All the worms with 2nā=ā50 chromosomes belonged to an already identified sibling species of T. The chromosome number distribution was consistent with the presence of at least six sympatric euploid chromosome complements (one diploid, one triploid, three tetraploids and one hexaploid), as confirmed by a FISH assay performed with an homologous 18S rDNA probe. To generate a robust phylogeny enabling the reconstruction of the evolutionary processes shaping the diversity of these sympatric lineages, we complemented our original 16S rRNA gene sequences with additional COI sequences. For each cocoon, we sequenced a fragment of the 16S rRNA from half of the sibling embryos and karyotyped the other half. To gain insights into the evolutionary events that generated this differentiation, we performed a cytogenetic analysis in parallel with a molecular assay. from the Lambro River (Milano, Italy), which we suggested comprise several cryptic species. In a molecular phylogenetic analysis, we detected substantial genetic variability among sympatric Tubifex spp. Tubifex tubifex is a widespread annelid characterized by considerable variability in its taxonomic characteristics and by a mixed reproductive strategy, with both parthenogenesis and biparental reproduction.
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