bioRxiv preprint doi: https://doi.org/10.1101/2020.02.20.957811; this version posted February 20, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Phylogeny of the stink bug tribe Chlorocorini (Heteroptera, 2 Pentatomidae) based on DNA and morphological data 3 4 Bruno C. Genevcius1*a, Caroline Greve2,3, Samantha Koehler4, Rebecca B. Simmons5, 5 David A. Rider3, Jocelia Grazia2 & Cristiano F. Schwertner1,6 6 7 1 – University of São Paulo (USP), Museum of Zoology, São Paulo, SP, Brazil. 8 2 – Federal University of Rio Grande do Sul (UFRGS), Department of Zoology, Porto 9 Alegre, RS, Brazil. 10 3 – North Dakota State University (NDSU), Department of Entomology, Fargo, ND, 11 United States of America. 12 4 - University of Campinas (UNICAMP), Department of Plant Biology, Campinas, SP, 13 Brazil. 14 5 - University of North Dakota (UND), Department of Biology, Grand Forks, ND, 15 United States of America. 16 6 – Federal University of São Paulo (UNIFESP), Department of Ecology and 17 Evolutionary Biology, Diadema, SP, Brazil. 18 19 * - corresponding author - [email protected] 20 a - current address: University of São Paulo, Department of Genetics and Evolutionary 21 Biology, São Paulo (SP), Brazil. 22 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.20.957811; this version posted February 20, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 23 ABSTRACT 24 Pentatomidae is the fourth largest family of true bugs, comprising nine subfamilies and 25 over 40 tribes. Few tribes in the family have been studied in a phylogenetic context, and 26 none of them have been examined using molecular data. Here, we conduct a 27 phylogenetic study of the tribe Chlorocorini (Pentatominae) combining 69 28 morphological characters and five DNA loci in a bayesian framework. The tribe stands 29 out as the most diverse tribe in the subfamily Pentatominae which occurs exclusively in 30 the New World. Chlorocorini was proposed as a probable monophyletic group based on 31 synapomorphies found on nearly all body parts, including the spined humeral angles of 32 the pronotum, a dorsal projection on the apices of each femora, the absence of an 33 abdominal spine and the presence of well-developed, paired projections in the male 34 genitalia (hypandrium). Here, we provide solid evidence that the tribe as currently 35 recognized is not monophyletic based both on DNA and morphological data. The 36 genera Arvelius Spinola and Eludocoris Thomas were consistently placed outside of the 37 Chlorocorini, while the remaining genera were found to form a monophyletic group. 38 Furthermore, all morphological diagnostic characters for the tribe were homoplastic, 39 except for the developed hypandrium. Lastly, we also provide a preliminary glimpse of 40 main phylogenetic relationships within the Pentatomidae, which indicate that most of 41 the included subfamilies and tribes are not monophyletic. Our results suggest that the 42 current classification of Pentatomidae is not completely adequate to reflect its 43 evolutionary history, and we urge for a complete phylogeny of the family. 44 45 Key-words: classification, molecular, Neotropics, phylogenetics, stink bugs, taxonomy 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.20.957811; this version posted February 20, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 46 INTRODUCTION 47 Pentatomidae corresponds to the fourth largest family of true bugs (Hemiptera, 48 Heteroptera). With nearly 5,000 species and over 900 genera, pentatomids are 49 distributed in all terrestrial biomes, except Antarctica (Grazia et al., 2015). They exhibit 50 a plethora of anatomical and behavioral characteristics that make the group interesting 51 models to approach evolutionary and ecological questions. Examples of these features 52 include a variety of feeding habits (Weirauch et al., 2018), aposematism (Paleari, 2013), 53 exaggerated sexual traits (McLain, 1981), and parental care (Requena et al., 2010), 54 among others. However, evolutionary studies addressing these topics are practically 55 unfeasible with pentatomids due to the absence of phylogenetic hypotheses for major 56 groups. While the position of the Pentatomidae was secondarily explored in studies 57 focusing on other pentatomoids (e.g. Wu et al. 2016; Liu et al. 2019), our knowledge 58 about the lineages that compose the family and the relationships among them remain 59 elusive. 60 The family is currently divided into nine to ten subfamilies, depending on the 61 classification hypothesis (Schuh & Slater, 1995; Grazia et al., 2015; Rider et al., 2018). 62 Most subfamilies are arguably monophyletic as they exhibit sets of unique and 63 remarkable anatomical features not present in any other group (Rider, 2000). For 64 example, the Asopinae show a range of elaborations in the head and mouthparts that 65 most likely represent a single-origin adaptation to their preying habits (Parveen et al., 66 2015). The exception is the most diverse subfamily, Pentatominae, whose monophyly 67 has been broadly questioned (Grazia et al., 2008a, 2015). The classification within this 68 group has been called “chaotic” (Rider, 2000), currently comprising over 40 tribes that 69 encompass all genera that do not fit in the other subfamilies. Few tribes of Pentatomidae 70 were studied in a phylogenetic context (e.g. Campos and Grazia 2006; Bernardes et al. 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.20.957811; this version posted February 20, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 71 2009; Schwertner and Grazia 2012), and none of these utilize molecular data. Therefore, 72 the current classification is mostly based on morphological comparisons of traditional 73 characters whose reliability for identifying natural groups has rarely been assessed. The 74 availability of molecular data for species of Pentatomidae has increased over recent 75 years; however, taxon sampling is extremely biased to Asian and European species (e.g. 76 Yuan et al. 2015; Wu et al. 2016; Liu et al. 2019). Establishing phylogenetic 77 relationships for the pentatomid fauna of the New World is considered paramount for 78 developing a more accurate classification for the family, and for better understanding 79 the evolution of stink bugs. 80 Among the tribes of Pentatominae, Chlorocorini stands out as the most diverse 81 tribe in the subfamily which occurs exclusively in the New World (Rider et al., 2018). 82 The tribe comprises 77 species organized into eight genera: Arvelius Spinola (17 spp.), 83 Chlorocoris Spinola (24 spp), Chloropepla Stål (14 spp.), Eludocoris Thomas (1 sp.), 84 Fecelia Stal (4 spp.), Loxa Amyot and Serville (10 spp.), Mayrinia Horváth (3 spp.), 85 and Rhyncholepta Bergroth (4 spp.). Chlorocoris is the most diverse genus, and it is the 86 only one devided into sub-genera: Chlorocoris (Arawacoris), Chlorocoris (Chlorocoris) 87 and Chlorocoris (Monochrocerus). Several authors previously suggested close 88 relationships among some of these genera, formerly placing them in the tribe 89 Pentatomini (Becker & Grazia, 1971; Rolston & McDonald, 1984). Stål (1868) 90 described Chloropepla and keyed it together with Chlorocoris and Loxa; later, he also 91 included Fecelia in his key (Stål, 1872). More recently, Grazia (1968, 1976) suggested 92 that Chlorocoris, Chloropepla, Loxa, Mayrinia and Fecelia are related based on the 93 general morphology (e.g. body coloration, head and general body shape, highlighting 94 the presence of spined humeral angles and a dorsal apical projection on each femur as 95 the main diagnostic characters) (Fig. 1); however, these femoral projections are not 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.20.957811; this version posted February 20, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 96 found in Chlorocoris (Eger, 1978; Thomas, 1985). The genera Arvelius, Eludocoris and 97 Rhyncholepta were later added to the group based on the presence of at least some of 98 the morphological features described as characteristics of the tribe, such as the humeral 99 angles projected and the tapering apexes of the juga (Becker & Grazia, 1971; Thomas, 100 1992; Greve et al., 2013; Kment et al., 2018; Rider et al., 2018). However, it has been 101 only recently that these eight genera have been formally recognized as a distinct tribe 102 within the Pentatominae (Rider et al., 2018). 103 The tribe has been considered to be monophyletic based on several 104 characteristics found on nearly all body parts (Greve et al., 2013; Rider et al., 2018). 105 For example, some of the purported defining characters are the triangular head, the 106 spined humeral angles on the pronotum, the dorsal projections on the apices of the 107 femora, the absence of an abdominal spine, and the presence of a well-developed pair of 108 projections (the so called hypandrium) in the male genital capsule. There are also 109 additional features that, in combination, are diagnostic for species of the Chlorocorini: a 110 depressed body, anterior pronotal margins with conspicuous denticles, short ostiolar 111 rugae, and a medially-carinate mesosternum (Becker & Grazia, 1971; Greve et al., 2013; 112 Rider et al., 2018).