To address the limited number of mitochondrial genomes (mitogenomes) in the subfamily Steganinae (Diptera: Drosophilidae), we assembled 12 complete mitogenomes for six representative species in the genus Amiota and six representative species in the genus Phortica. We performed a series of comparative and phylogenetic analyses for these 12 Steganinae mitogenomes, paying special attention to the commonalities and differences in the D-loop sequences. Primarily determined by the lengths of the D-loop regions, the sizes of the Amiota and Phortica mitogenomes ranged from 16,143–16,803 bp and 15,933–16,290 bp, respectively. Our results indicated that the sizes of genes and intergenic nucleotides (IGNs), codon usage and amino acid usage, compositional skewness levels, evolutionary rates of protein-coding genes (PCGs), and D-loop sequence variability all showed unambiguous genus-specific characteristics and provided novel insights into the evolutionary implications between and within Amiota and Phortica. Most of the consensus motifs were found downstream of the D-loop regions, and some of them showed distinct genus-specific patterns. In addition, the D-loop sequences were phylogenetically informative as the data sets of PCGs and/or rRNAs, especially within the genus Phortica.

The Stegana (Steganina) shirozui species group is mainly distributed in East Asia. In the present study, the molecular phylogeny of the S. shirozui group was investigated based on mitochondrial (COI and ND2) and nuclear (28S rRNA) markers. The resulting trees support the S. shirozui group as monophyletic and indicate that in this group, species associated with closer affinities show higher structural homogeneity in male genitalia. Molecular species delimitation assess most species limits and recognize four new species in the S. shirozui group from south-west China: S. alianya sp. nov., S. diodonta sp. nov., S. zebromyia sp. nov. and S. zopheria sp. nov. One new synonym was also recognized. Additionally, three typical male genital characters of the S. shirozui group were placed on the molecular phylogenetic framework. The outcome of both divergence-time estimation and ancestral area reconstruction suggests that the S. shirozui group likely originated in south-west China in the Middle Miocene.

Phylogenetic relationships of the two largest species groups in the genus Amiota [the Amiota alboguttata group (29 known and six new species) and the Amiota basdeni group (17 known and three new species)] were reconstructed using two mitochondrial gene sequences (COI and ND2). The paraphyly of the A. alboguttata group was identified based on molecular evidence. The monophyletic A. basdeni group is found to be nested in the A. alboguttata group, rendering the latter paraphyletic. Automatic barcode gap discovery and Bayesian phylogenetics and phylogeography methods were used to assess species limits of the A. alboguttata and A. basdeni groups. The results indicate that most analysed species could be delimited clearly, including nine new species (Amiota beama sp. nov., Amiota cyclophylla sp. nov., Amiota flormontana sp. nov., Amiota obtusa sp. nov., Amiota planiceps sp. nov., Amiota scrobicula sp. nov., Amiota jianjuni sp. nov., Amiota tentacula sp. nov. and Amiota xinglaii sp. nov.), except for some closely related morphospecies. Furthermore, based on the highly biased distributions of these two groups, southwestern China is hypothesized to be a possible centre of origin and diversification for Amiota in East Asia.

Phylogenetic analysis of DNA sequences of three mitochondrial (COI, COII and ND2) and six nuclear (28S, atpA, CAD, EF2, HSPA4 and TUBA1) gene regions recovered a novel lineage (Crypsistegana subgen.n.) and lends support to the monophyly of the subgenus Oxyphortica. Integrating molecular and morphological evidence, we revise the taxonomic status of Oxyphortica, reclassify species of uncertain affinity into four new species groups (adentata, geisson, polystylata and subconvergens) and place five new species discovered from southern China in Crypsistegana subgen.n. and two species groups (geisson and polystylata) of Oxyphortica, respectively. To evaluate the performance of individual genes in phylogenetic inference, the phylogenetic informativeness profiles were generated. The results reveal mitochondrial loci possess much greater phylogenetic signal to resolve species-level relationships, while nuclear loci perform better at the subgenus level within the genus Stegana. In addition, both tectonic and climatic events appear to have played pivotal roles in the diversification of Oxyphortica. The present distribution pattern of this subgenus might have been shaped from a rapid adaptive radiation in Southwest China as a secondary area of endemicity.

A total of 58 (eight known and 50 new) species of the subgenus Stegana (Steganina) from China were surveyed and (re)described: S. (S.) bacilla Chen & Aotsuka, 2004, S. (S.) belokobylskiji Sidorenko, 1997, S. (S.) hirticeps Wang, Gao, & Chen, 2013, S. (S.) izu Sidorenko, 1997, S. (S.) kanmiyai Okada & Sidorenko, 1992, S. (S.) masanoritodai Okada & Sidorenko, 1992, S. (S.) maymyo Sidorenko, 1997, stat. rev., S. (S.) nigripes Zhang & Chen, 2015, S. (S.) alafoliacea Zhang & Chen, sp. nov., S. (S.) baoxing Li & Chen, sp. nov., S. (S.) bibarbata Li & Chen, sp. nov., S. (S.) bimai Cui & Chen, sp. nov., S. (S.) cinereipecta Zhang & Chen, sp. nov., S. (S.) cardua Cui & Chen, sp. nov., S. (S.) cordhirsuta Wang & Chen, sp. nov., S. (S.) cornuta Li & Chen, sp. nov., S. (S.) cucullata Li & Chen, sp. nov., S. (S.) cultella Cui & Chen, sp. nov., S. (S.) curvitabulata Cui & Chen, sp. nov., S. (S.) daiya Cui & Chen, sp. nov., S. (S.) dendrophila Zhang & Chen, sp. nov., S. (S.) flabella Li & Chen, sp. nov., S. (S.) flavipes Li & Chen, sp. nov., S. (S.) formosa Zhang & Chen, sp. nov., S. (S.) fusca Li & Chen, sp. nov., S. (S.) fuscipes Li & Chen, sp. nov., S. (S.) glaucopalpula Cui & Chen, sp. nov., S. (S.) haba Zhang & Chen, sp. nov., S. (S.) hirticlavata Cui & Chen, sp. nov., S. (S.) iaspidea Zhang & Chen, sp. nov., S. (S.) idiasta Cui & Chen, sp. nov., S. (S.) kanda Cui & Chen, sp. nov., S. (S.) labao Li & Chen, sp. nov., S. (S.) lancang Li & Chen, sp. nov., S. (S.) latifoliacea Wang & Chen, sp. nov., S. (S.) liusanjieae Li & Chen, sp. nov., S. (S.) magniflava Cui & Chen, sp. nov., S. (S.) mailangang Li & Chen, sp. nov., S. (S.) marenubila Cui & Chen, sp. nov., S. (S.) menghai Zhang & Chen, sp. nov., S. (S.) menglian Li & Chen, sp. nov., S. (S.) minutiflava Li & Chen, sp. nov., S. (S.) multiprocera Li & Chen, sp. nov., S. (S.) nayun Li & Chen, sp. nov., S. (S.) nigridentata Wang & Chen, sp. nov., S. (S.) nigripalpula Cui & Chen, sp. nov., S. (S.) otphylla Cui & Chen, sp. nov., S. (S.) radiciflava Zhang & Chen, sp. nov., S. (S.) rava Cui & Chen, sp. nov., S. (S.) sciophila Li & Chen, sp. nov., S. (S.) septencolorata Li & Chen, sp. nov., S. (S.) serrata Zhang & Chen, sp. nov., S. (S.) silvestrella Zhang & Chen, sp. nov., S. (S.) simola Cui & Chen, sp. nov., S. (S.) yani Li & Chen, sp. nov., S. (S.) yixiang Zhang & Chen, sp. nov., S. (S.) zaduo Cui & Chen, sp. nov., and S. (S.) zhuoma Cui & Chen, sp. nov. We also provided a complete list of Chinese Steganina species together with their geographical distributions. In addition, the majority of currently available DNA barcode (partial sequence of the mitochondrial cytochrome c oxidase subunit I (COI) gene) sequences of this subgenus (435 sequences of 102 spp.) were employed in a molecular analysis for species delimitation. Taken together, morphology- and molecular-based species delimitation results reached a consensus for an overwhelming majority of these Steganina species (98 of 102 spp.).