This paper provides the first DNA sequences and phylogenetic insight into the Central Asian genus Eodorcadion. We used four molecular markers (COI, CAD, ITS2, and Histone 3) and investigated COI genetic distances to verify phylogenetic position of closely related taxa endemic to Mongolia of the ‘Eodorcadion intermedium species-group’. Histone3 data are presented for the first time for Cerambycidae species. We also designed new PCR primers for better amplification of the Cerambycidae COI barcode region. Morphology of all targeted taxa was examined by means of scanning electron microscopy. Our study showed that while there were very few nucleotide differences among COI sequences of three closely related taxa, such that they shared one haplotype, two of them were grouped separately in the all-data tree, and all three were morphologically distinguishable. Conversely, there was a clear barcode gap between E. intermedium intermedium and all the remaining taxa, including E. intermedium kozlovi. Based on the phylogeny, they belong to two different species-groups; thus, E. kozlovi is herein restored to specific rank. Using Bayesian analysis, we contrasted the COI-based tree with the one supported by nuclear data and showed that COI-only data are not sufficient to resolve the phylogeny of the recently derived flightless groups such as Dorcadionini. We conclude that the Eodorcadion intermedium species-group is a polyphyletic species assemblage, established based on the pattern of the elytral stripes, which may be a case of parallel evolution driven by ecological adaptation.

DNA barcoding has revolutionized how we discover, identify, and detect species. A substantial foundation has been established with millions of mitochondrial cytochrome c oxidase I sequences freely available for eukaryotes. However, issues with COI ranging from uniparental inheritance and small genetic population sizes to nuclear and asymmetric introgression can impede its use. We propose using CAD as the “nuclear barcode” to complement the COI barcode and ameliorate these concerns. We focused on beetles from taxonomically diverse species-level studies that used COI and CAD. An ambiguous barcode gap was present between intra- and interspecific genetic distances in CAD and COI; this led to difficulty with automated gap detection methods. We found pseudogenes, problematic population structure, introgression, and incomplete lineage sorting represented in the COI data. A CAD gene tree illuminated these cryptic problems. Placement tests of species and outgroups using distance-based tree building were largely successful for CAD, demonstrating its phylogenetic signal at the species and genus levels. Species placement issues were typically unique to one locus, allowing for recognition of misdiagnosis. We conclude that a CAD barcode is a valuable tool for beetle diagnostics, metabarcoding, and faunistic surveys.

The flightless tribe Dorcadionini is one of the most species-rich and taxonomically challenging groups in the family Cerambycidae. Eodorcadion is one of five genera included in Dorcadionini, and one of three distributed in Central Asia. The current classification of the genus is based on taxonomic works pre-dating the application of cladistic methods. We used molecular data from one mitochondrial and three nuclear markers (COI, CAD, ITS2, and Histone 3) for 70 specimens in 23 species and subspecies, representing all subgenera and most species groups of Eodorcadion to build its first phylogeny. By including some taxa of Central Asian Dorcadion and Lamia textor (type genus of Lamiini and Lamiinae), we assessed the monophyly of the tribe. The results of our analyses are the first to fully support the synonymy of Dorcadionini with Lamiini and confirm the morphology-inferred monophyly of Eodorcadion. Consequently, Lamiini = Dorcadionini syn. nov. Moreover, based on the obtained phylogeny and morphological differences, we diagnosed and subsequently described two new subgenera of Eodorcadion: Altaidorcadion Karpiński subgen. nov. and Arenodorcadion Karpiński subgen. nov., while the currently recognized subgenus Humerodorcadion stat. nov. was elevated to the genus rank. Our phylogeny is remarkably consistent with results previously obtained by other authors that used endophallic morphology.

As defining various taxonomic ranks, especially at and within a specific level, can often be difficult, we used an evolutionarily young group of Central Asian beetles, the genus Anoplistes, to examine the limits between species, subspecies and ecotype. The molecular data (COI genetic distance, haplotype network and species delimitation) and detailed morphology (using SEM) of taxa assigned to the rank of species, subspecies and ecotype were used to verify their taxonomic status and attempt to more precisely establish the boundaries between these concepts. The minimum threshold for the COI genetic distance between species was found to be approximately 3%, while at the threshold of 4%–5% the morphology left no doubt as to the species distinctiveness. The threshold of approximately 2% was accepted as adequate for a subspecies rank, whereas intraspecific variability among individuals of the same subspecies (including its ecotypes) ranged from 0% to 2%. A newly presented colour/elytral pattern form, possibly another ecotype of A. halodendri distributed in western Mongolia, exemplifies adaptation to different environmental conditions via ecological speciation. Our results provide support that ecotypes can form phylogenetically distinct clades. Based on the results of the haplotype network, it can be tentatively concluded that the sand ecotype was the incipient form from which further ecotypes and subspecies developed.