A total of 583 individuals of E. octoculata were sampled between 2007 and 2009 in 18 freshwater environments in Poland: rivers, streams, channels, lakes and ponds, differing in terms of their bottom substrate (Table 1). Leeches were sampled manually from stones and with sweep-net from macrophytes and silt. Animals were preserved in 10% ethyl alcohol and after one hour in 80% ethyl alcohol. Selected animals were photographed under magnification c. 100 ×, alive and after preservation. On the basis of the photographic data-base, the sampled individuals were classified into five morphological forms (morphotypes), distinguished mainly on the basis of the area covered by dark pigment on the dorsal surface of the post-clitellar region (measured on somites 14-15) (Fig. 1a-f). These morphological forms are characterised as follows:
- very dark (Fig. 1a): more than 90% of the dorsal surface is covered by dark pigment, very rarely the body is completely black on the dorsal side (forma localis - Pawłowski, 1936). Only small spots around papillae arranged in regular rows lack the dark pigment. Ventral surface is coloured dark-grey. The body of an adult specimen is typically large, even to 70 mm in length - this form is the least frequent of all forms.
- dark (Fig. 1b): 60-90% of dorsal surface is covered by dark pigment. Light spots around papillae, arranged in rows have different size but are separated (forma atomaria - Pawłowski, 1936). Ventral surface light (not pigmented). Sometimes the background of the body is red-brownish in colour. The body of adult specimens does not reach 70 mm in length.
- medium (Fig. 1c): 35-60% of dorsal surface is covered by dark pigment. Light, multishaped spots around papillae arranged irregularly are merged (forma typica - Pawłowski, 1936) and typically exceed the borders between annuli. Ventral surface light. The body of adult specimens does not reach 70 mm in length.
- light (Fig. 1d): 10-35% of dorsal surface is covered by dark pigment. Dark, pigmented areas between papillae are joined together (forma pallida - Pawłowski, 1936). Ventral surface light. The body of adult specimens always shorter than 70 mm. Individuals of this form are relatively difficult to distinguish from the type 3.
- very light (Fig. 1e): less than 10% of dorsal surface is covered by dark pigment, sometimes body completely unpigmented (forma pallida - Pawłowski, 1936), pale-ochre coloured in live specimens. Dark spots and relatively large patches, if present, localised mid-dorsally and separated. Sometimes the colour of the body is greenish and darker on the clitellum. Ventral surface light. The body of adult specimens typically large, even to 70 mm.
The differences between different types of environments and between different types of bottom substrates in proportion to particular morphological forms and in mean proportion of pigmented area on the dorsal surface were determined. Statistical significance of the differences in proportions was tested with Chi2 tests, while those in the mean pigment coverage of the body was tested with one-way ANOVA and post-hoc Tukey’s test.