Zoological Journal of the Linnean Society, 2015. With 6 figures 1bs_bs_query Integrative insect taxonomy based on morphology, 2bs_bs_query 3bs_bs_query mitochondrial DNA, and hyperspectral 4bs_bs_query 5bs_bs_query reflectance profiling 6bs_bs_query 1 2 1 7bs_bs_query 1 bs_bs_query YANG WANG †, CHRISTIAN NANSEN † and YALIN ZHANG * 8bs_bs_query 1 9bs_bs_query Key Laboratory of Plant Protection Resources and Pest Management, Ministry of Education, 10bs_bs_query Entomological Museum, Northwest A&F University, Yangling, Shaanxi 712100, China 2 11 bs_bs_query Department of Entomology and Nematology, UC Davis Briggs Hall, Room 367, Davis, CA, USA 12bs_bs_query 13bs_bs_query Received 23 June 2015; revised 6 October 2015; accepted for publication 7 October 2015 14bs_bs_query 15bs_bs_query Integrative taxonomy is considered a reliable taxonomic approach of closely related and cryptic species by inte- 16bs_bs_query grating different sources of taxonomic data (genetic, ecological, and morphological characters). In order to infer 17bs_bs_query the boundaries of seven species of the evacanthine leafhopper genus Bundera Distant, 1908 (Hemiptera: Cicadellidae), 18bs_bs_query an integrated analysis based on morphology, mitochondrial DNA, and hyperspectral reflectance profiling (37 spec- 19bs_bs_query tral bands from 411–870 nm) was conducted. Despite their morphological similarities, the genetic distances of the 20bs_bs_query cytochrome c oxidase subunit I (COI) gene among the tested species are relatively large (5.8–17.3%). The species- 21bs_bs_query 2 specific divergence of five morphologically similar species (Bundera pellucida and Bundera spp. 1–4) was revealed bs_bs_query 22bs_bs_query in mitochondrial DNA data and reflectance profiling. A key to identifying males is provided, and their morpho- 23bs_bs_query logical characters are described. Average reflectance profiles from the dorsal side of specimens were classified based 24bs_bs_query on linear discriminant analysis. Cross-validation of reflectance-based classification revealed that the seven species 25bs_bs_query could be distinguished with 91.3% classification accuracy. This study verified the feasibility of using hyperspectral 26bs_bs_query imaging data in insect classification, and our work provides a good example of using integrative taxonomy in studies 27bs_bs_query of closely related and cryptic species. 28bs_bs_query 29bs_bs_query © 2015 The Linnean Society of London, Zoological Journal of the Linnean Society, 2015 30bs_bs_query doi: 10.1111/zoj.12367 31bs_bs_query 32bs_bs_query ADDITIONAL KEYWORDS: 16S rDNA – classification – DNA barcoding – Hemiptera – hyperspectral imaging 33bs_bs_query – leafhopper – method – species delineation. 34bs_bs_query 35bs_bs_query INTRODUCTION taxonomy consists of integrating different data types 50bs_bs_query for species delineation, and it is becoming widely ac- 51bs_bs_query 36bs_bs_query Given the limited availability of taxonomic expertise cepted in modern taxonomy (Glaw & Vences, 2002; 52bs_bs_query 37bs_bs_query for many diverse and complex groups of insects, methods Dayrat, 2005; Will et al., 2005; Padial et al., 2010; 53bs_bs_query 38bs_bs_query are needed to: (1) partially automate the initial screen- Schlick-Steiner et al., 2010; Yeates et al., 2011; Riedel 54bs_bs_query 39bs_bs_query ing and separation of insect species; and (2) develop et al., 2013; Bluemel et al., 2014; Miraldo et al., 2014). 55bs_bs_query 40bs_bs_query complementary and synergistic methods to improve the As part of integrative taxonomy, molecular tech- 56bs_bs_query 41bs_bs_query performance of existing procedures for the delinea- niques are widely used, including DNA barcoding 57bs_bs_query 42bs_bs_query tion and identification of closely related and cryptic (Hebert, Ratnasingham & deWaard, 2003; Hajibabaei 58bs_bs_query 43bs_bs_query 3 species. Regarding the latter aim, overlapping char- bs_bs_query et al., 2006; Rivera & Currie, 2009; Robinson et al., 2009; 59bs_bs_query 44bs_bs_query acter variation within and among species is well docu- Hebert, deWaard & Landry, 2010; Park et al., 2011; 60bs_bs_query 45bs_bs_query mented (Will, Mishler & Wheeler, 2005). Integrative Astrin et al., 2012; Alex Smith et al., 2013) and the use 61bs_bs_query 46bs_bs_query of other regions of mitochondrial DNA (Dietrich, 62bs_bs_query 47bs_bs_query Whitcomb & Black, 1997; Barco et al., 2013; Allegrucci 63bs_bs_query 48bs_bs_query *Corresponding author. E-mail: [email protected] bs_bs_query 49bs_bs_query †These authors contributed equally to this work. et al., 2014; Yang et al., 2014) and nuclear genes (Rokas 64 © 2015 The Linnean Society of London, Zoological Journal of the Linnean Society, 2015 1 2 Y. WANG ET AL. 1bs_bs_query et al., 2002; Danforth, Lin & Fang, 2005; Germain et al., three species of minute juvenile egg parasitoids 57bs_bs_query 2bs_bs_query 2013; Gutierrez-Gutierrez et al., 2013; Taylor et al., 2013). (Trichogramma) developing inside moth host eggs could 58bs_bs_query 3bs_bs_query As examples of integrative molecular techniques in be accurately classified based on the reflectance pro- 59bs_bs_query 4bs_bs_query insect taxonomy, three cryptic species of Anania files acquired from the host eggs. There are also studies 60bs_bs_query 5bs_bs_query (Lepidoptera: Crambidae: Pyraustinae) from North in which reflectance profiling was used in the system- 61bs_bs_query 6bs_bs_query America were classified based on DNA barcoding and atics of fossil insects (Mietchen et al., 2005). Finally, 62bs_bs_query 7bs_bs_query morphology (Yang et al., 2012). Handoo et al. (2014) in- Luo, Wei & Nansen (2015) showed that hyperspectral 63bs_bs_query 8bs_bs_query tegrated data from nuclear ribosomal DNA genes, mor- imaging of forewing costae could be used to differen- 64bs_bs_query 9bs_bs_query phology, and morphometrics into a species classification tiate ‘mute’ cicadas from cicadas with tymbal sound 65bs_bs_query 10bs_bs_query of 18 stunt nematode species (Nematoda: production. 66bs_bs_query 11 bs_bs_query Telotylenchidae). In addition, there are numerous taxo- In this study, we compared and integrated three taxo- 67bs_bs_query 12bs_bs_query nomic studies in which ecological observations are in- nomic procedures: (1) classification based on tradition- 68bs_bs_query 13bs_bs_query tegrated into the analysis, including: host selection al insect morphology; (2) classification based on the 69bs_bs_query 14bs_bs_query (Bernardo et al., 2008; Chesters et al., 2012; Knee et al., cytochrome c oxidase subunit I ( COI) mitochondrial DNA 9 10 70bs_bs_query bs_bs_query bs_bs_query 15bs_bs_query 2012), geographical distribution (Rius & Teske, 2013; (mtDNA) gene and 16S ribosomal DNA (rDNA) gene 71bs_bs_query 16bs_bs_query Miraldo et al., 2014), biochemical characters (Falahee analyses; (3) reflectance-based classification. Bundera 11 72bs_bs_query bs_bs_query 17bs_bs_query & Angus, 2010; Fusu, 2010; Rezac et al., 2014), eco- Distant, 1908 belongs to the subfamily Evacanthinae, 73bs_bs_query 18bs_bs_query logical niche modelling (Raxworthy et al., 2007; Zhu and it is widely distributed in the Oriental Region. For 74bs_bs_query 19bs_bs_query et al., 2013), cross-breeding analysis (Sourassou et al., the purpose of this study, we had access to speci- 75bs_bs_query 20bs_bs_query 2012), and sound production (Marsh, 1999; Brown et al., mens collected in China and Thailand. This compre- 76bs_bs_query 21bs_bs_query 2006; Popple, 2013). Bluemel et al. (2014) confirmed hensive analysis confirmed seven distinct species of 77bs_bs_query 22bs_bs_query four cryptic species of the leafhopper genus Aphrodes Bundera. With a high level of morphological similar- 78bs_bs_query 23bs_bs_query (Hemiptera: Cicadellidae) as behaviourally, genetical- ity both in terms of external features and male geni- 79bs_bs_query 24bs_bs_query ly, and morphologically distinct species. talia (Figs 2–4) among some species of Bundera, this 80bs_bs_query 25bs_bs_query In recent years, there has been a growing interest genus is considered a taxonomically challenging group 81bs_bs_query 26bs_bs_query 4 in the use of imaging technologies in species classifi- and therefore highly suitable for a detailed study of 82bs_bs_query bs_bs_query 27bs_bs_query cation (Nansen & Elliot, 2016). Some applications are the complementarity of reflectance profiling as part of 83bs_bs_query 28bs_bs_query based on reflectance data acquired from only a few an integrative taxonomy procedure. A taxonomic re- 84bs_bs_query 29bs_bs_query (three) wide spectral bands measured with a digital vision of Bundera leafhoppers (Wang, Nansen & Zhang, 12 85bs_bs_query bs_bs_query 30bs_bs_query rgb camera (Arbuckle et al., 2001; Watson, O’Neill & unpubl. data) was not the objective of this study, but 86bs_bs_query 31bs_bs_query Kitching, 2003; Russell et al., 2007; Arribas et al., 2011; we provide reliable species delineation and a classi- 87bs_bs_query 32bs_bs_query Faria et al., 2014; Zhu & Zhang, 2014). Recently, Nguyen fication key for males of the seven species. We also 88bs_bs_query 33bs_bs_query et al. (2014) described an imaging system that can be use this analysis to discuss the potential of reflec- 89bs_bs_query 34bs_bs_query used to develop digitized three-dimensional models of tance profiling as part of integrative taxonomy. 90bs_bs_query 35bs_bs_query insect species. Such digitized models of insects are easy 91bs_bs_query 36bs_bs_query to share and store, and may therefore reduce the need MATERIAL AND METHODS 92bs_bs_query 37bs_bs_query for the shipment of specimens among taxonomists and NSECT SAMPLING