Discussion: 60

Our results suggest that the responses in aperture width and shell lip thickness do not appear to be correlated (Table 6, Figs. 5 and 6). We found that exposure and chromosome number had a significant effect on the response in shell lip thickness only. We also found that the site of origin nested within exposure explained 21% of the

variance for the response in aperture width while it did not contribute at all to the response in shell lip thickness. We believe this may partially explain the contradictory results of previous experiments on phenotypic plasticity that looked at the response in different traits. We observed that snails of the 2n = 26 karyotype showed the greatest average change in shell lip thickness in relation to shell length, followed by the 2n = 27, 29 and 32 karyotypes, respectively. We also found that the 2n = 26 and 27 karyotypes had a significantly greater increase in shell lip thickness than the 2n = 29 and 32 karyotypes. Given the correlation between chromosome number and wave exposure this could account for the previously observed greater plasticity in exposed shore morphs (Palmer 1990).

When we looked within the 2n = 27 karyotype that included two exposed sites and one protected site, however, we found that the snails from the protected site showed the greatest average response in shell lip thickness. This leads us to conclude that there is asymmetric plasticity in dogwhelks, but the plastic response is a product of both ecological and chromosomal factors. Because the karyotypes of the populations utilized by Etter (1988b) and Palmer (1990) were unknown it is possible that their contradictory

results were due to the influence of a chromosomal polymorphism on the plastic responses of the different shell traits. In European populations snails with the same number of chromosomes may be polymorphic for different paracentric inversions (Day et al. 1993, Pascoe and Dixon 1994). It is currently not known whether inversions are found in dogwhelk populations in Maine, but further cytogenetic analyses may explain why individuals of the same karyotype had different phenotypic responses. Further examination of the chromosomal differences between populations should shed more light on how ecological and cytological factors interact to yield the drastic morphological variation found in the dogwhelk.

Investigators have sought a relationship between the chromosomal

polymorphism, phenotypic variance, and ecological variation found in Nucella lapillus

since the discovery of the different chromosomal races. Exposed shore populations are typically dominated by individuals of the 2n = 26 karyotype while the 2n = 36 karyotype occurs nearly exclusively on protected shores (Bantock and Cockayne 1975, Kirby et al. 1994, Staiger 1957). Snails on exposed shores typically have relatively short shells with larger apertural openings and a large pedal surface area (Moore 1936, Kitching 1977). These animals are able to more tenaciously adhere to the substratum and are less likely to be dislodged on wave swept shores (Kitching et al. 1966). These individuals grow to greater maximum shell lengths and have more robust shells that are better able to withstand predation (Currey and Hughes 1982, Hughes and Elner 1979). Past studies have consistently found a correlation between chromosome number and wave exposure and have also found individuals of intermediate karyotypes occurring sympatrically alongside the extreme karyotypes on shores of intermediate exposure (Bantock and Cockayne 1975, Day et al. 1993, Hoxmark 1970, Staiger 1957).

Other investigators have focused on the connection between phenotypic plasticity and the ecological factors that potentially drive ecotypic variation in shell morphology and behavior (Etter 1988b, Freeman and Hamer 2009, Hughes and Taylor 1997, Palmer 1990, Large and Smee 2010, Trussell et al. 2003). Field and laboratory experiments have yielded contradictory results. A reciprocal transplant experiment showed greater phenotypic plasticity in the protected shore ecotype as snails reared on a protected shore subsequently transplanted to an exposed shore showed the greatest change in pedal surface area (Etter 1988b). A laboratory experiment that exposed one protected and one exposed population to water-borne crab predator cues elicited the greatest phenotypic response in snails from an exposed shore that had the greatest change in body weight, shell weight, apertural tooth height, and shell lip thickness (Palmer 1990). Thus we have data asserting there is asymmetric plasticity among ecotypes with dissenting opinions as to which ecotype is more plastic. Both studies utilized snails from a single exposed shore population and a single protected shore population, which confounds differences due to exposure and those due to site-specific factors. Furthermore, the laboratory experiment yielded the greatest response in the control treatment for exposed shore snails where the crab predator cue was altogether absent. This suggests the dogwhelks may instead be responding to the removal of wave action instead of the desired response to the crab predator cue. Wave action on exposed shores is thought to limit growth and maximum shell length and acts as a major selective force in these environments (Etter 1989, Kitching et al. 1966). While it is certainly possible that snails responded to the crab predator cue a possible response to the calmer conditions cannot be excluded.

Chromosomal studies have ignored phenotypic plasticity while phenotypic plasticity studies have ignored the possible role of the chromosomal polymorphism on phenotype and/or phenotypic plasticity in dogwhelks. In order to separate the affects of exposure, chromosome number, plasticity and site specific factors we performed a transplantation experiment involving snails from three protected and four exposed shores with karyotypes of 2n = 32, 29, 27, and 26. We, too, have found a correlation between chromosome number and wave exposure. Our results are consistent with past studies that have found that individuals with lower chromosome numbers are more common on wave exposed shores and that chromosome number increases with decreasing wave exposure (Staiger 1957, Kirby et al. 1994).

Chromosomal rearrangements can drive intraspecific phenotypic variation directly through the production of discrete phenotypes (Hatadani et al. 2004, Joron et al. 2011, Wilson et al. 2013) or indirectly by influencing the degree to which different

karyotypic races are phenotypically plastic (Ayala et al. 2014, Hatadani and Klaczko 2008, Macdonald et al. 1988). Chromosomal inversions and Robertsonian

translocations can act as pre-mating and post-mating isolating mechanisms. Chromosomal races may be reproductively isolated if the resulting morphological variation associated with karyotypic differences lead to physiological mating

incompatibilities or cause assortative mating where morphologically similar individuals preferentially mate with each other (Ayala and Coluzzi 2005, Hatadani et al. 2004). Even if members of different karyotypic races achieve copulation there are numerous potential complicating factors that may arise leading to offspring inviability.

Chromosomal rearrangements can result in monobrachial homology where two different chromosomes share a single homologous arm and can lead to complications during

chromosomal segregation or lead to hybrid sterility or inviability (Baker and Beckham 1986, Britton-Davidian et al. 2000). These complexes can lead to errors in segregation and result in aneuploidy. Thus, the occurrences of chromosomal inversions and rearrangements have been speculated to be a driving force in intraspecific divergence and ultimately speciation (Ayala and Coluzzi 2005, Baker and Beckham 1986, Faria and Navarro 2010, Feder et al. 2005, King 1998, White 1978, Michel et al. 2010).

In the case of chromosomal inversions recombination is suppressed in inverted regions and loci on these regions segregate as a single Mendelian allele or supergene (Joron et al. 2011 Rieseberg 2001, Michel et al. 2010). Inversions are thought to explain the morphological changes that accompany the range expansion across a variety of habitats in the sub-Saharan malaria vector Anopheles sp. (Ayala et al. 2001, Ayala and Coluzzi 2005, Rocca et al. 2009). Inversions on chromosome 2 in Drosophila

mediopunctata are responsible for the distinct abdominal markings that characterize the species and possibly led to assortative mating among similarly marked individuals (Hatadani et al. 2004). Chromosomal races may also differ in the degree and pattern of phenotypic plasticity as ahs been shown in the perennial plant species Stellaria longipes

(Macdonald et al. 1988).

The dogwhelk is a model organism for ecotypic variation and has been the focal species of classic ecological papers (Connell 1961, Menge 1976). Despite our extensive understanding of its ecology and documentation of its morphological variation there has been a tremendous lag-time in our efforts to integrate the chromosomal factors long speculated to influence ecotypic variation. The majority of studies concerned with chromosomal rearrangements and their roles in intraspecific divergence and in some cases speciation focus mostly on either small vertebrate species like mice, shrews,

gerbils, and lizards or insects. While we remain ignorant of the reproductive

consequences of the chromosomal polymorphism in Nucella lapillus and our therefore unable to postulate about potentially speciating populationsour data suggest that chromosomal rearrangements have played a role in shaping ecotypic variation through canalization of phenotypes and/or the magnitude of the plastic response(s) to

environmental variables. The karyotypes with reduced chromosome numbers have been shown to be more phenotypically plastic in this instance.

There are two prominent haplotypes of mitochondrial malate dehydrogenase (mMDH) whose frequencies vary clinally along a 6 km region of the southwestern peninsula of England (Kirby 2000 and 2004). These populations also show clinal variation for the polymorphism with individuals at the more exposed site having 26 chromosomes and those at the more protected site 32 chromosomes. mMDH-1 is very near to fixation in the population on the exposed shore while its frequency on the protected site is only 0.3. Kirby (2004) estimated the time of divergence between the two haplotypes to be between 100 and 144 million years ago, predating both the genus

Nucella and the family Muricidae and further speculated that the two haplotypes are potentially reflective of a coadapted gene complex. Our data are consistent with this suggestion. Elucidation of the role of the chromosomal polymorphism on phenotypic plasticity and evolution in the dogwhelk can be achieved through further karyotyping in conjunction with genetic analyses and field manipulations across different environmental settings. It is perhaps not surprising but nevertheless suggestive that the documentation of these phenomena in an increasing number of species suggests they are pervasive across taxa and act to produce an array of phenotypes adapted to a breadth of environments. Integrative studies that incorporate ecological and genetic factors can

only continue to expand our understanding of intraspecific phenotypic variation in model organisms and extend it to new taxa and systems.

Table  6:    REML whole model effects on aperture width and shell lip thickness. Asterisks indicate statistical significance.

Table  7:  ANOVA results for sites where 2n = 27 only, protected sites only, and exposed sites only with significance values and percent of variance due to each effect. Asterisks indicate statistical significance.

Figure  5:  Response in shell lip thickness for exposed (black diamonds) and protected (grey squares) sites by chromosome number.

  0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 26 27 28 29 30 31 32 C h an g e in s h el l l ip th ic kn es s (m m ) Chromosome number Exposed Protected

Figure  6:  Response  in  aperture  width  in  exposed  (black  diamonds)  and  protected  (grey  squares)   sites  by  chromosome  number.

-1 -0.5 0 0.5 1 1.5 2 26 27 28 29 30 31 32 C h an g e in a p er tu re w id th (m m ) Chromosome number Exposed Protected

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