# Unraveling Wing Shape Variation in Malaria Mosquitoes from the Arctic Edge: A Geometric Morphometric Study in Western Siberia

## TL;DR

This study uses wing shape analysis and genetic data to distinguish malaria-carrying mosquitoes in Western Siberia, where traditional methods fail due to similar appearances.

## Contribution

The study introduces geometric morphometrics of wing venation as a reliable method for differentiating cryptic malaria mosquito species and their hybrids.

## Key findings

- Wing shape and size differences were statistically significant among the three mosquito species and their hybrids.
- Hybrid mosquitoes showed intermediate wing morphology overlapping with their parental species.
- Landmarks on radial and medial veins were most important for species separation, and wing shape differences were not explained by size.

## Abstract

In Western Siberia, the Anopheles maculipennis subgroup includes three malaria vectors An. messeae, An. daciae, and An. beklemishevi, and their hybrids, which are difficult to tell apart using traditional traits such as egg exochorion. We combined molecular identification with geometric morphometrics of wing venation to evaluate differences in wing shape and size among species. We found statistically significant differences in wing shape and centroid size, with hybrids showing intermediate morphology and overlap with their parental species. Landmarks on the radial and medial veins contributed most to species separation. We did not evaluate environmental adaptation, and wing shape differences were not explained by size. These findings support the use of wing morphometrics, together with genetic data, to improve species delimitation and surveillance of malaria vectors in temperate regions.

In Russia, Western Siberia, Anopheles from maculipennis subgroup comprises three vector species: An. messeae, An. daciae, An. beklemishevi, and the hybrid between An. messeae and An. daciae (Anopheles m-d), which exhibit complex cryptic morphological traits. Traditional morphological methods, such as egg morphology and exochorion coloration, have proven insufficient for reliably distinguishing these closely related species due to overlapping characteristics and high intra-species variability. To overcome these limitations, geometric morphometrics (GM) has emerged as a powerful tool for analyzing cryptic morphology. This article focuses on wing venation patterns, where GM provides precise, quantitative data based on defined anatomical landmarks, enabling detailed assessment of size and shape variation among species. Procrustes ANOVA, principal component analysis (PCA), and canonical variate analysis (CVA) were employed to assess shape variation and species differentiation. Centroid size and its relationship to shape variation were examined using multivariate regression. Despite significant morphological differences, the overlap observed in hybrids (An. m-d) reflects their intermediate position between the parental species. Our analyses revealed significant differences in wing shape and size among An. messeae, An. daciae, An. beklemishevi, and their hybrids, with hybrids showing intermediate morphologies. Landmarks on radial and medial veins were the most consistent contributors to species separation. No evidence of static allometry was detected, and wing shape differences were not explained by size. These findings demonstrate that wing morphometrics, combined with molecular identification, provides a reliable framework for species delimitation and surveillance of malaria vectors in temperate regions.