Molecular Phylogeny of the Grassland Leafhopper Tribe Hecalini (Hemiptera: Cicadellidae: Deltocephalinae)

Molecular Phylogeny of the Grassland Leafhopper Tribe Hecalini (Hemiptera: Cicadellidae:... Abstract The phylogenetic status and relationships of the globally distributed grassland leafhopper tribe Hecalini were explored by analyzing a DNA sequence dataset comprising three gene regions (nuclear 28S rDNA, histone H3, 12S mtDNA) for 14 genera and 29 species plus 9 outgroups. Bayesian, maximum likelihood, and maximum parsimony analyses recovered similar trees and supported the monophyly of Hecalini and its two included subtribes, but excluded the Oriental genus Hecalusina He, Zhang & Webb, the position of which was poorly resolved among the included outgroup taxa. Within subtribe Hecalina, the analysis recovered two major lineages, one including only New World taxa and the other including a second New World clade nested within a larger lineage comprising taxa from the Afrotropical, Oriental, and Australian regions. The large, widely distributed genus Hecalus was not recovered as monophyletic. Species of this genus grouped into two separate, noncontiguous clades, one North American and the other comprising African, Australian, and Oriental species. The deltocephaline leafhopper tribe Hecalini (sensu Zahniser and Dietrich 2013, including Dorycephalini sensu Hamilton 2000 in part) is a diverse group of plant-sap-sucking insects that appear to feed exclusively on grasses and are common in temperate and tropical grasslands worldwide (Linnavuori 1957, 1975; Evans 1966; Hamilton 2000; Dietrich 2017). Hecalini are medium-sized to large-sized leafhoppers, all with some degree of dorsoventral flattening, but a few with the body greatly elongated, possibly mimicking the seeds of their host grasses (Fig. 1). The extensive morphological variation exhibited by species of Hecalini, particularly in head structure, previously resulted in members of the group being included in different tribes or subfamilies (reviewed by Hamilton 2000, Zahniser and Dietrich 2013). According to the current morphology-based concept of the tribe, supported at least in part by a previous molecular phylogenetic study of Deltocephalinae overall (Zahniser and Dietrich 2013), Hecalini differ from other deltocephaline leafhoppers in having the ocelli closer to the eyes than the laterofrontal sutures. The head is produced and often flattened, sometimes to the point of having the crown concave in lateral view, giving rise to the common names ‘spoon-bill leafhoppers’ and ‘shovel-headed leafhoppers’ (Hamilton 2000). This feature, in addition to cryptic coloration that is green, yellow, or brown, occasionally with red or orange linear markings, makes these insects well camouflaged when resting on the stems or inflorescences of their grass hosts. This group contains monophagous and polyphagous taxa, although all known hosts are grasses (Poaceae). Although most species appear to have relatively narrow host associations, a few widespread taxa are found on grasses that are distantly related (Hamilton 2000). Sexual dimorphism is common in Hecalini, particularly in size (females being significantly larger) but also in head shape (males often have the head less produced and flattened) and in the degree of wing development (females are often submacropterous or brachypterous). Fig. 1. View largeDownload slide Representatives of Hecalini. (A) Glossocratus afzelii (Zambia); (B) Attenuipyga delongi (USA: Texas); (C) New genus A (China); (D) Hecalus sp. (USA: Montana); (E) Hecalus pallescens (Australia); (F) Linnavuoriana arcuata (Australia); (G) Memnonia flavida (USA: Illinois); (H) Neohecalus magnificus (USA: Illinois); (I) Otamendiella linnavuorii (Argentina); (J) Spangbergiella felix (Argentina). Photographs: (A) J. N. Zahniser (used with permission); (B–J) C. H. Dietrich. Fig. 1. View largeDownload slide Representatives of Hecalini. (A) Glossocratus afzelii (Zambia); (B) Attenuipyga delongi (USA: Texas); (C) New genus A (China); (D) Hecalus sp. (USA: Montana); (E) Hecalus pallescens (Australia); (F) Linnavuoriana arcuata (Australia); (G) Memnonia flavida (USA: Illinois); (H) Neohecalus magnificus (USA: Illinois); (I) Otamendiella linnavuorii (Argentina); (J) Spangbergiella felix (Argentina). Photographs: (A) J. N. Zahniser (used with permission); (B–J) C. H. Dietrich. Currently, the tribe includes 24 genera and ca. 180 species divided between two subtribes: Glossocratina and Hecalina (Zahniser and Dietrich 2013, Dietrich 2017). Glossocratina includes only the genus Glossocratus Fieber, 1866, which is recognized by the keeled laterofrontal sutures and serrate second valvula (Zahniser and Dietrich 2013). This subtribe has an Old World distribution, excluding Australia. The other subtribe, Hecalina, now contains the remaining members of the tribe and is distributed worldwide. Hecalina are recognized by the unkeeled laterofrontal suture, ovipositor extending beyond the pygofer apex, and second valvula without dorsal teeth. Although most Hecalini genera are fairly restricted in distribution (often from only a single biogeographic region), the type genus, Hecalus Stål, 1864 is distributed worldwide and the predominantly North American genus Memnonia Ball, 1900 includes one species recorded from East Asia (Kwon and Lee 1979). Hecalus, as currently defined, is particularly diverse and widespread with over 50 valid species and occurs in all biogeographic regions, although it is sparsely represented in Australia and South America. Hamilton (2000) suggested that only the Holarctic species of this genus should be ‘definitely included’, reinstating two Old World genera, Linnavuoriella Evans, 1966 and Thomsoniella Signoret, 1880, that had been treated as synonyms of Hecalus by Morrison (1973). Although several morphology-based taxonomic treatments of Hecalini have been published (e.g., Linnavuori 1957, 1975; Evans 1966; Morrison 1973; Hamilton 2000; Dietrich 2017), these have focused exclusively on regional faunas and no attempt has been made to revise the classification of the group comprehensively or elucidate their phylogenetic relationships. The morphological and molecular phylogenies of Zahniser and Dietrich (2008, 2010, 2013) examined relationships among Deltocephalinae more broadly but included only three representatives of Hecalini, which did not consistently group in a single clade. Nevertheless, these placed Hecalus and Attenuipyga Oman, 1949 as sister groups, justifying the transfer of the New World ‘Dorycephalini’ (excluding the Palearctic type genus) to Hecalini. Relationships among other genera of this tribe have never been explored by explicit phylogenetic analysis. Most recognized genera are restricted to particular biogeographic realms and appear to be reasonably well defined morphologically (Hamilton 2000), but some, including the type genus Hecalus, which is distributed worldwide, are less well defined and doubtfully monophyletic (Linnavuori 1975, Hamilton 2000). This article reports results of the first detailed molecular phylogeny of Hecalini. Previous studies have included too few representatives of Hecalini to provide an adequate test of the monophyly of the tribe or to elucidate the phylogenetic relationships and status of included genera. Materials and Methods Specimen Acquisition, Taxon Sampling, and DNA Extraction The taxon sample, summarized in Appendix A (online only), included 35 specimens representing 31 hecaline species in 15 different genera (two undescribed) from all major grassland regions of the world. Several genera included in the tribe are known only from type material or museum specimens collected decades ago (e.g., Alospanbergia Evans, 1973; Annidion Kirkaldy, 1905; Cephalius Fieber, 1875; Clavena Melichar, 1902; Psegmatus Fieber, 1875; Reuteriella Signoret, 1879), and these could not be included. Where possible, multiple species were included for genera that occur in different biogeographic regions or for which the monophyly of the genus was questionable based on morphological criteria. Because the monophyly of Hecalini was not consistently recovered in the analyses of Zahniser and Dietrich (2013), eight taxa from six other grass-specialist Deltocephalinae tribes were included as outgroups to facilitate further testing of the monophyly of the tribe. These taxa were selected based on Zahniser and Dietrich (2013), which placed Hecalini confidently in a larger clade of grass-specialist Deltocephalinae but with uncertain position within that clade. In most cases, DNA was extracted from recently collected specimens preserved in 95% ethanol and stored at −20°C prior to extraction. In a few instances, the ethanol-preserved material was not available, so pinned museum specimens were used for DNA extraction (Appendix A [online only]). DNA was extracted from each specimen using a DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA) following a modified version of the manufacturer’s protocol ‘Total DNA from Animal Tissues’ that included lengthening the incubation period in step 2 to 36 h and decreasing the amount of Buffer AE in step 7 to 50 µl, which was repeated using a different 1.5-ml collection tube, rather than a single tube. Because Hecalini are relatively large leafhoppers, abdomens were removed for DNA extraction, whereas the rest of the insect was dry-mounted as a voucher specimen. Abdomens were punctured with two to four small holes to ensure that the extraction buffer permeated the specimen. After extraction, cleared specimens were placed in microvials with glycerin and stored with the point mounted thorax and head as voucher specimens. These specimens are deposited in the Illinois Natural History Survey Insect Collection. In general, fresh specimens yielded better-quality DNA, but sequences from pinned specimens were of high enough quality to be included in this study. PCR and DNA Sequencing A pilot study of eight genes (12S, 16S, histone H3, 28S, COI, COII, wingless, and arginine kinase) was first performed to select genes that amplified readily across the tribe and included characters informative of hecaline relationships. Following initial screening, three gene regions were amplified and sequenced for all taxa: 12S mtRNA (401 bp), 28S nRNA (2716 bp), and nuclear histone H3 (351 bp). All PCRs were 25 µL and used Taq polymerase (Promega, Madison, WI; see Appendices B and C [online only] for primers and reaction conditions). Products were submitted for high-throughput sequencing to the Roy G. Carver Biotechnology Center of the University of Illinois. Sequencher 4.8 was used to automatically assemble contigs (minimum match = 60; minimum overlap = 20), and each contig for a given gene was assembled into a single alignment and exported as FASTA file. FASTA files were aligned in SeaView 4.3.0 (Gouy et al. 2010) using the built-in version of MUSCLE (Edgar 2004) with all parameters set at default, except in the case of 12S that required a higher gap opening penalty in some regions where the original alignment resulted in high numbers of extraneous gaps. Resulting alignments were then adjusted by eye. DNA sequences are deposited in GenBank (accession numbers are provided in Appendix A [online only]). Phylogenetic Analysis ModelGenerator (nset=6) was used to select the best-fitting evolutionary model for each gene based on Akaike information criterion (AIC; Keane et al. 2006). Individual gene trees were initially inferred using BEAST (Drummond and Rambaut 2007), with Bayesian Markov-chain Monte Carlo runs of 20 million generations (with 25% burn-in) and default priors (Drummond and Rambaut 2007). Gene trees were then compared, and if no well-supported conflict was present, the data were combined for subsequent analysis with individual genes treated as separate data partitions, each with its own model/parameters in maximum likelihood and Bayesian analyses. Sequences were trimmed to exclude primer regions, but no other regions were removed, and gaps were treated as missing data. Phylogenies were inferred using parsimony (PAUP* 4.0b10; Swofford 2003) with all characters equally weighted, 10,000 random addition sequence replicates and TBR branch swapping. Maximum likelihood analyses were performed using GARLI (Zwickl 2006) with 10 independent runs, default settings, and an automated stop criterion if lnL score remained constant for 50,000 generations. Bayesian estimation was performed using MrBayes (Ronquist et al. 2012) with four runs, each with four chains, of 20 million generations, and BEAST with 40 million generations, tree prior = speciation: birth–death process. For both methods, the first 25% of generations were discarded as burn-in and log files were viewed in Tracer to ensure convergence was reached (Swofford 2003, Ronquist et al. 2012). Both Bayesian posterior probabilities and parsimony and maximum likelihood bootstrap values (1,000 replicates of 100 random addition sequences) were calculated to estimate branch support. The concatenated alignment and trees resulting from phylogenetic analysis will be available from TreeBASE (submission ID: 21696). Results PCR Amplification and Sequencing Alignment Most taxa were represented by complete sequences of all three genes although a few gene regions were not obtained for a few taxa (see Appendix A [online only]). Initial gene tree analysis revealed no significant gene tree conflict, so data were combined into a supermatrix for subsequent analyses. The final alignment included a total of 3,482 characters, of which 2,823 were constant, 220 were variable but parsimony-uninformative, and 439 were parsimony informative (Appendix A [online only] contains a complete summary by gene). Phylogenetic Analysis GTR + I + G was chosen as the best-fitting model for both histone H3 and 28S. For 12S, AIC slightly favored K81uf + G over HKY + G (a difference in AIC scores of less than 0.4, whereas the third most favored model was about 2.0 points worse). HKY + G was selected over K81uf + G by the other criteria ModelGenerator uses to rank models but the latter model cannot be implemented in MrBayes or BEAST so HKY + G was used. Tree topologies were largely consistent regardless of analytical technique although minor differences, particularly toward the tips of major branches, were found. In all cases, nodes that were incongruent among analyses received low (<50%) bootstrap support. Thus, only the consensus tree resulting from the partitioned Bayesian analysis is shown in Fig. 2. The monophyly of Hecalini (including Attenuipyga but excluding Hecalusina He, Zhang & Webb) was consistently recovered, although with equivocal branch support. Glossocratus (and therefore Glossocratina) was sister to the Hecalina and the monophyly of both subtribes received strong branch support. Fig. 2. View largeDownload slide Molecular phylogeny of Hecalini and outgroups from the partitioned Bayesian analysis of the concatenated DNA sequence dataset (12S, H3, and 28S) analyzed using BEAST. Numbers above branches are Bayesian posterior probabilities, whereas numbers below the branches are bootstrap values (parsimony/likelihood). Missing values indicate <0.80 posterior probability or <50% bootstrap support. Fig. 2. View largeDownload slide Molecular phylogeny of Hecalini and outgroups from the partitioned Bayesian analysis of the concatenated DNA sequence dataset (12S, H3, and 28S) analyzed using BEAST. Numbers above branches are Bayesian posterior probabilities, whereas numbers below the branches are bootstrap values (parsimony/likelihood). Missing values indicate <0.80 posterior probability or <50% bootstrap support. Within Hecalina there are two main clades, one containing only New World genera and the other globally distributed, including New and Old World taxa. Although four of the five genera for which more than one species was included were found to be monophyletic, Hecalus, the largest and most widely distributed genus, was polyphyletic, forming two distinct geographically restricted clades, one North American and the other including African, Asian, and Australian species. Attenuipyga, formerly placed in Dorycephalini but recently transferred to Hecalini (Zahniser and Dietrich 2013), was embedded within the Hecalina, and sister to a clade containing five strictly New World genera (Spangbergiella Signoret, 1879; Neohecalus Linnavuori, 1975; Dicyphonia Ball, 1990; Jiutepeca Linnavuori and DeLong 1978; and Otamendiella Dietrich, 2017). Dorycephalini (sensu stricto, including only the type genus), was firmly placed within the outgroup. One of the undescribed genera from Asia was sister to the clade of New World Hecalus + Memnonia Ball, whereas the other was sister to the clade comprising Old World genera Thomsoniella + Linnavuoriella. Discussion Taxonomic Implications Phylogenetic analysis of three gene regions yielded a well-resolved phylogeny (Fig. 2) that consistently recovered Hecalini as monophyletic and supported the status of subtribes Glossocratina and Hecalina as monophyletic sister groups. Thus, our results are consistent with the current tribal and subtribal classification (Zahniser and Dietrich 2013) with the exception of placement of the Oriental genus Hecalusina. This genus, which has not been previously included in any phylogenetic analysis, was sister to a clade comprising the African genus Drakensbergena Linnavuori (Drakensbergenini) and the Palearctic genus Dorycephalus Kouchakéwitch (Dorycephalini). Morphologically, Hecalusina is similar to Paradorydium Kirkaldy (Eupelicini: Paradorydiina), a genus not included in the present dataset but grouped with Drakensbergenini and Dorycephalini in the maximum likelihood analysis of Zahniser and Dietrich (2013). Both genera have the forewing veins elevated, the male pygofer with a digitiform dorsal lobe, the connective with anterior arms weakly divergent, and the aedeagus long, slender, and recurved, without a distal process. Unfortunately, because we did not include Paradorydium in our dataset and the relationships between Hecalusina and other outgroup taxa received low branch support, we cannot provide phylogenetic confirmation of the correct tribal placement of Hecalusina. Within Hecalini, with the exception of Hecalus, all genera for which more than one representative was included were recovered as monophyletic. Most of these genera have previously been well characterized morphologically, so these results are not surprising. Nevertheless, because we were only able to include a small fraction of the known species, more detailed analyses will be necessary to provide a comprehensive tree for the entire tribe and conduct more robust tests of the phylogenetic status of recognized genera. Because Hecalus was not recovered as monophyletic and the type species of Hecalus, H. paykulli Stål (morphologically similar to the included African species H. aurora Linnavuori), is a member of the Old World Hecalus clade, our results suggest that the taxonomic concept of this genus should be restricted to include only Old World species and that a new genus should be erected to include the New World species of Hecalus. Because Hecalus is a large genus comprising >50 described species and our dataset included only a small fraction of the known species, such a reclassification of the genus would be best undertaken within the context of a more detailed taxonomic revision. Biogeographic Implications Taxa occurring in the same biogeographic realm tended to group together in our phylogenies, suggesting that continental-scale geographic isolation was an important driver in the early diversification of Hecalini. Glossocratina, a strictly Old World lineage was consistently recovered as sister to Hecalina comprising both New and Old World lineages. One of the two large clades of Hecalina, comprising the North American genus Attenuipyga and its sister group (Fig. 2), is restricted to the New World. South American species of this lineage (Otamendiella linnavuorii Dietrich and two species of Spangbergiella, one of which, S. vulnerata (Uhler), is widespread in both North and South America) occupy relatively derived positions within the clade, suggesting that the clade diversified in the warm temperate grasslands of North America before subsequently dispersing into South America. The second major lineage of Hecalina includes both New and Old World species. Within this clade, the New World species of Hecalus and Memnonia form a single lineage derived from within a grade comprising the Old World taxa (Linnavuoriella, Thomsoniella, Parabolocratalis Evans, 1955; Hecalus in part, and two undescribed Asian genera), suggesting that the common ancestor of New World Hecalus and Memnonia dispersed into the New World from Asia. A detailed and explicit biogeographic analysis of this and two additional unrelated lineages of grassland Auchenorrhyncha (deltocephaline leafhopper tribes Paralimnini + Deltocephalini and the fulgoroid family Caliscelidae) was undertaken by Catanach (2013) and will be published separately. Supplementary Data Supplementary data are available at Annals of the Entomological Society of America online. Acknowledgments Fieldwork by the authors that yielded many of the included specimens was facilitated by M. M. Cigliano, M. I. Catalano, P. Lozada, M. M. Yang, H. T. Shih, Y. Zhang, and G. Moya-Raygoza. Additional ethanol-preserved specimens were provided by J. Cryan, K. Hill, M. Irwin, B. Morris, M. Sharkey, M. Stiller, D. Takiya, and J. Zahniser. Sequencing was funded by two H. H. Ross Memorial Awards to T.A.C. from the Illinois Natural History Survey and a France M. and Harlie M. Clark Research Support Grant from the University of Illinois Urbana-Champaign, School of Integrative Biology. This work is modified from Chapter 4 of a doctoral dissertation submitted by T.A.C. to the Graduate College of the University of Illinois (Catanach 2013). References Cited Catanach, T. A. 2013. Biogeography and phylogenetics of grassland Auchenorrhyncha . Ph.D. dissertation, University of Illinois, Urbana, p. 133. http://hdl.handle.net/2142/46689 (accessed 14 October 2017). Dietrich, C. H. 2017. South American leafhoppers of the tribe Hecalini (Hemiptera: Auchenorrhyncha: Cicadellidae: Deltocephaline). Entomol. Am . 122: 398– 404. Drummond, A. J., and Rambaut A.. 2007. BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evol. Biol . 7: 214. Google Scholar CrossRef Search ADS PubMed  Edgar, R. C. 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res . 32: 1792– 1797. Google Scholar CrossRef Search ADS PubMed  Evans, J. W. 1966. The leafhoppers and froghoppers of Australia and New Zealand (Homoptera: Cicadelloidea and Cercopoidea). Mem. Aust. Mus . 12: 1– 347. Google Scholar CrossRef Search ADS   Gouy, M., Guindon S., and Gascuel O.. 2010. SeaView version 4: a multiplatform graphical user interface for sequence alignment and phylogenetic tree building. Mol. Biol. Evol . 27: 221– 224. Google Scholar CrossRef Search ADS PubMed  Hamilton, K. G. A. 2000. Five genera of New-World “shovel-headed” and “spoon-bill” leafhoppers (Hemiptera: Cicadellidae: Dorycephalini and Hecalini). Can. Entomol . 132: 429– 503. Google Scholar CrossRef Search ADS   Keane, T. M., C. J. Creevey, M. M. Pentony, T. J. Naughton, and Mclnerney J. O.. 2006. Assessment of methods for amino acid matrix selection and their use on empirical data shows that ad hoc assumptions for choice of matrix are not justified. BMC Evol. Biol . 6: 29. Google Scholar CrossRef Search ADS PubMed  Kwon, Y. J., and Lee C. E.. 1979. Revision of the tribe Hecalini Distant from Korea. Nat. Life Southeast Asia  9: 41– 48. Linnavuori, R. 1957. The Neotropical Hecalinae (Hom. Cicadellidae). Suom. Hyönteistiet. Aikaka . 23: 133– 143. Linnavuori, R. 1975. Revision of the Cicadellidae (Homoptera) of the Ethiopian region, III. Deltocephalina, Hecalini. Acta Zool. Fenn . 143: 1– 37. Morrison, W. P. 1973. A revision of the Hecalinae (Homoptera: Cicadellidae) of the Oriental region. Pac. Insects  15: 379– 438. Oman, P. W. 1949. The Nearctic leafhoppers: a generic classification and check list. Mem. Entomol. Soc. Wash . 3: 1– 253. Ronquist, F., M. Teslenko, P. van der Mark, D. L. Ayres, A. Darling, S. Höhna, B. Larget, L. Liu, M. A. Suchard, and Huelsenbeck J. P.. 2012. MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst. Biol . 61: 539– 542. Google Scholar CrossRef Search ADS PubMed  Swofford, D. L. 2003. PAUP*. Phylogenetic Analysis Using Parsimony (*and Other Methods), version 4 . Sinauer Associates, Sunderland, MA. Zahniser, J. N., and Dietrich, C. H. 2008. Phylogeny of the leafhopper subfamily Deltocephalinae (Insecta: Auchenorrhyncha: Cicadellidae) and related subfamilies based on morphology. Syst. Biodivers . 6: 1– 24. Google Scholar CrossRef Search ADS   Zahniser, J. N., and Dietrich, C. H. 2010. Phylogeny of the leafhopper subfamily Deltocephalinae (Hemiptera: Cicadellidae) based on molecular and morphological data with a revised family-group classification. Syst. Entomol . 35: 489– 511. Google Scholar CrossRef Search ADS   Zahniser, J. N., and Dietrich, C. H. 2013. A review of the tribes of Deltocephalinae (Hemiptera: Auchenorrhyncha: Cicadellidae). Eur. J. Taxon . 45: 1– 211. Zwickl, D. J. 2006. Genetic algorithm approaches for the phylogenetic analysis of large biological sequence datasets under the maximum likelihood criterion . Ph.D. dissertation, The University of Texas at Austin. © The Author(s) 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of the Entomological Society of America Oxford University Press

Molecular Phylogeny of the Grassland Leafhopper Tribe Hecalini (Hemiptera: Cicadellidae: Deltocephalinae)

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Abstract

Abstract The phylogenetic status and relationships of the globally distributed grassland leafhopper tribe Hecalini were explored by analyzing a DNA sequence dataset comprising three gene regions (nuclear 28S rDNA, histone H3, 12S mtDNA) for 14 genera and 29 species plus 9 outgroups. Bayesian, maximum likelihood, and maximum parsimony analyses recovered similar trees and supported the monophyly of Hecalini and its two included subtribes, but excluded the Oriental genus Hecalusina He, Zhang & Webb, the position of which was poorly resolved among the included outgroup taxa. Within subtribe Hecalina, the analysis recovered two major lineages, one including only New World taxa and the other including a second New World clade nested within a larger lineage comprising taxa from the Afrotropical, Oriental, and Australian regions. The large, widely distributed genus Hecalus was not recovered as monophyletic. Species of this genus grouped into two separate, noncontiguous clades, one North American and the other comprising African, Australian, and Oriental species. The deltocephaline leafhopper tribe Hecalini (sensu Zahniser and Dietrich 2013, including Dorycephalini sensu Hamilton 2000 in part) is a diverse group of plant-sap-sucking insects that appear to feed exclusively on grasses and are common in temperate and tropical grasslands worldwide (Linnavuori 1957, 1975; Evans 1966; Hamilton 2000; Dietrich 2017). Hecalini are medium-sized to large-sized leafhoppers, all with some degree of dorsoventral flattening, but a few with the body greatly elongated, possibly mimicking the seeds of their host grasses (Fig. 1). The extensive morphological variation exhibited by species of Hecalini, particularly in head structure, previously resulted in members of the group being included in different tribes or subfamilies (reviewed by Hamilton 2000, Zahniser and Dietrich 2013). According to the current morphology-based concept of the tribe, supported at least in part by a previous molecular phylogenetic study of Deltocephalinae overall (Zahniser and Dietrich 2013), Hecalini differ from other deltocephaline leafhoppers in having the ocelli closer to the eyes than the laterofrontal sutures. The head is produced and often flattened, sometimes to the point of having the crown concave in lateral view, giving rise to the common names ‘spoon-bill leafhoppers’ and ‘shovel-headed leafhoppers’ (Hamilton 2000). This feature, in addition to cryptic coloration that is green, yellow, or brown, occasionally with red or orange linear markings, makes these insects well camouflaged when resting on the stems or inflorescences of their grass hosts. This group contains monophagous and polyphagous taxa, although all known hosts are grasses (Poaceae). Although most species appear to have relatively narrow host associations, a few widespread taxa are found on grasses that are distantly related (Hamilton 2000). Sexual dimorphism is common in Hecalini, particularly in size (females being significantly larger) but also in head shape (males often have the head less produced and flattened) and in the degree of wing development (females are often submacropterous or brachypterous). Fig. 1. View largeDownload slide Representatives of Hecalini. (A) Glossocratus afzelii (Zambia); (B) Attenuipyga delongi (USA: Texas); (C) New genus A (China); (D) Hecalus sp. (USA: Montana); (E) Hecalus pallescens (Australia); (F) Linnavuoriana arcuata (Australia); (G) Memnonia flavida (USA: Illinois); (H) Neohecalus magnificus (USA: Illinois); (I) Otamendiella linnavuorii (Argentina); (J) Spangbergiella felix (Argentina). Photographs: (A) J. N. Zahniser (used with permission); (B–J) C. H. Dietrich. Fig. 1. View largeDownload slide Representatives of Hecalini. (A) Glossocratus afzelii (Zambia); (B) Attenuipyga delongi (USA: Texas); (C) New genus A (China); (D) Hecalus sp. (USA: Montana); (E) Hecalus pallescens (Australia); (F) Linnavuoriana arcuata (Australia); (G) Memnonia flavida (USA: Illinois); (H) Neohecalus magnificus (USA: Illinois); (I) Otamendiella linnavuorii (Argentina); (J) Spangbergiella felix (Argentina). Photographs: (A) J. N. Zahniser (used with permission); (B–J) C. H. Dietrich. Currently, the tribe includes 24 genera and ca. 180 species divided between two subtribes: Glossocratina and Hecalina (Zahniser and Dietrich 2013, Dietrich 2017). Glossocratina includes only the genus Glossocratus Fieber, 1866, which is recognized by the keeled laterofrontal sutures and serrate second valvula (Zahniser and Dietrich 2013). This subtribe has an Old World distribution, excluding Australia. The other subtribe, Hecalina, now contains the remaining members of the tribe and is distributed worldwide. Hecalina are recognized by the unkeeled laterofrontal suture, ovipositor extending beyond the pygofer apex, and second valvula without dorsal teeth. Although most Hecalini genera are fairly restricted in distribution (often from only a single biogeographic region), the type genus, Hecalus Stål, 1864 is distributed worldwide and the predominantly North American genus Memnonia Ball, 1900 includes one species recorded from East Asia (Kwon and Lee 1979). Hecalus, as currently defined, is particularly diverse and widespread with over 50 valid species and occurs in all biogeographic regions, although it is sparsely represented in Australia and South America. Hamilton (2000) suggested that only the Holarctic species of this genus should be ‘definitely included’, reinstating two Old World genera, Linnavuoriella Evans, 1966 and Thomsoniella Signoret, 1880, that had been treated as synonyms of Hecalus by Morrison (1973). Although several morphology-based taxonomic treatments of Hecalini have been published (e.g., Linnavuori 1957, 1975; Evans 1966; Morrison 1973; Hamilton 2000; Dietrich 2017), these have focused exclusively on regional faunas and no attempt has been made to revise the classification of the group comprehensively or elucidate their phylogenetic relationships. The morphological and molecular phylogenies of Zahniser and Dietrich (2008, 2010, 2013) examined relationships among Deltocephalinae more broadly but included only three representatives of Hecalini, which did not consistently group in a single clade. Nevertheless, these placed Hecalus and Attenuipyga Oman, 1949 as sister groups, justifying the transfer of the New World ‘Dorycephalini’ (excluding the Palearctic type genus) to Hecalini. Relationships among other genera of this tribe have never been explored by explicit phylogenetic analysis. Most recognized genera are restricted to particular biogeographic realms and appear to be reasonably well defined morphologically (Hamilton 2000), but some, including the type genus Hecalus, which is distributed worldwide, are less well defined and doubtfully monophyletic (Linnavuori 1975, Hamilton 2000). This article reports results of the first detailed molecular phylogeny of Hecalini. Previous studies have included too few representatives of Hecalini to provide an adequate test of the monophyly of the tribe or to elucidate the phylogenetic relationships and status of included genera. Materials and Methods Specimen Acquisition, Taxon Sampling, and DNA Extraction The taxon sample, summarized in Appendix A (online only), included 35 specimens representing 31 hecaline species in 15 different genera (two undescribed) from all major grassland regions of the world. Several genera included in the tribe are known only from type material or museum specimens collected decades ago (e.g., Alospanbergia Evans, 1973; Annidion Kirkaldy, 1905; Cephalius Fieber, 1875; Clavena Melichar, 1902; Psegmatus Fieber, 1875; Reuteriella Signoret, 1879), and these could not be included. Where possible, multiple species were included for genera that occur in different biogeographic regions or for which the monophyly of the genus was questionable based on morphological criteria. Because the monophyly of Hecalini was not consistently recovered in the analyses of Zahniser and Dietrich (2013), eight taxa from six other grass-specialist Deltocephalinae tribes were included as outgroups to facilitate further testing of the monophyly of the tribe. These taxa were selected based on Zahniser and Dietrich (2013), which placed Hecalini confidently in a larger clade of grass-specialist Deltocephalinae but with uncertain position within that clade. In most cases, DNA was extracted from recently collected specimens preserved in 95% ethanol and stored at −20°C prior to extraction. In a few instances, the ethanol-preserved material was not available, so pinned museum specimens were used for DNA extraction (Appendix A [online only]). DNA was extracted from each specimen using a DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA) following a modified version of the manufacturer’s protocol ‘Total DNA from Animal Tissues’ that included lengthening the incubation period in step 2 to 36 h and decreasing the amount of Buffer AE in step 7 to 50 µl, which was repeated using a different 1.5-ml collection tube, rather than a single tube. Because Hecalini are relatively large leafhoppers, abdomens were removed for DNA extraction, whereas the rest of the insect was dry-mounted as a voucher specimen. Abdomens were punctured with two to four small holes to ensure that the extraction buffer permeated the specimen. After extraction, cleared specimens were placed in microvials with glycerin and stored with the point mounted thorax and head as voucher specimens. These specimens are deposited in the Illinois Natural History Survey Insect Collection. In general, fresh specimens yielded better-quality DNA, but sequences from pinned specimens were of high enough quality to be included in this study. PCR and DNA Sequencing A pilot study of eight genes (12S, 16S, histone H3, 28S, COI, COII, wingless, and arginine kinase) was first performed to select genes that amplified readily across the tribe and included characters informative of hecaline relationships. Following initial screening, three gene regions were amplified and sequenced for all taxa: 12S mtRNA (401 bp), 28S nRNA (2716 bp), and nuclear histone H3 (351 bp). All PCRs were 25 µL and used Taq polymerase (Promega, Madison, WI; see Appendices B and C [online only] for primers and reaction conditions). Products were submitted for high-throughput sequencing to the Roy G. Carver Biotechnology Center of the University of Illinois. Sequencher 4.8 was used to automatically assemble contigs (minimum match = 60; minimum overlap = 20), and each contig for a given gene was assembled into a single alignment and exported as FASTA file. FASTA files were aligned in SeaView 4.3.0 (Gouy et al. 2010) using the built-in version of MUSCLE (Edgar 2004) with all parameters set at default, except in the case of 12S that required a higher gap opening penalty in some regions where the original alignment resulted in high numbers of extraneous gaps. Resulting alignments were then adjusted by eye. DNA sequences are deposited in GenBank (accession numbers are provided in Appendix A [online only]). Phylogenetic Analysis ModelGenerator (nset=6) was used to select the best-fitting evolutionary model for each gene based on Akaike information criterion (AIC; Keane et al. 2006). Individual gene trees were initially inferred using BEAST (Drummond and Rambaut 2007), with Bayesian Markov-chain Monte Carlo runs of 20 million generations (with 25% burn-in) and default priors (Drummond and Rambaut 2007). Gene trees were then compared, and if no well-supported conflict was present, the data were combined for subsequent analysis with individual genes treated as separate data partitions, each with its own model/parameters in maximum likelihood and Bayesian analyses. Sequences were trimmed to exclude primer regions, but no other regions were removed, and gaps were treated as missing data. Phylogenies were inferred using parsimony (PAUP* 4.0b10; Swofford 2003) with all characters equally weighted, 10,000 random addition sequence replicates and TBR branch swapping. Maximum likelihood analyses were performed using GARLI (Zwickl 2006) with 10 independent runs, default settings, and an automated stop criterion if lnL score remained constant for 50,000 generations. Bayesian estimation was performed using MrBayes (Ronquist et al. 2012) with four runs, each with four chains, of 20 million generations, and BEAST with 40 million generations, tree prior = speciation: birth–death process. For both methods, the first 25% of generations were discarded as burn-in and log files were viewed in Tracer to ensure convergence was reached (Swofford 2003, Ronquist et al. 2012). Both Bayesian posterior probabilities and parsimony and maximum likelihood bootstrap values (1,000 replicates of 100 random addition sequences) were calculated to estimate branch support. The concatenated alignment and trees resulting from phylogenetic analysis will be available from TreeBASE (submission ID: 21696). Results PCR Amplification and Sequencing Alignment Most taxa were represented by complete sequences of all three genes although a few gene regions were not obtained for a few taxa (see Appendix A [online only]). Initial gene tree analysis revealed no significant gene tree conflict, so data were combined into a supermatrix for subsequent analyses. The final alignment included a total of 3,482 characters, of which 2,823 were constant, 220 were variable but parsimony-uninformative, and 439 were parsimony informative (Appendix A [online only] contains a complete summary by gene). Phylogenetic Analysis GTR + I + G was chosen as the best-fitting model for both histone H3 and 28S. For 12S, AIC slightly favored K81uf + G over HKY + G (a difference in AIC scores of less than 0.4, whereas the third most favored model was about 2.0 points worse). HKY + G was selected over K81uf + G by the other criteria ModelGenerator uses to rank models but the latter model cannot be implemented in MrBayes or BEAST so HKY + G was used. Tree topologies were largely consistent regardless of analytical technique although minor differences, particularly toward the tips of major branches, were found. In all cases, nodes that were incongruent among analyses received low (<50%) bootstrap support. Thus, only the consensus tree resulting from the partitioned Bayesian analysis is shown in Fig. 2. The monophyly of Hecalini (including Attenuipyga but excluding Hecalusina He, Zhang & Webb) was consistently recovered, although with equivocal branch support. Glossocratus (and therefore Glossocratina) was sister to the Hecalina and the monophyly of both subtribes received strong branch support. Fig. 2. View largeDownload slide Molecular phylogeny of Hecalini and outgroups from the partitioned Bayesian analysis of the concatenated DNA sequence dataset (12S, H3, and 28S) analyzed using BEAST. Numbers above branches are Bayesian posterior probabilities, whereas numbers below the branches are bootstrap values (parsimony/likelihood). Missing values indicate <0.80 posterior probability or <50% bootstrap support. Fig. 2. View largeDownload slide Molecular phylogeny of Hecalini and outgroups from the partitioned Bayesian analysis of the concatenated DNA sequence dataset (12S, H3, and 28S) analyzed using BEAST. Numbers above branches are Bayesian posterior probabilities, whereas numbers below the branches are bootstrap values (parsimony/likelihood). Missing values indicate <0.80 posterior probability or <50% bootstrap support. Within Hecalina there are two main clades, one containing only New World genera and the other globally distributed, including New and Old World taxa. Although four of the five genera for which more than one species was included were found to be monophyletic, Hecalus, the largest and most widely distributed genus, was polyphyletic, forming two distinct geographically restricted clades, one North American and the other including African, Asian, and Australian species. Attenuipyga, formerly placed in Dorycephalini but recently transferred to Hecalini (Zahniser and Dietrich 2013), was embedded within the Hecalina, and sister to a clade containing five strictly New World genera (Spangbergiella Signoret, 1879; Neohecalus Linnavuori, 1975; Dicyphonia Ball, 1990; Jiutepeca Linnavuori and DeLong 1978; and Otamendiella Dietrich, 2017). Dorycephalini (sensu stricto, including only the type genus), was firmly placed within the outgroup. One of the undescribed genera from Asia was sister to the clade of New World Hecalus + Memnonia Ball, whereas the other was sister to the clade comprising Old World genera Thomsoniella + Linnavuoriella. Discussion Taxonomic Implications Phylogenetic analysis of three gene regions yielded a well-resolved phylogeny (Fig. 2) that consistently recovered Hecalini as monophyletic and supported the status of subtribes Glossocratina and Hecalina as monophyletic sister groups. Thus, our results are consistent with the current tribal and subtribal classification (Zahniser and Dietrich 2013) with the exception of placement of the Oriental genus Hecalusina. This genus, which has not been previously included in any phylogenetic analysis, was sister to a clade comprising the African genus Drakensbergena Linnavuori (Drakensbergenini) and the Palearctic genus Dorycephalus Kouchakéwitch (Dorycephalini). Morphologically, Hecalusina is similar to Paradorydium Kirkaldy (Eupelicini: Paradorydiina), a genus not included in the present dataset but grouped with Drakensbergenini and Dorycephalini in the maximum likelihood analysis of Zahniser and Dietrich (2013). Both genera have the forewing veins elevated, the male pygofer with a digitiform dorsal lobe, the connective with anterior arms weakly divergent, and the aedeagus long, slender, and recurved, without a distal process. Unfortunately, because we did not include Paradorydium in our dataset and the relationships between Hecalusina and other outgroup taxa received low branch support, we cannot provide phylogenetic confirmation of the correct tribal placement of Hecalusina. Within Hecalini, with the exception of Hecalus, all genera for which more than one representative was included were recovered as monophyletic. Most of these genera have previously been well characterized morphologically, so these results are not surprising. Nevertheless, because we were only able to include a small fraction of the known species, more detailed analyses will be necessary to provide a comprehensive tree for the entire tribe and conduct more robust tests of the phylogenetic status of recognized genera. Because Hecalus was not recovered as monophyletic and the type species of Hecalus, H. paykulli Stål (morphologically similar to the included African species H. aurora Linnavuori), is a member of the Old World Hecalus clade, our results suggest that the taxonomic concept of this genus should be restricted to include only Old World species and that a new genus should be erected to include the New World species of Hecalus. Because Hecalus is a large genus comprising >50 described species and our dataset included only a small fraction of the known species, such a reclassification of the genus would be best undertaken within the context of a more detailed taxonomic revision. Biogeographic Implications Taxa occurring in the same biogeographic realm tended to group together in our phylogenies, suggesting that continental-scale geographic isolation was an important driver in the early diversification of Hecalini. Glossocratina, a strictly Old World lineage was consistently recovered as sister to Hecalina comprising both New and Old World lineages. One of the two large clades of Hecalina, comprising the North American genus Attenuipyga and its sister group (Fig. 2), is restricted to the New World. South American species of this lineage (Otamendiella linnavuorii Dietrich and two species of Spangbergiella, one of which, S. vulnerata (Uhler), is widespread in both North and South America) occupy relatively derived positions within the clade, suggesting that the clade diversified in the warm temperate grasslands of North America before subsequently dispersing into South America. The second major lineage of Hecalina includes both New and Old World species. Within this clade, the New World species of Hecalus and Memnonia form a single lineage derived from within a grade comprising the Old World taxa (Linnavuoriella, Thomsoniella, Parabolocratalis Evans, 1955; Hecalus in part, and two undescribed Asian genera), suggesting that the common ancestor of New World Hecalus and Memnonia dispersed into the New World from Asia. A detailed and explicit biogeographic analysis of this and two additional unrelated lineages of grassland Auchenorrhyncha (deltocephaline leafhopper tribes Paralimnini + Deltocephalini and the fulgoroid family Caliscelidae) was undertaken by Catanach (2013) and will be published separately. Supplementary Data Supplementary data are available at Annals of the Entomological Society of America online. Acknowledgments Fieldwork by the authors that yielded many of the included specimens was facilitated by M. M. Cigliano, M. I. Catalano, P. Lozada, M. M. Yang, H. T. Shih, Y. Zhang, and G. Moya-Raygoza. Additional ethanol-preserved specimens were provided by J. Cryan, K. Hill, M. Irwin, B. Morris, M. Sharkey, M. Stiller, D. Takiya, and J. Zahniser. Sequencing was funded by two H. H. Ross Memorial Awards to T.A.C. from the Illinois Natural History Survey and a France M. and Harlie M. Clark Research Support Grant from the University of Illinois Urbana-Champaign, School of Integrative Biology. This work is modified from Chapter 4 of a doctoral dissertation submitted by T.A.C. to the Graduate College of the University of Illinois (Catanach 2013). References Cited Catanach, T. A. 2013. Biogeography and phylogenetics of grassland Auchenorrhyncha . Ph.D. dissertation, University of Illinois, Urbana, p. 133. http://hdl.handle.net/2142/46689 (accessed 14 October 2017). Dietrich, C. H. 2017. South American leafhoppers of the tribe Hecalini (Hemiptera: Auchenorrhyncha: Cicadellidae: Deltocephaline). Entomol. Am . 122: 398– 404. Drummond, A. J., and Rambaut A.. 2007. BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evol. Biol . 7: 214. Google Scholar CrossRef Search ADS PubMed  Edgar, R. C. 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res . 32: 1792– 1797. Google Scholar CrossRef Search ADS PubMed  Evans, J. W. 1966. The leafhoppers and froghoppers of Australia and New Zealand (Homoptera: Cicadelloidea and Cercopoidea). Mem. Aust. Mus . 12: 1– 347. Google Scholar CrossRef Search ADS   Gouy, M., Guindon S., and Gascuel O.. 2010. SeaView version 4: a multiplatform graphical user interface for sequence alignment and phylogenetic tree building. Mol. Biol. Evol . 27: 221– 224. Google Scholar CrossRef Search ADS PubMed  Hamilton, K. G. A. 2000. Five genera of New-World “shovel-headed” and “spoon-bill” leafhoppers (Hemiptera: Cicadellidae: Dorycephalini and Hecalini). Can. Entomol . 132: 429– 503. Google Scholar CrossRef Search ADS   Keane, T. M., C. J. Creevey, M. M. Pentony, T. J. 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Suchard, and Huelsenbeck J. P.. 2012. MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst. Biol . 61: 539– 542. Google Scholar CrossRef Search ADS PubMed  Swofford, D. L. 2003. PAUP*. Phylogenetic Analysis Using Parsimony (*and Other Methods), version 4 . Sinauer Associates, Sunderland, MA. Zahniser, J. N., and Dietrich, C. H. 2008. Phylogeny of the leafhopper subfamily Deltocephalinae (Insecta: Auchenorrhyncha: Cicadellidae) and related subfamilies based on morphology. Syst. Biodivers . 6: 1– 24. Google Scholar CrossRef Search ADS   Zahniser, J. N., and Dietrich, C. H. 2010. Phylogeny of the leafhopper subfamily Deltocephalinae (Hemiptera: Cicadellidae) based on molecular and morphological data with a revised family-group classification. Syst. Entomol . 35: 489– 511. Google Scholar CrossRef Search ADS   Zahniser, J. N., and Dietrich, C. H. 2013. A review of the tribes of Deltocephalinae (Hemiptera: Auchenorrhyncha: Cicadellidae). Eur. J. Taxon . 45: 1– 211. Zwickl, D. J. 2006. Genetic algorithm approaches for the phylogenetic analysis of large biological sequence datasets under the maximum likelihood criterion . Ph.D. dissertation, The University of Texas at Austin. © The Author(s) 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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Annals of the Entomological Society of AmericaOxford University Press

Published: Mar 1, 2018

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