The first divergence time estimation of the subfamily Stenogastrinae (Hymenoptera: Vespidae) based on mitochondrial phylogenomics Pan Huang a , James M. Carpenter b , Bin Chen a , Ting-Jing Li a, ⁎ a Chongqing Key Laboratory of Vector Insects, Institute of Entomology and Molecular Biology, College of Life Science, Chongqing Normal University, Chongqing 401331, People's Republic of China b Division of Invertebrate Zoology, American Museum of Natural History, Central Park West at 79th Street, New York, NY 10024, USA abstract article info Article history: Received 8 March 2019 Received in revised form 28 June 2019 Accepted 28 June 2019 Available online 30 June 2019 In this study, the mitochondrial genomes of three Stenogastrinae species, Eustenogaster scitula, Liostenogaster nitidipennis and Parishnogaster mellyi were sequenced and annotated, and a total of 16 vespid mtgenomes are comparatively analyzed. Our results indicate that codon usage bias is mainly dominated by mutational pressure, and affected only slightly by natural selection. Selective pressure analysis of protein-coding genes (PCGs) shows that the highest evolutionary rate is present in NADH complex I, and the lowest in cox1. Compared with the re- ported mtgenomes of other Vespidae, in Stenogastrinae, trnH is shifted to a new position. Phylogenetic analyses are performed using Bayesian method and Maximum Parsimony. Phylogenetic analysis further confirms that the Stenogastrinae is the sister group of all remaining Vespidae. Divergence time of Stenogastrinae from other Vespidae is estimated at ~ 166 Mya. Our results also support that eusociality evolved twice in the family Vespidae. © 2019 Elsevier B.V. All rights reserved. Keywords: Hymenoptera Stenogastrinae Mitochondrial genome Phylogeny Divergence time 1. Introduction The subfamily Stenogastrinae in the family Vespidae is a group of primitively eusocial wasps, common in some South-east Asian countries and including 72 species in seven genera [1–3]. De Saussure included Ischnogaster (junior synonym of Stenogaster) in Vespiens [4], Bingham included Ischnogaster in Vespidae [5]. Ashmead erected Ischnogasterinae, which was included in Eumenidae [6]. Bequaert was first to use Stenogastrinae [7]. From 1902 to 1962, it was debated how many subfamilies belong to the family Vespidae, but the Stenogastrinae was always treated as a subfamily. Carpenter discussed the taxonomic system of Vespidae based on morphological characteristics and pro- posed the Stenogastrinae as one of six subfamilies within a single family Vespidae [1], which has been adopted by most scholars in the past 35 years. During this period, Schmitz and Moritz proposed that Polistinae + Vespinae and Eumeninae had closer relationship than Stenogastrinae based on mitochondrial 16S and nuclear 28S rDNA phy- logenetic analysis [8]. However, the view of Schmitz and Moritz was rejected by combined analysis of molecular sequence data with avail- able morphological and behavioral characteristics [9], which supported traditional vespid relationships. Hines et al. inferred the same phyloge- netic relationships as Schmitz and Moritz from a set of four nuclear encoded genes (18S and 28S rDNA, abdominal-A and RNA polymerase II) and a significantly improved taxon sampling [8,10]. This result indi- cated that Stenogastrinae was sister to all other Vespidae [10]. It was later contradicted by Pickett and Carpenter [11]. However, Peters et al. and Bank et al. recovered Stenogastrinae as the sister group of all re- maining Vespidae in the phylogenetic analysis of transcriptomes [12,13]. Recently, Piekarski et al. also supported the relationship of Stenogastrinae as the sister to all other Vespidae [14]. The Stenogastrinae is today considered to be the sister of the rest of Vespidae based on transcriptome and other molecular studies, but there are no phenotypic data supporting this relationship. In current research, Hymenoptera was estimated to have started di- versifying between the Carboniferous and the Triassic (~280 Mya) [12]. There has been no research about divergence times of Stenogastrinae and other Vespidae. Given that there is high diversity in the biology of Vespidae comprising solitary, subsociality and eusociality [15], the fam- ily Vespidae was recognized as one of the few groups that could eluci- date the transitional stages of social evolution [10,16]. Therefore, the phylogeny of Vespidae in the present study may provide a framework for testing how eusocial groups originate and evolve [14]. In all insects, mtgenomes vary from 15 kb to 18 kb in length and con- tain 13 protein-coding genes (PCGs), two ribosomal RNA genes (rRNAs), 22 transfer RNA genes (tRNAs) and a control region (CR, also called A + T-rich region) [17–19]. Owing to matrilineal inheritance, rare genetic recombination and rapid evolutionary rates, mtgenomes have been a useful molecular marker in insect phylogenetic research [20–22]. Up to date, there is a lack of reported Stenogastrinae mtgenomes or any estimation of divergence times for Vespoidea. In this study, three mtgenomes of Eustenogaster scitula (E. scitula), Liostenogaster nitidipennis (L. nitidipennis) and Parishnogaster mellyi (P. mellyi) were sequenced and annotated, providing the first International Journal of Biological Macromolecules 137 (2019) 767–773 ⁎ Corresponding author. E-mail address: ltjing1979@hotmail.com (T.-J. Li). https://doi.org/10.1016/j.ijbiomac.2019.06.239 0141-8130/© 2019 Elsevier B.V. All rights reserved. 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