bs_bs_banner Zoological Journal of the Linnean Society, 2015, 174, 473–486. With 9 figures A revised classification of the genus Matrona Selys, 1853 using molecular and morphological methods (Odonata: Calopterygidae) XIN YU1*†, JUNLI XUE1†, MATTI HÄMÄLÄINEN2, YANG LIU1 and WENJUN BU1* 1Institute of Entomology, College of Life Sciences, Nankai University, Tianjin 300071, China 2Naturalis Biodiversity Center, P.O. Box 9517, 2300 RA, Leiden, The Netherlands Received 31 July 2014; revised 27 January 2015; accepted for publication 27 January 2015 An extensive review of the genus Matrona is presented based on mitochondrial (COI) and nuclear (ITS) se- quences from 150 samples which cover all the known taxa of this genus. The separation of two main clades (oreades group: M. oreades, M. corephaea and M. taoi; basilaris group: M. basilaris, M. nigripectus, M. cyanoptera, M. ja- ponica and M. annina) is strongly supported. The classification of all traditional recognized species is confirmed. The Hainan population separates very well from mainland M. basilaris populations, which is also confirmed by geometric morphometric analysis of wing shape. Given the implications of the molecular analysis the genus Matrona is grouped into two subgenera: subgen. Matrona (type species M. basilaris) and Divortia subgen. nov. (type species M. oreades). A new species, M. (M.) mazu sp. nov., from Hainan is described. Brief taxonomic notes on the nine recognized species of the genus are given. Lectotype designations of M. basilaris and M. nigripectus are published. © 2015 The Linnean Society of London, Zoological Journal of the Linnean Society, 2015, 174, 473–486. doi: 10.1111/zoj.12253 ADDITIONAL KEYWORDS: damselfly – geometric morphometric – lectotype designation – new species – new subgenus – phylogenetics. INTRODUCTION Matrona basilaris Selys, 1853, M. nigripectus Selys, 1879, M. japonica Förster, 1897, M. cyanoptera Matrona Selys, 1853 is a genus of rather large, con- Hämäläinen and Yeh, 2000, M. oreades Hämäläinen, spicuous and attractively coloured East Asian Yu and Zhang, 2011, M. corephaea Hämäläinen et al., calopterygid damselflies. In the Asian mainland their 2011, M. taoi Phan & Hämäläinen, 2011 and M. annina range extends from Meghalaya in the west to Vietnam Zhang & Hämäläinen, 2012. in the south and to Hebei province in the north. In Although morphological characters suggested enough addition, Matrona species inhabit Hainan and Taiwan evidence to place M. oreades, M. corephaea and M. taoi islands and Okinawa and Amami islands in Japan in their own species group within the genus (Fig. 1). Less than 15 years ago only one species (Hämäläinen et al., 2011; Phan & Hämäläinen, 2011; (M. basilaris) with two named and one unnamed Zhang & Hämäläinen, 2012), the phylogenetic rela- subspecies was known, but now eight species are tionships of all taxa within Matrona have not then been recognized (Hämäläinen in Karjalainen & Hämäläinen, studied. Three of the taxa (M. basilaris, M. nigripectus 2013). They are listed here in chronological and M. cyanoptera) have been included in molecular order: studies by Bybee et al. (2008), Dumont et al. (2005), Dumont, Vierstraete & Vanfleteren (2007), Dumont, Vierstraete & Vanfleteren (2010) and Guan et al. (2012), which show Matrona closely related to *Corresponding authors. E-mail: [email protected]; [email protected] Atrocalopteryx Dumont et al., 2005 and Neurobasis Selys, †These authors contributed equally to this work. 1853. © 2015 The Linnean Society of London, Zoological Journal of the Linnean Society, 2015, 174, 473–486 473 474 X. YU ET AL. Figure 1. Approximate distribution ranges of known Matrona species. The present phylogenetic analysis is based on analy- MATERIALS AND METHODS sis of the mitochondrial gene cytochrome c oxidase MATERIAL ACQUISITION subunit 1 (COI) and combined nuclear gene of the ri- bosomal ITS1–5.8S–ITS2 region (ITS). Wing shape and A total of 188 samples were studied, 139 for molecu- colour have been subjected to intense sexual selec- lar research and 63 for wing shape analysis (Support- tion pressure in calopterygids and are thus more im- ing Information: S1). The samples were mainly collected portant than genitalia and caudal appendages in by the first author (X.Y.), with the exception of a few classification (Cordoba-Aguilar & Cordero-Rivera, 2005; specimens received from foreign colleagues. Paratypes Eroukhmanoff et al., 2009; Outomuro, Bokma & of both M. oreades and M. corephaea were included in Johansson, 2012; Outomuro, Adams & Johansson, 2013). the molecular analysis. All the specimens have been Morphometric analysis of calopterygid wing shape can deposited in the collections of the Institute of Ento- yield useful information (Sadeghi, Adriaens & Dumont, mology, College of Life Sciences, Nankai University, 2009; Outomuro et al., 2013). The present study is the China. Voucher specimens were preserved in 75% first to apply morphometric analysis to wings of Matrona ethanol immediately upon collection in the field. In most to confirm the real division between closely related popu- cases, one or two legs were isolated from the samples. lations within the a priori basilaris group. Occasionally, dried specimens were used to obtain DNA Our molecular results support two monophyletic clades data, although this was difficult. In addition, the fol- of the genus Matrona, suggesting the division into two lowing sequences were obtained from GenBank, new subgenera Divortia and Matrona. We also de- M. annina (JX852701.1, JX852702.1), M. cyanoptera scribe a new species, M. mazu sp. nov., which is sup- (AJ459205.1), M. nigripectus (AJ459206.1) and M. ja- ported by our molecular and morphological assessments. ponica (AB706444.1, JAB706443.1, AB706442.1, © 2015 The Linnean Society of London, Zoological Journal of the Linnean Society, 2015, 174, 473–486 REVISED CLASSIFICATION OF MATRONA 475 AB706441.1, AB706440.1, AB706439.1 and AB706438.1). settings and subsequently corrected manually to ensure For wing shape analysis hind wings of a subsample the peak figure of each mutation loci was credible. of specimens were detached and photographed with a Nikon D700 digital camera. In addition, for the taxonomic analysis of the genus, PHYLOGENETIC ANALYSES the third author (M.H.) studied numerous Matrona Subsequent analyses were performed using optimality specimens in various museums and private collec- criteria including neighbour-joining (NJ), maximum- tions. These include 19 specimens of M. mazu sp. nov. likelihood (ML) and the Bayesian inference algo- kept in his private collection, the syntype series of rithm (BI) to resolve the phylogenetic relationships. M. basilaris and M. nigripectus at IRSN (Brussels), and The distance method NJ tree was derived using representative paratypes of all other Matrona taxa BioEdit7.2.0 based on the Kimura two-parameter model presently recognized as valid, with the exception of with setting transition/transversion ratio = 2.0. ML M. japonica. analysis was performed using RAxML v8.0.0 (Stamatakis, 2014). Final ML tree searches were con- ducted under the GTR+I+Gmodel for both COI and DNA EXTRACTION AND AMPLIFICATION ITS selected as appropriate models of sequence evo- Total genomic DNA was extracted using the protocol lution as implemented through ModelTest3.7 (Posada of the UniversalGen DNA Kit (Beijing ComWin Biotech). & Crandall, 1998). Bootstrap analyses were per- Small doses were used as a template for PCR ampli- formed with the rapid algorithm. We obtained boot- fication. Each PCR amplification was performed in 50- strap support for each node from 1000 rapid bootstrap μL reaction mixes containing 6 μL 10× LA PCR BufferII pseudoreplicates with every fifth tree used as start- (Mg2+ Plus), 6 μL dNTP Mixture (2.5 mM), 2.5 U TaKaRa ing point for subsequent ML optimization on the origi- LA Taq (TaKaRa Biotechnology), 1 μL of each primer nal dataset. Bayesian phylogenetic analyses were (10 μM), 2 μL DNA template and 33.7 μL distilled water. performed using MrBayes v3.1.2 (Huelsenbeck & ITS was amplified with the primers detailed by Dumont Ronquist, 2001) with GTR+I+Gmodel for COI and et al. (2010), namely Vrain2F (5′-CTTTGTACAC GTR + I for ITS. MrModeltest v2.3 (Nylander, 2004) ACCGCCCGTCGCT-3′) and Vrain2R (5′-TTTCACTC was used to explore appropriate substitution models GCCGTTACTAAGGGAATC-3′). Newly designed primers for both COI and ITS. All the acquired trees were set COIf1 (5′-GRGCATGRGCAGGWATAGTNG-3′) and to 10 million generations and for every 1000 genera- COIr1 (5′-GGGTAGTCTGARTATCGTCGNGGT-3′) were tions the chain was sampled. The Markov chain Monte used for amplification of COI. The PCR cycling pro- Carlo (MCMC) process was run over four parallel chains, cedure was 2 min at 95 °C followed by 35 cycles of de- one cold and three incrementally heated. Theoretical- naturation at 95 °C for 30 s, annealing temperature ly, it could not be stopped until the average standard at 56 °C (ITS) or 52 °C (COI) for 30 s, and extension deviation of split frequencies was down to < 0.01 and at 72 °C for 1 min, with a final single extra exten- was discarded as burn-in. Convergence diagnostic was sion step at 72 °C for 8 min. All PCR products were determined with Tracer 1.5 (Rambaut & Drummond, visualized via 1% agarose gel electrophoresis and am- 2007). Trees sampled after burn-in of the first 25% of plifications were purified using a gel extraction kit each run from the four runs were combined and used (Sangon Biotech), then sent to commercial companies to construct a 50% majority rule consensus tree. Trees (BGI TechSolutions or GENEWIZ) for sequencing based were displayed with TreeView v1.6.6 (Page, 1996) and on Sanger’s chain termination method. Fragments which FigTree v1.4.0 (Rambaut, 2012). Four sequences, failed in direct sequencing were cloned into a TA- HLJH01 (Atrocalopteryx atrata [Selys, 1853]), cloning vector, pMD-18T (TaKaRa), and transformed HaNWZS01 (Neurobasis chinensis (Linnaeus, 1758)), into competent Escherichia coli DH5α. Putative clones SCYA05 Vestalaria velata (Ris, 1912) and SCYA06 containing the PCR fragments were selected and se- (Vestalaria velata), were used as outgroups.