Genetically-based population differences in acid tolerance and potential for local adaptation
Adopting common-garden experimentation, we found that wild alevins from an acidified river (TUSK) had higher survival at acidified pH than either farmed (FARM) or wild (STEW) alevins originating from nonacidified sources. TUSK alevins also survived cumulatively longer than FARM or STEW alevins within the range of pH found in the Tusket River (e.g. pH = 4.6–4.9). The higher survival of TUSK alevins under conditions of their local environment is one prerequisite of local adaptation (Kawecki and Ebert 2004). Indeed, the spring hatching period in Atlantic salmon is potentially the most critical season for survival, as alevins are exposed to abrupt pH reductions from melting snow and ice runoff (Daye and Garside 1979; Lacroix 1985).
On the other hand, more definitive support for local adaptation, which we did not find, might have been provided if: (i) FARM and STEW alevins survived better than TUSK alevins at higher, nonacidified pH levels; and similarly, (ii) TUSK alevins performed better at lower (pH = 4.6–5.2) than higher pH (pH = 5.7–7.0) (Kawecki and Ebert 2004). Additionally, we detected no differences in parr mortality or growth between cross-types that were attributable to the pH normally encountered by wild TUSK parr. Nonetheless, less severe fitness effects were expected at this stage relative to the alevin stage, as alevins are more sensitive to low pH than parr in Atlantic salmon (Daye and Garside 1977, 1979; Lacroix 1989). Given that local adaptation usually entails a physiological cost in environments where it is not needed (Kawecki and Ebert 2004), our overall results cannot conclusively provide evidence that adaptive genetic variation exists in TUSK salmon for tolerating acidity.
It would, however, be premature for several reasons to conclude that adaptive genetic variation relating to pH does not exist. First, comparisons of the performance of TUSK salmon at different pH levels (acidified/nonacidified) or relative to FARM/STEW salmon at nonacidified pH might not be definitive tests of local adaptation. Salmon only require acid tolerance in freshwater stages (e.g. juvenile/spawning) because seawater stages (subadult/adult) of all populations are exposed to a similarly high and relatively homogeneous sea pH of 7.5–8.4. By default then, salmon from acidified rivers (e.g. TUSK) are exposed to, and thus require tolerance to, both acidic and nonacidic pH (although not necessarily both at any one particular stage). This could account for the apparent lack of constraint on TUSK genotypes at higher pH. Secondly, and similarly, the wider range of pH to which salmon from acidified rivers are exposed should favor the evolution of adaptive phenotypic plasticity (Hutchings 2004; Kawecki and Ebert 2004). It could be that pH adaptation in TUSK salmon relates more to tolerating a range of pH than a specific pH per se. Third, while our study focused on stages that have previously shown sensitivities to low pH in Atlantic salmon (Daye and Garside 1977; Lacroix 1985), adaptive genetic variation might exist at earlier or later, unexamined life history stages, such as during embryonic development or the parr-smolt transformation (Smith and Haines 1995). Finally, our experimentation may have failed to mimic specific environmental conditions related to pH in which adaptive genetic variation is expressed (Kawecki and Ebert 2004). For instance, our pH exposure trials did not incorporate interactions between acidity and heavy metals, such as aluminum, which can affect the toxicity of pH (Lacroix 1989, 1992). Nonetheless, these latter interactions are unlikely to have affected our results because Southern Upland Rivers have high levels of dissolved organic matter which decrease the toxicity of heavy metals (Farmer et al. 1980; Lacroix 1985).
F1 versus the F2 generation of farmed-wild interbreeding
Interbreeding between divergent populations often generates F1 heterosis followed by hybrid breakdown in the F2 or later recombinant generations (Edmands 2007). A salient and contrasting result of our study is that F1 TUSK × FARM hybrids showed reduced performance relative to parental populations at acidified pH, whereas we found limited evidence for reduced performance in TUSK × FARM backcrosses, and F2 TUSK × FARM hybrids performed better or equally well to TUSK salmon. Several explanations, relating both to the genetic characteristics of salmon and our experimental design, might account for these discrepancies.
F1 outbreeding depression is normally attributable to a disruption of local adaptation (via extrinsic interactions between genes and the environment), underdominance, or epistatic interactions (Edmands 1999, 2007). These mechanisms may act concurrently, but our study was not designed to disentangle which of them might explain the observed reduction in fitness in F1 TUSK × FARM hybrids. Nevertheless, reduced F1 hybrid performance relative to parental populations was highly environmentally-dependent and only detectable as the pH became more acidic. This suggests that extrinsically-based disruption of local adaptation was involved. However, we emphasize that only TUSK males and FARM females were used to generate our F1 hybrids. Reciprocal F1 hybrids (TUSK female × FARM male) may not experience as great a reduction in fitness at acidified pH as the F1 hybrids in our study. On the other hand, available data suggest that mating between wild males and farmed females may be more representative of what takes place in the wild (Fleming et al. 2000).
In contrast to F1 hybrids, we found limited evidence for reduced performance in TUSK × FARM backcrosses, and F2 TUSK × FARM hybrids occasionally performed better than, but most often equally well to, TUSK salmon. The general lack of F2 outbreeding depression might suggest that co-adapted gene complexes related to acid tolerance do not exist in salmon, at least at the life history stages examined. Or, perhaps there has been insufficient time to evolve tightly-linked co-adapted gene complexes given Atlantic salmon only colonized the Southern Upland region 12 000 years ago after the last glaciation (Pielou 1991). Yet alternative explanations might explain the lack of F2 outbreeding depression and complicate interpretations of the mechanisms underlying hybrid fitness.
For example, salmonids are well-known for exhibiting pronounced maternal effects in many of the traits evaluated here (alevin size, yolk sac size, size at hatch, parr growth). These maternal effects can be due to either environmental or genetic causes, or both (e.g. Einum and Fleming 2000; McClelland et al. 2005; Perry et al. 2005). All females used to generate the 2005 crosses in our study were raised under common environmental conditions except that for logistical reasons, cross-types had to be kept in individual, separate holding tanks from 6 months postexogenous feeding onwards. Thus, we cannot entirely discount the possibility that tank effects might have led to environmentally-driven maternal effects. These in turn could have affected comparisons of the performance of certain cross-types relative to one another that were based on different generations of interbreeding (e.g. F1 versus F2 hybrids).
We believe it is more likely, however, that maternal effects with a genetic basis could have influenced hybrid fitness. For instance, if first-generation interbreeding led to F1 hybrid females with heterosis, maternal heterosis might have masked negative fitness effects in their F2 hybrid offspring (Tave et al. 1990; Falconer and MacKay 1996). Interestingly, the mean diameter (±1 SE) of F1 TUSK × FARM hybrid female eggs was slightly larger than TUSK females (5.99 ± 0.29 vs 5.85 ± 0.34 mm) or FARM females (5.46 ± 0.27 mm), despite an intermediate body length of F1 TUSK × FARM hybrid females (TUSK = 53.4 ± 1.2 cm; F1 TUSK × FARM = 59.4 ± 0.5 cm; FARM = 64.5 ± 0.6 cm). Accordingly, F2 alevins derived from F1 TUSK × FARM females had larger yolk sacs than any cross-type, including TUSK alevins (data not shown), and importantly, yolk sac volume had a significant influence on alevin survival in our analyses. Similarly, F1 TUSK × FARM hybrid fitness may have been affected because only FARM females were used to generate them, but under common environmental conditions, FARM females produced smaller eggs with smaller yolk sacs than TUSK females.
Many studies of interbreeding between divergent populations that find F1 heterosis and F2 hybrid breakdown are also based on diploid organisms, yet salmonids are residual tetraploids and some gene loci are still duplicated (Allendorf and Thorgaard 1984). The larger number of loci involved in genetic interactions than in a diploid organism might diminish fitness effects in F2 hybrids (Etterson et al. 2007; McClelland and Naish 2007), particularly under greater environmental stress (Edmands 2007), as was observed at acidified pH. Similarly, whereas diploids are expected to exhibit the greatest amount of heterosis after one generation of interbreeding (Falconer and MacKay 1996), heterosis in other polyploids is not fully attained until later generations (Bingham et al. 1994). Later generation heterosis in polyploids might also appear elevated if considerable inbreeding existed within parental populations (Etterson et al. 2007). This is a further possibility in our study given that the TUSK salmon population is small (<100–250 annual spawning adults; Amiro et al. 2000; DFO 2002) and that farmed salmon strains can exhibit reduced genetic diversity (Hutchings and Fraser 2008).
Fitness comparisons made in this study were also initiated after salmon embryos had hatched. At earlier embryonic stages, concurrent work suggests that a partial inviability might exist in F2 TUSK × FARM hybrids as they have reduced survival relative to parental populations (D. J. Fraser and J. A. Hutchings, unpublished data). Our study, therefore, used only the remaining F2 gene combinations that survived the hatching period, and on average, these genotypes might have had superior fitness to either parental population at the stages examined. Finally, on a related note, we point out that spawning salmon may preferentially spawn in upwelling areas that have higher pH (Lacroix 1992). If such areas exist within acidified rivers, and if salmon selectively use them, then the adverse effects from farmed-wild interbreeding documented here at the alevin stage might be buffered somewhat in the wild.
Conservation and management implications
Marked differences in pH between Southern Upland Rivers and the ancestral source river of regional farmed salmon provided a benchmark for evaluating the risk posed to small and declining fish populations from interbreeding with their escaped farmed counterparts. We showed that wild salmon inhabiting acidified rivers had higher survival at acidified pH than farmed salmon or F1 farmed-wild hybrids, the hybrids that will be most commonly generated in the wild. Interbreeding also resulted in maladaptive (i.e. survival-reducing) changes to the reaction norms for acid tolerance in F1 hybrids. It is unlikely that these fitness reductions were due to advertent/ inadvertent selection during the farming process per se, but rather to the ancestral characteristics of the farmed individuals. The transfer and production of these farmed individuals into different geographical regions than where they originated then sets the stage for interbreeding of potentially maladapted farmed individuals with wild individuals when the former escape (Hutchings and Fraser 2008). For mitigating the effects of farmed-wild interbreeding, our results are thus directly relevant to ongoing debates regarding the use of farmed strains derived from local or nonlocal wild populations relative to where the farming is taking place (Hutchings and Fraser 2008). They are also relevant for considering the scale at which a farmed strain can be considered ‘local’.
We also found, however, that later generation (F2, BC1) farmed-wild hybrids generally performed equally well, if not better than, wild salmon at acidified pH. Furthermore, we did not find definitive evidence for the existence of adaptive genetic variation relating to pH in wild salmon. These results have two implications. First, divergent mechanisms likely affect the performance of farmed-wild hybrids between F1 and later generations. Secondly, our results provided some evidence both for and against the hypothesis that repeated farmed-wild interbreeding may lead to a dilution of adaptive genetic variation and potentially affect the persistence of wild populations.
We caution, nevertheless, that although our results do not point to one clear answer, this should not be used as justification for societal or governmental inaction with respect to mitigating the potentially negative impacts of aquaculture on wild species. First, our study focused on the response of only a few traits related to pH. Adaptive genetic variation in wild salmon, or outbreeding depression in multi-generational farmed-wild hybrids, could exist at other, unexamined traits. Secondly, we were logistically unable to examine the lifetime performance of wild, farmed and multi-generational hybrid salmon. Interestingly, in the only study that has done so to date, later generation (F2, BC1) farmed-wild hybrids exhibited similar, equal or greater fitness at embryo to smolt stages relative to wild Atlantic salmon, but lower overall lifetime success (see McGinnity et al. 2003). Thirdly, while later generation (F2, BC1) farmed-wild hybrids in our study exhibited equal if not superior fitness relative to wild salmon, their generation ultimately depends on the survival of F1 hybrids. Our results suggest that F1 hybrid survival may be much poorer relative to wild salmon. Fourthly, even if true F2 farmed-wild hybrids were produced in the wild, at present it cannot be ruled out that outbreeding depression in fishes may be generated in F3 or later generations after further recombination. For instance, this has been observed in some plant and invertebrate studies (Edmands 1999; Fenster and Galloway 2000). Clearly then, the generality of our findings as they pertain to other animals, fishes or salmonid populations awaits further studies of (i) the genetic architecture underlying fitness in multi-generational hybrids at a variety of traits; (ii) the lifetime fate of hybrids in the wild; and (iii) the degree to which multi-generational, farmed-wild interbreeding influences overall wild population growth rates or productivity.