Contributions to Zoology, 80 (1) – 2011Pawel Koperski; Rafal Milanowski; Agnieszka Krzyk: Searching for cryptic species in Erpobdella octoculata (L.) (Hirudinea: Clitellata): discordance between the results of genetic analysis and cross-breeding experiments

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Field study

The proportions of morphological forms of leeches sampled from different types of bottom substrate were significantly different. Light and very light individuals dominated on stone bottom, while dark and very dark individuals were more frequent among silt and submerged plants. These differences were more significant in flowing waters than in lakes. The proportions of morphological forms on the stone bottom were significantly different from the silt/plant bottom (Chi2 test, p<0.01) (Table 1). Mean area of the individuals’ dorsal surface of the body covered by the dark pigment was different in four types of environments when tested with ANOVA and post-hoc Tukey’s test (p = 0.05) (running waters-stone bottom and standing waters-stone bottom - as homogenous group A and running waters-silt/plant bottom and standing waters-silt/plant bottom as homogenous group B).

Laboratory experiments on reproduction success

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As a result of experimental mating of 84 pairs, 492 cocoons were laid from which 2039 offspring hatched. Because of high mortality in first few days after hatching, 1193 individuals (58.5%) remained alive after 5 days and only 210 individuals (10.3%) remained alive to the moment when the determination of the morphological form was possible (15-20 days). 10 pairs of these individuals selectively chosen for mating produced 16 offspring (generation F2). The colour pattern of the offspring was not strictly determined by that of their parents - ca. 17% of the offspring belonged to a different morphological form than both identical parents, and 3% of them were highly different from the parents (Table 2).

The reproduction success differed between parental pairs belonging to different morphological forms (Table 3). It was significantly higher for morphologically identical parents than that of parents differing in morphology when measured by the mean number of cocoons, mean number of offspring hatched and mean number of offspring which survived after 5 days per pair (Wilcoxon’s test, p<0.01) but not when measured by mean number of offspring per cocoon (Fig. 3). Higher reproductive success of morphologically similar parents suggests the existence of weak reproductive barriers between groups of individuals.


Table 2. Percentages of morphological forms in offspring of morphologically similar and morphologically different parents. The number of parental pairs added.


Table 3. Reproduction success of parent pairs E. octoculata differed in terms of morphology expressed as the number of offspring per pair with SD and median values. The number of parental pairs added.


Fig. 3. Reproduction success of morphologically similar and morphologically different parent pairs of E. octoculata expressed as different demographic parameters with SD. * denotes significant difference for ‘within forms’ vs ‘between forms’ comparison (Wilcoxon’s test).

Analysis of DNA sequences

If there were reproductive barriers between morphological forms, we would expect these to be reflected in fixed differences between these sequences. No repeatable differences in COI and ITS sequences between morphological forms of E. octoculata were found. The lack of polymorphism in these regions clearly shows that no reproductive barriers between morphological forms exist. The results of the analysis of genetic similarity with AP-PCR suggest that diversity is strongly related to geographical distance among populations: individuals collected in the same water body are more similar one to another than to individuals collected in other water bodies (Fig. 4). Individuals most similar in terms of AP-PCR analysis were collected at the nearest sampling sites: the Wadąg lake, Święcek and Wisła rivers. Geographical distance, however, does not explain all differences in mean genetic similarity between individuals collected in compared water-bodies - the best fit regression explaining the relation between geographical distance and UPGMA genetic similarity is described by the exponential equation:

y = 0.1385 x0.4066 (with relatively weak value of regression coefficient R2 = 0.4012)

where x is geographical distance between sampling sites (in km) and y is UPGMA similarity between a pair of individuals.


Fig. 4. Biplot of non-hybrid MDS presenting genetic distance (AP-PCR method) between individuals of E. octoculata differed in terms of morphology (empty circles - light form, grey circles - medium form, black circles - dark form, black squares - very dark form) sampled in various environments (marked by ovals).

It is clearly visible that genetic similarity between individuals is not related to their classification into 5 morphological forms. The genetic distance between individuals compared within particular environments was significantly lower than between those compared between environments at p<0.0001 (Table 4). Contrary to that, genetic distance between individuals compared within particular morphological forms did not differ from those compared between forms. Coefficient of variance calculated for values of genetic distance between individuals compared within particular environments was 5.6 times higher than for those compared between environments, which might be explained by the hypothesis that individuals collected in certain environments (e.g. in the Wisła river) are much more genetically diverse than in the other (e.g. in lake Wadąg).


Table 4. Mean genetic distance (AP-PCR method) between groups of individuals differed in terms of morphology and sampled in various environments with SD and coefficient of variance for these groups. Result of comparisons between the genetic distance of analysed groups of individuals with T-test are also added as p values.