Adaptive divergence is often a compromise between the opposing forces of selection and gene flow. The tension between these forces is especially strong in the case of ecotypic differentiation, in which populations in close proximity may become locally adapted to different selective pressures in the presence of persistent gene flow. An ongoing challenge is to establish the degree and scale of ecotypic differentiation, empirical questions that must be tackled on a taxon by taxon basis. We pursue an analysis of ecotypic differentiation using FST-QST comparison and test a hypothesis of divergence within and between ecotypes. We further identify candidate selective agents using direct and indirect observations of predation events.
The statistical comparison of population differentiation at quantitative traits (QST) and neutral molecular markers (FST) provides a powerful test for the role of selection in phenotypic divergence (Lande, 1992; Spitze, 1993; Merilä & Crnokrak, 2001; McKay & Latta, 2002). Departures from neutral expectations can be used to distinguish between a history dominated by diversifying (QST > FST) as opposed to stabilizing selection (QST < FST). In applications in a wide range of taxa, a priori expectations of the FST-QST relationship have been used both to detect and assay modes of selection acting on quantitative traits (Palo et al., 2003; Wong et al., 2003; Cano et al., 2004) and to test specific hypotheses about local selection (Baker, 1992; Waldmann & Andersson, 1998; Gomez-Mestre & Tejedo, 2004). Here, we test the power of the FST-QST approach by asking whether it can resolve the role of selection in promoting differentiation among populations within ecotypes, as well as between ecotypes. The success of this test may indicate whether the FST-QST approach will generally be able to assess the role of selection in truly subtle instances of phenotypic differentiation.
In our study system, populations of the terrestrial garter snake (Thamnophis elegans) in the Eagle Lake basin of Lassen Co., California show ecotypic differentiation on a scale of several kilometres and in the presence of gene flow high enough to override drift (up to 1.8 effective migrants per generation) at neutral microsatellite markers (Bronikowski & Arnold, 1999; Manier & Arnold, 2005; Sparkman et al., 2007). Previous research has documented ecotypic differences in reproduction, growth and survival between populations along the rocky shoreline of Eagle Lake and those inhabiting the densely vegetated surrounding meadows (Bronikowski & Arnold, 1999). The life history differences constitute a syndrome that may be driven by higher predation rates at the lakeshore. These populations grow faster, reproduce at an earlier age and have larger litters but suffer higher adult mortality than meadow populations. Common garden experiments have demonstrated a genetic basis for the difference in growth rate (Bronikowski, 2000).
Here, we focus on differences in colouration that appear to increase crypticity in rocky lakeshore and grassy meadow environments. To visual predators, the muted colours of lakeshore snakes (dull yellow or tan stripes on a grey background colour) tend to match the rocky substrate of the lakeshore, whereas the meadow snake colour pattern (yellow or orange stripes on a black background) closely resembles dead rushes that litter the shallow meadow substrates (Fig. 1). The difference in colouration between lakeshore and meadow ecotypes may be a result of differential selection for crypticity (Kephart, 1981).
We also examined six scale counts for adaptive differences between T. elegans ecotypes. Vertebral number (measured using ventral and subcaudal scale counts) can vary between different habitats as a function of a snake's ability to utilize substrate irregularities or ‘push-points’ for propulsion during locomotion (Jayne, 1988; Gasc et al., 1989; Kelley et al., 1997). In thickly vegetated habitats with a higher density of push-points, T. elegans and other snake populations have fewer vertebrae, whereas rocky habitats that have fewer push-points support populations with more vertebrae (Klauber, 1941; Kelley et al., 1997; Arnold & Phillips, 1999). Because lakeshore habitats provide fewer push-points than meadow habitats, we expected to see more body and tail vertebrae in lakeshore than in meadow T. elegans. The other scalation traits are likely to reflect a snake's ability to ingest large prey, with high values for these traits promoting extended cranial kinesis (infralabial, supralabial and postocular scale counts) and midsection elasticity (midbody scale count). Because diet studies indicate that lakeshore snakes generally eat larger prey items (fish) than meadow snakes (anuran larvae, leeches; Kephart & Arnold, 1982; Kephart, 1982), we expect selection for ability to swallow larger prey in lakeshore populations and hence higher scale counts. Both scale counts and colouration have been shown to be under selection in these and other populations of snakes (Arnold, 1988; Arnold & Bennett, 1988; Brodie, 1992; King, 1993a; Lindell et al., 1993), making these traits good candidates for our study.
We also ask whether selection has played a role in promoting the more subtle differences in colouration and morphology that appear to characterize populations within each of the two ecotypes. Meadow populations in our system, for example, appear to differ subtly in colouration, such that some populations have a higher incidence of yellow as opposed to orange dorsal stripes. Likewise, meadow populations occupy habitats that differ slightly in water depth, seasonal patterns of drying, phenology and composition of vegetation, as well as in prey availability (Kephart, 1982; Manier & Arnold, 2006). Similar small differences in snake colouration and habitat characteristics can be seen from one lakeshore population to the next. One of our goals is to assess the statistical reality of these impressions of morphological differentiation within ecotypes and to determine whether they might represent responses to selection.
We used estimates of neutral divergence at microsatellite loci to determine whether colouration and scalation traits have experienced diversifying selection, especially between the two ecotypes. We can reject neutrality as an explanation of population differentiation in quantitative traits if QST≠FST. Thus, QST > FST suggests diversifying selection, whereas QST < FST suggests stabilizing selection towards the same optimum in different populations (Lande, 1992; Spitze, 1993). Because each ecotype is represented by multiple populations, we can also determine the importance of differentiation within and between ecotypes to FST and QST. Based on our hypothesis of ecotypic differentiation, we expect QST estimates to far exceed FST in ventral scale counts and colouration. We expect that this population differentiation is largely a consequence of strong divergence between ecotypes (QCT >> FCT), with some contribution because of slight divergence among populations within ecotypes (QSC > FSC). We used parallel, three-level analysis of variance for microsatellite alleles and for quantitative traits to test these hypotheses. We also present evidence from direct observations of predation and analysis of culmen impressions on snakes that implicate avian predators as the selective agents responsible for this adaptive differentiation.