| Summary: | The Eurasian malaria vector <i>Anopheles messeae</i> is a widely spread and genetically diverse species. Five widespread polymorphic chromosomal inversions were found in natural populations of this mosquito. A cryptic species, <i>Anopheles daciae,</i> was differentiated from <i>An. messeae</i> by the presence of several nucleotide substitutions in the Internal Transcribed Spacer 2 (ITS2) region of ribosomal DNA. However, because of the absence of a high-quality reference cytogenetic map, the inversion polymorphisms in <i>An. daciae</i> and <i>An. messeae</i> remain poorly understood. Moreover, a recently determined heterogeneity in ITS2 in <i>An. daciae</i> questioned the accuracy of the previously used Restriction Fragment Length Polymorphism (RFLP) assay for species diagnostics. In this study, a standard-universal cytogenetic map was constructed based on orcein stained images of chromosomes from salivary glands for population studies of the chromosomal inversions that can be used for both <i>An. messeae</i> and <i>An. daciae.</i> In addition, a new ITS2-RFLP approach for species diagnostics was developed. Both methods were applied to characterize inversion polymorphism in populations of <i>An. messeae</i> and <i>An. daciae</i> from a single location in Western Siberia in Russia. The analysis demonstrates that cryptic species are remarkably different in their frequencies of chromosomal inversion variants. Our study supports previous observations that <i>An. messeae</i> has higher inversion polymorphism in all autosomes than the cryptic species <i>An. daciae</i>.
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