RESULTS

3. 1 Phylogenetic analysis
The CO1 -based phylogenetic analysis recovered two distinct clusters, which were called ‘Tsushima’ and ‘Kuroshio’ lineages after the corresponding ocean currents in the snails’ native range (Figure 2G). The Hacheon (Korea) and Nemuro (Japan) individuals were part of the Tsushima lineage, while the Matsushima (Japan) and Elkhorn Slough (USA) individuals belonged to the Kuroshio lineage. These lineage assignments agree with a previous study that identified the region of Japan most likely to be the source of the introduced North American population (Miura, Torchin, Kuris, Hechinger, & Chiba, 2006).

3.2 Multimodel inference and model averaging

Based on the criteria of Z -scores > 0 andP -values < 0.05, the average parameter estimates indicated that all the model terms were more or less positively related to changes in snail movement distance, in which the es term was the most important, followed by o (relative importance = 98%), es × o (89%), and li (85%) (Table 1). This result is also supported by the AICc scores (low to high) of the models that included es, o, and li (models i, ii, and iii, Table 2). In particular, the multimodel inference test indicated that the most parsimonious model (based on the lowest AICc score, 3416.97) was es × o + li (model i). In accordance with the ΔAICc ≤ 6 cut-off rule, the second and third best-fit models were es × o (model ii) and es × li + o (model iii). These three models respectively received 76%, 14%, and 8% of the total weight wi . All the other models, which omitted or included lo and p, received higher AICc scores and lower weights, indicating that these models are not important in describing the locomotion of salt-stressed snails. Therefore, the multimodel analysis supports the conclusion that the geographic distribution-origin and genetic composition substantially influence the movement distance of snails in response to salinity stress.

3.3 Locomotor performance changes upon variables

The locomotor experiments showed that the intertidal snailBatillaria attramentaria from different locations was able to acclimate to a range of salinity from 13 to 43 PSU. From these experiments, we observed significant locomotion differences between the snail groups exposed to 13 PSU and other treatments of 23, 33, and 43 PSU (LMMModelxi,F ES (1, 278) = 99.47, P< 0.0001, Table 3). Particularly, they significantly reduced movement distance when transferred from normal salinity condition of 33 PSU to acute low salinity of 3 PSU but not significantly changed their movement distance when transferred to moderately changed salinities of 23 and 43 PSU (Figure 3A). Post-hoc tests using the Tukey post-hoc criterion for significance indicated that snails exposed to very low salinity (13 PSU) moved significantly less than the other treatment groups ( d 23PSU-13PSU = 0.43 ± 0.04 m1/2, d 33PSU-13PSU = 0.48 ± 0.04 m1/2, d 43PSU-13PSU = 0.45 ± 0.04 m1/2, P < 0.0001, Appendix 1: Table A2, Figure 3A). While, the moderately stressed snails (exposed to 23 and 43 PSU) moved slightly less than the control group (exposed to 33 PSU) but this difference was not statistically significant (d 33PSU-23PSU = 0.05 ± 0.04 m1/2 and P = 0.5688,d 33PSU-43PSU = 0.03 ± 0.04 m1/2and P = 0.8637, and d 43PSU-23PSU = 0.02 ± 0.04 m1/2 and P = 0.9559).
Notably, when considering all treatment groups, we observed that the origin factor had significant impacts on locomotor pace of the snails with F O (1, 278) = 31.68 and P< 0.0001 (Model xii). Subsequent post-hoc test of this analysis indicated that the native populations moved significantly more than the introduced population (d Native-Introduced = 0.28 ± 0.05 m1/2, P < 0.0001, Appendix 1: Table A2, Figure 3B). Though, we did not record any significant differences in movement distance between the two CO1 -lineages (LMMModelxv,F li (1, 278) = 0.84, P = 0.3587, Table 3) with d Tsushima-Kuroshio = 0.03 ± 0.04 m1/2 and P = 0.3597. Besides, we also found significant locomotion differences based on location and population (LMMModel xiii,F lo (1, 278) = 28.53, P< 0.0001 and LMMModel xiv,F p (1, 278) = 5.28, P = 0.0223, Table 3). Subsequent Tukey post-hoc tests revealed that differences in locomotor responses among native snail populations were not statistically significant (d Korea-Japan = 0.06 ± 0.04 m1/2 and P = 0.3198,d Hacheon-Nemuro = -0.03 ± 0.05 m1/2 and P = 0.9426,d Hacheon-Matsushima = -0.09 ± 0.05 m1/2 and P = 0.2163,d Nemuro-Matsushima = -0.06 ± 0.05 m1/2 and P = 0.5226), but differences between native snail locations and the introduced location were significant (d Korea-USA = 0.24 ± 0.06 m1/2and P = 0.0001, d Japan-USA = 0.30 ± 0.05 m1/2 and P < 0.0001,d Hacheon-Elkhorn Slough = 0.24 ± 0.06 m1/2 and P = 0.0001,d Matsushima-Elkhorn Slough = 0.33 ± 0.06 m1/2 and P < 0.0001,d Nemuro-Matsushima = 0.27 ± 0.06 m1/2 and P < 0.0001, Appendix 1: Table A2).
A linear mixed-effect model test of the best model (model i) showed that the interaction of Origin (o) and Salinity Exposure (es) (LMMModel i,F es × o (1, 275) = 11.59, P = 0.0008) was significant, and so was the effect of Lineage (li) (LMMModel i,F li (1, 275) = 5.38, P = 0.0211, Table 3). This result corresponds to the significant es × o interaction and li term in the model outputs (Zes × o = 3.41, P < 0.001, Table 1). However, when implemented separately, only es and o significantly influenced the movement distance of the snails independently (LMMModel xi,F ES (1, 278) = 99.47, P< 0.0001 and LMMModel xii,F O (1, 278) = 31.68, P< 0.0001, Table 3), while in contrast, li did not (LMMModel xv,F li (1, 278) = 0.84, P = 0.36, Table 3). Detailed differences in locomotion under the effect of the interaction es × o + li estimated by Tukey post-hoc test can be found in Appendix 1: Table A3.

3.4 Variation in shell length with distribution and CO1 lineage

We conducted a two-way ANOVA to examine the effect of geographic distribution and genetic composition on the shell length of all 280 individuals included in the locomotor analyses. We confirmed that introduced B. attramentaria individuals were significantly longer than native ones (F o (1, 278) = 133.5,P value < 2e-16, Appendix 1: Table A4A and Figure A2). Simple main effect analyses showed that the average shell length of introduced snails was 31% longer (lN ative = 2.14 cm,l Introduced = 3.12 cm, Appendix 1: Table A4B). These analyses also revealed that snails from different locations or populations also exhibited significant differences in shell length withF l (2, 277) = 193.7, p< 2e-16 andF P (3, 276) = 195.1, p< 2e-16 (Appendix 1: Table A4A). In particular, the snail individuals from Korea were smallest and follow by the Japan and the USA populations (l Korea(Hacheon) = 1.68 cm,l Japan = 2.38 cm, in whichl Nemuro = 2.62 cm andl Matsushima = 2.13 cm, andl USA = 3.12 cm, Appendix 1: Table A4B). Furthermore, shell length also significantly varied with lineageF li (1, 278) = 19.42; p = 1.5e-05 with l Tsushima = 2.15 cm and l Kuroshio = 2.62 cm, respectively (Appendix 1: Table A4A and B), which is not surprising considering that one of the two lineages includes the introduced (larger) individuals.