#ZooKeys ZooKeys 1177: 147-165 (2023) DOI: 10.3897/zookeys.1177.102811 Research Article Description of three new species of Benedictus (Coleoptera, Chrysomelidae, Galerucinae, Alticini) from China, with comments on their biology and modified ethanol traps for collecting flea beetles Yongying Ruan'®, Alexander S. Konstantinov2®, Albert F. Damaska?®, Lihao Zheng*, Jun Chen®®, Ziye Meng" non oF Wo DY — Plant Protection Research Center, Shenzhen Polytechnic, Shenzhen 518055, China Systematic Entomology Laboratory, USDA, Smithsonian Institution, PO. Box 37012, National Museum of Natural History, Washington, DC 20013-7012, USA Department of Zoology, Faculty of Science, Charles University, Viniéna 7, 128 00 Prague, Czech Republic Guang’an Vocational and Technical College, Guang’an 638000, China Key Laboratory of Zoological Systematics and Evolution, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China Research Center of Buckwheat Industry Technology, Guizhou Normal University, Guiyang, Guizhou, 550025, China Corresponding authors: Yongying Ruan (yongyingruan@hotmail.com); Ziye Meng (ziyemeng@outlook.com) OPEN Qaccess Academic editor: Caroline Chaboo Received: 3 March 2023 Accepted: 24 April 2023 Published: 30 August 2023 ZooBank: https://zoobank. org/72997BC7-B301-4EAB-95FB- 3B6FD839B1F4 Citation: Ruan Y, Konstantinov AS, Damaska AF, Zheng L, Chen J, Meng Z (2023) Description of three new species of Benedictus (Coleoptera, Chrysomelidae, Galerucinae, Alticini) from China, with comments on their biology and modified ethanol traps for collecting flea beetles. In: Chaboo CS, Schmitt M (Eds) Research on Chrysomelidae 9. ZooKeys 1177: 147-165. https://doi.org/10.3897/ zookeys.1177.102811 Copyright: © Yongying Ruan et al. This is an open access article distributed under terms of the CCO Public Domain Dedication. Abstract The diversity and biology of the moss and leaf litter-inhabiting flea beetles are still poorly known. In this study, three new species of Benedictus are described from China: Benedictus fuanensis Ruan & Konstantinov, sp. nov., Benedictus quadrimaculatus Ruan & Konstantinov, sp. nov., and Benedictus wangi Ruan & Konstantinov, sp. nov. Comments on their biology are given. Benedictus quadrimaculatus has a highly unusual morphological feature not reported before in flea beetles: black spots on the abdominal tergites that are visible through the elytra. Traditional and modified ethanol traps were tested and proven useful for collecting leaf litter- and moss-inhabiting flea beetles. Based on our tests, eight traps could collect one specimen each day in the testing sites in Fujian Province; three traps could collect one specimen each day in the testing sites in Guangdong Province. Key words: Diversity, flea beetles, leaf litter, pan trap, pitfall trap, taxonomy Introduction Benedictus Scherer, 1969 consists of 26 species prior to this study, of which eight species are known from China. Benedictus species occur in Oriental Re- gion and Papua New Guinea, and the adults are usually wingless and inhabit moss cushions and leaf litter (Sprecher-Uebersax et al. 2009). The most recent studies on Benedictus include the taxonomic revisional work by Sprecher-Ueber- sax et al. (2009) which reported 25 species of the genus, and the description of a new species by Damaska and Aston (2019). Benedictus is morphologically allied to Microcrepis Chen and Loeblaltica Scherer (Sprecher-Uebersax et al. 2009). Recent molecular phylogenetic studies (Damaska et al. 2022; Douglas et al. 2023) revealed that flea beetles from multiple and often distant lineages adapted to moss and leaf litter habitats. Damaska et al. (2022) revealed that Benedictus belongs to the Manobia generic group, which contains the moss-inhabiting ge- nus Benedictoides and leaf surface-living genera Aphthonoides Jacoby, Manobia 147 Yongying Ruan et al.: New species of Benedictus Jacoby, and Phyllotreta Chevrolat. Despite the studies mentioned above, the true diversity and the biology of the moss-inhabiting species of Benedictus are still poorly known. In this work, we describe three new species from China and pro- vide insights into their biology. We also provide a key to the 11 species occurring in China. The feeding habit and living environment of Benedictus fuanensis sp. nov. are found to be very similar to that of Cangshanaltica fuanensis Ruan et al. (2022). In Fujian province, they were found in the same location and share the same host plant Hypnum plumaeforme Wilson (Hypnaceae). When reared in the laboratory, they both feed on the distal ends of moss branches of the host plant. Moss and leaf litter-inhabiting flea beetles are usually collected by the tradi- tional Berlese funnel (e.g., Konstantinov et al. 2013; Linzmeier and Konstantin- ov 2020; Konstantinov and Linzmeier 2020) and the fan-driven Berlese funnel (Ruan et al. 2020). In Damaska and Konstantinov (2016), specimens were col- lected by simply beating semi-dry moss surfaces and cushions on standing and fallen trees. Moss and leaf litter sifting technique was used to concentrate the samples and thus speed up the extraction process. However, Berlese fun- nels usually require electricity and a suitable room to accommodate them. In this study, we test the ethanol trap for collecting moss and leaf litter inhabiting flea beetles. This method may enhance our abilities to collect these groups of flea beetles and contribute to revealing their diversity and biology. Materials and methods Morphological and taxonomic methods Observations of the habitus and diagnostic characters of flea beetles were made using the Nikon SMZ645 stereomicroscope and Nikon OPTIPHOT mi- croscope. Genitalia with the last few abdominal tergites were separated using sharp insect pins attached to plastic sticks. The tissues surrounding the ae- deagus were cleared. Female genitalia and accompanying structures (the last tergites) were immersed in a hot 10% NaOH solution for 30 s (or the appropri- ate time required to soften irrelevant tissue). The extra tissues surrounding the genitalia were carefully removed using insect pins. For photography, the female genitalia were mounted on slides with glycerine; male genitalia were glued to paper card points. Digital images were taken with a Canon D800 camera at- tached to Canon MP-E 65-mm lens or microscope lens. Morphological terminology follows Ruan et al. (2019). Specimen labels are cit- ed verbatim. Ninety-two specimens were assembled for this study based on mu- seum collections and our fieldwork. Abbreviations for insect collections. IZCAS: Institute of Zoology, Chinese Academy of Sciences, Beijing, China. SZPT: Plant Protection Research Center, Shenzhen Polytechnic, Shenzhen, Guangdong, Chi- na. Field-collected and lab-reared specimens are deposited in SZPT and IZCAS. Rearing methods Benedictus fuanensis sp. nov. were reared and observed in the laboratory environment. Rearing methods mainly follow those used for Cangshanaltica fuanensis Ruan, Konstantinov & Damaska, 2022 (see Ruan et al. 2020). Trans- parent plastic rearing containers (15 cm x 7 cm x 5 cm) were selected and ZooKeys 1177: 147-165 (2023), DOI: 10.3897/zookeys.1177.102811 148 Yongying Ruan et al.: New species of Benedictus placed in a north-facing room to avoid direct sunlight. Two small openings were carved and sealed with non-woven fabrics, allowing for air to circulate and preventing other organisms from coming into the container. A thick layer of moist paper towel was placed at the bottom of the container to maintain proper humidity and avoid larvae from drowning in water drops; a thin layer of soil was placed above the paper towel to provide nutrition for the host plant; fresh host plant moss was collected and placed loosely above the soil layer. Distilled water was sprayed on the moss once a day to maintain humidity us- ing a small spraying device. Ethanol traps Two types of ethanol pan traps (as ethanol traps hereinafter) were used: a regu- lar one to collect dead specimens (Fig. 8A—C) and a modified one for collecting live specimens (Fig. 8D, E). The modified trap consists of the following compo- nents: 1) a plastic container such as a plate or a bowl; 2) ethanol dipped sponge (or paper towel) placed on the bottom of the container; 3) the upper opening of the bowl is sealed by plastic film leaving a narrow opening in the middle for beetles to crawl in. The plastic film forms a slope with a central opening at the bottom. Usually, the flea beetles would either stay close to the ethanol-dipped sponge or be trapped at the higher part of the plastic film. Ethanol traps were usually placed close to concentrations of moss, leaf litter, or liverworts. Sometimes moss or leaf litter on the ground was slightly excavat- ed to accommodate the ethanol traps. Test 1. In this test, 35 modified ethanol traps (Fig. 8D, E) were placed in three moist and moss-abundant sites in a village near Fuan City, Fujian Province. Each site is approximately 100 m2. The experiment lasted 26 days (including five rainy days) in January and February 2021. The ethanol traps were refreshed, and the specimens were collected each day. The number of flea beetles collect- ed was counted each day. Test 2. In this test, 37 traditional ethanol traps (Fig. 8A—C) were used. They were placed in three sites in the Che-ba-ling nature reserve, Guangdong Province, in June 2021. The ethanol traps were refreshed, and the specimens were collect- ed each day. The experiment lasted for three days (including one rainy day). Results Taxonomy Genus Benedictus Scherer, 1969 Benedictus Scherer, 1969: 99. Type species: Benedictus elisabethae Scherer, 1969, by original designation. Himalalta Medvedev, 1990: 42. Type species: Himalalta brevicornis Medvedev, 1990 (= Benedictus leoi Scherer, 1989). Synonymised by Sprecher-Uebersax et al. 2009: 476. Distribution. China (Fujian, Guangdong, Hongkong, Sichuan, Yunnan, Tibet), India, Nepal, Thailand, Philippines, Papua New Guinea. ZooKeys 1177: 147-165 (2023), DOI: 10.3897/zookeys.1177.102811 149 Yongying Ruan et al.: New species of Benedictus Key to Chinese Benedictus species 1 10 Elytral punctures shallow and tiny, arranged in barely perceptible striae... 1 Se 3 aa B. sichuanensis Sprecher-Uebersax et al., 2009 Elytral punctures deep and large, arranged in well-developed striae......... 2 Transverse antebasal groove of pronotum poorly defined, shallow, barely Visible! and wihoutlarge- DUNC UMeS i). decent ok Hemmant neteeecneethes 3 Transverse antebasal groove of pronotum well defined and deep; if shal- low, then marked by a row of much deeper and larger punctures ............. 5 Pronotum and elytra dark chestnut-brown, apex of aedeagus broadly FOUVAG CQ esteeceusucvcesy orev censeden Oe B. kurbatovi Sprecher-Uebersax et al., 2009 Pronotum pale brown or yellowish; elytra usually as pale as pronotum, but sometimes slightly darker; apex of aedeagus narrow, not broadly round- Ventral surface of aedeagus with relatively sharp ridge stretching from Dasal Opening tO-aDICAal 2B. cec.cia v