Reevaluation of Blapimorpha and Opatrinae

Reevaluation of Blapimorpha and Opatrinae : addressing a major phylogeny-classification gap in darkling beetles ( Coleoptera: Tenebrionidae: Blaptinae ) Marcin Kamiński, Ryan Lumen, Kojun Kanda, Dariusz Iwan, M. Andrew Johnston, Gael Kergoat, Patrice Bouchard, Xing Long Bai, Xiu Min Li, Guo Dong Ren, et al.

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Marcin Kamiński, Ryan Lumen, Kojun Kanda, Dariusz Iwan, M. Andrew Johnston, et al.. Reeval- uation of Blapimorpha and Opatrinae : addressing a major phylogeny-classification gap in darkling beetles ( Coleoptera: Tenebrionidae: Blaptinae ). Systematic Entomology, Wiley-Blackwell, In press, 10.1111/syen.12453. hal-02952228

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Distributed under a Creative Commons Attribution - NonCommercial| 4.0 International License Systematic Entomology (2020), DOI: 10.1111/syen.12453

Reevaluation of Blapimorpha and Opatrinae: addressing a major phylogeny-classiﬁcation gap in darkling beetles (Coleoptera: Tenebrionidae: Blaptinae)

MARCIN J. KAMINS´ K I1,2 ,RYANLUMEN1, KOJUN KANDA3, DARIUSZ IWAN2, M. ANDREW JOHNSTON4 , GAEL J. KERGOAT5 , PATRICE BOUCHARD6, XING LONG BAI7,XIU MIN LI7, GUO DONG REN7 andAARON D. SMITH1 1Department of Entomology, Purdue University, West Lafayette, Indiana, USA, 2Zoological Museum, Museum and Institute of Zoology, Polish Academy of Sciences, Warszawa, Poland, 3USDA Systematic Entomology Laboratory, c/o Smithsonian Institution, National Museum of Natural History, Washington, District of Columbia, USA, 4Biodiversity Knowledge Integration Center, Arizona State University, Tempe, Arizona, USA, 5CBGP, INRAE, CIRAD, IRD, Montpellier SupAgro, University Montpellier, Montpellier, France, 6Canadian National Collection of Insects, Arachnids and Nematodes, Agriculture and Agri-Food Canada, Ottawa, Ontario, Canada and 7The Key Laboratory of Zoological Systematics and Application, College of Life Sciences, Institute of Life Science and Green Development, Hebei University, Baoding, PR China

Abstract. The taxonomic concepts of Blapimorpha and Opatrinae (informal and traditional, morphology-based groupings among darkling beetles) are tested using molecular phylogenetics and a reassessment of larval and adult morphology to address a major phylogeny-classification gap in Tenebrionidae. Instead of a holistic approach (family-level phylogeny), this study uses a bottom-up strategy (tribal grouping) in order to define larger, monophyletic lineages within Tenebrioninae. Sampling included representatives of 27 tenebrionid tribes: Alleculini, Amarygmini, Amphidorini, Blaptini, Bolitophagini, Branchini, Cerenopini, Coniontini, Caenocrypticini, Dendarini, Eulabini, Helopini, Lagriini, Melanimini, Opatrini, Pedinini, Phaleriini, Physogasterini, Platyno- tini, Platyscelidini, Praociini, Scaurini, Scotobiini, Tenebrionini, Trachyscelini, Triboli- ini and Ulomini. Molecular analyses were based on DNA sequence data from four non-overlapping gene regions: carbamoyl-phosphate synthetase domain of rudimentary (CAD) (723 bp), wingless (wg) (438 bp) and nuclear ribosomal 28S (1101 bp) and mitochondrial ribosomal 12S (363 bp). Additionally, 15 larval and imaginal characters were scored and subjected to an ancestral state reconstruction analysis. Results revealed that Amphidorini, Blaptini, Dendarini, Pedinini, Platynotini, Platyscelidini and Opatrini form a clade which can be defined by the following morphological features: adults – antennae lacking compound/stellate sensoria; procoxal cavities externally and internally closed, intersternal membrane of abdominal ventrites 3–5 visible; paired abdominal defensive glands present, elongate, not annulated; larvae – prolegs enlarged (adapted for digging); ninth tergite lacking urogomphi. To accommodate this monophyletic grouping (281 genera and ∼4000 species), the subfamily Blaptinae sens. nov. is resurrected. Prior to these results, all of the tribes within Blaptinae were classified within the polyphyletic subfamily Tenebrioninae. The non-monophyletic nature of Terebrion- inae has already been postulated by previous authors, yet no taxonomic decisions were

Correspondence: Marcin J. Kaminski,´ Purdue University, Department of Entomology, 901 W. State Street, West Lafayette, IN 47907, U.S.A.; and Zoological Museum, Museum and Institute of Zoology, Polish Academy of Sciences, Wilcza 64, 00-679 Warszawa, Poland. E-mail: [email protected]

made to fix its status. The reinstatement of Blaptinae, which groups ∼50% of the for- mer Tenebrioninae, helps to clarify phylogenetic relations among the whole family and is the first step towards a complete higher-level revision of Tenebrionidae. The Central Asian tribe Dissonomini (two genera, ∼30 species) was not included in Blaptinae due to a lack of representatives in the performed phylogenetic analyses; however, based on morphological features, the tribe is listed as a potential addition to the subfamily.

Introduction Cerenopini Horn, Dissonomini Medvedev, Eulabini Horn, Opa- trini, Pedinini, Platyscelidini Lacordaire, Scaurini, Scotobi- Darkling beetles (Tenebrionidae Latreille) represent one of the ini Solier (representing Tenebrioninae), Branchini LeConte, most diverse insect families, with over 20 000 described species Coniontini Waterhouse, Caenocrypticini Koch, Lachnogyini (Matthews et al., 2010). As a result of their worldwide distri- Seidlitz, Physogasterini Lacordaire, Praociini Eschscholtz (rep- bution and common xerophily, darkling beetles have been used resenting Pimeliinae), Phaleriini Blanchard, Trachyscelini Blan- in a variety of biogeographic studies (Fattorini, 2002a,b; Fat- chard (representing Diaperinae). torini & Fowles, 2005; Condamine et al., 2013; Kaminski,´ 2015; Watt (1974) largely retained the concept of Blapimorpha in his Kaminski´ et al., 2018; Johnston, 2019). Currently, the fam- seminal revision of the subfamilial classification of Tenebrion- ily is subdivided into 10 subfamilies: the nine proposed in idae (excluding the pimeliine tribes Coniontini, Caenocrypticini, Matthews et al. (2010) and Bouchard et al. (2011): Alleculinae Physogasterini, Praocini and Branchini). He noted that the larval Laporte, Diaperinae Latreille, Lagriinae Latreille, Nilioninae characters were likely sufficient to support the phylogenetic Oken, Phrenapatinae Solier, Pimeliinae Latreille, Stenochiinae distinctiveness of this assemblage; only because of the lack of Kirby, Tenebrioninae Latreille and Zolodininae Watt plus Kuhi- reliable adult characters did he not give it subfamily status. Other tangiinae Medvedev considered previously as tribe of Pimeli- authors have criticized the concept of Blapimorpha, stating that inae but erected as subfamily by Nabozhenko & Sadeghi (2017). the group is defined by evolutionarily convergent features related However, this classification system is far from stable, and it to soil-dwelling larvae (Doyen, 1972; Schulze, 1969). Iwan & is not supported by available phylogenetic data (Doyen & Becváˇ rˇ (2000) were the last to discuss the concept of Blapimor- Tschinkel, 1982; Kergoat et al., 2014; Kanda, 2017). pha. Based on newly available larval material and a comprehen- Both molecular and morphological data point to Tenebrion- sive study of the literature, they refuted some of the diagnostic inae as one of the most problematic subfamilies among dark- features proposed by Skopin (1962) and Watt (1974). For ling beetles. The diversity of this grouping is estimated to be instance, they showed that the bipartite tarsungulus occurs only about 8000 species, which are distributed in 30 morphologically in some Opatrini Brullé and Pedinini Eschscholtz, and there- diverse tribes (Bouchard et al., 2011; Kaminski´ et al., 2019a). fore it cannot be used to unambiguously define Blapimorpha. According to Kergoat et al. (2014), Tenebrioninae is para- They concluded that, although they recognized Blapimorpha as phyletic with regard to Alleculinae, Diaperinae, Phrenapatinae monophyletic, no reliable larval characters were ever proposed and Stenochiinae; a similar scenario was also presented by to define the group. Only the most general definition wassus- Kanda (2017). This reflects the fact that, for the majority of its tained, i.e., the absence of urogomphi and enlarged, prominent, history, Tenebrioninae has been treated as a ‘dumping-ground’ digging forelegs with tubercles and asymmetrical chaetotaxy. for the whole family and is largely defined by the absence of The concept of Opatrinae (opatrinoid beetles) is much apomorphies that characterize the other subfamilies (Watt, 1974; more commonly recognized among tenebrionid workers ver- Doyen, 1989). A number of informal groupings have been pro- sus Blapimorpha (Koch, 1956; Medvedev, 1968; Doyen & posed within the heterogeneous assemblage of tribes currently Lawrence, 1979; Iwan, 2001, 2004, 2006; Aalbu et al., 2002; in Tenebrioninae. In this study, two of these concepts, ‘Blapi- Johnston, 2019). In the current classification of Tenebrionidae, morpha’ (sensu Skopin, 1964) and the ‘opatrine lineage’ (sensu species of Opatrinae are included in Tenebrioninae, but as Doyen & Tschinkel, 1982) are investigated. noted by Iwan (2001), this group is abiit, non obiit (dead, The concept of Blapimorpha was first proposed by but not forgotten). Nineteen years later, the situation has not Skopin (1962, 1964), to group tribes characterized by the changed. Although it is not listed as a separate subfamily in the following larval features: absence of urogomphi; enlarged currently accepted classification of darkling beetles (Bouchard forelegs with spines (on ventral side) and asymmetrical chaeto- et al., 2011; Bousquet et al., 2018), Opatrinae informally exists taxy, presumably modified for digging; bipartite tarsungulus. in the collections and minds of many tenebrionid workers Skopin (1962, 1964) mentioned that this group includes the (Kaminski´ pers. obsrv.). Opatrinoid beetles are distinguished majority of tribes listed in Gebien’s (1937, 1939, 1940, 1941 from other Tenebrioninae by the deeply emarginate clypeus , 1942a,b) system, from Scaurini Billberg up to Cryp- (Reitter, 1917). Although the exact composition varies with ticini Brullé, except Pimeliini Latreille. Therefore, his con- different researchers, Opatrinae usually includes a restricted cept of Blapimorpha includes the following currently recog- list of tribes from Blapimorpha. The most commonly listed nized tribes (Table 1): Amphidorini LeConte, Blaptini Leach, tribes are (Iwan & Becváˇ r,ˇ 2000; Iwan, 2006): Dendarini,

Table 1. List of the genera included in the phylogenetic analysis, with their tribal and subfamilial affiliation

Subfamily Tribe Sampled genera

In-group: Blapimorpha sensu Skopin (1964), except for Dissonomini and Lachnogyini Blaptinae stat nov. (formerly Tenebrioninae) Amphidorini Eleodes (10), Embaphion, Lariversius, Neobaphion, Trogloderus Blaptini Agnaptoria (2), Blaps (3), Blaptogonia, Gnaptor, Gnaptorina (4), Prosodes (2) Dendarini Dendarus (3), Heliopates (3), Hoplarion (2), Litoborus (3), Meglyphus (2), Melambius (2), Melansis (2), Minorus (3), Oreomelasma, Phylan (2), Zadenos (5) Opatrini Adoryacus, Ammobius, Blapstinus (2), Caedius, Gonocephalum (2), Opatrum, Parastizopus, Planostibes, Sulpius, Ulus Pedinini Anaxius, Drosochrus (2), Leichenum (4), Micrantereus (2), Pedinus (4) Platynotini Alaetrinus (2), Atrocrates (2), Crypticanus, Eurynotus (3), Gonopus, Melanopterus, Monodius, Oncotus (3), Schelodontes, Sebastianus, Styphacus, Trigonopus, Zidalus Platyscelidini Bioramix (2), Myatis, Oodescelis (3), Platyscelis (2), Somocoelia Diaperinae Phaleriini Phaleromella Trachyscelini Trachyscelis Pimeliinae Branchini Branchus Caenocrypticini Caenocrypticus Coniontini Eusattus Physogasterini Physogaster Praocini Praocis Tenebrioninae Cerenopini Argoporis, Cerenopus Eulabini Apsena, Eulabis Melanimini Cheirodes Scaurini Scaurus Scotobiini Scotobius Out-group: Tribes never affiliated with Blapimorpha or Opatrinae Alleculinae Alleculini Hymenorus Lagriinae Lagrinii Statira Tenebrioninae Amarygmini Pimelionotus Bolitophagini Bolitotherus Helopini Helops Tenebrionini Rhinandrus, Tenebrio, Zophobas Triboliini Tribolium Ulomini Uloma

Note: Numbers in parentheses indicate the number of included OTUs representing a given genus.

Melanimini Seidlitz, Pedinini, Platynotini, Platyscelidini resulted in a diverse dataset of DNA sequences which when and Opatrini. combined, provide a unique opportunity to test the historically The concepts of Blapimorpha and Opatrinae have not been morphologically based concepts of Blapimorpha and Opatrinae formally evaluated since their original examination(s) provided with molecular data. The aim of this paper is to integrate these above, and have largely been based upon morphology. Addition- data within a phylogenetic framework. ally, while the more recent molecular phylogenies of Kergoat et al. (2014) and Kanda (2017) tested some of the higher level (i.e., subfamily) problems within Tenebrionidae, their scope and Materials and methods taxon sampling did not provide enough data to test the concepts of Blapimorpha and Opatrinae by themselves. Recent phy- Taxon sampling logenetic efforts based on molecular data from independent research groups from China, France, Poland and the United In order to test the monophyly and composition of the ‘Blapi- States have provided some insights into the relationships within morpha/Opatrinae’ clade, ingroup taxa representing as many some of the most commonly included tribes within Blapimorpha as possible tribes prescribed initially by Skopin (1964) in and Opatrinae: Amphidorini (Johnston, 2019), Blaptini (Con- Blapimorpha were included. In particular, 14 representatives damine et al., 2011, 2013; Soldati et al., 2017), Platyscelidini of Amphidorini, 13 of Blaptini, 28 of Dendarini, 13 of Opa- (Bai et al., 2019), Dendarini, Pedinini, Platynotini and Opatrini trini, 13 of Pedinini, 19 of Platynotini and nine of Platyscelidini (Kaminski´ et al., 2018, 2019a; Lumen et al., 2020). This has (Table 1). The following tribes were represented by at least one

© 2020 The Royal Entomological Society, Systematic Entomology, doi: 10.1111/syen.12453 4 M. J. Kaminski´ et al. representative: Branchini, Cerenopini (two species), Conion- PCR products were subsequently checked by 1% agarose gel tini, Caenocrypticini, Eulabini (two species), Melanimini, Pha- electrophoresis and sequencing was performed at GENEWIZ leriini, Physogasterini, Praociini, Scaurini, Scotobiini and Tra- Biotech Co., Ltd. (Suzhou, China) using the same primers as chyscelini (Table 1, Supplementary Material S1). Representa- in the PCR. tives of the following tribes were not included in the analyses Second taxonomic subset (remaining taxa). DNA was due to the lack of well-preserved specimens: Dissonomini and extracted from specimens preserved in 95% ethanol and Lachnogyini. However, their potential affiliation to the group recently pinned specimens using DNeasy Blood & Tissue Kits of interest based on morphological traits is discussed below. (Qiagen, Germantown, MD, USA) following the manufacturer’s Outgroups from other tribes and/or subfamilies previously not protocols. Specimens were disarticulated into three parts (head, affiliated with Blapimorpha were also included to test the bound- thorax and abdomen) and inserted into a buffer for proteinase K aries and affinities of historically included groups that have digestion following protocols in Kanda et al. (2015). Polymerase since been assigned to other subfamilies (Supplementary Mate- chain reactions were performed using ExTaq (Takara, Mountain rial S1): Pimelionotus lugens (Fåhraeus) (Amarygmini Gis- View, CA, USA). Clean-up, quantification and sequencing were tel), Tribolium castaneum (Herbst) (Triboliini Gistel), Tenebrio performed by the University of Arizona’s Genetics Core Facility molitor L., Rhinandrus sp., and Zophobas atratus (Fabricius) (UAGC). Cleaned PCR products were sequenced on a 3730XL (Tenebrionini Latreille), Helops sp. (Helopini Latreille), Uloma DNA Analyzer (Applied Biosystems, Foster City, CA, USA). tenebrionoides (White) (Ulomini Blanchard), Bolitophagus sp. (Bolitophagini Kirby), Hymenorus sp. (Alleculini Laporte) and DNA analyses Statira pluripunctata Horn (Lagriini Latreille). All sequences of Dendarini, Pedinini, Platynotini and Opa- Sequences were aligned in Mesquite 3.61 (Maddison & Mad- trini were generated during previous phylogenetic projects dison, 2019) with MAFFTv. 7.130b (Katoh & Standley, 2013) (Kaminski´ et al., 2018, 2019a,b; Lumen et al., 2020), using the L-INS-i method. All alignments were concatenated as were selected Amphidorini (Johnston, 2019). Other into a single matrix (2625 bp) for phylogenetic analyses (Supple- sequences were newly generated for this study or were mentary Material S3). Data partitions and models of sequence sequenced as part of Kanda’s (2017) unpublished disser- evolution for Bayesian phylogenetic analyses (BI) were assessed tation. Previously unpublished sequence data are available in Partitionfinder v. 2.1.1 (Lanfear et al., 2017) implemented on GenBank (MT661944-MT661969; MT663963-MT664040; on the CIPRES Science Gateway 3.3 (Miller et al., 2010), MT647841-MT6478880; Supplementary Material S1). Voucher with the concatenated dataset initially partitioned by gene and specimens are deposited in the institutional collections of the codon position (for protein coding genes). Models were com- authors. pared using the greedy algorithm, the unlinked option for branch lengths, and the Bayesian information criteria (BIC). Bayesian analyses were run through CIPRES using MrBayes (v. 3.2.7a) DNA extraction and sequencing (Ronquist et al., 2012). Two independent runs were performed, each with four chains. Analyses were run for 20 million gener- Since the concatenated matrix analysed here is the result of ations, and parameters were sampled every 1000 generations. a collective international effort, the extraction and sequenc- A burnin fraction of 25% was used, and convergence was ing methods vary between the two included taxonomic sub- checked by visualising parameters in Tracer v. 1.7.1 (Rambaut sets, i.e., Blaptini + Platyscelidini (first subset) and remain- et al., 2018). Maximum likelihood (ML) analysis was conducted ing taxa (second subset). Laboratory procedures used for par- in IQ-TREE v. 1.6.10 (Nguyen et al., 2015) on the CIPRES Sci- ticular subsets are described below. However, regardless of ence Gateway. The run was performed with edge-proportional used extraction and sequencing methodology four gene regions partition models (-spp). Branch support was estimated with 1000 were amplified for all included taxa using PCR protocols and ultrafast bootstrap replicates (Minh et al., 2013), using the ‘bnni’ primers given in Kanda et al. (2015): nuclear protein-coding approach to reduce the risk of overestimating support values genes carbamoyl-phosphate synthetase domain of rudimentary (Hoang et al., 2018), and an increased value of maximum num- (CAD) (723 bp), wingless (wg) (438 bp), and nuclear riboso- ber of iterations to stop (−nm 10 000). Models of sequence evo- mal 28S (1101 bp) and mitochondrial ribosomal 12S (363 bp). lution for ML analysis were assessed in IQ-TREE prior to phy- Details were also presented in Supplementary Material S2. logenetic analysis (Supplementary Material S4). In discussing These regions were chosen because when concatenated, they support for obtained relationships, the following abbreviations recovered a strongly supported phylogeny for relationships for are used: UFB ultrafast bootstrap; PP Bayesian posterior prob- tribes traditionally included within the concept of Blapimorpha ability. Node support is defined as low/weak (UFB = 70–80, (i.e., Dendarini, Pedinini, Platynotini and Opatrini (Kaminski´ PP = 0.90–0.94), moderate (UFB = 81–95, PP = 0.95–0.97), et al., 2018, 2019a; Lumen et al. 2020)). or strong/high (values above those previously mentioned). First taxonomic subset (Blaptini + Platyscelidini). DNA was extracted from leg muscles using EZNA® Insect DNA Kits Supporting morphological analysis (Omega Bio-tek, USA). Polymerase chain reactions (PCR) were performed with standard settings for primer sequences A comparative study of selected morphological features was and thermocycler procedures from Kanda et al. (2015). The performed within Skopin’s (1964) Blapimorpha in order to

© 2020 The Royal Entomological Society, Systematic Entomology, doi: 10.1111/syen.12453 Reinstatement of the subfamily Blaptinae 5 assess the obtained phylogenetic hypotheses (Supplementary (Figs 1, 2). However, the following tribes were recovered Material S5). Analysed characters were selected based on a liter- together regardless of the used inference method: Amphi- ature search, which included the following publications: (Koch, dorini, Blaptini, Dendarini, Opatrini, Pedinini, Platynotini and 1956; Skopin, 1962, 1964; Medvedev, 1968; Doyen, 1972; Platyscelidini (Fig. 1). Support for this clade varied with the Watt, 1974; Doyen & Tschinkel, 1982; Iwan & Becváˇ r,ˇ 2000; used inference method from weak in the ML analysis (UFB: Aalbu et al., 2002; Johnston, 2019; Kaminski´ et al., 2019a, b). 68) to high in the BI (PP: 1.0). In the ML analyses, represen- The distribution of characters within groups was verified by tatives of Helopini were recovered sister to Blaptinae; how- referring to specimens preserved in the following collections: ever, the support for this topology was weak (UFB: 56). Fur- Ditsong National Museum of Natural History (Pretoria, South thermore, on the ML consensus tree the Blaptinae+Helopini Africa), Museum and Institute of Zoology, Polish Academy of clade is sister to Scaurini; although the support for this group- Sciences (Warsaw, Poland), Field Museum (Chicago, USA), ing is negligible (UFB: 33). On the other hand, in the Bayesian Purdue Entomological Research Collection, Purdue Univer- topology Blaptinae is sister to a clade containing representa- sity (West Lafayette, USA), Hebei University Museum (Hebei, tives of Alleculini, Amarygmini, Helopini, Lagriini, Phaleriini, China). In order to trace evolutionary patterns of morphological Scaurini, Trachyscelini and Ulomini (Fig. 2). The branch sup- features, a maximum parsimony ancestral state reconstruction port for the clade containing Blaptinae and all those tribes is was conducted in Mesquite 3.61 using a simplified phylogenetic weak (PP: 0.8). topology obtained in MrBayes (Fig. 3; Supplementary Material Within Blaptinae both inference methods recovered con- S5). Photographs were taken using a Canon 1000D body and flicting topologies. The differences concerned relations within Canon EF 100 mm f/2.8 Macro USM lens. SEM images were the opatrinoid clade (Fig. 1). Namely, Opatrini was recov- acquired with a Hitachi S-3400 N at the Museum and Institute ered sister to Pedinini+Platynotini in the Bayesian analysis of Zoology, Polish Academy of Sciences. (PP: 1.0 for the Pedinini+Platynotini+Opatrini clade). While in the maximum likelihood analysis it was rendered sister to all other members of the ‘opatrinoid’ clade (UFB: 70 for Results Dendarini+Pedinini+Platynotini). Relations within the ‘blaptoid’ clade remained unchanged regardless of the used infer- Morphological analysis ence method. Branch support for all major phylogenetic lineages within this clade was high in both analyses (Fig. 1). The analysis performed here enabled a morphologically con- Within the majority of tribes representing Blaptinae, the scious interpretation of the phylogenetic results based on molec- recovered topologies did not vary across inference methods ular data. Fifteen larval and imaginal characters were scored and (Fig. 2; Supplementary Material S4). The main difference subjected to an ancestral state reconstruction analysis (Supple- concerned the phylogenetic status of Pedinus Latreille, which mentary Material S4). This enabled delimitation of the newly was rendered paraphyletic in the ML analysis, and monophyletic reinstated subfamily Blaptinae (see below), and rejection of with high support (PP: 1.0) in the Bayesian analysis. Some slight some previously used synapomorphies as diagnostic for opatri- topology deviations were also found within Amphidorini. The noid beetles (e.g., deeply emarginate clypeus) (Reitter, 1917). subfamily Tenebrioninae was recovered as non-monophyletic by The character analysis revealed that Amphidorini, Blaptini, both used inference methods (Fig. 2). Dendarini, Opatrini, Pedinini, Platynotini and Platyscelidini (referred here as Blaptinae or Blapimorpha sensu novo)can Discussion all be clearly delimited from the other Tenebrionidae by the following combination of adult (a) and larval (b) features: (a) Concept of Blaptinae antennae lacking compound/stellate sensoria (Fig. 3F); procoxal cavities closed externally and internally, abdomen with inter- Although some molecular-based phylogenetic studies have sternal membrane of abdominal ventrites 3–5 (see Aalbu et al. been conducted on Tenebrionidae (Kergoat et al., 2014; 2002); paired abdominal defensive glands present, elongate, not Kanda, 2017), this is one of the first to address a particular annulated; (b) prolegs enlarged (adapted for digging) (Fig. 3A); hypothesis formulated by previous authors based on mor- ninth tergite lacking urogomphi (Fig. 3C). On the other hand, phological data. The polyphyletic nature of Tenebrioninae no synapomorphies were recovered for this diverse taxonomic was already postulated by previous analyses of higher-level grouping. Analyses also implied a close relation between Blapti- classification problems within darkling beetles (e.g., Doyen nae and Helopini. However, both lineages are clearly distin- & Tschinkel, 1982; Kergoat et al., 2014). However, no offi- guishable in both adult and larval stages (Fig. 3). Details are cial changes to classification have been made to address the presented in the discussion. issue. The main reason for the lack of classification is likely insufficient data in both morphological (shallow coverage of intertribal diversities) and molecular (poor branch sup- Molecular phylogeny port for deeper nodes) studies. Instead of a holistic approach (family-level phylogeny), this study uses a bottom-up strategy The monophyly of Blapimorpha as defined by Skopin (1962, (tribal grouping) in order to define large monophyletic lineages 1964) was not supported by any of the used inference methods within Tenebrioninae.

Fig 1. Cladograms illustrating phylogenetic relationships with the subfamily Blaptinae. Simplified consensus topologies recovered in Bayesian (left) and ML analyses (right). Triangle height corresponds to sampling effort (wider/larger triangle = more taxa sampled). Branch support values: Bayesian posterior probability (PP: 0.0–1.0) and maximum likelihood ultrafast bootstrap percentage (BP: 0–100). For clarity Opatrini is highlighted with asterisks. [Colour figure can be viewed at wileyonlinelibrary.com].

The recent designation of the ‘opatrinoid’ clade Doyen’s radical concept did not allow for partial scenarios. As (Dendarini+Pedinini+Platynotini+Opatrini) by Kaminski´ revealed here, the similar morphology of Amphidorini, Blaptini, et al. (2019a) has provided an interesting starting point for the Dendarini, Pedinini, Platynotini, Platyscelidini and Opatrini lar- present investigation, as that phylogenetic grouping contains vae was likely inherited from a common ancestor (Fig. 3A). This >30% of the species diversity within Tenebrioninae. However, set of tribes is largely convergent with the concept of Opatrinae the set of outgroups employed by Kaminski´ et al. (2019a) was (Iwan & Becváˇ r,ˇ 2000). Taking into consideration the taxonomic insufficient to decide if the ‘opatrinoid’ clade warranted subfam- diversity of this tribal grouping (281 genera, ∼4000 species; ily status. As such, the concept of Blapimorpha (Skopin, 1962; ∼50% of Tenebrioninae species), the recovery of this evolu- Watt, 1974; Iwan & Becváˇ r,ˇ 2000) seemed to be a valuable tionary lineage significantly clarifies phylogenetic relationships lead. Especially when considering the phylogenetic analyses within the family Tenebrionidae. In order to fix the concepts conducted by Kanda (2017) suggested a sister relation between presented here around a discrete, monophyletic grouping, res- the ‘opatrinoid’ clade and Amphidorini+Blaptini. urrection of the subfamily Blaptinae Leach (type genus Blaps It is not surprising that Skopin’s (1964) concept (original tribal Fabricius; = Opatrinae junior synonym) is proposed (Fig. 2). composition) of Blapimorpha was not supported by the presently This decision also highlights the need for extensive revisionary conducted phylogenetic analyses, as the group initially consisted changes in darkling beetle classification, as the current subfamil- of all tenebrionid tribes with soil-dwelling larvae. As hypothe- ial classification does not accurately reflect phylogenetic rela- sized by Doyen (1972), in the majority of those tribes certain tionships within the family (Doyen & Tschinkel, 1982; Kergoat features (enlarged prolegs) evolved convergently and should not et al., 2014; Kanda, 2017). In other words, the growing phy- be used as synapomorphies (see also Schulze, 1969). However, logeny/classification gap should be addressed in order to provide

Fig 2. Detailed phylogeny of Blaptinae. Presented topology is the majority rule consensus of post-burn-in trees obtained in Bayesian analysis of the concatenated CAD, wg, 12S and 28S matrix. Posterior probabilities are displayed above branches in red. Taxa marked with light grey represent subfamilies outside Tenebrioninae. Morphological diversity of the subfamily (A–T): Platynotini (A–D): Platynotus (A), Notocorax (B), Alaetrinus (C), Anomalipus (D); Pedinini (E–H): Diestecopus (E), Pedinus (F), Emyon (G), Leichenum (H); Opatrini (I–L): Blapstinus (I), Heterotarsus (J), Planostibes (K), Psammogaster (L); Dendarini (M–N): Litoborus (M), Heliopates (N); Blaptini (O–P): Blaps (O), Prosodes (P); Platyscelidini (Q–R): Bioramix (Q), Oodescelis (R); Amphidorini (S–T): Embaphion (S), Eleodes (T). [Colour figure can be viewed at wileyonlinelibrary.com].

Fig 2. Continued. [Colour figure can be viewed at wileyonlinelibrary.com].

© 2020 The Royal Entomological Society, Systematic Entomology, doi: 10.1111/syen.12453 Reinstatement of the subfamily Blaptinae 9 a more natural classification system (Franz, 2005). Future stud- relationship with other tribes sampled in this study (Scaurini, ies should challenge the status of Blaptinae by investigating its Ulomini, Amarygmini) would require more sampling from these relations with other larger evolutionary lineages among Tenebri- groups, and is outside the purview of this study. onidae. Ulomini can be separated from all the tribes of Blaptinae by the following adult features: dorsoventrally flattened antennae bearing stellate/placoid sensillae on terminal antennomeres and an Morphological delimitation of Blaptinae exposed pygidium (tergite VII) (Matthews & Bouchard, 2008). Available larval descriptions for Ulomini are scarce; how- The limits of Blaptinae have been comprehensively tested ever, those available emphasize differing morphology com- here (except for Dissonomini and Lachnogyini) by contrasting pared to Blaptinae (e.g., shortened prothoracic legs compared traditional morphology-based hypotheses with molecular data to meso/meta thoracic legs) (Hayashi, 1964, 1966, 1968). Inves- (Figs 1, 2; Supplementary Material S4). Additionally, some tigating the relation between Helopini, Ulomini and Blaptinae of the tribes representing Blapimorpha sensu Skopin (1962, is one of the most promising avenues to further clarify phyloge- 1964) have already been classified within different subfam- netic relations within Tenebrionidae (Tschinkel & Doyen, 1980; ilies based on discrete characters by previous workers or, Doyen & Tschinkel, 1982; Purchart & Nabozhenko, 2012; outside of convergent traits, are otherwise extremely divergent Fig. 3). from Blaptinae morphologically (e.g., Lachnogyini and Tra- Although some of the known larval stages of Scaurini chyscelini) (Ferrer, 2003; Masumoto et al., 2012; Nabozhenko (namely Herpiscius sommeri Solier) show strong morpho- & Purchart, 2017). However, in order to assess the reliability of logical resemblance to Blaptinae larvae, this phenomenon the acquired phylogenetic topologies (Figs 1, 2), a discussion should be interpreted as a result of evolutionary convergence of morphological characters of some presently and historically (Schulze, 1969). Adult representatives of Scaurini can be sep- closely recovered tribes within Tenebrioninae (Amarygmini, arated from those of Blaptinae by a number of morpholog- Helopini, Scaurini and Ulomini) is conducted here. It should ical characters (see Berry, 1973), of which the most promi- be underlined that the relationships between and among these nent is the elongation of the head behind the eyes. Blapti- groups are outside the scope of this work. nae differs from Amarygmini by having symmetric aedeagal A close affiliation between representatives of Blaptinae and tegmina (Bremer & Lillig, 2014). Both Amarygmini and Scau- Helopini has been postulated by many authors based on different rini additionally differ from Blaptinae in the presence of stel- datasets. Tschinkel & Doyen (1980) noted that Amphidorini, late sensoria on the apical antennomeres, which Blaptinae lacks Helopini and Opatrini share a trend toward bilobed reservoirs (Medvedev, 1977). of the abdominal defensive glands (with no common volume between the reservoirs). In the phylogenetic analyses conducted by Doyen & Tschinkel (1982) Helopini and Ulomini were Phylogenetic relationships within Blaptinae frequently closely recovered as sister to ‘Opatrini’ – an OTU largely corresponding to Blaptinae as defined here. Further- The ‘opatrinoid’ clade of Blaptinae was recovered differently more, Purchart & Nabozhenko (2012) indicated that the larvae than in previous studies (Kaminski´ et al., 2019a). In the results of Helopini possess slightly enlarged and elongate prolegs, presented here, Platynotini was rendered sister to Pedinini, which might indicate a close affiliation between it and Blapti- and the relationship between Dendarini, Playtnotini+Pedinini nae. However, the delimitation selected here for the subfamily and Opatrini changed depending on the inference method used separates it from Helopini mainly by larval morphology. As (Fig. 1). However, similar to Kaminski´ et al. (2019a), relation- revealed by Purchart & Nabozhenko (2012), helopine larvae ships below the tribal level were relatively stable. The topol- possess an extremely short ninth tergite bearing long urogomphi ogy in the ML analyses, despite having lower support than the overlapping segment VIII, and segment VIII bearing spines Bayesian analysis, better converges upon morphological trends on the dorsal side (see also Nabozhenko & Gurgenidze, 2006; within the ‘opatrinoid’ clade. While there are some outliers Matthews & Lawrence, 2015). These features are not present within specialized Namibian genera (e.g., Periloma Gebien, in Blaptinae (Medvedev, 1968, 2001; Yu et al., 2000; Smith Psammogaster Koch) (Schulze, 1963), there is a trend within et al., 2014; Kaminski´ et al., 2019b). Additionally, Tschinkel opatrine larvae to have many spines on the terminal abdominal and Doyen (1982) clustered Helopini, Ulomini and ‘Opa- segments (Kaminski´ et al., 2019b). Similar abdominal struc- trini’ using characters that are widespread and plesiomorphic tures are also observed in the ‘blaptoid’ clade (Fig. 3B). Con- within Tenebrionidae (e.g., hindwing flecks, mandibular molae versely, the rest of the ‘opatrinoid’ clade (Pedinini, Dendarini structure and ‘short’ ovipositor coxite 1), which they also noted. and Platynotini) show a trend towards a reduction of these spines The analyses conducted here, regardless of inference method, (Kaminski´ et al., 2019b). Furthermore, representatives of Den- recovered Helopini outside the ‘urogomphiless’ clade (Fig. 3C) darini, Pedinini and Platynotini (except Eurynotina Mulsant and of Blaptinae. Additionally, features of the ovipositor separating Rey) all possess aedeagal clavae (Fig. 3D). These morphologi- Helopini and Blaptini were listed by Tschinkel & Doyen (1980). cal trends lend further support to the ML topology. At this point, While this does not rule out a sister relationship with Blaptinae, it should be noted that the phylogenetic relationships within there is enough evidence here to support Helopini’s exclusion the ‘opatrinoid’ clade have not been ultimately fixed. Future from Blaptinae. Clarifying its position within Tenebrioninae and studies employing NGS datasets will likely resolve the current

Fig 3. Evolution of selected morphological features in Blaptinae and selected related tenebrionid tribes: (A) structure of larval prolegs, 0: not enlarged, 1: enlarged; (B) spine arrangement on larval ninth tergite, 0: arrangement different than following, 1: >8 short spines present (most cases), 2: 4–6 elongate spines present (most cases) (see Kaminski´ et al., 2019b for exceptions); (C) larval ninth tergite, 0: elongate, without urogomphi; 1: short and equipped with urogomphi (Purchart & Nabozhenko, 2012); (D) aedeagal clavae, 0: absent; 1: present (see Kaminski´ et al., 2019a for exceptions); (E) defensive glands, 0: with common volume, 1: without common volume (after Tschinkel & Doyen, 1980); (F) antennal stellate sensoria, 0: absent; 1: present (see Medvedev, 1977). Character optimizations analysed using the maximum parsimony method in Mesquite. SEM images illustrate chosen states used in morphological analysis. Tribal assignment: Guilda (Dendarini), Eurynotus (Platynotini), Pedinus (Pedinini), Scaurus (Scaurini). [Colour figure can be viewed at wileyonlinelibrary.com]. ambiguity in these relationships. On the other hand, the ‘blap- Other potential Blaptinae toid’ clade was consistently recovered as monophyletic with a stable topology regardless of the analyses employed (Fig. 1). The Central Asian tribe Dissonomini (two genera, ∼30 These results present an interesting biogeographic scenario in species) bears a striking superficial resemblance to Blaptini respect to the distributions of the Nearctic/Neotropical (Amphi- and Platyscelidini as adults (Fig. 4A); however, larval charac- dorini) and Palearctic (Blaptini and Platyscelidini) ‘blaptoid’ ters align with the ‘opatrinoid’ clade (i.e., Pedinini). Specif- beetles. This exact topology was predicted by Medvedev (2001) ically, adults have a reduced scutellum and ‘blaptoid’ dorsal based on an examination of morphological data. From a tax- habitus reflecting a closer affiliation with Blaptini/Platyscelidini onomic perspective, the delimitation of Amphidorini is unam- (Medvedev, 1968). Furthermore, the dilated pro- and mesotar- biguous based on the presence of aedeagal clavae in the males someres indicates a close relationship between Dissonomini and and undivided ovipositor coxites in the females of all Amphi- Platyscelidini (Medvedev, 1968). At the same time, Dissono- dorini genera (Peña, 1971; Doyen & Lawrence, 1979; John- mini larvae possess similar morphology of the last abdomi- ston, 2016; Lumen et al., 2020), while distinction between Blap- nal segment to some Pedinini – the presence of four enlarged tini and Platyscelidini is more complicated, as the two tribes are apical spines (Medvedev, 1968). Platyscelidini can be distin- much more closely related (Fig. 2). The most reliable features for guished from Dissonomini by the anterior margin of epis- separating Blaptini and Platyscedilini concern their tarsal struc- toma lacking a notch in the middle; visible membrane between ture (see identification key). labrum and epistome in dorsal view; eyes not narrowed by

Fig 4. Morphology of Dissonomus sp. (Tenebrioninae: Dissonomini): dorsal habitus (A); ventral side of head (B); protrochanter (C); aedeagal tegmen (D).. [Colour figure can be viewed at wileyonlinelibrary.com]. expanding temples/genae; middle part of the mentum lacking cannot be assigned to any of the currently recognized tribes of longitudinal keel (Fig. 4B); outer margins of epipleura reaching the ‘blaptoid’ clade (Amphidorini, Blaptini, Platyscelidini). As sutural angle, or interrupted at middle or before apex of ely- larvae of Stenolamus are currently unknown, at this time it is tra (Medvedev, 1968; Abdurakhmanov & Nabozhenko, 2011). impossible to decide if this genus should be classified within Additionally, Dissonomini differs from Pedinini by lacking Blaptinae. aedeagal clavae (Fig. 4D), and having an elongate basal por- tion of the aedeagal tegmen (short in Pedinini) (Kaminski´ Taxonomy & Iwan 2017). Superficially, representatives of Dissonomini resemble Opatrini, from which they can be differentiated by hav- Subfamily Blaptinae Leach ing non-opatrinoid protrochanters (see Iwan & Kaminski´ 2016) (Fig. 4C), and the already mentioned structure of larval ninth ter- =Opatrinae Brullé gite, as the majority of Opatrini possess several shorter spines Diagnosis : Adults: antennae lacking compound/stellate sen- on the dorsal margins (Kaminski´ et al., 2019b). To conclude, soria (Fig. 3F); procoxal cavities externally and internally at this point the exact phylogenetic placement of Dissonomini closed, intersternal membrane of abdominal ventrites 3–5 cannot be assessed based on morphology alone. Due to a lack of visible; paired abdominal defensive glands present, elongate, ethanol-preserved specimens in this study, representatives of this not annulated. Larvae: prolegs enlarged (adapted for digging) tribe were not included in the phylogenetic analysis performed (Fig. 3A); ninth tergite lacking urogomphi (Fig. 3C). here. For this reason, Dissonomini is not officially incorporated Tribal composition : Amphidorini, Blaptini, Dendarini, Opa- within Blaptinae. trini, Pedinini, Platynotini and Platyscelidini. The subtribal and Future studies should also aim to resolve the status of the generic classification is presented in Table 2. genus Stenolamus Gebien, which in some of the previous clas- Key to the tribes of subfamily Blaptinae based on adults sification concepts constituted a separate subtribe Stenolam- (compiled from Kaszab, 1940; Johnston et al., 2015; ina Koch within Opatini (Koch 1956; Bouchard et al., 2011). Kaminski´ et al., 2019a): However, based on the structure of male terminalia and pro- tochanter Stenolamus was excluded from the ‘opatrinoid’ clade 1. Gulawithstridulatorysurface(Fig.5A) ...... (Dendarini, Pedinini, Platynotini and Opatrini) by Iwan (2004), ...... Platynotini. who did not propose an exact taxonomic placement for it. - Gula smooth or covered with irregular rugosities ...... Furthermore, based on morphological evidence, Stenolamus ...... 2.

Table 2. Subtribal and generic classification of the newly formulated darkling beetle subfamily Blaptinae

Tribe Subtribal/generic composition

Amphidorini LeConte Eleodes Eschscholtz, Eleodimorpha Blaisdell, Embaphion Say, Lariversius Blaisdell, Neobaphion Blaisdell, Nycterinus Eschscholtz, (7 genera) Trogloderus LeConte Blaptini Leach Blaptina Leach (27 genera) Ablapsis Reitter, Blaps Fabricius, Coelocnemodes Bates, Dila Fischer von Waldheim, Dilablaps Bogatchev, Hoplitoblaps Fairmaire, Medvedevia Chigray, Nalepa Reitter, Protoblaps Medvedev, Thaumatoblaps Kaszab & Medvedev Gnaptorina Medvedev Gnaptor Brullé Gnaptorinina Medvedev Agnaptoria Reitter, Asidoblaps Fairmaire, Belousovia Medvedev, Blaptogonia Medvedev, Colasia Koch, Gnaptorina Reitter, Itagonia Reitter, Montagona Medvedev, Nepalindia Medvedev, Pseudognaptorina Kaszab, Sintagona Medvedev, Tagonoides Fairmaire, Viettagona Medvedev & Merkl Prosodina Skopin Prosodes Eschscholtz, Tagona Fischer von Waldheim Remipedellina Semenov Remipedella Semenov Dendarini Mulsant & Rey Dendarina Mulsant & Rey (38 genera) Bioplanes Mulsant, Dendarophylan Español, Dendarus Dejean, Heliopates Dejean, Litoboriolus Español, Litororus Reitter, Meglyphus Motschulsky, Microphylacinus Iwan, Kaminski´ & Aalbu, Micrositus Mulsant & Rey, Neoisocerus Bouchard, Lawrence, Davies & Newton, Phylacinus Fairmaire, Phylan Dejean, Phylanmania Ferrer, Pythiopus Koch Melambiina Mulsant & Rey Allophylax Bedel, Bermejoina Español, Gridelliopus Koch, Guildia Antoine, Hadroderus Koch, Haemodus Gebien, Hanstroemium Koch, Hoplarion Mulsant & Rey, Lasioderus Mulsant & Rey, Litoborus Mulsant & Rey, Melambius Mulsant & Rey, Melansis Wollaston, Melasmana Strand, Minorus Mulsant & Rey, Orarabion Leo & Liberto, Oreomelasma Español, Otinia Antoine, Peyerimhoffius Koch, Psammoardoinellus Leo, Pseudemmallus Koch, Silvestriellum Koch, Tragardhus Koch, Zadenos Laporte de Castelnau, Zoutpansbergia Koch Opatrini Brullé (117 genera + 6 Ammobiina Desbrochers des Loges incertae sedis) Adavius Mulsant & Rey, Ammidium Erichson, Ammobius Guérin-Méneville, Amphithrixoides Bouchard & Löbl, Asiocaedius Medvedev & Nepesova, Brachyidium Fairmaire, Caediexis Lebedev, Caedius Mulsant & Rey, Clitobius Mulsant & Rey, Coeloecetes Blair, Corinta Koch, Cornopterus Koch, Cyptus Gerstaecker, Diaderma Koch, Dilamus Jacquelin du Val, Emmalus Erichson, Falsammidium Koch, Falsocaedius Español, Freyula Koch, Hadrodes Wollaston, Helenomelas Ardoin, Mateuina Español, Messoricolum Koch, Moragacinella Español, Nesocaedius Kolbe, Perithrix Fairmaire, Platyprocnemis Español & Lindberg, Plesioderes Mulsant & Rey, Prodilamus Ardoin, Proscheimus Desbrochers des Loges, Psammestus Reichardt, Pseudoleichenum Ardoin, Raynalius Chatanay, Tarphiophasis Wollaston, Trigonopoda Gebien, Weisea Semenov Blapstinina Mulsant & Rey Aconobius Casey, Ammodonus Mulsant & Rey, Austrocaribius Marcuzzi, Blapstinus Sturm, Cenophorus Mulsant & Rey, Conibiosoma Casey, Conibius LeConte, Cybotus Casey, Diastolinus Mulsant & Rey, Goajiria Ivie & Hart, Hummelinckia Marcuzzi, Nevisia Marcuzzi, Nocibiotes Casey, Notibius LeConte, Platylus Mulsant & Rey, Tonibiastes Dejean, Tonibius Casey, Trichoton Hope, Ulus Horn, Xerolinus Ivie & Hart Heterotarsina Blanchard Diphyrrhynchus Fairmaire, Heterocheira Lacordaire, Heterotarsus Latreille, Scymena Pascoe Neopachypterina Bouchard, Löbl & Merkl Amblysphagus Fairmaire, Eupachypterus Kiirejtshuk, Nabozhenko & Nel, Neopachypterus Bouchard, Löbl & Merkl, Pseudolamus Fairmaire Opatrina Brullé Anatrum Reichardt, Brachyesthes Fairmaire, Caediomorpha Blackburn, Ephalus LeConte, Eumylada Reitter, Falsolobodera Kaszab, Gonocephalum Solier, Hadrophasis Ferrer, Jintainum Ren, Melanesthes Dejean, Melanocoma Wollaston, Mesomorphus Miedel, Myladina Reitter, Opatroides Brullé, Opatrum Fabricius, Penthicinus Reitter, Penthicus Faldermann, Phelopatrum Marseul, Polycoelogastridion Reichardt, Reichardtiellina Kaszab, Scleropatroides Löbl & Merkl, Scleropatrum Reitter, Sinorus Mulsant & Revelière, Sobas Pascoe, Socotropatrum Koch, Tidiguinia Español, Trichosternum Wollaston, Wolladrus Iwan & Kaminski´ Sclerina Lacordaire Eurycaulus Fairmaire, Palaeosclerum Nabozhenko & Kirejtshuk, Platynosum Mulsant & Rey, Sclerum Dejan Stizopina Lacordaire Adoryacus Koch, Amathobius Gebien, Blacodatus Koch, Blenosia Laporte de Castelnau, Calaharena Koch, Crististibes Koch, Eichleria Kaminski,´ Eremostibes Koch, Helibatus Mulsant & Rey, Luebbertia Koch, Microstizopus Koch, Namazopus Koch, Nemanes Fairmaire, Parastizopus Gebien, Periloma Gebien, Planostibes Gemminger & Harold, Psammogaster Koch, Sphaerostibes Koch, Stizopus Erichson, Sulpius Fairmaire, Syntyphlus Koch Incertae sedis: Hovarygmus Fairmaire, Pachymastus Fairmaire, Penichrus Champion, Pocadiopsis Fairmaire, Scleroides Fairmaire, Trigonopilus Fairmaire Pedinini Eschscholtz (19 genera) Helopinina Lacordaire Ametrocera Fåhraeus, Anaxius Fåhraeus, Aptila Fåhraeus, Asidodema Koch, Blastarnodes Koch, Diestecopus Solier, Drosochrus Erichson, Micrantereus Solier, Nicandra Fairmaire, Oncopteryx Fairmaire, Oncosoma Westwood, Piscicula Robiche, Psectes Hesse Leichenina Mulsant Apsheronellus Bogatchev, Leichenum Dejean Pedinina Eschscholtz Cabirutus Strand, Colpotinus Fairmaire, Loensus Lucas, Pedinus Latreille

Table 2. Continued

Tribe Subtribal/generic composition

Platynotini Mulsant & Rey (72 Eurynotina Mulsant & Rey genera) Byrrhoncus Koch, Capidium Koch, Colophonesthes Koch, Eurynotus Kirby, Heteropsectropus Kaszab, Hirtograbies Koch, Isoncophallus Koch, Menederes Solier, Menederopsis Koch, Ograbies Péringuey, Oncotus Solier, Phaleriderma Koch, Phylacastus Fairmaire, Psectropus Solier, Schyzoschelus Koch, Stridigula Koch Platynotina Mulsant & Rey Adamus Iwan, Alaetrinus Iwan, Amblychirus Koch, Anchophthalmops Koch, Anchophthalmus Gerstaecker, Angolositus Koch, Anomalipus Latrielle, Atrocrates Koch, Atrocrypticanus Iwan, Bantodemus Koch, Clastopus Fairmaire, Claudegirardius Iwan, Colpotinoides Kaszab, Crypticanus Fairmaire, Doyenus Iwan, Ectateus Koch, Eleoselinus Kaminski,´ Eucolus Mulsant & Rey, Eviropodus Koch, Glyptopteryx Gebien, Gonopus Latrielle, Hovademus Ardoin, Lechius Iwan, Madobalus Fairmaire, Melanocratus Fairmaire, Melanopterus Mulsant & Rey, Menearchus Carter, Monodius Koch, Nesopatrum Gebien, Notocorax Dejean, Opatrinus Dejean, Parabantodemus Iwan, Paraselinus Kaminski,´ Penthicoides Fairmaire, Phallocentrion Koch, Phymatoplata Koch, Platyburak Iwan, Platyburmanicus Iwan, Platycolpotus Iwan, Platynotoides Kaszab, Platynotus Fabricius, Pokryszkiella Iwan, Pseudoblaps Guérin, Pseudonotocorax Iwan, Pteroselinus Kaminski,´ Rugoplatynotus Kaszab, Schelodontes Koch, Sebastianus Iwan, Selinopodus Koch, Selinus Mulsant & Rey, Stenogonopus Gebien, Styphacus Fairmaire, Trigonopus Mulsant & Rey, Upembarus Koch, Zidalus Mulsant & Rey, Zophodes Fåhraeus Platyscelidini Lacordaire (8 Bioramix Bates, Microplatyscelis Kaszab, Myatis Bates, Oodescelis Motschulsky, Platyscelis Latreille, Somocoelia Kraatz, genera) Somocoeloplatys Skopin, Trichomyatis Schuster

Fig 5. Diagnostic characters for different tribes of the subfamily Blaptinae: Platynotini, stridulatory gula (A); Opatrini, ‘opatrinoid’ trochanter (B); Platynotini, ‘pedinoid’ trochanter (C); Dendarini, subdivided coxites of ovipositor (D); Amphidorini, undivided coxites of ovipositor (E); Pedinini, laterally situated palpifer (F); Dendarini, apically situated palpifer (G). [Colour figure can be viewed at wileyonlinelibrary.com].

2. Protrochanter with elongate base (Fig. 5B) ...... Supplementary Material S3: Nexus-format matrix of ...... Opatrini. molecular data spanning four non-overlapping gene regions - Protrochanter without elongate base (Fig. 5C) ...... 3. (carbamoyl-phosphate synthetase domain of rudimentary 3. Aedeagaltegmenwithclavae(Fig.3D) ...... 4. (CAD) (723 bp), wingless (wg) (438 bp), and nuclear ribo- - Aedeagal tegmen without clavae ...... 6. somal 28S (1101 bp) and mitochondrial ribosomal 12S 4. Coxites of ovipositor not subdivided into lobes (Fig. 5E) (363 bp)...... Amphidorini - Coxites of ovipositor subdivided into lobes (usually 4 Supplementary Material S4: Maximum likelihood topol- plates)(Fig.5D)...... 5. ogy obtained in IQ-TREE analysis. 5. Middle part of mentum extending laterally, completely covering lateral wings; palpifer well developed, situated Supplementary Material S5: Morphological characters apically on the basistipes (Fig. 5G) ...... Dendarini. used in the ancestral state reconstruction. - Mentum with lateral wings well visible; palpifer smaller than basistipes, situated laterally (Fig. 5F) ...... Pedinini. Acknowledgements 6. Male pro- and mesotarsi more-or-less strongly expanded; tomentose ventraly (yellow setae). Projection between Funding was provided by the Polish National Science Centre (Sonata 7 Project 2014/13/D/NZ8/02428) and the NSF ARTS tarsalclawswithbristles ...... Platyscelidini. Program (DEB #1523605 and DEB #2009247). We are grateful - Tarsi not expanded in males; rarely tomentose ventrally. to Ruth Müller for her hospitality during our visits to Transvaal Projection between tarsal claws without bristles ...... Museum, Pretoria. Rolf Aalbu for assistance in estimating ...... Blaptini. species richness of some Tenebrioninae tribes. Luna Grey for SEM photographs of stellate sensoria in Scaurini. Przemysław Szymroszczyk for photograph of Dissonomus. Mention of trade Conclusions names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA. The USDA is an equal opportunity provider and employer. The authors • The concept of Blaptinae (=Opatrinae; Blaphimorpha sensu declare there are no conflicts of interest. Moreover, there are nov.) has traditionally been recognized by many previous no disputes over the ownership of the data presented in the researchers; however, the formal designation of this group paper and all contributions have been attributed appropriately, was previously suppressed due to a lack of concrete evidence. via coauthorship or acknowledgement, as appropriate to the situa • Amphidorini, Blaptini, Dendarini, Pedinini, Platynotini, tion. Platyscelidini and Opatrini form a clade supported by new molecular data and morphological features discussed here. To accommodate this monophyletic grouping (281 genera Data availability statement and ∼ 4000 species) the subfamily Blaptinae is resurrected. • Two main clades were recovered within Blaptinae, the ‘blap- The data that supports the findings of this study are available in toid’ clade (Amphidorini, Blaptini, Platyscelidini) and ‘opa- the supplementary material of this article. trinoid’ clade (Dendarini, Pedinini, Platynotini and Opatrini). Topology of the ‘blaptoid’ clade was stable across different inference methods, while the relations with the ‘opatrinoid’ References clade were not ultimately fixed. • The placement of Dissomini in reference to Blaptini is Aalbu, R.L., Triplehorn, A., Campbell, J.M., Brown, K.W., Somerby, uncertain, although probable based on morphological traits. R.E. & Thomas, D.B. (2002) 106. Tenebrionidae Latreille 1802. • The subfamily Tenebrioninae is not monophyletic. American Beetles. Volume 2. Polyphaga: Scarabaeoidea through Curculionoidea (ed. by R.H. Arnett, M.C. Thomas, P.E. Skelley and J.H. Frank), pp. 463–509. CRC Press, Boca Raton. Abdurakhmanov, G.M. & Nabozhenko, M.V.(2011) Keys and catalogue Supporting Information to darkling beetles (Coleoptera: Tenebrionidae s. str.) of the Caucasus and south of European part of Russia, Moscow, KMK Scientific Press. Additional supporting information may be found online in 361pp. the Supporting Information section at the end of the article. Bai, X.L., Li, X.M. & Ren, G.D. (2019) A review of the genus Oode- scelis Motschulsky, 1845 (Coleoptera: Tenebrionidae: Platyscelidini) Supplementary Material S1: Specimens used in this study from China. Zootaxa, 4656, 401–430. with coding data, corresponding GenBank accession num- Berry, R.L. (1973) The Cerenopini and Eulabini, two tribes previously bers. included in the Scaurini (Coleoptera: Tenebrionidae). Annals of the Entomological Society of America, 66, 70–77. Supplementary Material S2: Thermocycler profiles and Bouchard, P., Bousquet, Y., Davies, A.E. et al. (2011) Family-group PCR primers used in this study. names in Coleoptera (Insecta). ZooKeys, 88, 1–972.

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