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Results
Morphometry
next sectionRaw data. The PCA based on raw data shows a strong size-dependency of most characters, with the first axis (Eigenvalue = 12.0) explaining 54.6% of the variation and the second one (Eigenvalue = 3.52) only 16.0% (Fig. 2A). All characters of the Iberian population were size-dependent (except LA and PS), while the only size-dependent characters in the Congolese population were WL, NS, WWP, VCL, DLS and VCS (Table 1A). The size-relationship in the PCA plot was also more evident for the Iberian population, whose specimens showed a wider size-range representativeness than the Congolese ones. Nevertheless, the two populations clearly form separate groups in the PCA plot, and turned to be significantly different (ANOSIM, Global R = 0.435, significance level = 0.1%).
Fig. 2. Principal Component Analyses plots. a. Based on raw data. b. Based on size independent data. c. Based on measurement proportions. |
Table 1. Relationships with worm size (as body width with parapodia) in Oxydromus okupa sp. nov., with bold characters indicating significant differences. A. Morphometric measurements. B. Taxonomically relevant proportions. Measurement abbreviations as in Fig. 1 . Coeff: Pearson correlation coefficient; p: significance level. |
The intra-population average distances for the Iberian and Congolese worms were 29.9% and 7.10%, respectively, whilst the inter-population dissimilarity was 48.67%. PS, LA and PP, and NS, WL and PP most contributed to the Iberian and Congolese intra-population similarity, respectively, whilst LA, PS and PP most contributed to the inter-population dissimilarity (SIMPER, Table 2).
Table 2. List of the ten most contributing raw measurements to the intra-population similarities and inter-population dissimilarity based on the SIMPER analyses. Measurement abbreviations as in Fig. 1. Av.Value: Average value; Av.Sq.Dist: average square distance; Sq.Dist/SD: square distance divided by standard deviation; Contrib%: percentage of contribution; Cum.%: cumulative percentage of contribution. |
All averaged character measurements showing significant differences were higher in the Iberian than in the Congolese population, except for NS and DLS, with the most remarkable difference being at LA (one-way ANOVA, Table 3).
Table 3. Comparative table of the raw morphometric measurements (µm) in the Iberian and Congolese populations. Differences expressed as percentages, with bold characters indicating significant differences (according to Benjamini & Hochberg, 1995) that may be higher (normal text) or lower (italics) in the Iberian than in the Congolese population, respectively. Measurement abbreviations as in Fig. 1. Min: minimum; Max: maximum; Mean: average ± standard deviation; F: Fisher’s F index; p: significance level. |
Based on raw data, the discriminant function included the variables DLS, PP, PNCLL and LA (Table 4). This function correctly classified the 100% of individuals from both localities (Fig. 3) and the success probability in the cross validation was of 98%.
Table 4. Results of the discriminant analyses based on raw data, size-independent data and measurement proportions. F: Fisher’s F index; Lambda: Rao approach to the Wilks’ Lambda test; p: significance level; Coeff.: Standardized coefficients for the variables included in the inter-population discriminant functions, arranged in a decreasing order, according to their contribution to the inter-population discrimination; Congo, Iberian: coefficients of the selected variables in the classification functions for the Congolese and Iberian populations, respectively. Intercept: Intercept of the classification functions for each dataset. Measurement abbreviations as in Fig. 1. |
Size-independent data. For the purpose of this analysis, and taking into account that the size range was well represented in the Iberian population, only LA and PS raw data were considered as size-independent (Table 1A). The PCA plot based on size-independent data revealed again two clearly different groups corresponding to the two studied populations (Fig. 2B). Despite the axes being less representative than those obtained for the raw data (Eigenvalues = 6.61 and 3.49, variation explained = 31.5% and 16.6%, respectively for axis 1 and 2), the two populations were more clearly distinguishable and showed more significant differences than the raw data (ANOSIM, global R = 0.539, significance level = 0.1%).
Based on these size-independent characters, the intra-population average distance within the Iberian and Congolese populations was 21.0% and 11.1%, respectively, whilst the inter-population distance was 50.3%. PS, DAPE and LA, and WL, NS, VCS, and PP most contributed to the Iberian and Congolese intra-population similarity, respectively, whilst the Congolese vs. Iberian dissimilarity was mainly explained by LA, DAPE, DLS, PP, and PS (SIMPER, Table 5).
Table 5. List of the ten most contributing size-independent measurements to the intra-population similarities and inter-population dissimilarity based on the SIMPER analyses. Measurement abbreviations as in Fig. 1. Av.Value: Average value; Av.Sq.Dist: average square distance; Sq.Dist/SD: square distance divided by standard deviation; Contrib%: percentage of contribution; Cum.%: cumulative percentage of contribution. |
When comparing the averaged character measurements, all those showing significant differences were higher in the Iberian than in the Congolese population (particularly, LA, PS, PP and DAPE), except for NS, WWP, DLL, and DLS in particular (one-way ANOVA, Table 6).
Table 6. Comparative table of the size-independent morphometric measurements in the Iberian and Congolese populations. Differences expressed as percentages, with bold characters indicating significant differences (according to Benjamini and Hochberg, 1995) that may be higher (normal text) or lower (italics) in the Iberian than in the Congolese population. Measurement abbreviations as in Fig. 1. Min: minimum; Max: maximum; Mean: average ± standard deviation ; F: Fisher’s F index; p: significance level. |
Based on size-independent data, the discriminant function included the variables WL, NS, DLS, PP, PNCLL, and LA (Table 4). This function correctly classified the 100% of individuals from both localities (Fig. 3) and the success probability in the cross validation was 100%.
Character proportions. Nine of the 20 character proportions analysed showed significant negative (n = 7) or positive (n = 2) correlations with size, indicating allometric relationships in the Iberian population, while only one was significantly negatively correlated with size in the case of the Congolese population (Table 1B).
In the PCA based on character proportions, Axes 1 (eigenvalue = 5.38) and 2 (eigenvalue = 3.13) explained 26.9% and 15.6% of the variation, respectively. The PCA plot also highlighted a marked clustering for the individuals of the two populations under study (Fig. 2C), the results being slightly less discriminant than the previous ones but equally highly significant (ANOSIM, Global R = 0.421, significance level = 0.1%).
The average intra-population distance for the Iberian and Congolese populations were 21.4% and 11.1%, respectively, whilst the average inter-population distance was 46.0%. DAPE/HL, DCSS/DLS, and LA/HL, and WL/WW, NS/WW, DCSS/DLS, and LA/LH were the most informative proportions for the intra-population similarity in Iberian and Congolese worms, respectively, whilst the intra-population dissimilarity was mainly explained by DCSL/DLL, LA/HL, NS/NW, and DAPE/HL (SIMPER, Table 7).
Table 7. List of the ten most contributing measurement proportions to the intra-population similarities and inter-population dissimilarity based on the SIMPER analyses. Measurement abbreviations as in Fig. 1. Av.Value: Average value; Av.Sq.Dist: average square distance; Sq.Dist/SD: square distance divided by standard deviation; Contrib%: percentage of contribution; Cum.%: cumulative percentage of contribution. |
All averaged character proportions showing significant differences, except for NS/WW and WWP/WW, were higher in the Iberian than in the Congolese population, most of them with differences higher than 10% and particularly higher than 20% in the case of DCSS/DLS (one-way ANOVA, Table 8).
Based on character proportions, the discriminant function included the variables WL/WW, NS/WW, DCSS/DLS, PS/HL, and DAE/DPE (Table 4). This function correctly classified the 100% of individuals from both localities (Fig. 3) and the success probability in the cross validation was 96%.
Population size-structure
A total of 275 worms were collected during this study, 246 infesting S. plana (241 during the seasonal monitoring, five in January 2013), 26 in M. pellucida (plus three outside the host, but in the same container) (Table 9). Sizes ranged from 1.8 to 3.6 mm. Small-sized worms seem to be better represented during late autumn, but also in winter, while large worms were more or less constantly present throughout the study period, except during late winter and early spring (Fig. 4).
Table 9. Oxydromus okupa sp. nov. Synthesis of the monthly captures, total and infested number of hosts, percentage of ripe females, and prevalence during the study period. |
Ripe females occurred during the whole period except in April 2012, and were always among the largest size-classes (2.2 to 3.6 mm) (Fig. 4). The highest percentages occurred in mid spring and summer, being August 2011 the single month during which the proportion ripe females vs. non-sexed adults sexes was 1:1 (Fig. 5; Table 9). The lowest percentages occurred in autumn and winter (Fig. 5; Table 9).
Fig. 5. Oxydromus okupa sp. nov. Monthly prevalence of ripe females vs. total prevalence in the infested Scrobicularia plana. |
A total of 6,917 specimens of S. plana and 39 of M. pellucida were collected (Table 9). Length in S. plana ranged from 20 to 40 mm, with the exception of one infested host measuring 40.7 mm. However, bivalves with intermediate lengths (i.e., 26-36 mm) occurred during the whole year, being always the most abundant and also the most infested ones (Fig. 6). The most balanced size class frequency distribution occurred in September 2011. In M. pellucida, the size-class range was restricted to 20-30 mm length, and the most infested ones were slightly smaller (20-28 mm) than in S. plana (Fig. 6).
Overall, there was a non-significant size correlation between O. okupa sp. nov. and the host S. plana (Pearson coefficient = 0.123, p = 0.067). The monthly trends were also non-significant (Pearson coefficient = -0.375 to 0.351, p = 0.103 to 0.818), except for a positive correlation in April 2012 (Pearson coefficient = 0.870, p = 0.011). Conversely, O. okupa sp. nov. / M. pellucida symbiont-host pairs (26) collected in January 2013 showed a significant, positive size correlation (Pearson coefficient = 0.400, p = 0.021).
Infestation characteristics
The studied population of O. okupa sp. nov. showed a seasonal variability in prevalence (Fig. 5; Table 9). The highest percentages occurred during late autumn-mid winter. The highest peak was in December 2011 (i.e. 19.2%), in coincidence with the highest number of symbionts (Table 9). Intermediate prevalences occurred in November 2011 (i.e. 7.2%), January 2012 (i.e. 8.2%) and February 2012 (i.e. 6.1%). The remaining prevalences were always < 5% and the lowest one occurred in April 2011 (i.e. 1.6%). The intensity of the infestation was always a single worm per host. In the case of the specimens associated with M. pellucida, the prevalence was 66.7% (Table 9), reaching 89.3% in hosts > 20 mm long (Fig. 6).
There was a non-significant correlation between the percentage of ripe females and prevalence in S. plana along the studied period (Pearson coefficient = -0.407, p = 0.168). However, the percentage of ripe females was, on average, significantly higher (one-way ANOVA, F = 9.253, p = 0.011) in the warm than in the cold period, reaching 31.0±4.8% and 7.5±6.1%, respectively, while the prevalence showed exactly the contrary pattern, 3.2±1.3% vs. 8.9±1.7% (one-way ANOVA, F = 7.066, p = 0.022) (Fig. 5).