar. We are grateful to I. G. Pola (University of Oviedo, Spain) for his collaboration in laboratory tasks. Samples of donor populations of rainbow trout, brown trout and Atlantic salmon were generously provided by R. Simmons (Department of Animal Science, UC Davis, U.S.A.), U. Schliewen (Zoologische Staatssammlung Munchen, Germany) and F. Brautigan and N. Kramer, respectively. This study was supported by the National Spanish Grant MCYT REN2003-00303. E.G.V. received a Grant from the Spanish Government (PR2004-0084) for visiting the University of Massachusetts. F.J. was supported by a Hatch grant from the University of Massachusetts. References Allendorf, F. W. & Lundquist, L. L. (2003). Introduction: population biology, evolution, and control of invasive species. Conservation Biology 17, 24 30. Arribere, M. A., Ribeiro Guevara, S., Sanchez, R. S., Gil, M. I., Roman Ross, G., Daurade, L. E., Fajon, V., Horvat, M., Alcalde, R. & Kestelman, A. J. (2003). Heavy metals in the vicinity of a chlor-alkali factory in the Upper Negro River ecosystem, Northern Patagonia, Argentina. Science of the Total Environment 301, 187 203.

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