Effectiveness of ISSR markers for determination of the Aneura pinguis cryptic species and Aneuramaxima

• Genetic isolation between Aneura pinguis cryptic species was confirmed.
• Cryptic species of A. pinguis and Aneura maxima differ in ISSR profiles.
• ISSR markers can be used for identification of Aneura species.

Aneura pinguis and Aneura maxima belong to the simple thalloid liverworts. Previous isozyme studies revealed that A. pinguis is a complex of cryptic species difficult to distinguished based on morphology. In the present study four cryptic species of the A. pinguis complex and A. maxima were examined by means of ISSR method to assess genetic variation and to develop species-specific markers. Eight ISSR primers used generated 460 bands, of which 453 were polymorphic. The highest values of resolving power 28.4 and marker index 18.1 were noted for primer 835 (AG)8-YC, while polymorphism information content for primer 842 (GA)7-AYG. The total gene diversity (HT) based on polymorphic loci was 0.284 for A. pinguis and 0.06 for A. maxima. ISSR markers supported existence of cryptic species in A. pinguis and showed genetic isolation between them. Species-specific bands were found for all studied cryptic species of A. pinguis and A. maxima, thus ISSRs can be used for their identification. A. maxima clearly differ from all the A. pinguis cryptic species in each amplified ISSR primer. The AMOVA conducted for the A. pinguis complex showed that most of genetic variation (ΦPT 0.586) was present among species.