The process of ecological speciation consists of three components; an ecologically based source of divergent selection, a mechanism of reproductive isolation, and a genetic link between divergent selection and reproductive isolation (Rundle & Nosil, 2005). In this study, we examined two of these components, an ecological source of divergent selection (salinity) and the importance of three types of reproductive isolation (ecological, behavioural and genic isolation).
Adaptation to salinity
Our results provide evidence for ecological divergence between L. goodei and L. parva, and the nature of this divergence roughly reflects their distributions across salinity. Lucania goodei is more common in fresh water, whereas L. parva is more common in brackish water. Based on the results of this field study, we hypothesized that local adaptation to salinity exerts an ecological source of divergent selection on L. goodei and L. parva. Consistent with this hypothesis, we found differential effects of salinity on survival to adulthood and adult body size when animals were reared outside in stock tanks. Lucania parva had reduced survival to adulthood in fresh water (0 ppt), whereas L. goodei had reduced growth at 8 ppt and reduced hatching success at 15 and 20 ppt. However, these results do not explain why L. parva is frequently found in freshwater populations. In our study, L. parva was found in two freshwater sites (Lower Bridge and Three-Fingers). Furthermore, in a review of museum records, R.C. Fuller and L. Noa (unpublished data) found that 23% of L. parva populations were in fresh water.
One possible explanation for this discrepancy is that the animals in our stock tanks not only had to survive to adulthood, but they also had to overwinter, and thus, be able to withstand colder temperatures. Work on Fundulus heteroclitus suggests that enzymes critical to osmoregulation perform poorly in cold temperatures (Kidder et al., 2006). Similarly, in an experiment investigating the relative effects of salinity, temperature and food, Trexler et al. (1990) demonstrated that female sailfin mollies (P. latipinna) had the slowest growth when raised in cold temperatures and at low salinities. Similarly, sailfin mollies maintained in field cages usually had higher overwinter survival in salt water compared with fresh water (Trexler et al., 1992). This suggests that an interaction between salinity and temperature may be the critical abiotic factors that differentiate L. goodei and L. parva. This also potentially explains L. parva’s presence in many freshwater springs and spring fed rivers (Homosassa Springs: Herald & Strickland, 1949; Foster, 1967; Silver Springs: Hubbs & Allan, 1943). In addition to having high levels of dissolved ions, freshwater springs are remarkably constant in temperature year round (21 °C). Hence, springs may be tolerable for L. parva during winter. The idea that a combination of salinity and temperature are the critical abiotic variables distinguishing L. goodei and L. parva is also in keeping with early verbal hypotheses on the original divergence between these two taxa. Duggins et al. (1983) suggested that these two groups may have diverged in the Pleiocene when one population invaded fresh water during a time of heightened global cooling. Proper phylogenetic work is needed to establish the direction of the invasion (i.e. marine to fresh water vs. fresh water to marine) as well as the timing of the divergence.
The trade-off across salinity for L. goodei and L. parva is all the more striking when one considers that our experimental design precluded competition between the two species. Earlier work by Dunson & Travis (1991) found evidence for differential effects of salinity on competition between L. goodei and L. parva. In their experiment, they raised L. goodei and L. parva either alone or in combination at 0 and 15 ppt salinity and measured survival and growth. Lucania goodei grew faster in 0 ppt than in 15 ppt and the difference between 0 and 15 ppt increased when L. goodei was raised in competition with L. parva. Lucania parva also grew faster in 0 ppt than in 15 ppt, but this pattern reversed when raised in competition with L. goodei. The main result was that salinity (0 vs. 15 ppt) altered the effects of competition between L. goodei and L. parva. Hence, the trade-off in survival across salinity may be even greater in nature than that found in this study.
Mechanisms of reproductive isolation
This study provides strong evidence for the role of behavioural isolating mechanisms in the maintenance of the two species, L. goodei and L. parva. Heterospecific pairs had a longer latency to spawn and produced fewer eggs than conspecific pairs of either L. goodei or L. parva. This strong pattern emerged using a relatively conservative one-way choice test where animals were given a choice between spawning (with whomever they were paired) and not spawning at all (see Houde, 1997; Coyne & Orr, 2004 for discussions of one-way choice tests; see Nagel & Schluter, 1998; Boughman, 2001; Boughman et al., 2005 for stickleback experiments using one-way choice tests). In 2005, we isolated gravid females and reproductive males in aquaria at the height of the spawning season. In 2006, we isolated gravid females at the start of the spawning season. We typically have great success in getting animals to spawn at these times. Despite this, many hybrid pairs had a long latency to spawn and some failed to spawn at all indicating strong prezygotic isolation.
The evolutionary mechanism via which behavioural isolation arose between L. goodei and L. parva is unknown. Given that the two species occur in sympatry in some areas, reinforcement (selection against forming hybrids in areas of sympatry) is a possibility. However, given that we found no evidence for reinforcement, this would require the spread of genes conferring behavioural isolation from sympatric populations to allopatric populations for both species. Another possibility is that sexual selection has occurred independently in the two lineages and resulted in the evolution of different signals and preferences. The critical issue is determining the extent to which ecological divergence between the two species contributed to behavioural isolation. Another critical assumption is that the behavioural isolation between the two species is the result of genetic differentiation. Because this experiment was performed with wild-caught animals, we cannot rule out the possibility that learning (i.e. environmental effects) has also contributed to behavioural isolation.
The actual mechanism that creates prezygotic isolation in Lucania is currently unknown. Given that L. goodei and L. parva differ in body size, male colouration and male courtship, we assume that a critical cue is missing which makes animals reluctant to mate. However, we currently do not know whether prezygotic isolation is a function of male behaviour, female behaviour or both. Another possible scenario is that heterospecifics do mate, but then subsequently cannibalize their eggs. Egg cannibalism is high in L. goodei (Fuller & Travis, 2001) and appears to be important in reproductive isolation in other external fertilizers (Albert & Schluter, 2004). Although we cannot rule out this alternative, we find it unlikely. We found no eggs in the spawning substrates of many heterospecific pairs, meaning that they would have had to quickly eat all of their eggs after every spawning.
This work adds to the growing body of literature indicating the importance of behavioural isolation in teleost fish (Endler & Houde, 1995; Seehausen et al., 1997; Rundle & Schluter, 1998; Ishikawa & Mori, 2000; Albert & Schluter, 2004; Alexander & Breden, 2004). In sticklebacks, reproductive isolation has arisen between benthic and limnetic forms irrespective of lakes (i.e. whether the morphs recently shared an ancestor) (Rundle et al., 2000), and this isolation is primarily dependent on differences in body size between the two morphs that have arisen due to ecological divergence (McKinnon et al., 2004; Boughman et al., 2005) and secondarily on differences in colouration (Boughman, 2001; McKinnon et al., 2004; Boughman et al., 2005). Similarly, in some cichlid species, reproductive isolation appears to depend on the operation of visual cues (Seehausen et al., 1997) where deterioration of environmental cues (e.g. lake eutrophication and the reduced transmission of light) is associated with increased hybridization.
Our study found no evidence for reinforcement (i.e. increased behavioural isolation in areas of sympatry). The lack of reinforcement may be due to the fact that sympatric populations are not stable over long enough periods of time for selection to produce increased levels of behavioural isolation. In this study, the zone of sympatry fluctuated where some populations were sympatric at one census period, but allopatric at another census period. Continued long-term censuses of these sites will indicate the degree to which these populations are consistently sympatric.
This study also provides little evidence for genic isolation in the F1 hybrid stage. Instead we found high hatching success and high survival to adulthood when raised in stock tanks at 2 ppt. The idea that genic isolation is not important in the early stages of speciation in teleosts is supported by two comparative studies (darters –Mendelson, 2003; sunfish –Bolnick & Near, 2005) as well as studies on individual species/population pairs (sticklebacks –Hatfield & Schluter, 1999; guppy –Alexander & Breden, 2004). However, genic isolation has been demonstrated numerous times in other groups (plants, Fishman & Willis, 2001; Brandvain & Haig, 2005; Lepidoptera, Presgraves, 2002 and Drosophila, see Coyne & Orr, 2004 for a review), and there are a few examples in teleosts as well. In whitefish (Coregonus), F1 and back-crossed animals have been shown to experience significantly higher mortality than either parental strain (Lu & Bernatchez, 1998; Rogers & Bernatchez, 2006). Similarly, a study on hybridization among guppy populations examined both the F1 and F2 stages and found evidence for reduced fitness in the F2 stage (Russell & Magurran, 2006). It has also been suggested that hybridization between marine and landlocked sticklebacks (G. aculeatus) produces sterile F1 males (Honma & Tamura, 1984).
The apparent lack of evidence for genic isolation in some fish groups may be due to the fact that: (1) only a small number of taxa have been studied; (2) some studies only examine the survival of embryos and ignore later reproductive stages; (3) survival in the F2 and back-crossed animals is often not measured due to the fact that many fish require at least a full year to attain reproductive maturity; and (4) survival under natural conditions is infrequently studied. These reservations apply equally to this work. We primarily investigated survival in F1 animals at early life-history stages. Although we were able to raise hybrids to adulthood at one salinity level, we were unable to examine their survival over the range of relevant salinities. Furthermore, we have not yet raised them under natural conditions where they would be exposed to predators and other stressful factors. Currently, we are investigating genic isolation in F2 and back-crossed animals.
Similarly, we found little evidence that F1 hybrids were differentially affected by salinity. At early life-history stages, we found high hatching success and larval survival of F1 animals at all salinities – even at the higher salinities where L. goodei had low hatching success. Unfortunately, we were unable to raise F1 animals to adulthood in stock tanks at all salinities where we found the greatest evidence for effects of salinity. Thus we were unable to test whether hybrid fitness varied across a range of salinities. This is unfortunate because ecologically dependent post-zygotic isolation is a unique prediction of ecological speciation (Rundle & Nosil, 2005). Environmentally dependent hybrid inferiority has been demonstrated in some systems (Jiggins et al., 2001). In G. aculeatus, F1 backcrosses to benthic parents have higher fitness in benthic habitats than F1 backcrosses to limnetic parents (Rundle, 2002) indicating that reduced hybrid fitness is dependent on ecological conditions in this group.
In conclusion, our study found strong evidence that L. goodei and L. parva are differentially adapted to salinity where L. goodei is very tolerant of fresh water and L. parva is tolerant of brackish water. However, we found this effect only when animals were raised to adulthood in stock tanks and forced to overwinter or when L. goodei eggs and fry were raised at salinities above 10 ppt. The interaction between salinity and temperature may be the critical factor that differentiates habitat space between L. goodei and L. parva. We also found strong evidence for prezygotic, behavioural isolation between L. goodei and L. parva. We found no evidence for genic isolation (i.e. hybrid inviability) in F1 animals. Hatching success, probability of surviving through the eating stage, and survival to adulthood were intermediate between the two parent species. However, we are currently investigating survival of F2 and back-crossed animals.