International Journal of Molecular Evolution and Biodiversity 2024, Vol.14, No.5, 208-218 http://ecoevopublisher.com/index.php/ijmeb 210 distinguishing between different aphid species and genera, especially in paleontological contexts where molecular data is unavailable. Additionally, the external morphology of aphids can be highly variable and influenced by environmental factors. For example, the European species of the genus Eulachnus exhibit significant morphological differences in head, antennae, legs, and dorsal chaetotaxy, which have been used to propose three distinct species groups: “agilis”, “brevipilosus”, and “cembrae” (Kanturski et al., 2015). These morphological differences are essential for accurate species identification and understanding the evolutionary relationships within the genus. 3.2 Advances in microscopic techniques Advances in microscopic techniques have significantly enhanced the resolution and accuracy of morphological studies in aphid taxonomy. Scanning electron microscopy (SEM) has been particularly valuable in revealing fine details of aphid morphology that are not visible with traditional light microscopy. For example, SEM methods were employed to study the external morphology of the European species of the genus Eulachnus, providing high-quality images that revealed detailed surface structures and morphological signs of cell deterioration (Kanturski et al., 2015). These high-resolution images allow for more precise comparisons between species and can uncover subtle morphological differences that are crucial for accurate classification. Furthermore, the use of cryo-SEM and HMDS preparation techniques has improved image quality by reducing surface tension and minimizing artifacts such as depressions and membrane blebs. These advancements in microscopic techniques have not only facilitated more detailed morphological studies but also enabled the discovery of new species and genera, as demonstrated by the identification of Tanyaulus caudisetula in mid-Cretaceous amber (Poinar, 2018). 3.3 Limitations of morphological taxonomy Despite the advancements in microscopic techniques, morphological taxonomy has its limitations. One major challenge is the phenotypic plasticity of aphids, where environmental factors can cause significant variations in morphology, leading to potential misidentifications. For instance, the rapid radiation and diversification of aphids, influenced by both abiotic and biotic factors, have resulted in incongruent molecular and morphological phylogenies (Owen and Miller, 2022). This discordance complicates the classification and understanding of evolutionary relationships among aphid species. Moreover, morphological traits alone may not be sufficient to resolve complex taxonomic questions, especially in cases of cryptic species or recent divergences. The reliance on morphological characteristics can also be problematic when dealing with incomplete or damaged specimens, such as those found in fossil records. In such cases, integrating molecular data with morphological studies can provide a more comprehensive and accurate approach to aphid taxonomy. For example, the use of genome and transcriptome data has been shown to clarify phylogenomic relationships among aphid subfamilies, despite the presence of gene tree discordance and introgression events (Owen and Miller, 2022). Therefore, while morphological approaches remain fundamental, they are most effective when complemented by molecular techniques. 4 Molecular and Genetic Approaches 4.1 DNA barcoding and phylogenetic analysis DNA barcoding has emerged as a powerful tool for the identification and classification of aphid species, leveraging the mitochondrial cytochrome c oxidase I (COI) gene as a standard marker. This method has proven effective in distinguishing between morphologically similar species and uncovering cryptic diversity. For instance, a comprehensive study on European aphids demonstrated that DNA barcoding could reliably identify 80% of species, even those difficult to distinguish morphologically (D’acier et al., 2014). Similarly, research on the subfamily Calaphidinae revealed significant cryptic diversity (Figure 1), with DNA barcoding identifying discrepancies between traditional taxonomy and genetic data in 21.7% of the species studied (Lee et al., 2017).
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