A Review of the Damselfly Genus Megalestes Selys, 1862 (Insecta: Odonata: Zygoptera: Synlestidae) Using Integrative Taxonomic Methods

Zootaxa 4851 (2): 245–270 ISSN 1175-5326 (print edition) https://www.mapress.com/j/zt/ Article ZOOTAXA Copyright © 2020 Magnolia Press ISSN 1175-5334 (online edition) https://doi.org/10.11646/zootaxa.4851.2.2 http://zoobank.org/urn:lsid:zoobank.org:pub:DCD23BAB-CBF9-48F0-90C9-E490803F0757

A review of the damselfly genus Megalestes Selys, 1862 (Insecta: Odonata: Zygoptera: Synlestidae) using integrative taxonomic methods

XIN YU1* & JUNLI XUE2 1College of Life Sciences, Chongqing Normal University, Chongqing, 401331, China 2Key Laboratory of Atherosclerosis in Universities of Shandong Province, Shandong First Medical University & Shandong Academy of Medical Sciences, Tai'an, China. https://orcid.org/0000-0003-0696-2596 *Corresponding author. �[email protected]; https://orcid.org/0000-0003-4172-7575

Abstract

We used morphological and molecular characters to review the classification of damselflies in the genus Megalestes Selys, 1862 (Zygoptera: Odonata). Several methods were used for this review, including morphology, cladistics, genetic distance analysis, and molecular taxonomy. Six species were newly defined as junior synonyms (Megalestes palaceus to Megalestes haui, Megalestes chengi to Megalestes micans, Megalestes discus to Megalestes micans, Megalestes raychoudhurii to M. micans, Megalestes maai to Megalestes riccii, and Megalestes tuska to M. riccii). The total species number in Megalestes was reduced from 18 to 12. The importance of an objective species diversity measure is discussed.

Keywords: integrative taxonomy, morphological and molecular, multimethods, synonyms

Introduction

Biodiversity is an important topic that incorporates taxonomy as a key component (Dijkstra 2016; Wilson 2017). Studies of species diversity require adequate taxonomic support. Methods such as DNA barcoding and integrative taxonomy have led to the discovery of many cryptic species, which has increased our knowledge of biodiversity (Hebert et al. 2003; Damm et al. 2010). However, even within well-known insect groups such as Odonata (dragonflies and damselflies), there may exist questionable taxa, and some species definitions appear to be problematic (Kosterin 2019; Yu et al. 2019). This situation has caused an artificially high level of biodiversity in some groups of Odonata and affected the assessment of the current level of species diversity (Kosterin 2019; Yu et al. 2019). Along with the search for new cryptic species, integrative taxonomy should play a role in examining existing but dubious species. Combining morphological and molecular data can often improve taxonomic classification (Yu et al. 2015; Ortiz & Francke 2016; Zheng et al. 2019; Eberle et al. 2019; Masonick & Weirauch 2020). The genus Megalestes Selys, 1862 (Zygoptera: Synlestidae) is a group of large-sized, but poorly known, damselflies distributed from northern India throughout Southeast Asia to mainland China (Fig. 1). Almost all Megal- estes adults look similar, being giant damselflies with a shining metallic green body color, hyaline wings, and an elongated abdomen (Fig. 2). It is impossible to distinguish species of this genus only from habitus photographs. The damselflies are easily spotted when resting on branches above spring waters in the mountains, since this group is only slightly vigilant and active. When disturbed, the insects usually only fly away for a short distance and then rest again. According to our observations, all species of Megalestes live exclusively in clear and slow running montane waters, and therefore, they could be good bioindicators of habitat quality. If Megalestes are present, then there is clean water nearby. To date, a total of 18 Megalestes species have been recorded globally (Paulson & Schorr 2020), of which 12 occur in China. After de Selys Longchamps (1862) described M. major, the type species of this genus, from “Patrie: Inde méridionale,” most Megalestes species have been discovered on the China mainland. Fraser (1926), Needham (1930), Navás (1934), Chao (1947, 1965), Chen (1947), Asahina (1956, 1969, 1985a,b), Lahiri (1979, 1987), Wilson & Reels (2003), Wilson (2004), Wilson & Xu (2007), Yu (2008), Zhou & Zhou (2008), Ka- rube (2014), Gyeltshen et al. (2017), and Phan & To (2018) have all published taxonomic research on this genus.

Accepted by M. Marinov: 21 Aug. 2020; published: 10 Sept. 2020 245 However, the taxonomy of Megalestes remains ambiguous. First, all species in Megalestes look similar. Most of the morphological characters are either convergent or gradually vary both spatially and temporally, making morphological species definitions difficult. Also, many Megalestes species have been inadequately studied. Figures and descriptions of some species were so simple and equivocal that later researchers were unable to correctly interpret them. For example, the dense setae on the surface of male paraprocts of M. riccii Navás, 1935 and M. micans Need- ham, 1930 is an important character that should be emphasized in both descriptions and drawings. However, this character was often neglected by researchers due to either being treated as a trivial decoration or to studying older specimens lacking all of their setae. Few early studies describe intraspecies variation. This information can only be acquired by examining a sufficient number of specimens from the entire range (very difficult). Several species of Megalestes were regarded as possible synonyms (Yu 2008), but there has been no revision of this genus. To examine whether there are valid and usable diagnostic morphological characters and to identify the best strategy for obtaining a robust classification for Megalestes, we conducted a revision based on both morphological and molecular data using multiple methods of analysis.

Figure 1. Distribution of the species of genus Megalestes.

Material and methods

Specimens. A total of 335 specimens of the genus Megalestes were examined. Among these, 60 were used for molecular analyses (Table 1). Eight specimens were chosen as the outgroup for phylogenetic analysis, including species of Lestes sponsa (Hansemann, 1823), Orolestes selysi McLachlan, 1895, Sinolestes edita Needham, 1930, and Sympecma paedisca (Brauer, 1877) (Table 1). Additionally, > 30 specimens were obtained, on loan, from the Institute of Zoology, Chinese Academy of Sciences, and the Shanghai Institute of Entomology, Chinese Academy of Sciences. The loaned specimens included type specimens of M. chengi, M. riccii, and M. omeiensis. Type specimens of M. irma were examined and photographed in the Natural History Museum, London, UK. Moreover, photos of diagnostic characters of type specimens of M. discus, M. haui, and M. tuska were also examined. These were provided by K.D.P. Wilson and Guanhong Huang from the Agriculture, Fisheries and Conservation Department of Hong Kong.

246 · Zootaxa 4851 (2) © 2020 Magnolia Press YU & XUE note ...... continued on the next page storage CQNU CQNU CQNU CQNU CQNU CQNU CQNU CQNU CQNU NKUM CQNU CQNU CQNU CQNU CQNU CQNU CQNU CQNU CQNU CQNU CQNU CQNU CQNU NKUM CQNU CQNU CQNU collector Toan Phan, Quoc Xin Yu, Xin Yu, Xin Yu, Xin Yu, Xue, Jun-li Chen, Bin Xue, Jun-li Xue, Jun-li Wei-bing Zhu, Xin Yu, Xin Yu, Xin Yu, Xin Yu, Xin Yu, Tong-yin Xie, Huan-huan Yang, Zhang, Hai-guang Zhang, Dan-li Xin; Pu, Xu-bo Yu, Xin Yu, Xin Yu, Xue, Jun-li Xue, Huai-jun Xin; Pu, Xu-bo Yu, Xin; Pu, Xu-bo Yu, Xin; Pu, Xu-bo Yu, altitude 923 1080 1500 1180 886 820 1425-1723 1000 1183 1300 1350 1370 1250 900-1100 900-1100 900-1200 850 650 850 978-1140 1183 1200 1200 1100 date 20120615 20110725 20050712 20050713 20050714 20160713 20170704 20140629 20160702 20040528 20040820 20040822 20040824 20040825 20040826 20130812 20140725 20140725 20140727 20090526 20130818 20130820 20160719 19990717 20130723 20130723 20130725 ietnam V Xa Lang, Tay Giang, Quang Nam, Tay Xa Lang, locality Sichuan, China Yaan, Bifengxia, Gansu, China Wen, Bikou, Gansu, China Wen, Bikou, Gansu, China Wen, Bikou, Chengkou, Chongqing, China Chengkou, Chongqing, China Dabieshan, Huanggang, Hubei, China China Yunnan, Daguan, Zhaotong, Daozhen, Zunyi, Guizhou, China Daozhen, Zunyi, Guizhou, China Daozhen, Zunyi, Guizhou, China Daozhen, Zunyi, Guizhou, China Daozhen, Zunyi, Guizhou, China Daozhen, Zunyi, Guizhou, China Daozhen, Zunyi, Guizhou, China Dujiangyan, Sichuan, China Dujiangyan, Sichuan, China Dujiangyan, Sichuan, China Emeishan, Sichuan, China Emeishan, Sichuan, China Emeishan, Sichuan, China Guangyuan, Sichuan, China Hefeng, Hubei, China Hongya, Meishan, Sichuan, China Hongya, Meishan, Sichuan, China Hongya, Meishan, Sichuan, China Sex m m 2m m m m 4m m 3m, 2f 1m, 1f m 5m 3m 13m, 4f 2m, 1f f 1m, 1f 3m, 3f 1f m f 6m, 1f 9m, 2f 1m, 1f m m m V N01 MeA ID/Accession numbers HBHGd01 MeO-1 SCEM01 List of examined and analyzed specimens. Specimens with ID numbers were used for molecular analysis, and L indicates larva. List of examined and analyzed specimens. Specimens with ID numbers were used for molecular analysis, L 1. a ble Species In group australis distans T

A review of the damselfly genus Megalestes Selys, 1862 Zootaxa 4851 (2) © 2020 Magnolia Press · 247 note ...... continued on the next page storage CQNU CQNU CQNU IZAS IZAS CQNU CQNU CQNU CQNU CQNU CQNU CQNU CQNU CQNU CQNU CQNU CQNU CQNU IZAS CQNU CQNU CQNU CQNU IEAS CQNU CQNU CQNU collector Xin; Pu, Xu-bo Yu, Xue, Jun-li Xin Yu, ? Chai, Huai-cheng Xin Yu, Xin Yu, Xin Yu, Song, Chao Xue, Jun-li Liu, Hua-xi Sun, Xi Xin Yu, Xin Yu, Gui-jiang Yang, Li, Xiu-rong Li, Xiu-rong Xue, Jun-li Chai, Huai-cheng Xin Yu, Xin Yu, Xin Yu, Xin Yu, Liu, Zu-yao Toan Phan, Quoc Xin Yu, Xin Yu, altitude 1100 1086 800 800 850 1340 800 800 1000 1312 1214 900 690 1200 1200 1047 900 799 1200 1200 1200 2100 837 837 date 20130725 20160723 20110720 19570812 19830916 20050803 20110720 20110720 20150727 20160726 20140811 20100813 20100607 20100607 20100811 20130806 20130807 20160710 19830803 20150806 20110809 20110809 20110805 19880707 20140522 20150801 20150801 ietnam V Sapa, Lao Cai, locality Hongya, Meishan, Sichuan, China Pengzhou, Chengdu, Sichuan, China Qingchengshan, Sichuan, China Qingchengshan, Sichuan, China Qingchengshan, Sichuan, China Qingchengshan, Sichuan, China Qingchengshan, Sichuan, China Qingchengshan, Sichuan, China Qingchengshan, Sichuan, China Qingchengshan, Sichuan, China Shangluo, Shaanxi, China Suiyang, Zunyi, Guizhou, China Suiyang, Zunyi, Guizhou, China Suiyang, Zunyi, Guizhou, China Suiyang, Zunyi, Guizhou, China Suiyang, Zunyi, Guizhou, China Suiyang, Zunyi, Guizhou, China Dazhou, Sichuan, China Wanyuan, Sichuan, China Wenchuan, Yingxiu, Zhangjiajie, Hunan, China Damingshan, Guangxi, China Damingshan, Guangxi, China Damingshan, Guangxi, China Leishan, Guizhou, China Xiaodanjiang, Leishan, Guizhou, China Xiaodanjiang, Leishan, Guizhou, China Sex 7m, 1f 7m,1f m m 2m 7m, 4f m 8m, 1f 2m 3m 2m, 2f m L L m 2m m 4m m 2m m m m m 2m m m ID/Accession numbers SCQC01 GZKKR01 GZKKS02 GXNNh01 GXNNh02 MePLGSH01 MePLGSH02 BLE 1. (Continued) TA Species haui

248 · Zootaxa 4851 (2) © 2020 Magnolia Press YU & XUE note paratypes ...... continued on the next page storage CQNU CQNU CQNU CQNU CQNU CQNU CQNU CQNU CQNU CQNU CQNU NKUM NKUM CQNU BMNH CQNU CQNU CQNU CQNU CQNU CQNU CQNU CQNU CQNU collector Xin Yu, Xin Yu, Xin Yu, Xin Yu, Xin Yu, Xin Yu, Xin Yu, Xin Yu, Xin Yu, Xin Yu, Xin Yu, Ying Tian, Wen-jun Bu, Xin Yu, F.C. Fraser, Chen, Jin Chen, Jin Chen, Jin Chen, Jin Hämäläinen, Matti Hämäläinen, Matti Hämäläinen, Matti Hämäläinen, Matti Xin Yu, altitude 1600 1600 1250 1550 600 1193 1300 277 900 750 1100 1100 1000 1500-1800 1800 1820 1820 1820 1135 1250 1135 1250 1200 date 20070801 20070801 20070728 20070730 20070731 20140505 20160811 20070726 20130422 20070813 20160801 20050730 20050731 20070803 19240529 20120822 20120821 20120821 20120821 20001010 20120509 20001009 20120508- 10 20130813 locality Fengyangshan, Lishui, Zhejiang, China Fengyangshan, Lishui, Zhejiang, China Fengyangshan, Lishui, Zhejiang, China Fengyangshan, Lishui, Zhejiang, China Fengyangshan, Lishui, Zhejiang, China Anhui, China Huangshan, Jingning, Lishui, Zhejiang, China Longquamshan, Lishui, Zhejiang, China Ninghai, Ningbo, Zhejiang, China Qingliangfeng, Zhejiang, China Ningbo, Zhejiang, China Yuyao, Simingshan, Zhejiang, China Wuyanling, Zhejiang, China Wuyanling, Zhejiang, China Wuyanling, Sikhim Gandtor, China Yunnan, Anning, Kunming, China Yunnan, Anning, Kunming, China Yunnan, Anning, Kunming, China Yunnan, Anning, Kunming, Thailand Doi Suthep, Chiang Mai, Doi Inthanon, Chiang Waterfall, Siriphum Thailand Mai, Doi Inthanon, Chiang Waterfall, Siriphum Thailand Mai, Doi Inthanon, Chiang Waterfall, Siriphum Thailand Mai, China Yunnan, Gejiu, Tabai, Sex m f m 2f 1m,1f L 2m m L f m m f m 2m, 1f m m m 1f 11m, m m 1m, 1f 6m 1f m - ID/Accession numbers ZZLS01 ZZLS02 AHHSu01 ZJNBu01 MeK-1 YNKM01 YNKM02 MeK-2 MeKYN GJ01 BLE 1. (Continued) TA Species heros irma kurahashii

A review of the damselfly genus Megalestes Selys, 1862 Zootaxa 4851 (2) © 2020 Magnolia Press · 249 note all types of M. chengi including the holotype paratype of M. chengi ...... continued on the next page storage CQNU CQNU CQNU CQNU CQNU CQNU CQNU CQNU CQNU CQNU CQNU CQNU CQNU CQNU CQNU CQNU CQNU CQNU IZAS IEAS CQNU CQNU CQNU CQNU CQNU collector Xin Yu, Chen, Jin Chen, Jin Chen, Jin Xin Yu, Xin Yu, Tong-yin Xie, Tong-yin Xie, Hämäläinen, Matti Chen, Jin Chen, Jin Chen, Jin Chen, Jin Chen, Jin Xue, Jun-li Xue, Jun-li Xin Yu, Xin Yu, Chao, Hsiu-fu Chao, Hsiu-fu Xin Yu, Xin Yu, Xin Yu, Xin Yu, Xin Yu, altitude 1200 1155 1155 1155 1200 1850 1000 1000 2454 2454 1800 1800 1800 1020 1020 1590 663 1060 1600 1600 1550 1550 1600 date 20130813 20120531 20120531 20120531 20130813 20060810 20140720 20140720 19861010 20120812 20120816 20120605 20120605 20120605 20160725 20160725 20060821 20110810 19450811 19450816 20070801 20070731 20070729 20070730 20070801 locality China Yunnan, Gejiu, Tabai, China Yunnan, Gejiu, Tabai, China Yunnan, Gejiu, Tabai, China Yunnan, Gejiu, Tabai, China Yunnan, Gejiu, Tabai, China Yunnan, Tengchong, Nagarkot, Nepal Nagarkot, Nepal Sulphur springs[Sulphur springs], Dehra Dun, India China Yunnan, Ailaoshan, Jingdong, China Yunnan, Ailaoshan, Jingdong, China Yunnan, Anning, Kunming, China Yunnan, Anning, Kunming, China Yunnan, Anning, Kunming, Sichuan, China Yaan, Bifengxia, Sichuan, China Yaan, Bifengxia, China Yunnan, Dali, Damingshan, Guangxi, China Dazhulan, Shaowu, Fujian, China Dazhulan, Shaowu, Fujian, China Fengyangshan, Lishui, Zhejiang, China Fengyangshan, Lishui, Zhejiang, China Fengyangshan, Lishui, Zhejiang, China Fengyangshan, Lishui, Zhejiang, China Fengyangshan, Lishui, Zhejiang, China Sex f m L 1m, 1f 2m, 1f m m m m m 5m,1f m m 3m m m m m 7m m m f m 2m 3m,2f - ID/Accession numbers MeKYN GJ02 YNGJk01 YNGJu01 Nepal01 Nepal02 YNANm01 MeMiYA01 MeMiYA02 MeMLS01 MeMLS02 BLE 1. (Continued) TA Species kurahashii major micans

250 · Zootaxa 4851 (2) © 2020 Magnolia Press YU & XUE note ...... continued on the next page storage CQNU CQNU CQNU CQNU CQNU CQNU CQNU IZAS CQNU IZAS IEAS CQNU CQNU IEAS CQNU NKUM CQNU CQNU CQNU CQNU CQNU CQNU CQNU CQNU CQNU CQNU CQNU collector Xue, Jun-li Xue, Jun-li Xin Yu, Jie Yang, Xue, Jun-li Hämäläinen, Matti Xin; Pu, Xu-bo Yu, Shu-yong Wang, Xin Yu, ? Liu, Zu-yao Xin Yu, Li, Min Liu, Zu-yao Xin Yu, Zheng, Le-yi Liu, Hua-xi Zhang, Jia-qing Zhang, Jia-qing Zhang, Jia-qing Hua, Ji-meng Xin Yu, Xin Yu, Xue, Jun-li Xue, Jun-li Xue, Jun-li Xue, Jun-li altitude 1200 1200 1200 1200 978-1140 1900 1100 2400 1194 1550 1100 1540 2000 1515 1663 1900 1080 1178 1178 855 1800 1850 2120 1047 1047 1047 1860 date 20140810 20140810 20140806 20140810 20160720 20090520 20130725 19840826 20160819 19560511 19880701 20150730 20090511 19960809 20110728 19960524 20140730 20140630 20140630 20140629 20060809 20060811 20060812 20160710 20160710 20160710 20160704 ietnam V Hoang Lien, Sapa, Lao Cai, locality Guadun, Nanping, Fujian, China Guadun, Nanping, Fujian, China Guadun, Nanping, Fujian, China Guadun, Nanping, Fujian, China Guangyuan, Sichnuan, China Hongya, Meishan, Sichuan, China China Yunnan, Jingding, Lanping, Jinggangshan, Jiangxi, China China Yunnan, Jinping, Leishan, Guizhou, China Leishan, Guizhou, China China Yunnan, Longling, Baoshan, Luanchuan, Henan, China Luding, Ganzi, Sichuan, China China Yunnan, Pingbian, Shangluo, Shaanxi, China Shangluo, Shaanxi, China Shangluo, Shaanxi, China Shanyang, Shangluo, Shaanxi, China China Yunnan, Tengchong, China Yunnan, Tengchong, China Yunnan, Tengchong, Dazhou, Sichnuan, China Wanyuan, Dazhou, Sichnuan, China Wanyuan, Dazhou, Sichnuan, China Wanyuan, China Yunnan, Zhaotong, Yiliang, Sex m m m m m m 2m 3m 2m m 4m L m m m m m m f m m m m m m ID/Accession numbers FJWYc01 FJWYc02 FJWYc03 FJWYc04 SCMSm01 YNBSu01 SAXSL01 SAXSLm01 MeMiDZ01 MeMiDZ02 MeMiZT01 BLE 1. (Continued) TA Species micans

A review of the damselfly genus Megalestes Selys, 1862 Zootaxa 4851 (2) © 2020 Magnolia Press · 251 note Paratype Paratype Holotype ...... continued on the next page storage CQNU CQNU CQNU CQNU CQNU CQNU NKUM CQNU CQNU IZAS IZAS IZAS IZAS CQNU CQNU CQNU CQNU CQNU CQNU CQNU CQNU CQNU CQNU CQNU IEAS IZAS IEAS collector Xue, Jun-li Xue, Jun-li Xin Yu, Xin Yu, Xin Yu, Xin Yu, Ling, Zuo-pei Xin Yu, Xin Yu, Xing-chi Yang, Xing-chi Yang, Xing-chi Yang, Zong-yuan Wang, Xin Yu, Xin; Pu, Xu-bo Yu, Xin; Pu, Xu-bo Yu, Xin; Pu, Xu-bo Yu, Xin Yu, Xin Yu, Liang-jong Wang, Liang-jong Wang, Xin Yu, Xin Yu, Xin Yu, Jin, Xingbao O. Piel O. Piel altitude 1860 1860 2180 2100 2180 2180 2800 850 850 580-1100 580-1100 580-1100 800 1100 1100 1100 1314 1314 1314 970 date 20160704 20160704 20060722 20060720 20060722 20060722 19790814 20130820 20130820 19550609 19550613 19550620 19570608 20090526 20130725 20130725 20130725 20110806 20110806 20060722 20060722 20160812 20160812 20160812 19810907 19330913 19350503 locality China Yunnan, Zhaotong, Yiliang, China Yunnan, Zhaotong, Yiliang, China Yunnan, Yuanjiang, China Yunnan, Yuanjiang, China Yunnan, Yuanjiang, China Yunnan, Yuanjiang, China Yunnan, Lijiang, Yulong, Emeishan, Sichuan, China Emeishan, Sichuan, China Emeishan, Sichuan, China Emeishan, Sichuan, China Emeishan, Sichuan, China Emeishan, Sichuan, China Emeishan, Sichuan, China Hongya, Meishan, Sichuan, China Hongya, Meishan, Sichuan, China Hongya, Meishan, Sichuan, China Damingshan, Guangxi, China Damingshan, Guangxi, China China Taiwan, Hualian, China Taiwan, Hualian, Jingning, Lishui, Zhejiang, China Jingning, Lishui, Zhejiang, China Jingning, Lishui, Zhejiang, China Jinxiu, Guangxi, China Lushan, Jiangxi, China Lushan, Jiangxi, China Sex m 10m,1f m 2m m 5m,2f m L L m f m f 2m m m m m f m m m m 2m m 1m, 1f 2m, 2f ID/Accession numbers MeMiZT02 YNYJm01 SCEMu01 SCEMu02 SCMSo01 SCMSo02 MeT-1 MeT-2 MeM-1 MeM-2 MeRZJJN01 MeRZJJN02 BLE 1. (Continued) TA Species omeiensis riccii

252 · Zootaxa 4851 (2) © 2020 Magnolia Press YU & XUE note storage CQNU CQNU CQNU CQNU CQNU CQNU CQNU CQNU CQNU CQNU CQNU CQNU CQNU CQNU CQNU CQNU collector Xin Yu, Xin Yu, Xin Yu, Xin Yu, Wen-chi Yeh, Wen-chi Yeh, Xin Yu, Xin Yu, Xin Yu, Xin Yu, Xin Yu, Xin Yu, Xue, Junli Xin Yu, Zhang, Xu Zhang, Xu altitude 1200 1000 1000 1000 1035 1035 500 100 100 700 772 277 993 993 date 20070810 20070803 20070803 20070803 20160717 20160717 20150807 20150807 20100816 20110821 20110821 20140505 20140108 20130422 20070827 20070827 locality Zhejiang, China Tianmushan, Zhejiang, China Wuyanling, Zhejiang, China Wuyanling, Zhejiang, China Wuyanling, China Taiwan, Yilan, China Taiwan, Yilan, Zhangjiajie, Hunan, China Zhangjiajie, Hunan, China Heilongjiang, China Wudalianchi, Liangfengjiang, Guangxi, China Liangfengjiang, Guangxi, China Anhui, China Huangshan, Tangkou, Hainan, China Wuzhishan, Ningbo, Zhejiang, China Asuhai, Inner Mongolia, China Asuhai, Inner Mongolia, China Sex m m m 1m,1f m m m m m m L m L m m m ID/Accession numbers MeRi-1 MeRi-3 MeMaa01 MeMaa02 MeRZJJ01 MeRZJJ02 LeSWDL02 GXNN02 GXNN03 AHHS02 HNWZM01 NbNh01 SmPHHH01 SmPHHH02 BLE 1. (Continued) TA Species riccii Out group Lestes sponsa selysi Orolestes selysi Orolestes Sinolestes edita Sinolestes edita Sinolestes edita Sympecma paedisca Sympecma paedisca

A review of the damselfly genus Megalestes Selys, 1862 Zootaxa 4851 (2) © 2020 Magnolia Press · 253 Thirteen of the currently 18 recognized species were included in our combined morphological and molecular revision (australis, chengi, distans, haui, heros, kurahashii, maai, major, micans, omeiensis, riccii, tuska, palaceus). The other species were revised based on morphology only (discus, gyalsey, irma, lieftincki, raychoudhurii).

Abbreviations of Collections CQNU Chongqing Normal University, Chongqing, China NKUM Nankai University, College of Life Sciences, Tianjin, China IZAS Institute of Zoology, Academia Sinica, Beijing, China IEAS Shanghai Institute of Entomology, Academia Sinica, Shanghai, China BMNH Natural History Museum, London, UK

Abbreviations of figures To avoid confusion with the multiple figures prepared as illustrations for the present study and citations of figures from literature, we capitalise the first letter (‘Fig.’) for figures in this paper but use the lower case letter (‘fig.’) in literature references to figures published elsewhere.

Morphological characters All photos of living individuals were taken in the field before or just after collection using a digital camera (Nikon D3200, Thailand). Specimens were examined and dissected under a Zeiss V8 stereomicroscope (Germany) in the laboratory. Character photos were taken using a Zeiss V20 (Germany) microphotography system. S = abdominal segment (s). The nomenclature of wing venation followed Rehn (2003). In total, 19 morphological characters were studied, which covered all the features previously used in taxonomic studies of Megalestes. These characters were: 1) presence of postocular spots, 2) color of middle lobe of pronotum, 3) color of anterior and posterior lobes of pronotum, 4) color and shape of synthorax carinal fork, 5) area of green color between humeral suture and metathoracic pleural suture, 6) stripe on metathoracic pleural suture, 7) color of metaposternum, 8) color of wing, 9) color of abdomen S3–7, 10) color of cercus, 11) shape of basal tooth of cercus, 12) dilation of the end of cercus, 13) presence of basal disc of cercus, 14) color and shape of the basal disc of cercus, 15) shape of basal tooth on dorsal paraproct, 16) shape of paraproct end, 17) shape of male genital ligula, 18) presence of swelling on dorsal side of S10, and 19) shape of the mesostigmal plate.

DNA extraction and amplification Total genomic DNA was extracted using the protocol of the UniversalGen DNA Kit (Beijing ComWin Biotech Co., Ltd.). Small doses were used as a template for polymerase chain reaction amplification (PCR). Each PCR amplification was performed in 50 µl reaction mixes containing 6 μl 10×LA PCR Buffer II (Mg2+ Plus), 6 μl dNTP Mixture (2.5 mM), 2.5 U TaKaRa LA Taq (TaKaRa Biotechnology Co., Ltd., Dalian, China), 1 µl each of primer (10 μM), 2 μl DNA template, and 33.7 μl distilled water. The combined nuclear gene of the ribosomal ITS1–5.8S–ITS2 region (ITS) was amplified with the primers proposed in Dumont et al. (2010), viz. Vrain2F (5’-CTTTGTACACACC- GCCCGTCGCT-3’) and Vrain2R (5’-TTTCACTCGCCGTTACTAAGGGAATC-3’). Primers designed by Yu et al. (2015) were used to amplify the target mitochondrial gene COI (cytochrome oxidase I) sequences, namely COIf1 (5’-GRGCATGRGCAGGWATAG TNG-3’) and COIr1(5’-GGGTAGTCTGARTATCGTCGNGGT-3’). The PCR cycling procedure was 2 min at 95°C followed by 35 cycles of denaturation at 95°C for 30 s, annealing at a tem- perature of 56°C (ITS) or 52°C (COI) for 30 s, and extending at 72°C for 1 min, with a final single extra extension step at 72°C for 8 min. All PCR products were visualized by 1% agarose gel electrophoresis, and amplifications were purified using a gel extraction kit (Sangon Biotech Co., Ltd., Shanghai, China), then sent to commercial com- panies (BGI TechSolutions Co., Ltd., and GENEWIZ Co. Ltd.) for sequencing based on Sanger’s Chain Termina- tion Method. Fragments that failed in direct sequencing were cloned into a TA-cloning vector pMD-18T (TaKaRa) and transformed into competent E. coli DH5α. Putative clones containing the PCR fragments were selected and sequenced.

Sequence analyses Sequences obtained were edited and assembled in BioEdit v7.2.0 (Hall 1999). Alignments of protein-coding genes were translated into amino acids using MEGA v6.06 (Tamura et al. 2013) to detect frameshift mutations and pre-

254 · Zootaxa 4851 (2) © 2020 Magnolia Press YU & XUE mature stop codons, which may indicate the presence of pseudogenes. Sequences were aligned using the ClustalX version 2.1 program package (http://www.clustal.org/) with default settings and were subsequently corrected manu- ally to ensure that the peak figure of each mutation locus was credible. Genetic distances of the analyzed COI and ITS gene fragments between Megalestes species were constructed using MEGA v6.06 based on the Kimura two- parameter model.

Phylogenetic analyses Subsequent analyses were performed using optimality criteria, including maximum likelihood (ML) and Bayesian inference (BI) algorithms to resolve the phylogenetic relationships. Maximum likelihood analysis was performed using RAxML v8.0.0 (Stamatakis 2014). Final ML tree searches were conducted under the GTR+I+G model for both COI and ITS. These were selected as appropriate models of sequence evolution, as implemented through Mod- elTest 3.7 (Posada & Crandall 1998). We obtained bootstrap support for each node from 1,000 rapid bootstrap pseu- doreplicates, with every fifth tree used as a starting point for subsequent ML optimization on the original dataset. Bayesian phylogenetic analyses were performed using MrBayes v3.2.6 (Ronquist et al. 2011) with the GTR+I+G model for both COI and ITS, which were explored by MrModeltest v2.3 (Nylander 2004). A combined dataset of COI + ITS was also analyzed via the Bayesian method. All the acquired trees were set to 10 million generations, and every 1000 generations the chain was sampled. The Markov Chain Monte Carlo (MCMC) process was run over four parallel chains, one cold and three incrementally heated. Convergence diagnostics were determined with Tracer 1.5 (Rambaut & Drummond 2007). Trees sampled after a burn-in of the first 25% of each run from the four runs were combined and used to construct a 50% majority rule consensus tree. Trees were displayed with FigTree v1.4.0 (Rambaut 2012).

Molecular taxonomic analyses Automatic Barcode Gap Discovery (ABGD) (http://wwwabi.snv.jussieu.fr/public/abgd/) and Molecular Operation- al Taxonomic Units (MOTU) were used to test species delimitations based on all molecular data. The values of X and Nb bins are the main parameters in ABGD (Puillandre et al. 2012). In order to gain stable results, the value scope 1.0–1.3 for X and 10–20 for Nb bins was tested for ITS, and 1.0–1.5 for X and 20–30 for Nb bins were tested for COI. MOTU division was performed in jMOTU (Jones et al. 2011). Parameter Multiple was chosen with value ranges from 1 to 50, and the Low BLAST identity filter was set to 97. All other parameters were left as default.

Species delimitation results

Morphological study Most of the morphological characters that we studied were unstable, either changing with age (features 1−3) or varying within populations (features 4−9, 18, 19). Color patterns were especially unstable and unreliable due to intraspecies variation (Fig. 2). Features on caudal appendages (features 10–16) and genital ligula (feature 17) were confirmed as good diagnostic features that could be used in males of all species, even though small variations ex- isted. OTUs delimitated on the basis of these two kinds of characters included nine species: australis, distans, haui, heros, kurahashii, major, micans, omeiensis, and riccii (Fig. 8). We have provided 46 photos of appendages (Figs. 3–4), 40 photos of ligula (Fig. 5), and drawings of the lateral view of male paraprocts (Fig. 6).

Genetic distances analysis Two datasets consisting of 64 sequences of COI (1023 bp) and 59 sequences of ITS (975 bp) were obtained. The compared pairwise genetic distances of species based on COI (0.05±0.029, range 0.007–0.11) were distinctly larger than those based on ITS (0.025±0.013, range 0.0–0.059). Delimitations on the basis of genetic distances of ITS were similar separation based on morphological data (Fig. 8) when the gap was set to 0.01 (Table S1, Support- ing information). Results based on COI were not comparable with the results based on morphological or ITS data (Fig. 8). For example, the genetic distance between distinct morphological species M. kurahashii and M. haui was only 0.013±0.003, whereas that for the closely related morphological species M. riccii and M. maai was as large as 0.045±0.006. Nevertheless, we set a standard gap of 0.03 for COI (Hebert et al. 2003), since the results were still able to provide classification information (Table S2, Supporting information).

Phylogeny The delimitation derived from the phylogeny (BI and ML analyses) based on ITS data was generally coincident with that based on morphological characters. The ML tree gave a complete resolving phylogeny (Fig. S2) implying a delimitation similar to the one based on morphological data. The BI tree, although trichotomic, implied a delimitation identical to the one based on morphological data (Fig. S1). Topologies based on COI data in both BI and ML trees were almost identical (Figs. S3 and S4), which implied a delimitation different from that based on morphological data. Megalestes micans was not recovered as a monophyletic species due to irregular scatter into a large clade that enclosed most other species. Within this clade, different groups of M. micans were sisters to M. major, M. palaceus, M. kurahashii, M. riccii, M. maai, and M. chengi. Megalestes kurahashii was also not recovered as a monophyletic species but was split into two groups widely separated from each other (Figs. S3 and S4). Megalestes omeiensis, M. heros, M. major, and M. australis were recovered as monophyletic species (Figs. S3 and S4). The results produced by the combined COI+ITS dataset were generally similar to the COI results (Fig. S5).

Molecular taxonomic analyses A stable result for the partition based on the ITS data was obtained via ABGD analysis, with value scopes of X (1.0–1.3) and Nb bins (10–20). A total of 13 groups were defined with P=0.001–0.0028 (Figure S6). However, the results based on COI were variable. After testing large value ranges for X (1.0–1.5) and Nb bins (20–30), a relatively stable result was achieved. There were three groups with P=0.001–0.013 (Figure S7). In the jMOTU analysis, the optimal division was eight clusters with a MOTU discrimination of 14–16 bp (1.45%–1.65%; Figure S8) for the ITS data set and nine clusters with a MOTU discrimination of 12–24 bp (1.15%–2.55%; Fig. S9) for COI.

Discussion and conclusions

Morphology Megalestes differs from related genera not only in its isolated distribution but also in possessing a series of special morphological characters, such as the position of mf (joining point of RP1 and RP3 veins) and structures of the caudal appendages (see the details at the beginning of the section “Taxonomic considerations in males of Megalestes”). The > 30% proportion of synonyms in Megalestes (of the 18 described species) is high. The main reasons for this situation are: 1) failure to examine sufficient individual specimens from different populations, and 2) neglecting specimen variation due to aging in studies that used only morphology. In 90 specimens from > 20 populations of M. micans, we found large variation in body color patterns (features 1–9) and morphological characters such as the basal disc of the cercus (feature 13), shape of the basal tooth on the dorsal paraproct (feature 15), and the shape of the mesostigmal plate (feature 19). Chao (1965) noted that feature 2

256 · Zootaxa 4851 (2) © 2020 Magnolia Press YU & XUE (color of the middle lobe of the pronotum, one of the main differences between M. micans and M. chengi) in teneral individuals of M. chengi was yellow color (same as M. micans). Chao had noted variation due to aging, but he only examined specimens from one population. We found that in one of three males from Ailaoshan, Yunnan, and in two of seven males from Fengyangshan (Zhejiang), feature 2 was green (viz. in Chao’s words, the same as M. chengi). All specimens from Leigongshan, Guizhou and Jinggangshan, Jiangxi were also green in feature 2. The other main difference between M. micans and M. chengi according to Chao (1965) was that the base of the basal tooth of the paraproct (feature 15) is slightly, but not greatly, bulged in M. chengi. This is also a non-diagnostic character since both states (slightly and greatly bulged) occur in the Fengyangshan (Zhejiang) population (Fig. 6i–j). Feature 13 (presence of basal disc of cercus), formerly thought unique to M. discus (Wilson 2004), is common in M. micans, which varies gradually both in color and shape among populations (and perhaps also with age) (Fig. 3k–l). Chao (1965), Asahina (1985b), and Phan & To (2018) all consider feature 19 (shape of mesostigmal plate) as a diagnostic character, although in their papers, figures of this feature are different for the same species. We found that both features 19 and 1 vary among individuals from the same population. With the aid of molecular data, we found that the shape of the paraproct with the long setae on its apex (feature 16) was a consistent character (Figs. 4k–l, 6i–j) as well as the shape of the genital ligula (Fig. 5i, m, w, x, ab) in M. micans. The “yellow cerci” group (including M. riccii, M. maai, and M. tuska) has bright yellow cerci, in sharp contrast to other species (Fig. 3). Wilson & Reels (2003) noted that M. tuska has its cerci blackish brown at the base, which is different from the entirely yellow base of M. riccii. However, both types of individuals were found in the Jingn- ing (Zhejiang) population along with a gradually changing body color (Fig. 2). We suggest that the large number of individuals and special ambiance (open and easy to observe) of the Jingning group allowed us to determine that the color of the cerci in M. riccii varies with age (Fig. 2). We also have specimens from other populations (Zhangjiangjie (Hunan), Lushan (Jiangxi), and Wuyanling (Zhejiang)), but none of these show this variation. The specimens are either entirely yellow or dark at the base of the cerci, possibly since only a few individuals at a time can be observed in each of these populations.

Molecular phylogeny Analysis based on ITS yielded good species delimitation when there was a traditional classification as a reference (Fig. 7). For instance, the large clade (((M. maai + M. riccii) + (M. riccii + M. tuska)) + (M. chengi + M. micans )) was well supported (BPP= 0.69, MLB=73), and within this clade, ((M. maai + M. riccii) + (M. riccii + M. tuska)) and (M. chengi + M. micans) formed two distinct clades (MLB=42 and 63, respectively), which indicated two distinct species. The first clade shows a topology as ((M. maai + M. riccii) + (M. riccii + M. tuska)). Since each of the two subclades included samples of M. riccii from the same population (Zhangjiajie, Hunan), we can only treat the first clade as a single species. The second clade will not be monophyletic if the subclade of M. chengi is excluded (Fig. 7); therefore, M. chengi and M. micans should be considered as the same species. The result of COI-based phylogenetic analysis (mtDMA) (Figs. S3–S4) differed from that based on ITS (nDNA) (Figs. S1–S2). This may be an example of the so-called mito-nuclear discordance (Toews & Brelsford 2012), which has been previously reported in Odonata (Dow et al. 2015; Yu et al. 2015; Xue et al. 2019) and also in other insects (Eberle et al. 2019). However, the COI result, although not fully coinciding with the final delimitation in our study, still provides useful information for species such as M. omeiensis and M. heros. In these groups, the mito-nuclear discordance (for Megalestes possibly by introgression) seems to be limited.

Morphology and molecular data combined In practice, we treated the results from each of the above methods as having equal weights in species delimitation. The MOTU supported by the most evidence was defined as a valid species (Fig. 8). Morphological characters, although limited (only caudal appendages and genital ligula), provided robust verification of the classification that was completely coincident with the final decision (Fig. 8). This shows that taxonomy based on these two characters can yield creditable results. Phylogenetic analysis in this study, although failing to give a perfect resolution of the evolutionary history of Megalestes, provided corroboration of species delimitation (Fig. 7). Delimitation based on genetic distances, ABGD, or jMOTU alone cannot yield a complete and clear conclusion. However, each of these contributed to the final decision, especially the results based on ITS (Fig. 8). Accordingly, we condensed the genus Megalestes into 12 species.

Megalestes is a group of large damselflies. The body length usually ranges from 65 to 80 mm, and the wing length ranges from 30 to 45 mm. Wings are obviously petiolate and usually outspread at 180° when resting. Wing venation is relatively sparse, with the mf close to arc rather than to nodus. Structures of caudal appendages and genital ligula are simple, with cerci usually longer and more robust than the paraprocts. All species in Megalestes have the same ecological and biological features. They occur in slowly moving montane springs or brooks with dense vegetation. Since the number of studied specimens was large, we placed all of the information concerning “Material examined” into Table 1.

Megalestes australis Karube, 2014 (Figs. 3a; 4a; 5a, n; 6a)

Megalestes australis Karube, 2014: 73–76, fig. 3 [Bach Ma National Park, Thua Thien Hue, Vietnam]; Phan & To 2018: 1–6, figs. 7–9, 14–18, 25–27 [Tay Giang, Quang Nam; Bach Ma National Park, Thua Thien Hue, Vietnam].

This is a relatively large Vietnam endemic species, somewhat resembling M. heros and M. distans in the shape of appendages but unique in the shape of the genital ligula with relatively long and thin horns (Fig. 4a, n). Its species status was supported by molecular analyses (Fig. 7).

Megalestes distans Needham, 1930 (Figs. 3c, d; 4c, d; 5b, o; 6b)

Megalestes distans Needham, 1930: 231–232 [Guangxi, Sichuan]; Chao 1965: 189–190, figs. 9–11 [Emeishan, Sichuan, China]; Asahina 1969: 192–197, figs. 1–8 [Hubei, Sichuan].

This is a large, endemic Chinese species with a relatively wide distribution range, including Chongqing, Gansu, Guizhou, Henan, Hubei, Hunan, Jiangxi, Sichuan, and Yunnan. The body size and color pattern are quite consistent throughout all populations. Cerci are special with the apex extended and the basal prominence long, cylindrical, foot-like (Fig. 3c–d). The end of the genital ligula has terminal lateral lobes largely divergent and up-curved at the tips (Fig. 5o). The basal teeth on paraprocts varied slightly among populations (Fig. 4c–d). Needham (1930) stated that his specimens came from both Guangxi and Sichuan. We checked through his original descriptions and could not find any localities belonging to Guangxi, but the species is present in Sichuan with Yu Chi Valley as the type locality. Wilson & Reels (2003) reported several specimens from Guangxi. However, the figures and descriptions were not consistent with our specimens nor were they like those from Needham (1930), Chao (1965), or Asahina (1969). Therefore, we do not believe that M. distans occurs in Guangxi.

Megalestes gyalsey Gyeltshen, Kalkman & Orr, 2017 (Fig. 6c)

Megalestes gyalsey Gyeltshen et al., 2017: 588–594, figs. 3, 4a–b [Trongsa, Bhutan].

We have not examined any specimens of this species, since its description is newly published, and the species is restricted to its type locality in Bhutan. However, according to the original description and good quality photos, the unique shape of the paraproct (quite long, strong, directly backward pointed and without a basal tooth) suggests that it is a valid species (cf. Figure 4 in Gyeltshen et al. 2017).

258 · Zootaxa 4851 (2) © 2020 Magnolia Press YU & XUE Figure 3. Caudal appendages of males (dorsal view). a) australis; b) irma, paratype; c) distans, from Yaan, Sichuan; d) distans from Dashahe, Guizhou; e) heros; f) omeiensis, paratype; g) kurahashii; h) major; i) haui; j) “palaceus”; k) micans, bd: basal disk; l) “chengi”; m) riccii; n) “maai”; o) “tuska,” from Damingshan, Guangxi; p) “tuska,” from Jinxiu, Guangxi. (Scale bar = 0.5 mm).

Megalestes haui Wilson & Reels, 2003 (Figs. 3i; 4i; 5g–h, u–v; 6f)

Megalestes haui Wilson & Reels, 2003: 256–257, figs. 46–57 [Shiwandashan, Guangxi, China]; Karube 2014: 74, fig. 2 [Tam Dao, Vinh Phu, Vietnam]; Phan & To 2018: 1–6, figs. 4–6, 12–13, 22–24 [Hoang Lien National Park, Lao Cai, Vietnam]. Megalestes palaceus Zhou & Zhou, 2008: 1–2, figs. 1–5 [Xiaodanjiang, Leishan, Guizhou, China]. Syn. nov.

We examined eight specimens (Table 1), including specimens collected from the type locality of M. palaceus and holotype photos of M. haui that were provided by Dr. Guanhong Huang from the Agriculture, Fisheries and Con- servation Department of Hong Kong. Characters of caudal appendages, genitalia and ligula, and body color pattern are coincident among all these individuals. In the molecular phylogeny, M. haui and M. palaceus nested in the same clade with strong support (BPP= 0.8) and cannot be further separated (Fig. 7). Most independent evidence supports

A review of the damselfly genus Megalestes Selys, 1862 Zootaxa 4851 (2) © 2020 Magnolia Press · 259 classifying these two as the same species (Fig. 8). Therefore, we believe M. palaceus is a junior synonym of M. haui. This species is known to occur in Guangxi and Guizhou in China and in northern Vietnam.

Figure 4. Caudal appendages of males (lateral view). a) australis; b) irma, paratype; c) distans, from Yaan, Sichuan; d) distans from Qingchengshan, Chengdu, Sichuan; e) heros; f) omeiensis, paratype; g) kurahashii; h) major; i) haui; j) “palaceus”; k) micans; l) “Chengi,” paratype; m) riccii; n) maai; o) tuska, from Damingshan, Guangxi; p) tuska, from Jinxiu, Guangxi. (Scale bar = 0.5 mm).

Megalestes heros Needham, 1930 (Fig. 3e; 4e; 5c, p; 6g)

Megalestes heros Needham, 1930: 229–230 [Fujian, Sichuan]; Asahina 1985b: 11, 13, figs. 32–33 [Guadun, Fujian]. Megalestes suensoni Asahina, 1956: 213–216, figs. 12–14 [Tien-Mu-Shan, Chekiang]; Chao 1965, proposed a junior synonym.

This is a large, endemic Chinese species mainly occurring in Fujian and Zhejiang. Needham (1930) did not provide any figures for this species. Asahina (1956) described M. suensoni on base of a female specimen from Tianmushan, Zhejiang. After carefully studying another female specimen collected from the type locality of M. suensoni Chao (1965) believed it was a junior synonym of M. heros, which was finally accepted by Asahina (1985b). Chao (1965)

260 · Zootaxa 4851 (2) © 2020 Magnolia Press YU & XUE also doubted one of the type specimen of M. heros (the teneral male from Sichuan) was true of this species. We have explored Sichuan for many years and have never found M. heros, but the similar-sized species M. omeiensis is present. We agree with Chao (1965) to believe that Needham confused these two species in his original study.

Megalestes irma Fraser, 1926 (Figs. 3b; 4b; 5f, s; 6l)

Megalestes irma Fraser, 1926: 32–33 [Gandtor, Sikkim]; Asahina 1985b: 5–8, figs. 11–18 [Sikkim; Nepal].

The first author examined the type specimens (2 m#m#, 1 f# Gandtor Sikhim, 5000–6000 ft, 1924-V-29, Fraser leg.) of this species and recorded the photos at BMNH in July 2017. This is a large species with paraprocts appearing unique (Figs. 4b, 6l) in having the basal tooth bent forward, which coincides with the description in Fraser (1926). The ligula resembles that of M. australis but without the terminal lateral lobes (Fig. 5f, s). The photos of M. irma in Gyeltshen et al. (2017) were actually of M. micans, which can be easily identified by the shape of the paraprocts and the basal tooth of the cerci (cf. Fig. 4e–f, in Gyeltshen et al., 2017, for detailed discussion please see “Megalestes micans Needham, 1930”). This species is known only from Sikkim and Nepal.

Megalestes kurahashii Asahina, 1985 (Figs. 3g; 4g; 5t; 6d)

Megalestes kurahashii Asahina, 1985a: 2–11 [Thailand]; Asahina 1985b: 12–13 [Assam, India].

This is the first formal record of this species from China. It is unique in that the shape of the paraproct apex is flat and broad, decorated with some strong setae giving the appearance of a brush. To date, this species has only been reported from India, Thailand, and Yunnan, China. However, it is quite possible that it may also occur in Myanmar and Vietnam.

Megalestes lieftincki Lahiri, 1979

Megalestes lieftincki Lahiri, 1979: 122–125, figs. 1–4 [Tahlia, Arunachal Pradesh, India]; Asahina 1985b: 11–12, figs. 27–31 [Salari, Kameng, Assam, India].

We did not examine any specimens of this species, since they are difficult to access. According to the figures in the original description in Lahiri (1979), this species resembles M. heros. Asahina (1985b) put character figures of M. lieftincki together with M. heros but provided no figure of the ligula for heros. Asahina (1985b) did not provide a key, and some emphasized characters, such as “disk-shaped” basal tooth of cerci with “bush of hairs” on the apex, actually exist in both species. Only one distinct character separates M. lieftincki from M. heros if the drawings in Lahiri (1979) and Asahina (1985b) are credible, that is, the dorsal side of S10 is flat (rare in Megalestes) in M. lieftincki but swelling in M. heros. Megalestes heros is limited to the Fujian and Zhejiang provinces of China which is about 2000 km away from the type locality of M. lieftincki. This makes it unlikely that M. lieftincki is conspecific with M. heros for which reason we regard it to be a good species. Nonetheless a more detailed description, if possible combined with genetic data, would be valuable.

Megalestes major Selys, 1862 (Figs. 3h; 4h; 5e, r; 6e)

Megalestes major: Asahina, 1985b: 2–5, 14–16, figs. 1–10 [India; Nepal]; Gyeltshen et al. 2017: 593, figs. 4c–d [Bhutan].

This is the type species of Megalestes, the only species that has an inner lateral tooth in the middle of the cerci (Fig. 3h). Its paraprocts are almost spherical, covered with dense setae, with an acute dorsal base tooth pointed directly

A review of the damselfly genus Megalestes Selys, 1862 Zootaxa 4851 (2) © 2020 Magnolia Press · 261 upwards (Fig. 6e). Megalestes major is distributed in India, Nepal, and Bhutan, and it only occurs on the west side of the Himalayas.

Megalestes micans Needham, 1930 (Figs. 3k–l; 4k–l; 5i, m, w–x, ab; 6i–j)

Megalestes micans Needham, 1930: 230–231 [Sichuan]; Asahina 1985b: 8–10; Karube 2014: 73, fig. 1 [Sapa, Lao Cai, Viet- nam]; Phan & To 2018: 1–6, figs. 1–3, 10–11, 19–21 [Hoang Lien National Park, Lao Cai, Vietnam]. Megalestes chengi Chao, 1947: 15–25, figs. 2, 4, 6, 8, 10–11, 14, 16–17, 21; Chao 1965: 190–191; Asahina 1985b: 9–12. Syn. nov. Megalestes discus Wilson, 2004: 424–427, figs. 4–8, [Mangshan, Hunan, China]; Wilson & Xu 2007: 102–103, “Nanling, Guangdong,” key to species. Syn. nov. Megalestes raychoudhurii Lahiri, 1987: 54–56, figs. 131–132, 298–299, 519 [Meghalaya, India]. Syn. nov. Megalestes irma [nec. Fraser, 1926]: Gyeltshen et al., 2017: 588–594, fig. 4e–f [Bhutan].

This is the most widespread species of Megalestes, occurring throughout South China (Fujian, Guangdong, Guangxi, Guizhou, Henan, Hunan, Jiangxi, Sichuan, Yunnan, and Zhejiang) extending to Vietnam, Bhutan, and India. It is also the only species distributed on both sides of the Himalayas. The diagnostic character for this mid-sized species is the base of the basal tooth on the paraproct bulged, covered in dense setae (Fig. 6i-j). This species is closely related to M. riccii, and this was confirmed by molecular analysis (Fig. 7). In the description of M. chengi, Chao (1947) only compared his material with M. heros, a totally different species. Later, Chao (1965) stated that the differences between M. chengi and M. micans included the middle portion of the middle lobe of the pronotum green, not yellow (feature 2), and the base of the basal tooth of the paraproct slightly, but not greatly, bulged (feature 15). However, all these characters occur in intraspecies variation (Discus- sion section). Although both Chao (1965) and Asahina (1985b) have emphasized that M. chengi was very similar to M. micans, neither author confirmed that they are really the same species. We examined eight of the type specimens of M. chengi deposited in IZAS and IEAS including the holotype and found no evident morphological differences with M. micans. The molecular analyses also suggested that these two are the same species (Fig. 7). Therefore, based on both morphological and molecular evidence (Fig. 8), we confirm that M. chengi is a junior synonym of M. micans. As we have previously discussed, diagnostic characters of M. discus cannot separate it from M. micans. Wil- son & Xu (2007) mentioned that M. discus have “inferior appendages without a basal pair of stout spines or robust teeth,” which is incorrect, and “occipital margin with/without transverse yellow spot” (feature 1) supports our opin- ion that this character is not stable. We examined the diagnostic character photos of the paratype of discus (1 m#, Mangshan (near Babaoshan), Shikengkong, S Hunan, China, 26-VI-2000, K.D.P. Wilson leg.), which were kindly sent by K. D. P. Wilson, and found that all the characters are consistent with M. micans. No further information on M. raychoudhurii has been published after its original description. According to the features of caudal appendages and genital ligula in the original description and figures, there is no difference between M. raychoudhurii and M. micans. We believe that both discus and M. raychoudhurii are junior synonyms of M. micans.

Megalestes omeiensis Chao, 1965 (Figs. 3f; 4f; 5d, q; 6h)

Megalestes omeiensis Chao, 1965: 192 [Emeishan, Sichuan]

A large-sized species restricted to the western edge of the Sichuan Basin that has not been studied further since its original description. Chao (1965) indicated the holotype (m#, Emeishan, Sichuan, 1955-vi-20, 580–1100 m, Yang Xingchi leg.) but did not attach a type tag to the specimen. Thus, for a long time, the holotype was unknown until the first author found it as well as two paratypes in IZAS and attached type tags. This species resembles M. heros as they are both the largest species in this genus and have a similar appearance. However, it is easy to distinguish them, in that the terminal lateral lobes of the genital ligula are small and curly in M. omeiensis but very large, like wings, in M. heros.

262 · Zootaxa 4851 (2) © 2020 Magnolia Press YU & XUE Figure 5. Genital ligula of males (lateral view) (a–m) and ventral view (n–ab). a) australis; b) distans; c) heros; d) omeiensis, paratype; e) major; f) irma, paratype; g) “palaceus”; h) haui; i) “Chengi”; j) maai; k) riccii; l) tuska; m) micans; n) australis, Hr: horn; o) distans, Tlb: terminal lateral lobe; p) heros; q) omeiensis, paratype; r) major; s) irma, paratype; t) kurahashii; u) “palaceus”; v) haui; w) micans, from Jinping, Yunnan; x) “Chengi”; y) maai; z) riccii, Blb: basal lateral lobe; aa) tuska, Mlb: middle lateral lobe; ab) micans, from Fengyangshan, Zhejiang. (Scale bar = 0.2 mm).

A review of the damselfly genus Megalestes Selys, 1862 Zootaxa 4851 (2) © 2020 Magnolia Press · 263 Figure 6. Line drawing of male paraprocts (lateral view). a) australis; b) distans; c) gyalsey; d) kurahashii; e) major; f) haui; g) heros; h) omeiensis; i) micans; j) micans; k) riccii; l) irma.

Megalestes riccii Navás, 1935 (Figs. 3m–p; 4m–p; 5j–l, y–aa; 6k; 8)

Megalestes riccii Navás, 1934: 89–90, fig. 55, “Kuling, Kiang-si” [Lushan, Jiangxi]; Chao 1962: 27–28, figs. 9–12; Chao 1965: 188–189, figs. 1–3. Megalestes maai Chen, 1947: 27, figs. 1–4 [Taiwan]; Chao 1965: 189, figs. 5–8 [Taiwan]. Syn. nov. Megalestes tuska Wilson & Reels, 2003: 258–259, figs. 52–63, [Damingshan, Guangxi]. Syn. nov.

This is an intermediate-sized, pale-colored species, usually with a very bright yellow color pattern, especially when young (Fig. 2). The most conspicuous character of M. riccii is the yellow-colored cerci. This species is distributed in Guangxi, Hunan, Jiangxi, Taiwan, and Zhejiang. There were three named species of Megalestes that have yellow-colored cerci, M. riccii, M. maai, and M. tuska, all endemic to China. Megalestes maai is restricted to Taiwan. We examined a series of type specimens of M. riccii deposited in IZAS and IEAS, as well as photos of the holotype of M. tuska, kindly provided by Dr. Guanhong Huang from the Agriculture, Fisheries and Conservation Department of Hong Kong. After studying our series of specimens, including four specimens of M. maai kindly provided by Mr. Wen-chi Yeh and Dr. Liang-jong Wang from Taiwan, we found no distinct differences among these three. Molecular analysis also implied that they were

264 · Zootaxa 4851 (2) © 2020 Magnolia Press YU & XUE the same species (Fig. 7). Chao (1965) compared a single male of M. maai from Taiwan with type specimens of M. riccii and the results indicated that M. maai was different from M. riccii in the longer wings and the obtuse tip of the basal tooth on the paraproct. However, among our series of specimens, the body sizes of some Zhejiang individuals (M. riccii) were larger than those from Taiwan (M. maai), and the shape of the paraproct basal tooth tip varied randomly. Wilson & Reels (2003) mentioned that M. tuska has “sharply pointed, bifurcated” tips of the basal tooth on the paraproct. Again, as discussed above, there is also intraspecies variation since our specimens show that both M. tuska and M. riccii can have the tips obvious or not, and “bifurcated” or not. One individual from Jinxiu (very near the type locality of M. tuska) even has a very long and pointed tip (Fig. 4p). Thus, we believe that M. maai and M. tuska are both junior synonyms of M. riccii.

Figure 7. Phylogenetic reconstruction from ITS data modified based on BI and ML trees, with both posterior probabilities (right) and bootstrap values (left) shown.

A review of the damselfly genus Megalestes Selys, 1862 Zootaxa 4851 (2) © 2020 Magnolia Press · 265 Figure 8. Molecular and morphology-based species delimitation of Megalestes. M: morphological character method; lg: genital ligula; app: caudal appendages, including cerci and paraprocts; GD: genetic distance; TC: traditional classification.

Key to male Megalestes

1 Color of cercus yellow (Fig. 3m); genital ligula with large middle lateral lobes (Fig. 5z) and obvious basal lateral lobes (Fig. 5aa)...... riccii - Color of cercus black or dark brown; genital ligula without obvious basal lateral lobes, middle lateral lobes relatively small or absent...... 2 2 Paraproct without basal tooth, straight and strong, longer than 1/2 the length of the cercus (Fig. 6c)...... gyalsey - Paraproct with basal tooth, never straight, seldom as long as 1/2 the cercus length...... 3 3 Horns present at the end of genital ligula (Fig. 5n)...... 4 - Horns absent at the end of genital ligula, or only having lateral lobes ...... 7 4 Distinct inner lateral tooth present at the middle of cercus (Fig. 3h), paraproct almost spherical (Figs. 4h; 6e) ...... major

266 · Zootaxa 4851 (2) © 2020 Magnolia Press YU & XUE - No lateral tooth at middle of cercus, apex of paraproct produced, not spherical...... 5 5 Paraproct apex truncate, extended transversely, serrated with some hairs, a brushlike appearance; basal tooth large with end pointed (Figs. 4g; 6d); end of the genital ligula narrow, with the margin rounded (Fig. 5t)...... kurahashii - Paraproct apex pointed; end of the genital ligula broad with the margin trapezoidal...... 6 6 Basal tooth of paraproct robust, longer than 1/2 the entire length of paraproct, with end pointed forward (Figs. 4b; 6l); genital ligula with obvious terminal lateral lobes (Fig. 5s)...... irma - Basal tooth of paraproct very small, shorter than 1/3 the entire length of paraproct, with end pointed upward (Figs. 4a, 6a); genital ligula without terminal lateral lobes (Fig. 5a, a1)...... australis 7 Dorsum of 10th abdomen segment normal, not swelling ...... lieftincki - Dorsum of 10th abdomen segment more or less swelling (Fig. 4d)...... 8 8 Paraproct short with apex obtuse, covered with long, dense hairs that entirely cloak the apex; basal tooth of paraproct strong with base bulged and apex very acute pointed upward or a little forward (this character varied to some degree, cf. Figs. 4k and l and 6i and j); end of genital ligula modestly broad, elongate, and concave like a spoon; modest middle lateral lobes present but no basal and terminal lobes (Fig. 5w and x)...... micans - Paraproct produced back-upward with apex pointed or a little blunt; basal tooth of paraproct conical; end of genital ligula not elongate like a spoon; terminal lateral lobe present but basal or middle lobes absent...... 9 9 Apex of paraproct somewhat blunt with sparse hairs mainly on the tip (Fig. 4i and j); end of genital ligula horizontal, broad, margin truncated, with small terminal lateral lobes (Fig. 5u and v)...... haui - Apex of paraproct pointed with sparse hairs mainly on the rear side; end of genital ligula not at horizontal level ...... 10 10 Apex of the cercus extended; basal prominence long, cylindrical with the end foot-like (Figs. 3c–d and 4c–d); basal tooth of paraproct long and strong, at least as long as the produced paraproct apex (Figs. 3c and d; 4c and d); end of genital ligula narrow, slightly spoon-like, with terminal lateral lobes divergent largely, up-curved at tips (Fig. 5o)...... distans - Apex of the cercus normal; basal prominence short, flat, somewhat rectangular; end of genital ligula not spoon-like...... 11 11 End of genital ligula broad, terminal lateral lobes very large and bent forward like wings (Fig. 5p)...... heros - End of genital ligula narrow, terminal lateral lobes small and wrapping genital end tightly (Fig. 5q)...... omeiensis

Conclusion

Taxonomy today should include a variety of methods, including molecular delimitation, DNA barcoding, phylogenetic analysis, and geometric morphometrics to reduce subjectivity. This is especially important when studying challenging groups that include either cryptic species or intricate, difficult taxonomic problems. The sample sizes studied should be large enough to avoid subjective mistakes due to information deficiency. In this study, we used a conservative stance to initially treat all the successively varied species groups as one species. We suggest that future taxonomic studies should include as many lines of evidence as possible to help assess the validity of biodiversity estimates. Taxonomy, as a traditional and fundamental category of science, should be increasingly rigorous to serve other research practices.

Acknowledgements

We are grateful of Drs. Wen-qi Yeh, Liang-jong Wang, Quoc Toan Phan and Mr. Jin Chen for granting important specimens, of Dr. Benjamin Price for helping to check type specimens from the British Natural History Museum. We thank Drs. Gexia Qiao and Weibing Zhu for loaning specimens, Drs. K.D.P. Wilson and Guan-hong Huang for providing photos of type specimens. We thank Dr. Martin Schorr for providing important literatures. We also thank Dr. Matti Hämäläinen for kindly reviewing the maunscript, and reviewers and editors for their valuable remarks and suggestions. This project was supported by the Chongqing basic research and frontier exploration special project (No. cstc2018jcyjAX0415) and the grant of Ministry of Science and Technology of China (No. 2015FY210300).

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Supporting Information

Table S1. Estimates of Evolutionary Divergence over Sequence Pairs between species with the Kimura two-parameter (K2P) substitution model using MEGA 6 on base of ITS; values lower than 0.01 are shown in red.

Table S2. Estimates of Evolutionary Divergence over Sequence Pairs between species with the Kimura two-parameter (K2P) substitution model using MEGA 6 based on COI; values lower than 0.03 are shown in red.

Figure S1. Phylogenetic reconstruction from ITS data, BI tree with posterior probabilities.

Figure S2. Phylogenetic reconstruction from ITS data, ML tree with bootstrap values.

Figure S4. Phylogenetic reconstruction from COI data, ML tree with bootstrap values.

Figure S5. Phylogenetic reconstruction from data ITS+COI, BI tree with bootstrap values.

Figure S6. ABGD analysis based on ITS data.

Figure S7. ABGD analysis based on COI data.

Figure S8. MOTU analysis based on ITS data.

Figure S9. MOTU analysis based on COI data.