Zootaxa 4969 (1): 101–118 ISSN 1175-5326 (print edition) https://www.mapress.com/j/zt/ Article ZOOTAXA Copyright © 2021 Magnolia Press ISSN 1175-5334 (online edition) https://doi.org/10.11646/zootaxa.4969.1.5 http://zoobank.org/urn:lsid:zoobank.org:pub:DAA2B420-B2E3-45C2-8F6E-33F85857B3FD

Molecular phylogeny of the family (, ) reveals rampant paraphyly and convergence of traditionally used taxonomic characters

SUNDUS ZAHID1,2, RICARDO MARIÑO-PÉREZ2,3 & HOJUN SONG2* 1Department of Zoology, Hazara University, Mansehra, Pakistan �[email protected]; https://orcid.org/0000-0001-8986-3459 2Department of Entomology, Texas A&M University, College Station, TX, USA 3Department of Ecology & Evolutionary Biology, University of Michigan, Ann Arbor, MI, USA �[email protected]; https://orcid.org/0000-0002-0566-1372 *Corresponding author. �[email protected]; https://orcid.org/0000-0001-6115-0473

Abstract

The grasshopper family Pyrgomorphidae is one of the most colorful orthopteran lineages, and includes biologically fascinating and culturally important species. Recent attempts to reconstruct the phylogeny of this family have resulted in a large degree of conflicts between a morphology-based study and a molecular-based study, mainly due to convergent morphological traits that affected phylogenetic reconstruction. In this study, a molecular phylogeny of Pyrgomorphidae based on 32 ingroup species and mitochondrial genome data is proposed, which is used to test the monophyly of the taxonomic groupings used in the current classification scheme. Using the ancestral character state reconstruction analyses and character mapping, we demonstrate that some of the morphological characters, including the male genitalia, which were considered to be taxonomically important, have evolved convergently across the phylogeny. We discuss the discrepancies between our phylogeny and the previous studies and propose an approach to establish a natural classification scheme for Pyrgomorphidae.

Key word: paraphyly, convergence, male genitalia, mitochondrial genome

Introduction

The grasshopper family Pyrgomorphidae is among the most recognizable orthopteran lineages in the world. Some members of the family are well known for their vibrant color patterns, elaborate sculptures, and large sizes, which collectively make the common name gaudy quite suitable (Mariño-Pérez & Song, 2018) (Fig. 1). Several of these colorful pyrgomorph species are known to be aposematic and feed on toxic plants (Rowell, 1967; Yang et al., 2019), and capable of sequestering plant secondary metabolites as defensive chemicals by foaming or squirting (Whitman, 1990). Most pyrgomorph species are, however, cryptically colored and not known to be chemically defended (Mariño-Pérez & Song, 2018). Some pyrgomorph species are important agricultural pests that cause considerable economic damages (COPR, 1982) while other species are culturally important as they are consumed by humans (Cerritos & Cano-Santana, 2008). Pyrgomorphidae is characterized by the presence of a groove in the fastigium (Kevan & Akbar, 1964) and distinctive male phallic structures, such as the cingulum extending around to the ventral side, medially directed endophallic apodemes, and the ejaculatory sac opening to the genital chamber (Eades, 2000). Because of these clear morphological synapomorphies, the monophyly of Pyrgomorphidae has not been questioned, and recent phylogenetic studies have consistently demonstrated the monophyly of the family (Eades, 2000; Mariño-Pérez & Song, 2018, 2019; Song et al., 2015; Song et al., 2020). This family is the sole member of the superfamily Pyrgomorphoidea, which is sister to the superfamily (Song et al., 2015; Song et al., 2020). These two superfamilies diverged in the early Cretaceous (Mariño-Pérez & Song, 2019; Song et al., 2020) and the lineage

Accepted by D. Rentz: 12 Apr. 2021; published: 10 May 2021 101 diversification within Pyrgomorphidae took place throughout the Cretaceous and well into the Cenozoic (Mariño- Pérez & Song, 2019). Currently, two subfamilies, Orthacridinae and , are recognized with 31 tribes, 148 genera, and 488 valid species (Cigliano et al., 2021).

Figure 1. Diversity of Pyrgomorphidae. A. Psedna nana (Rehn, 1953) (Australia: Western Australia); B. Atractomorpha sp. (Papua New Guinea: New Britain); C. concinna (Walker, 1871) (Australia, NSW); D. histrio Gerstaecker, 1884 (Mexico, Oaxaca); E. elegans (Thunberg, 1815) (South Africa, KZN); F. Dictyophorus spumans (Thunberg, 1787) (South Africa: KZN). Photo credit: (A, B, C, E, F) Hojun Song; (D) Ricardo Mariño-Pérez.

The current taxonomic classification of Pyrgomorphidae is mostly based on Kevan’s lifelong work (Kevan, 1952a, 1952b, 1963, 1966a, 1966b, 1968a, 1968b, 1969, 1970, 1982, 1990; Kevan & Akbar, 1964; Kevan et al., 1969, 1970, 1971, 1972, 1974; Kevan et al., 1975; Kevan & Chen, 1969; Kevan & Hsiung, 1985, 1989; Kevan et al., 1964). Kevan & Akbar (1964) provided a detailed account of the systematic history of the family, but expressed that it was difficult to subdivide the family into clear groups due to a high degree of morphological convergence and parallelism among different lineages as well as a large number of lineages that are uniquely distinct in terms of both external morphology and male phallic structures. A similar sentiment was expressed earlier by Dirsh (1961) who attempted to produce a preliminary classification of Acridomorpha. Nevertheless, Kevan and colleagues established two informal groups, ‘A’ and ‘B’ and recognized 31 tribes which were arranged into 10 series, with the Series I to IV belonging to the group ‘A’, and the Series V-X belonging to the group ‘B’ based on their extensive study of external morphology and male genitalia (Kevan & Akbar, 1964; Kevan et al., 1969, 1970, 1971, 1972, 1974; Kevan et al., 1975). Later, Otte (1994), in his synonymic catalogue of Orthoptera, elevated the informal groups ‘A’ and ‘B’ to the subfamily level and recognized Orthacridinae and Pyrgomorphinae, respectively, but provided no justification for this action. However, the taxonomic validity of these groupings, especially whether they represent monophyletic groups or not, has not been rigorously tested. While a number of modern phylogenetic studies on Orthoptera included various members of Pyrgomorphidae in a broader context (Fenn et al., 2008; Flook et al., 1999, 2000; Flook & Rowell, 1997; Rowell & Flook, 1998; Song et al., 2015), two recent studies have explicitly focused on resolving the phylogeny of this family. The first study was a cladistic analysis based on 28 out of 31 current recognized tribes and 119 morphological characters (Mariño-Pérez & Song, 2018). This work was the first explicit test of Kevan’s scheme (Kevan & Akbar, 1964;

102 · Zootaxa 4969 (1) © 2021 Magnolia Press Zahid et al. Kevan et al., 1969, 1970, 1971, 1972, 1974; Kevan et al., 1975). It found the monophyly of the Group ‘A’ or the subfamily Orthacridinae, but found the Group ‘B’ or the subfamily Pyrgomorphinae as paraphyletic. For the 10 series, only the Series X (consisting of Pyrgomorphini and Chrotogonini) was found to be monophyletic, while the remaining series were paraphyletic. This study revealed that the currently accepted taxonomic classification for Pyrgomorphidae would require a comprehensive revision to accurately reflect the phylogeny. Instead of two subfamilies, Mariño-Pérez & Song (2018) recovered four monophyletic groups within Pyrgomorphidae, which were supported by different numbers of synapomorphies. However, the Bremer support values for these four clades were either 1 or 2, suggesting relatively weak support for these lineages. The second study was a molecular phylogeny of Pyrgomorphidae aiming at addressing the origin of the New World species (Mariño-Pérez & Song, 2019). This work used mitochondrial genomes and four nuclear genes and included 25 pyrgomorph species, including representatives of all four tribes present in the New World: Sphenariini, Ichthiacridini, Ichthyotettigini, and Omurini. This work robustly demonstrated that ancestral Pyrgomorphidae independently colonized the New World from the Old World at least three times. While testing Kevan’s scheme was not the aim of their study, the molecular phylogeny did result in a strikingly different topology than their earlier morphological phylogeny (Mariño-Pérez & Song, 2018) and failed to recover Orthacridinae as a monophyletic group. In discussing the discrepancies between the two studies, they attributed to the rampant morphological convergence within Pyrgomorphidae that could have caused confusion when coding morphological characters and states (Mariño-Pérez & Song, 2019). In this context, we aim to critically re-examine the utility of morphological characters originally used by Kevan and colleagues to establish the current classification scheme in light of a more expanded molecular phylogeny. Specifically, we test the monophyly of Kevan’s Groups ‘A’ and ‘B’ and the concept of Kevan’s series (Kevan & Akbar, 1964; Kevan et al., 1969, 1970, 1971, 1972, 1974; Kevan et al., 1975), as well as the monophyly of four clades recovered by Mariño-Pérez & Song (2018) using a molecular phylogenetic analysis based on mitochondrial genome, as well as the ancestral character state reconstruction analyses. We highlight the problem of the current taxonomic classification and suggest a possible direction to achieve a natural classification for this fascinating group of .

Materials and methods

Taxon and character sampling We sampled a total of 35 taxa, which included 3 outgroups, representing 3 families (, , and ) and 32 ingroups representing the Pyrgomorphidae. The ingroup taxa covered both of the subfamilies (Orthacridinae and Pyrgomorphinae) and 17 out of 31 currently recognized tribes. We included partial or complete mitochondrial genome (mtgenome) data for all 35 taxa. Of these, the sequence data for 29 taxa were previously generated by us (Leavitt et al., 2013; Mariño-Pérez & Song, 2019; Song et al., 2020) and those for 5 taxa were obtained from Genbank. We newly generated mtgenome data for Rubellia nigrosignata for this study via Illumina shotgun sequencing, following the protocols previously described in Song et al. (2018). Detailed information about taxon sampling and Genbank accession numbers are shown in Table 1.

Phylogenetic analyses For mitochondrial protein-coding genes, we aligned based on the conservation of reading frames by first translating them into amino acids and aligning them individually in MUSCLE (Edgar, 2004) using default parameters in Geneious Prime® 2020.2.4 (Biomatters, Ltd.). We aligned ribosomal RNA genes (12S and 16S) in MAFFT (Katoh & Standley, 2013) using the E-INS-i setting also in Geneious. We then concatenated these 15 alignments into a single matrix using SequenceMatrix (Vaidya et al., 2011), and divided the data into a total of 41 data blocks (13 protein-coding genes divided into individual codon positions and 2 rRNAs). We used PartitionFinder2 (Lanfear et al., 2017) to find the best-fit partitioning scheme and the models of nucleotide substitution for each partition using the “greedy” algorithm with branch lengths estimated as “linked”. We performed a maximum likelihood (ML) analysis and a Bayesian analysis on the partitioned dataset (13,724 aligned bp and 35 taxa with 11 partitions). For the ML analysis, we used IQ-TREE multicore version 1.6.10 on XSEDE (Extreme Science and Engineering Discovery Environment, https://www.xsede.org) through the CIPRES Science Gateway (Miller et al., 2011). We performed a partitioned ML analysis with 1,000 bootstrap replications.

Molecular phylogeny of Pyrgomorphidae Zootaxa 4969 (1) © 2021 Magnolia Press · 103 . (2013) . (2013) . (1995) . (2020) . (2020) . (2007) et al et al et al et al et al et al ...... continued on the next page Leavitt Flook Leavitt Mariño-Pérez and Song (2019) Mariño-Pérez and Song (2019) Mariño-Pérez and Song (2019) Mariño-Pérez and Song (2019) Mariño-Pérez and Song (2019) Mariño-Pérez and Song (2019) Mariño-Pérez and Song (2019) Mariño-Pérez and Song (2019) Mariño-Pérez and Song (2019) Ding Mariño-Pérez and Song (2019) Mariño-Pérez and Song (2019) Mariño-Pérez and Song (2019) Song Mariño-Pérez and Song (2019) Mariño-Pérez and Song (2019) Mariño-Pérez and Song (2019) Song Mariño-Pérez and Song (2019) R eference number NC_020774 NC_001712 JX913764 MK531145-MK531153 MK514099 MK531214-MK531233 MK514096 MK531140-MK531144 MK514098 MK514097 MK531234-MK531254 MK514100 NC011824 MK514108 MK531154-MK531165 MK514106 MT011475, MT011522, MT011568, MT011702, MT011744 MK514105 MK514109 MK514195 MT011429, MT011474, MT011521, MT011567, MT011614, MT011701, MT011743, MT011791 MK514103 G enbank Accession Mariño-Pérez and Song (2018) G roup Clade A Clade A Clade A Clade A Clade A Clade A Clade A Clade A Clade A Clade D Clade D Unplaced Clade B Clade B Clade B Clade D Clade D Clade D Clade B Kevan’s section A-III A-III A-III A-III A-III A-III A-III A-III A-I B-VIII B-X B-V B-VII B-VII B-V B-IX B-IX B-IX B-VI sp. sp. Species Prionotropis hystrix Ichthiacris rehni Sphenacris crassicornis Ichthyotettix mexicanus Piscacris robertsi Pyrgotettix pueblensis Sphenotettix nobilis Caprorhinus sphenarioides Psedna nana Atractomorpha sinensis hemipterus Desmoptera Dictyophorous spumans Parapetasia femorata Monistria discrepans Algete brunneri Jaragua oviedensis Omura congrua morbillosus L entula callani L ocusta migratoria Tribe Ichthiacridini Ichthiacridini Ichthyotettigini Ichthyotettigini Ichthyotettigini Ichthyotettigini Orthacridini Popoviini Psednurini Atractomorphini Chrotogonini Desmopterini Dictyophorini Dictyophorini Monistriini Omurini Omurini Omurini Phymateini Subfamily Orthacridinae Orthacridinae Orthacridinae Orthacridinae Orthacridinae Orthacridinae Orthacridinae Orthacridinae Orthacridinae Pyrgomorphinae Pyrgomorphinae Pyrgomorphinae Pyrgomorphinae Pyrgomorphinae Pyrgomorphinae Pyrgomorphinae Pyrgomorphinae Pyrgomorphinae Pyrgomorphinae Family Lentulidae Acrididae Pamphagidae Pyrgomorphidae Pyrgomorphidae Pyrgomorphidae Pyrgomorphidae Pyrgomorphidae Pyrgomorphidae Pyrgomorphidae Pyrgomorphidae Pyrgomorphidae Pyrgomorphidae Pyrgomorphidae Pyrgomorphidae Pyrgomorphidae Pyrgomorphidae Pyrgomorphidae Pyrgomorphidae Pyrgomorphidae Pyrgomorphidae Pyrgomorphidae E 1. TABL

104 · Zootaxa 4969 (1) © 2021 Magnolia Press Zahid et al. . (unpublished); . (1999); Ruiz- . (2020) . (2020) . (2020) . (2010) . (2007) et al et al et al et al et al et al et al ...... continued on the next page Song Song Mariño-Pérez and Song (2019) Flook Ruano Chapco and Contreras (2011); Fries Song Mariño-Pérez and Song (2019) Zhao Mariño-Pérez and Song (2019) This study Mariño-Pérez and Song (2019) R eference number MT011449, MT011495, MT011541, MT011589, MT011634, MT011675, MT011723, MT011764, MT011809, MT011853, MT011899, MT011979 MT011428, MT011473, MT011520, MT011566, MT011613, MT011657, MT011700, MT011742, MT011790, MT011832, MT011878, MT011960 MK514104 Z97616, Z97600, KM384875, KM384853, JF932467, EU031779, EU031778, EU031777, EU031776 MT011446, MT011492, MT011586, MT011631, MT011720, MT011761, MT011850, MT011896 MK514110 NC014450 MK514101 MW881859-MW881873 MK514107 G enbank Accession Mariño-Pérez and Song (2018) G roup Clade B Clade B Clade D Clade D Clade D Clade D Clade C Clade C Clade C Clade C Kevan’s section B-VI B-VI B-X B-X B-X B-X B-IX B-IX B-IX B-IX sp. sp. sp. Species pictus Ochrophlegma conica Stenoscepa Tanita Mekongiana xiangchengensis Prosphena scudderi Rubellia nigrosignata Tribe Phymateini Poekilocerini Pyrgomorphini Pyrgomorphini Pyrgomorphini Pyrgomorphini Sphenariini Sphenariini Sphenariini Sphenariini Subfamily Pyrgomorphinae Pyrgomorphinae Pyrgomorphinae Pyrgomorphinae Pyrgomorphinae Pyrgomorphinae Pyrgomorphinae Pyrgomorphinae Pyrgomorphinae Pyrgomorphinae TABL E 1. (Continued) Family Pyrgomorphidae Pyrgomorphidae Pyrgomorphidae Pyrgomorphidae Pyrgomorphidae Pyrgomorphidae Pyrgomorphidae Pyrgomorphidae Pyrgomorphidae Pyrgomorphidae

Molecular phylogeny of Pyrgomorphidae Zootaxa 4969 (1) © 2021 Magnolia Press · 105 . (2007); et al . (2020) et al . (2011); Huo Lv and Huang (2012); Zhang et al Cui and Huang (unpublished); Bai and Huang (unpublished) Mariño-Pérez and Song (2019) Song R eference number JQ301463, JQ283277, GQ421456, DQ365908, JQ065110 MK080200 MT011442, MT011487, MT011535, MT011581, MT011627, MT011669, MT011715, MT011756, MT011803, MT011845, MT011891, MT011933, MT011973 G enbank Accession Mariño-Pérez and Song (2018) G roup Clade C Clade D Clade B Kevan’s section B-IX B-VIII B-VII Species Yunnanites coriacea indica miliaris Tribe Sphenariini Tagastini Taphronotini Subfamily Pyrgomorphinae Pyrgomorphinae Pyrgomorphinae TABL E 1. (Continued) Family Pyrgomorphidae Pyrgomorphidae Pyrgomorphidae

106 · Zootaxa 4969 (1) © 2021 Magnolia Press Zahid et al. For the Bayesian analysis, we subjected the same partitioned dataset and used MrBayes v3.2.6 x64 (Ronquist et al., 2012) also on the CIPRES. We used default priors and applied a different model for each partition, as recommended by PartitionFinder2, and ran four runs with four chains each for 100 million generations, sampling every 1,000 generations. We plotted the likelihood trace for each run to assess convergence in Tracer (Rambaut & Drummond, 2003-2009), and discarded 25% of each run as burn-in. For both ML and Bayesian analyses, the resulting trees were visualized in FigTree (Rambaut, 2006-2009).

Character evolution To trace the evolution of morphological traits that have been used to distinguish between the two subfamilies within Pyrgomorphidae, we performed ancestral character state reconstruction analyses. Specifically, we coded three morphological characters that Kevan and Akbar (1964) emphasized. There were: (i) the shape of metasternal pits; (ii) overall body form; and (iii) the shape of the basal lobes of hind femur. For the shape of metasternal pits, we coded whether these structures were small and joined by two sutures or large and connected by one suture. For the body form, we coded whether it was robust, cylindrical or elongated, dorsoventrally depressed, laterally compressed, or fusiform. For the shape of the basal lobes of hind femur, we coded whether the upper lobe was shorter, as long as, or longer than the lower lobe. We performed ancestral character state reconstruction in a maximum likelihood framework using the ML tree. We fitted a continuous-time Markov chain (Mk) single-rate (ER) model to our data to infer character evolution using the R package phytools (Revell, 2012). The resulting graphs were exported as EPS files and modified in Adobe Illustrator 2019. In order to visualize whether male phallic structures of the closely related species are similar to each other, we redrew genitalia illustrations from Kevan’s publications (Kevan et al., 1969, 1970, 1971, 1972, 1974; Kevan et al., 1975) and mapped on to our ML tree. The illustrations were taken from the PDF version of the publications, and digitally manipulated in Adobe Photoshop CC2019.

Results

Phylogeny of Pyrgomorphidae Both ML analysis and Bayesian analysis resulted in identical topologies (Fig. 2). Nodal support values were generally high across the phylogeny. Our study recovered Pyrgomorphidae as monophyletic, but neither of the two subfamilies (or groups ‘A’ and ‘B’) was recovered as monophyletic. The subfamily Orthacridinae resulted in three lineages scattered across the phylogeny: (i) a single lineage including an Australian endemic genus Psedna; (ii) a clade consisting of the Old World group Caprorhinus and Colemania; and (iii) a clade consisting of the Mexican endemic genera, Sphenacris, Ichthiacris, Pyrgotettix, Sphenotettix, Ichthyotettix, and Piscacris. Considering Kevan’s series, we included multiple members of the Series III, V, VI, VII, VIII, IX, and X, and none of these series was recovered as monophyletic. Of the tribes for which we included multiple taxa, we recovered Dictyophorini, Omurini sensu Mariño-Pérez and Song (2019), and Pyrgomorphini as monophyletic. Sphenariini, Ichthiacridini, Ichthyotettigini, and Phymateini were found to be paraphyletic. When we compared our tree with the morphological phylogeny by Mariño-Pérez and Song (2018), we found a large number of discordances between the two in terms topology, and none of their four clades was recovered as monophyletic (Fig. 3).

Ancestral character state reconstruction We found that the three morphological characters that Kevan found useful for separating subfamilies all evolved multiple times across the phylogeny of Pyrgomorphidae (Fig. 4). In terms of the shape of the metasternal pits (Fig. 4A), the ancestral condition for the family was inferred to be the condition joined by two sutures, and the condition connected by one suture independently evolved three times through the phylogeny. In terms of the body form (Fig. 4B), it is not clear what the ancestral shape for the common ancestor of Pyrgomorphidae, but the analyses inferred that the most likely ancestral body form for most pyrgomorphs would be the fusiform body, from which robust or cylindrical body formed were derived independently multiple times. In terms of the shape of the basal lobes of hind femur (Fig. 4C), the ancestral condition for the family was the condition in which the upper lobe was shorter than the lower lobe. The variation of the length of the upper lobe evolved multiple times over the phylogeny.

Molecular phylogeny of Pyrgomorphidae Zootaxa 4969 (1) © 2021 Magnolia Press · 107 Figure 2. Molecular phylogeny of Pyrgomorphidae based on mitochondrial genome data. Branches are color-coded according to the currently recognized subfamily. Letters in bracket next to each terminal corresponds to Kevan’s Group and Series. Bootstrap support values from the maximum likelihood tree and posterior probability values from the Bayesian analyses are shown.

108 · Zootaxa 4969 (1) © 2021 Magnolia Press Zahid et al. Figure 3. Comparison between morphological phylogeny by Mariño-Pérez & Song (2018) (left) and our molecular phylogeny (right).

Figure 4. Ancestral character state reconstruction of three morphological traits that were considered important, based on the molecular phylogeny. A. The shape of metasternal pits; B. Body form; C. The shape of basal lobe of hind femur.

Molecular phylogeny of Pyrgomorphidae Zootaxa 4969 (1) © 2021 Magnolia Press · 109 Male genitalia evolution When we mapped the male phallic structures on the resulting molecular phylogeny, the following qualitative patterns emerged (Fig. 5). The shape of cingulum and aedeagus was extremely variable across the phylogeny and no two genera had similar shapes. For epiphallus, phylogenetically divergent lineages often had a similar overall shape. The degree of morphological disparity among closely related species varied. For example, the phallic structures of the members of Omurini were superficially similar to each other, while those of Pyrgomorphini were very divergent from each other.

Discussion

Phylogeny of Pyrgomorphidae Our study proposes novel relationships among major lineages within Pyrgomorphidae, which show a number of conflicts with previous studies based on morphology. Because we have included only a small fraction of the known genus diversity in the family, we limit our comments on the most obvious conflicts that deserve some elaborations. Our study found the Australian endemic genus Psedna as the earliest diverging lineage and sister to the remaining Pyrgomorphidae. This grass-mimicking genus (Fig. 1A) belongs to the tribe Psednurini (Key, 1972), which Kevan et al. (1970) placed into the Series I, which they called “a miscellaneous assemblage of anomalous tribes, occurring in the Indo-Malayan and South Pacific regions.” Kevan et al. (1970) considered Psednurini as an aberrant and derived group within the family, and the morphological phylogeny by Mariño-Pérez and Song (2018) placed this genus deep within the phylogeny of Pyrgomorphidae as a member of Orthacridinae (their clade A). However, our study suggests that the tribe likely represents the ancestral and relic lineage within the family. The basal position of Psedna was previously recovered in Mariño-Pérez and Song (2019), which used mitochondrial genome data, as well as Song et al. (2020), which used transcriptome data. It remains to be seen whether other tribes that Kevan et al. (1970) placed into the Series I, on the basis of the shape of mouthparts, in which the galea of maxillae turned forward over the labrum (which they considered merely an adaptation of graminivorous feeding and of little phylogenetic significance), would be closely related to the Psednurini or whether this tribe will remain to be the sole basal lineage of the family. Another early diverging lineage that we recovered is the genus Chrotogonus. This belongs to the tribe Chrotogonini, a group of geophilous grasshoppers characterized by dorso-ventrally depressed body and collar- like prosternal process (Kevan, 1968c; Kevan et al., 1975). Kevan et al. (1975) were convinced of the affinity of Chrotogonini to Pyrgomorphini because they considered the phallic structures of these two tribes to be quite similar, and placed both in the Series X. The morphological phylogeny by Mariño-Pérez and Song (2018) also found Chrotogonini to be most closely related to the members of Pyrgomorphini, largely based on male genital characters. However, our study shows the basal placement of Chrotogonus, which is quite divergent from the Pyrgomorphini for which we included four taxa. Our finding demonstrates that the male phallic structures, which are mostly influenced by sexual selection (Arnqvist, 1997; Eberhard, 1985; Hosken & Stockley, 2004), can converge on similar morphology across phylogenetically divergent lineages, enough to confuse seasoned taxonomists. Kevan et al. (1974) placed the tribes Sphenariini and Omurini into the Series IX on the basis of the shape of cingulum, specifically based on strong inflections of the suprarami found in all genera included in this series. The tribe Sphenariini includes externally similar-looking grasshoppers with a strongly fusiform body form, but geographically disjunct groups occurring in Central America, China, East Africa, and Madagascar (Kevan et al., 1974; Mariño-Pérez & Song, 2019). On the other hand, Omurini includes only the South American lineages. Perez- Gelabert et al. (1995) described a new genus of Pyrgomorphidae from Dominican Republic (Jaragua), and placed it in Sphenariini. The morphological phylogeny by Mariño-Pérez & Song (2018) included four members of Sphenariini (Rubellia, Mekongiella, Prosphena, and Sphenarium) and one member of Omurini (Omura), and found Kevan’s Series IX to be paraphyletic because these two tribes did not group together. However, they did recover Sphenariini as monophyletic on the basis of triangular cerci and rectangular epiphallus. The molecular phylogeny by Mariño- Pérez & Song (2019) included six members of Sphenariini (Sphenarium, Prosphena, Mekongiana, Yunnanites, Mekongiella, and Jaragua) and one member of Omurini (Algete), and found Sphenariini to be paraphyletic. They found a monophyletic group consisting of the Central American genera Sphenarium and Prosphena, and another

110 · Zootaxa 4969 (1) © 2021 Magnolia Press Zahid et al. Figure 5a. Male genitalia mapped on to the molecular phylogeny. The figures were taken from Kevan’s works. Shown are the dorsal and lateral views of ecto and endophallus (left and center), and epiphallus (right). Scale is 1 mm, unless noted otherwise.

Molecular phylogeny of Pyrgomorphidae Zootaxa 4969 (1) © 2021 Magnolia Press · 111 Figure 5b. Male genitalia mapped on to the molecular phylogeny. The figures were taken from Kevan’s works. Shown are the dorsal and lateral views of ecto and endophallus (left and center), and epiphallus (right). Scale is 1 mm, unless noted otherwise.

112 · Zootaxa 4969 (1) © 2021 Magnolia Press Zahid et al. Figure 5c. Male genitalia mapped on to the molecular phylogeny. The figures were taken from Kevan’s works. Shown are the dorsal and lateral views of ecto and endophallus (left and center), and epiphallus (right). Scale is 1 mm, unless noted otherwise.

Molecular phylogeny of Pyrgomorphidae Zootaxa 4969 (1) © 2021 Magnolia Press · 113 monophyletic group consisting of the Chinese genera Mekongiana and Yunnanites, but these two clades were not grouped together. The phylogenetic position of the genus Mekongiella was weakly supported and unclear. They also found Algete and Jaragua to form a clade, and suggested that Jaragua should be placed in Omurini instead and Sphenariini. In the present study, we included five members of Sphenariini (Sphenarium, Prosphena, Mekongiana, Yunnanites, and Rubellia) representing those lineages from the Central America, China, and Madagascar. We also included three members of Omurini (Omura, Algete, and Jaragua). We found Omurini to be monophyletic, but found Sphenariini to be paraphyletic, scattered across the phylogeny. Specifically, Mekongiana and Yunnanites formed a clade and was closely related to Tagasta, Rubellia was closely related to Phymateus, and Sphenarium and Prosphena formed a clade and was sister to Pyrgomorphini. Our result strongly demonstrates that Kevan’s concept of Series IX was due to convergent traits, including the male genitalia characters.

Morphological convergence leads to rampant paraphyly Across various groups, modern phylogenetic studies have revealed time after time that traditional taxonomic groupings, such as family, subfamily, and tribe, are often recovered to be paraphyletic. In fact, this is a norm rather than an exception because the formalization of many classification schemes across insects either predated the advance of phylogenetic theories, or were established without much consideration of cladistic thinking. For some insect groups, however, recent molecular phylogenetic studies have revealed such a rampant degree of paraphyly across taxonomic levels that a major revision of the classification is inevitable. Specifically, familiar groups within Polyneoptera, such as Mantodea (Svenson & Whiting, 2004), Phasmatodea (Bradler et al., 2014), as well as some large families within Orthoptera, including (Mugleston et al., 2018), Gryllidae (Chintauan- Marquier et al., 2016), and Acrididae (Song et al., 2018), have been found to include a high number of traditional taxonomic groupings that are found to be paraphyletic. The common feature of these insect groups is that they are globally distributed, occupy similar environments in different continents, and convergently evolved similar external morphological traits across phylogenetically divergent lineages. Some of these convergent morphological traits are so convincing that taxonomists used them to formalize tribes, subfamilies, and even families. The use of these morphological characters for cladistic analyses without prior knowledge about the extent of convergence in these characters would potentially result in groupings united by homoplasies, and subsequently incorrect phylogenetic inferences. The present study clearly demonstrates that the current taxonomic classification of Pyrgomorphidae, one of the most colorful orthopteran families in the world, is severely affected by the extensive use of convergent morphological characters in defining taxonomic groupings. Kevan’s informal groups ‘A’ and ‘B’, which Otte (1994) elevated as the subfamilies Orthacridinae and Pyrgomorphinae, are both paraphyletic based on our molecular dataset. The external morphological characters that Kevan and colleagues used to define these two groups are convergent according to our ancestral character state reconstruction analyses (Fig. 4). It is interesting that Kevan & Akbar (1964) actually acknowledged the high degree of morphological convergence. They stated “due to the very considerable degree of convergence, parallelism and individuality, it is difficult to arrange the various tribes, subtribes, and genera in a really appropriate sequence.” It is actually well-known that ecomorph convergence is widespread across grasshoppers (Uvarov, 1977). For example, those species that are highly adapted to feed on grasses have independently evolved elongated bodies that mimic grasses. Those species that have ground-dwelling habits in a desert condition have similarly evolved dorsoventrally compressed body forms. Kevan and colleagues were certainly aware of this tendency of grasshoppers to convergently evolve similar body forms, and thus, they put a more emphasis on male phallic structures in inferring the phylogeny. One of the assumptions that Kevan and colleagues must have operated under was probably the idea that the natural selection that leads to ecomorph convergence of external characters would have little impact on the male genitalia because these are internal characters. In other words, they believed that the examination of male genitalia would lead to correct inference about the phylogenetic relationships. Our mapping of male phallic structures onto the phylogeny (Fig. 5) shows that similar genitalia morphology is found in phylogenetically divergent taxa. There are two possibilities for explaining this pattern. The first possibility is that similar male genital morphology can in fact evolve multiple times convergently. For example, the epiphalli of Chrotogonus and Pyrgomorpha are very similar despite their phylogenetic distances and a large amount differences in external morphology, and the only way to explain this pattern is convergent evolution. The second possibility is that male genitalia characters are actually morphologically conserved across the phylogeny, but sexual selection has resulted in exaggeration of

114 · Zootaxa 4969 (1) © 2021 Magnolia Press Zahid et al. genital morphology in certain lineages, but not all. This idea is difficult to test without a more extensive survey of genital morphology across the phylogeny. What is clear is that different genital components seem to follow different evolutionary trajectories because they are functionally different from each other. For example, the function of epiphallus is distinctly different from the function of cingulum (Woller & Song, 2017), and therefore, it is possible that two divergent taxa can have similar epiphalli, but very different cingulums, and vice versa. Our study clearly demonstrates that the notion of male genitalia containing “true” phylogenetic signal, as supposed by Kevan and colleagues, is flawed.

Towards the natural classification of Pyrgomorphidae In this study, we have highlighted the problems with the current and classification scheme for Pyrgomorphidae, which resulted from heavy reliance of morphological characters that appear to be prone to convergent evolution. Most of Kevan’s taxonomic groupings (Kevan & Akbar, 1964; Kevan et al., 1969, 1970, 1971, 1972, 1974; Kevan et al., 1975) have turned out to be paraphyletic in light of our molecular phylogeny (Fig. 4). However, this is not to say that Kevan’s works lack merits. In fact, his work was very detailed and thoughtful, and provides an excellent roadmap for the taxonomy of the family. The discrepancy between Kevan’s scheme and our molecular phylogeny simply indicates that Pyrgomorphidae is an ancient lineage and a particularly difficult group to classify based on the sole reliance on morphology. In cladistics, homoplasies are not necessarily bad, because certain characters can be globally homoplasious but locally synapomorphic (Wenzel & Siddall, 1999). For example, some of the morphological characters frequently used in Pyrgomorphidae taxonomy, such as the body form or the shape of epiphallus, have evolved multiple times across the phylogeny, but may be useful for uniting a particular group of genera. Our ancestral character state reconstruction analyses show that some of these convergent characters are indeed useful for grouping Ichthyotettigini and Ichthiacridini in Mexico (Fig. 4). In order to identify taxonomically useful and phylogenetically informative morphological traits, we think that we need to first build a robust phylogeny of the family based on extensive taxon sampling and a large amount of molecular data. Such a phylogeny will help us discern particular traits to be used for defining taxonomic groups that reflect the phylogeny, which will ultimately lead to the establishment of the natural classification of Pyrgomorphidae.

Acknowledgements

This work was supported by the International Research Support Initiative Program (IRSIP) of the Higher Education Commission of Pakistan to SZ; the Orthoptera Species File Grant ‘Enhancing digital content for Pyrgomorphidae (Orthoptera: Caelifera) in the Orthoptera Species File’ to HS and RMP; the Entomological Society of America, Systematics Evolution and Biodiversity Section (SysEB) Travel Award (2013) to RMP; Orthopterists’ Society Ted Cohn Research Fund (2014) to RMP; CONACYT (National Council of Science and Technology) scholarship #409158 to RMP; and the United States Department of Agriculture (Hatch Grant TEX0-1-6584) to HS.

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