c 2007 by Daniela Maeda Takiya. All rights reserved. SYSTEMATIC STUDIES ON THE SUBFAMILY (: CICADELLIDAE)

BY

DANIELA MAEDA TAKIYA B. Sc., Universidade Federal do Rio de Janeiro, 1998 M. Sc., Universidade Federal do Rio de Janeiro, 2001

DISSERTATION

Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Entomology in the Graduate College of the University of Illinois at Urbana-Champaign, 2007

Urbana, Illinois Abstract

The leafhopper subfamily Cicadellinae (=sharpshooters) includes approximately 340 genera and over 2,000

species distributed worldwide, but it is most diverse in the Neotropical region. In contrast to the vast majority

of (members of the family Cicadellidae), which are specialists on phloem or parenchyma fluids,

cicadellines feed on xylem sap. Because xylem sap is such a nutritionally poor diet, xylem specialists must

ingest large quantities of sap while feeding. They continuously spurt droplets of liquid excrement, forming

the basis for their common name. Specialization on xylem sap also occurs outside the ,

in members of the related superfamilies Cicadoidea () and Cercopoidea (spittlebugs) of the order

Hemiptera. Because larger with greater cibarial volume are thought to more easily overcome the

negative pressure of xylem sap, previous authors suggested that there may be a threshold of 8 mm above

which, the energetic cost of feeding is negligible. In chapter 1 the method of phylogenetic contrasts was used

to re-investigate the evolution of body size of Hemiptera and test the hypothesis that shifts to xylem feeding

were associated with an increase in body size. After correcting for phylogenetic dependence and taking

into consideration possible alternative higher-level phylogenetic scenarios, statistical analyses of hemipteran

body sizes did not show a significant increase in xylem feeding lineages. However, these results should

be viewed cautiously, because the lack of support for an increase in body size of xylem-feeders was the

strong negative contrast of supposedly xylem-feeding myerslopiids and the phloem-feeding ancestor of the

remaining Membracoidea. Additionally, the calculation of ancestral body sizes does not corroborate the

previous assumption of a size threshold, as all xylem-feeding ancestors, with the exception of cicadas, were

smaller than 8 mm.

Sharpshooters host two mutualistic bacterial endosymbionts to complement their poor diet. Chapter 2

addresses the question of whether these two dominant have undergone long-term codiversification

with their hosts. Twenty-nine leafhopper species, spanning six tribes with emphasis on sharpshooters,

were characterized for both Baumannia and Sulcia. Phylogenetic analysis were conducted based on the

16SrDNA from both bacteria and on COI, COII, 16SrDNA, and H3 for hosts. To test whether cospeciation occurred three statistical tests were conducted: a topology and maximum-likelihood-based

iii Shimoidara-Hasegawa test, a parsimony and event-based test (TreeFitter), and a parsimony and dataset-

based ILD test. A congruent evolutionary history of both Baumannia and Sulcia with their sharpshooter hosts is supported based on all (Baumannia) or most (Sulcia) statistical tests conducted here, suggesting a long-term association of these bacteria with their hosts.

Chapter 4 presents studies on the of St˚al,1869, the most economically important proconiine . These studies include a new designation of the type-species ( triangularis Fabricius,

1803) of the genus based on an original misidentification; transfer of Cicada triquetra Fabricius, 1803 to

Propetes Walker, 1851; designation of the lectotype of Tettigonia vitripennis Germar, 1821 and synonymy

of Tettigonia coagulata Say, 1832, making the former the appropriate scientific name for the glassy-winged

sharpshooter; a key to Brazilian species; description of the male of H. ignota Melichar; and description of a new species of Homalodisca from Northeastern Brazil.

Previous authors included in Cicadellinae different combinations of the tribes , Proconiini,

Mileewini, Errhomenini, Evacanthini, and Makilingiini. Incongruences in the classification were mainly due to the lack of robust phylogenetic hypotheses. A phylogenetic study on the Cicadellinae with emphasis on the tribe Proconiini is presented in the final three chapters. A morphological study based on 183 characters coded for 50 outgroup taxa and 121 ingroup species is presented along with a molecular study based on partial regions of COI, COII, 16S rDNA, and H3 gene sequences from 74 ingroup and 17 outgroup taxa.

Results support changes in the higher-level classification of Cicadellinae including the erection of the tribe

Oncometopiini based on previous members of the Proconiini; the treatment of Phereurhinini within the subfamily Cicadellinae; and the transfers of the following to genera (to appropriate Cicadellinae tribe or subfamily): Archeguina, Jilijapa, Namsangia, and Ochrostacta (Cicadellini), Ectypus (Proconiini), Pamplona

and Pamplonoidea (Oncometopiini), Homalogoniella (Phereurhinini), and Vidanoana (Mileewinae). Finally,

based on a combined analysis of both molecular and morphology, the origin of the egg-powdering behavior

and related sexually-dimorphic morphological characteristics were studied. Results suggest a single origin

of the egg-powdering behavior, possibly in the ancestor of Phereurhinini and Oncometopiini. Modifications

of the female hindlegs for scraping the brochosomes off onto the egg nests were also acquired once in the

ancestor of the Oncometopiini, while modifications on the female forewing setation for better anchoring of

brochosome pellets, seem to have been acquired multiple times. Multiple losses of the behavior and its

related associated traits occurred in various oncometopiine lineages.

iv This thesis is dedicated to my mother, Christina M. Takiya,

who once thought that Entomology was a dead science.

v “We are glorious accidents of an unpredictable process with no drive to complexity, not the expected results

of evolutionary principles that yearn to produce a creature capable of understanding the mode of its own

necessary construction.”

–Stephen J. Gould

“-Sadie: Daddy, why are starfish shaped like stars?

-Professor: That’s a very interesting question, Sadie. Functional adaptability and anatomical determina-

tion in biological systems is a fascinating issue that certainly warrants further investigation.

-Sadie: Does that mean you don’t know?

-Professor: It’s beyond the scope of my research. ”

–Jorge Cham @ phdcomics.com

“She’s filled with secrets. Where we’re from, the birds sing a pretty song, and there’s always music in the air.”

–The man from another place (Twin Peaks, written by Mark Frost)

vi Acknowledgments

After almost six years of working on this thesis I am indebted to many incredible people. In the first place to

Chris Dietrich and Roman Rakitov, whose interest in leafhopper evolution and biology made possible for me

to come to do my graduate studies in the University of Illinois and immensely broaden my research interests.

This opportunity came accompanied by field trips to Mexico, Peru, and Taiwan and my learning of new

techniques and research areas, which would not have been possible back home. Uncountable discussions on

leafhopper morphology were carried in our weekly meetings, which would not have been the same without

the input of my esteemed fellow lab members: Adam Wallner, Jamie Zahniser, Jesse Alberston, Natasha

Novikova, Suni Krishnankutty, and especially Dima Dmitriev, who set up the system for my sharpshooter

database and continuously improves the software.

Secondly, I would like to acknowledge my colleagues, who directly contributed in some of the papers

presented in this thesis. Gabriel Mejdalani (MNRJ) was a collaborator in CHAPTER 5, who definitely

molded myself with his own ideas of proconiine evolution, besides wholly supporting my decision to come

to the United States. Rodney Cavichioli’s (DZUP) enthusiasm towards sharpshooters is contagious and just

keeps me going on describing new species. He and Stu McKamey (USNM) were collaborators in CHAPTER

4. Stu’s grant on the revision of Homalodisca by the University of California Pierce’s Disease Research Grant

financed my flying to the Ukraine to study the type of H. vitripennis and take myself, Roman, and Jamie into

field trips to Costa Rica, Panama, and Venezuela, where material was collected and used in CHAPTER

6. Finally, Nancy Moran and Phat Tran (University of Arizona) whose interests in phytophagous insects endosymbionts were instrumental in producing the bacterial data studied in CHAPTER 3.

Support towards my research and career goals also came in various ways through many people. Mike

Wilson (National Museums and Galleries of Wales) allowed me to be a part of his incredible effort on imaging all sharpshooter species in the world, to be published as books sometime in the near future, and supported my re-analysis of his body size constraints ideas in CHAPTER 2. Brian Wiegmann (NCSU) allowed me the use of laboratory for trials of amplification of more useful gene regions in leafhoppers. Inspiration to conduct my research comes from amazing biologists who love their study groups with all their passion:

vii Adalberto Santos (Universidade Federal de Minas Gerais), Eduardo Venticinque (Wildlife Conservation

Society), Gustavo Graciolli (Universidade Federal do Mato Grosso do Sul), Jorge Nacimento (MNRJ), Jorge

L. Nessimian and Nelson Ferreira-Jr. (DZRJ), Lois O’Brien, Jeremy Miller (CAS), Martin Hauser (California

Department of Food and Agriculture), and Martin Villet (Rhodes University). Finally, I am indebted to my thesis committee, May Berenbaum, Jim Whitfield, Kevin Johnson, and Sydney Cameron, for being there in the past years for questions that I had, writing letters when needed, input on presentations given, teaching their interesting courses, and corrections of this thesis.

Illustrations and photographs by C. Dietrich, G. Mejdalani, M. Felix, and L. Costa (MNRJ), K. Arakawa

(USNM), M. Wilson and J. Turner (National Museums and Galleries of Wales, Cardiff), N. Scharff (Zoo- logical Museum, University of Copenhagen), P. Ceotto (Mus´eumnational d’Histoire naturelle, Paris), R.

Rakitov, and W. Azevedo-Filho (PUC, Porto Alegre) were used with in this thesis and are acknowledged herein and in the figure legend. I am thankful for curators and collection managers noted in the list of specimen depositories, who sent us leafhopper material to be studied or welcomed myself in their collections.

Information on the systematics of other Clypeorrhyncha lineages for CHAPTER 2 were shared by Jason

Cryan (NYSM), Matt Wallace (NCSU), Martin Villet (Rhodes University), and Max Moulds (Australian

Museum). Gathering of material preserved in ethanol for DNA sequencing for CHAPTER 5 would not be possible without the help of the following individuals: A. Hicks (University of Colorado Museum), B. Al- varado (INRENA, Peru), C. Bartlett (UDCC), C. Darling (ROM), C. Godoy (INBIO), D. Yanega (University of California at Riverside, USA), E. Virla (Conicet-PROIMI, Argentina), G. Moya-Raygoza (Universidad de Guadalajara, Mexico), G. Mejdalani (MNRJ), G. Logarzo (USDA-ARS, Argentina), H. T. Shih (Taiwan

Agricultural Research Institute, Taiwan), J. Cryan (New York State Museum, USA), J. Spotti-Lopes and

R. Marucci (Universidade de S˜aPaulo, Brazil), J. Grados, G. Lamas, C. Pe˜na, and T. Peque˜no (MUSM),

K.-W. Huang (National Museum of Natural Sciences, Taiwan), M. Sharkey and P. Freytag (UKL), M.-M.

Yang, J.-F. Tsai, P. Huang, and J.-Y. Liou (National Chunghsing University, Taiwan), M. Whiting (Brigham

Young University, USA), M. Irwin, M. Hauser, R. Rakitov, and V. Block (INHS), M. Stiller (Agricultural

Research Council, South Africa), N. Penny (CAS), P. Lozada and G. Solis (SENASA, Peru), S. McKamey,

T. Erwin, T. Henry, and W. Steiner (USNM), R. Whitcomb, R. Mizell III (University of Florida, USA), R.

Cavichioli (DZUP), T. Wood (UDCC), and V. Thompson (Kean University, USA).

A fellowship from Conselho Nacional de Desenvolvimento Cientfico e Tecnolgico (CNPq, Brazil) funded me through most of the duration of my Ph.D. studies. Additional support came through funding by National

Science Foundation grants DEB-0089671 to Chris Dietrich and DEB-9978026 to L. Deitz and Chris Dietrich.

A teaching assistantship through the Department of Entomology was provided during one semester. Steve

viii Taylor (INHS) supported me with a research assistantship for my last semester, showing me that cricket genitalia are not as complicated as I thought before. Furthermore, several small grants received from the

University of Illinois and Illinois Natural History Survey were used to complement this study in different ways. Those are listed in my curriculum vitae in the back of the thesis.

I take this opportunity to show my appreciation for additional people that make my life a happy and safe one. Josh Guffin shared everything with me, standing by my side at all times for the past couple years.

This thesis would not look as good if he didn’t advise me to using LATEX. Achilles Chirol, Ana Paula Penna, Barbara Feliciano, Carolina Spiegel, Daniel Buss, Daniela Pereira, Dimitri Rebello, Fernanda Kischinhevsky,

Guilherme Muniz, Gutemberg Gomes, Hana Paula Masuda, Juli˜aoNascimento, Paula Ceotto, Renata Roxo, and Rodrigo Valverde, however far away, have been here with me all along the way. New friends along this path kept me from an unbearable homesickness, will be missed in the future, but surely will not be forgotten,

Alejandro Valerio, Andrea Skoglund, Annie Ray, Bridget O’Neill, Charmaine Armitage, Cindy McDonnell,

Courtney McCusker, David Caplan, Emerson Lacey, Ian Kirwan, Jessica Antonia, Jamie Zahniser, Joana

Maria, John Kane, Lauren Kent, Letania Ferreira, Liz Graham, Martin Hauser, Nico Tzovolos, Pete Reagel,

Reed Johnson, Sandy No, and Sean Nowling. My family, mom Christina, gradmother Inˆes, nanny Dilc´ea, father Sim˜ao, stepmother Rosˆangela, and brothers Renan and Daniel have always been supportive of my decisions and always did their best to see my happiness. I would have never made it without all of your love.

ix Table of Contents

List of Tables ...... xiii

List of Figures ...... xiv

List of Abbreviations ...... xvi

List of Specimen Depositories ...... xviii

Chapter 1 Introduction ...... 1

Chapter 2 All Xylem Specialists Great and Small: Revisiting Minimum Body Size Constraints ...... 7 2.1 Introduction ...... 7 2.2 Materials and Methods ...... 9 2.2.1 Classification ...... 9 2.2.2 Feeding Categories ...... 9 2.2.3 Body Size Measurements ...... 11 2.2.4 Higher-Level Phylogenetic Hypotheses ...... 13 2.2.5 Lower-Level Phylogenetic Hypotheses ...... 15 2.2.6 Ancestral Character Optimizations ...... 19 2.2.7 Phylogenetic Independent Contrasts ...... 19 2.3 Results ...... 21 2.3.1 Constructed Supertrees ...... 21 2.3.2 Ancestral Feeding Niches ...... 21 2.3.3 Ancestral Body Sizes and Phylogenetic Contrasts ...... 21 2.4 Discussion ...... 22

Chapter 3 Codiversification of Sharpshooter Bacterial Endosymbionts ...... 31 3.1 Introduction ...... 31 3.2 Materials and Methods ...... 34 3.2.1 Taxon Sampling ...... 34 3.2.2 DNA Preparation ...... 35 3.2.3 PCR and Sequencing of Host Genes ...... 37 3.2.4 PCR and Sequencing of Symbiont Genes ...... 37 3.2.5 Alignments ...... 39 3.2.6 Phylogenetic Analyses ...... 39 3.2.7 Host-Symbiont Associations ...... 40 3.2.8 Evolutionary Rates of Bacterial 16S rDNA ...... 41 3.3 Results ...... 42 3.3.1 Distribution of Baumannia, Sulcia and Wolbachia among Hosts ...... 42 3.3.2 Host Trees ...... 43 3.3.3 Symbiont Phylogenies and Divergences ...... 43

x 3.3.4 Host-Symbiont Associations ...... 43 3.3.5 Evolutionary Rates of Bacterial 16S rDNA ...... 49 3.4 Discussion ...... 50 3.4.1 Codiversification in a Dual Symbiosis ...... 50 3.4.2 Nutritional Roles for Primary Symbionts and Implications for Host Diversification . . 51 3.4.3 Variable Rates of Evolution in Symbiont Sequences ...... 52 3.4.4 Wolbachia in leafhoppers ...... 53 3.4.5 Implications for Leafhopper Phylogenetics and Classification ...... 53 3.4.6 Conclusions ...... 54

Chapter 4 Taxonomy of the genus Homalodisca St˚al ...... 55 4.1 Introduction ...... 55 4.2 Materials and Methods ...... 57 4.3 Taxonomy ...... 57 4.3.1 Homalodisca St˚al...... 57 4.3.2 Homalodisca vitripennis Germar ...... 60 4.3.3 Propetes Walker ...... 63 4.3.4 Propetes triquetra (Fabricius) comb. nov...... 64 4.3.5 Notes on and New Records of Brazilian Homalodisca ...... 65 4.3.6 Homalodisca ignota Melichar ...... 65 4.3.7 Homalodisca spottii Takiya, Cavichioli et McKamey, sp. nov...... 70 4.3.8 Taxonomic Key to Brazilian Homalodisca species ...... 73 4.3.9 Additional Material Studied ...... 73 4.4 Conclusions ...... 74

Chapter 5 Morphological Analysis of the Proconiini ...... 75 5.1 Introduction ...... 75 5.2 Material and Methods ...... 75 5.2.1 Taxon Sampling ...... 75 5.2.2 Notes on Specimen Identifications ...... 76 5.2.3 Terminology ...... 80 5.2.4 Notes on Characters Used in Previous Studies ...... 82 5.2.5 Character Sampling and Assumptions ...... 85 5.2.6 Phylogenetic Analysis ...... 86 5.3 Results ...... 86 5.3.1 Morphological Matrix ...... 86 5.3.2 Morphological Characters and States ...... 96 5.3.3 Phylogenetic Analysis ...... 107 5.4 Discussion ...... 108 5.4.1 Changes in the Higher-Level Classification ...... 108 5.4.2 Changes in the Composition of Tribes ...... 114 5.5 Conclusions ...... 118

Chapter 6 Molecular Analyses of the Proconiini ...... 119 6.1 Introduction ...... 119 6.2 Materials and Methods ...... 119 6.2.1 DNA Sequence Data Gathering ...... 119 6.2.2 Alignment ...... 119 6.2.3 Phylogenetic Content ...... 123 6.2.4 Phylogenetic Analyses ...... 123 6.3 Results ...... 124 6.3.1 Phylogenetic Content of Datasets ...... 124 6.3.2 Phylogenetic Analyses ...... 125 6.4 Discussion ...... 131

xi 6.4.1 Topological Agreement with Morphological Analysis: Support for Classification Changes ...... 131

Chapter 7 Evolution of Egg-Powdering Behavior ...... 132 7.1 Introduction ...... 132 7.2 Materials and Methods ...... 134 7.2.1 Phylogenetic Analysis ...... 134 7.2.2 Character Optimization ...... 135 7.3 Results ...... 136 7.3.1 Phylogenetic Analyses ...... 136 7.3.2 Character Optimization ...... 136 7.4 Discussion ...... 141 7.4.1 Origin of the Egg-Powdering Behavior ...... 141 7.4.2 Gains, Losses, and Vestiges of Egg-Powdering Related Traits ...... 142 7.4.3 Multiple Losses of an Adaptive Behavior ...... 143 7.4.4 Conclusions ...... 145

References ...... 146

Curriculum Vitae ...... 169

xii List of Tables

1.1 Previous classifications of the subfamily Cicadellinae (= Tettigellidae, Tettigellinae) . . . . . 4

2.1 Feeding niches and body-size measurements of ...... 10 2.2 Feeding niches and body-size measurements of Archaeorrhyncha ...... 11 2.3 Feeding niches and body-size measurements of Cercopoidea (Clypeorrhyncha) ...... 12 2.4 Feeding niches and body-size measurements of Cicadoidea (Clypeorrhyncha) ...... 13 2.5 Feeding niches and body-size measurements of (Clypeorrhyncha) ...... 14 2.6 Feeding niches and body-size measurements of leafhoppers (Clypeorrhyncha) ...... 16 2.7 Body size ancestral reconstructions and phylogenetic constrasts of xylem and non-xylem feed- ing lineages ...... 26 2.8 Records of primary (*) or secondary (+) xylem sap feeding by “Homoptera” ...... 30

3.1 Material examined with accession numbers for and endosymbionts Candidatus Bau- mannia cicadellinicola and Candidatus Sulcia muelleri genes ...... 36 3.2 Oligonucleotide primer sequences ...... 38 3.3 Maximum likelihood pairwise distances among 16S rDNA sequences of selected Baumannia (A) and Sulcia (B) endosymbionts and related bacteria ...... 47 3.4 Maximum likelihood (-lnL) scores for pruned most-parsimonious (MP) and most likely (ML) topologies ...... 48 3.5 Results of TreeFitter analyses of coevolutionary events between leafhopper hosts and their two bacterial endosymbionts ...... 49

5.1 Species studied for the phylogenetic analysis of the Proconiini ...... 77 5.2 Matrix of morphological characters for the phylogenetic analysis of the Proconiini ...... 87

6.1 DNA sequences of Proconiini obtained ...... 120 6.2 Alignment of 16S rDNA of Proconiini ...... 121 6.3 Descriptive statistics on sequence data ...... 123

xiii List of Figures

1.1 Sharpshooter morphological diversity ...... 2 1.2 Relationships of Cicadellinae groups in previous parsimony analyses ...... 6

2.1 Alternative phylogenetic scenarios for higher-Level Euhemiptera ...... 15 2.2 Phylogeny of Prosorrhyncha...... 18 2.3 Phylogeny of Cercopoidea ...... 19 2.4 Phylogeny of Cicadoidea ...... 20 2.5 Phylogeny of Archaeorrhyncha ...... 22 2.6 Phylogeny of Membracoidea ...... 23 2.7 Phylogeny of ...... 24 2.8 Phylogeny of Membracidae ...... 25 2.9 Distribution of minimum ln body size of by feeding niche ...... 27

3.1 Sharpshooter bacteriomes ...... 33 3.2 Single maximum-likelihood phylogram for leafhopper hosts, based on the combined gene dataset (-lnL=19,451.13) ...... 44 3.3 Single maximum-likelihood phylogram for Baumannia symbionts and relatives in the γ- Proteobacteria, based on 16S rDNA sequences (-lnL = 9,175.09) ...... 45 3.4 Selected maximum-likelihood phylogram (one of three) for Sulcia symbionts and relatives in the Bacteroidetes phylum of Bacteria, based on 16S rDNA sequences (-lnL = 6,993.82) . . . . 46

4.1 The glassy-winged sharpshooter, Homalodisca vitripennis (Germar) ...... 59 4.2 Lectotype of Homalodisca vitripennis ...... 61 4.3 Dorsal habitus of type-specimens of Propetes Walker species ...... 64 4.4 Dorsal and lateral habitus of Homalodisca ...... 67 4.5 Homalodisca ignota Melichar, genital structures ...... 68 4.6 Homalodisca spottii sp. nov., genital structures ...... 71

5.1 Morphology of the head and thorax ...... 81 5.2 Morphology of the wings ...... 82 5.3 Morphology of the legs ...... 83 5.4 Morphology of the abdomen ...... 84 5.5 Morphological analysis: Strict consensus, part 1 ...... 109 5.6 Morphological analysis: Strict consensus, part 2 ...... 110 5.7 Morphological analysis: Optimized tree, part 1 ...... 111 5.8 Morphological analysis: Optimized tree, part 2 ...... 112 5.9 Morphological analysis: Optimized tree, part 3 ...... 113 5.10 Morphological analysis: Posterior likelihoods plot ...... 114 5.11 Morphological analysis: Clade support, part 1 ...... 115 5.12 Morphological analysis: Clade support, part 2 ...... 116

6.1 Molecules: Likelihood mapping diagrams ...... 124

xiv 6.2 Molecules: Posterior likelihoods plots ...... 125 6.3 COI: Bayesian consensus phylogram ...... 126 6.4 COII: Bayesian consensus phylogram ...... 127 6.5 16S: Bayesian consensus phylogram ...... 128 6.6 H3: Bayesian consensus phylogram ...... 129 6.7 Molecular: Bayesian consensus phylogram ...... 130

7.1 Egg-Powdering Behavior ...... 133 7.2 Egg-Powdering Behavior: Forewing setation dimorphism ...... 134 7.3 Egg-Powdering Behavior: Hindleg AV row setation dimorphism ...... 135 7.4 Combined: Posterior likelihood plots ...... 136 7.5 Combined: Bayesian consensus phylogram, part 1 ...... 137 7.6 Combined: Bayesian consensus phylogram, part 2 ...... 138 7.7 Combined: Bayesian consensus phylogram, part 3 ...... 139 7.8 Optimizations of Egg-Powdering Behavior and related morphological characteristics . . . . . 140 7.9 Egg brochosome diversity ...... 144

xv List of Abbreviations

α Significance level of statistical test -lnL Negative natural log-likelihood 16S Sequence encoding the ribosomal small subunit in non-eukaryotes ACCTRAN Accelerated character transformation ANOVA Analysis of Variance CAIC Comparative Analysis using Independent Contrasts [282] CI Consistency index [177]:

minimum number of steps CI = L

COI Sequence encoding the subunit I of the Cytochrome C oxidase enzyme COII Sequence encoding the subunit II of the Cytochrome C oxidase enzyme DELTRAN Delayed character transformation df Degrees of freedom DNA Deoxy-Ribonucleic Acid EF-1α Sequence encoding the alpha subunit of the elongation factor 1 protein EMIF Electronic monitoring of insect feeding F81 Felsenstein 1981 (model) [122]: variable base frequencies, all substitutions equally likely, PAUP* rate matrix: aaaaaa ftsZ Sequence encoding the ftsZ protein (prokaryotic analogue of eukaryotic tubulin) Γ Distribution model for rate variation across sites [383] GTR General time reversible (model) [300]: variable base frequencies, variable transversions, variable transitions, PAUP* rate matrix: abcdef

H0 Null hypothesis H3 Sequence encoding the Histone H3 protein hLRT Hierarchical Likelihood Ratio Test I Proportion of invariant sites

xvi ILD Incongruence length difference (test) [119] IUB International Union of Biochemistry JC Jukes-Cantor (model) [172]: equal base frequencies, all substitutions equally likely, PAUP* rate matrix: aaaaaa L Length: actual number of steps in a cladogram ME Minimum Evolution Mk1 Markov k-state 1 parameter (model) [191] ML Maximum likelihood MRP Matrix Representation with Parsimony [24, 285] NJ Neighbor-Joining [306] NNI Nearest neighbor interchange (branch swap) PCR Polymerase chain reaction rDNA Sequence encoding ribosomal RNA RELL Re-estimation of likelihoods RC Rescaled Consistency index [118]: RC = CI × RI RI Retention index [118]:

number of steps in unresolved cladogram − L RI = number of steps in unresolved cladogram − minimum number of steps

RNA Ribonucleic acid S-H Shimodaira and Hasegawa (test) [323] SPR Subtree pruning-regrafting (branch swap) SYM Symmetrical (model) [398]: equal base frequencies, variable transversions, variable transi- tions, PAUP* rate matrix: abcdef T3P Tamura 3-parameter (model): equal base frequencies, variable transversions, equal transi- tions, PAUP* rate matrix: abcaba Taq Thermus aquaticus (polymerase) TBR Tree-bisection and reconnection (branch swap) TrN Tamura-Nei (model) [350]: variable base frequencies, equal transversions, variable transi- tions, PAUP* rate matrix: abaaca TVM Transversion (model): variable base frequencies, variable transversions, equal transitions, PAUP* rate matrix: abcdbe wg Sequence encoding the wingless protein wsp Sequence encoding the Wolbachia surface protein

xvii List of Specimen Depositories

AMNH American Museum of Natural History, New York, U.S.A. (R. Schuh) BMNH Department of Entomology, The Natural History Museum, London, U.K. (M. Webb) BPBM Bernice P. Bishop Museum, Honolulu, U.S.A. CAS California Academy of Sciences, San Francisco, U.S.A. (N. Penny) CMNH Carnegie Museum of Natural History, Pittsburgh, U.S.A. DCMB Cole¸c˜aoEntomol´ogica, Departamento de Biologia, Universidade do Amazonas, Manaus, Brazil (N. Aguiar) DZUP Cole¸c˜aode Entomologia Pe. Jesus Santiago Moure, Departamento de Zoologia, Universi- dade Federal do Paran´a,Curitiba, Brazil (R. Cavichioli) EMEC Essig Museum of Entomology, University of California, Berkeley, U.S.A. (C. Barr) EPNC Escuela Polytecnica Nacional, Quito, Ecuador FMNH Field Museum of Natural History, Chicago, U.S.A. (V. Thompson) FSCA Florida State Collection of , Gainesville, U.S.A. (S. Halbert) IFNU Department of Zoology, Ivan Franko National University, Lviv, Ukraine (I. Shydlovsky and O. Holovachov) INBIO Instituto Nacional de Biodiversidad, Santo Domingo de Heredia, Costa Rica (C. Godoy) INHS Illinois Natural History Survey, Champaign, U.S.A. LACM Los Angeles County Museum of Natural History, Los Angeles, U.S.A. MMBC Department of Entomology, Moravian Museum, Brno, Czech Republic (I. Malenovsky) MNHN Mus´eumNational d’Histoire Naturelle, Paris, France (T. Bourgoin) MNRJ Museu Nacional, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil (G. Mej- dalani) MPEG Museu Paraense Em´ılioGoeldi, Bel´em, Brazil (A. Harada and O. Silveira) MTD Museum f¨ur Tierkunde, Dresden, Germany (R. Emmrich) MUSM Museo de Historia Natural, Universidad Mayor de San Marcos, Lima, Peru (P. Lozada) MZPW Museum and Institute of Zoology, Polish Academy of Sciences, Warsaw, Poland (J. Szwedo, T. Huflejt and M. Adamczewska)

xviii NCSU Department of Entomology, North Carolina State University, Raleigh, U.S.A. (L. Deitz and R. Blinn) NMW Naturhistorisches Museum Wien, Vienna, Austria (H. Zettel) OSAC Oregon State University, Corvallis, U.S.A. OSU Ohio State University, Columbus, U.S.A. (N. Johnson and L. Musetti) ROM Royal Ontario Museum, Toronto, Canada (C. Darling and B. Hubley) SEMC Snow Entomological Museum, University of Kansas, Lawrence, U.S.A. TAMU Department of Entomology, Texas A & M University, College Station, U.S.A. (E. Riley) TARI Taiwan Agricultural Research Institute, Taichung, Taiwan (H. T. Shih) UDCC Department of Entomology and Applied Ecology, University of Delaware, Newark, U.S.A. (C. Bartlett) UKL Department of Entomology, University of Kentucky, Lexington, U.S.A. (P. Freytag) UMO University Museum of Natural History, Oxford, U.K. UNAM Universidad Nacional Aut´onomade Mexico, Mexico, D.F., Mexico (H. Brailovsky) USNM United States National Museum of Natural History, Washington, D.C., U.S.A. (S. McK- amey)

xix Chapter 1

Introduction

The family Cicadellidae comprises phytophagous insects commonly known as leafhoppers. It contains ap- proximately 21,000 described species distributed worldwide [179], hence constitutes the largest family of the order Hemiptera (following classification proposed by Sorensen et al. [328]) and one of the ten largest insect families. Representatives of this family can be distinguished from the rest of the Membracoidea by the fol- lowing characteristics [79]: (1) pronotum not extending posteriorly to scutellar suture (except Signoretiinae,

Phlogisinae, and few Cicadellinae), (2) scutellum not strongly produced dorsally, (3) mesothorax with suture separating the anepisternum from katepisternum, (4) mesepisternum without hook-shaped process dorsally,

(5) metathoracic tibiae usually with four rows of large setae, and (6) integument clothed with brochosomes

(except some Iassinae, Ledrinae and Ulopinae). Brochosomes are proteinaceous particles, usually 0.2-2.0

µm in diameter, produced by glandular segments of the Malpighian tubules, and their most probable func- tion is protection against excessive humidity and sticking of honeydew to the body [286, 290]. Droplets of brochosome-containing fluid are liberated through the anus and spread throughout the body with setae of posterior legs in a behavior called anointing. After this fluid dries, leafhoppers spread these particles over the integument more homogeneously, again using setae of posterior legs, by means of a grooming behav- ior [289, 290].

In a phylogenetic analysis of higher groups of Membracoidea, based on morphological characters, Di- etrich & Deitz [87] considered the above mentioned character states (1) and (6) as synapomorphies of the

Cicadellidae family, but recent analyses indicate that Cicadellidae is probably paraphyletic with respect to

Membracidae and two other families [91,148,287]. The higher classification of Cicadellidae is very controversial. The number of subfamilies varies in the literature from 10 [147] to over 50 [249], with little agreement between authors (see [251] for a historical review). Such confusion in the classification is probably a consequence of the poor knowledge of the phylogenetic relationships among the numerous subfamilies and tribes, as well as of the large number of included species. Attempts to establish the relationships among higher cicadellid groupings based on morphological characters were published by Ross [304], Hamilton [147],

Dietrich [83], and Rakitov [287], and based on 28S rDNA sequences by Dietrich et al. [91].

1 Figure 1.1: Sharpshooter morphological diversity. (A) clarior, Oncometopiini.; (B) Abana gigas, Proconiini; (C) Phereurhinus sp., Phereurhinini; (D) Diedrocephala bimaculata, Cicadellini; (E) Rup- peliana episcopalis, Cicadellini; (F) Homalodisca vitripennis, Oncometopiini; (G) Parathona cayennensis, Cicadellini; (H) Tapajosa fulvopunctata, Oncometopiini; (I) Raphirhinus phosphoreus, Proconiini. (B) Pho- tographed by R. Rakitov, and (C) and (I) by C. Dietrich.

The subfamily Cicadellinae includes approximately 340 genera and over 2,000 species worldwide (Figure

1.1). They appear to be one of the most common and diverse leafhopper subfamilies in Neotropical rain- forests, not only in the canopy [93], but also in the seedling understory [22]. In contrast to the vast majority of leafhoppers, which are specialists on phloem or parenchyma fluids, cicadellines feed on xylem sap. This habit causes individuals to continuously spurt copious amounts of excrement while feeding, which is why they are commonly referred to as sharpshooters. Like several other xylem-sucking herbivores, cicadellines are mostly generalists feeding on different plant families [23, 256]. Also due to their xylem feeding habit, sharpshooters are not able to rely on mutualistic associations with ants or bees foraging for honeydew as a protection against predators and/or parasitoids, associations which are reported for some other leafhopper and several treehopper lineages [84, 89]. On the other hand, their large size and variable striking coloration

2 (in comparison to that of other leafhopper subfamilies) facilitate their use of visual cues as Batesian mimics against predators, cases of which have only been reported for this subfamily. Species of the Amazonian genus

Lissoscarta St˚al(tribe Cicadellini) have been reported mimicking epiponine wasps (Vespidae: Polistinae) and even showing a bluffing behavior when threatened, opening their wings to assume a wasp-like posi- tion and showing their constricted abdomen [35, 221]. The sexually dimorphic Propetes schmidti Melichar

(tribe Proconiini) from Southeastern Brazil is also reported to be a Batesian mimic of two distinct epiponine

M¨ullerian mimicry rings, with males and females having different models [346]. Mejdalani et al. [223] recently reported the first case of mimicry involving a non-wasp model –Teletusa limpida (Signoret) is a Batesian mimic of megachilid bees, which are also part of a mimicry ring in Southeastern Brazil.

The delimitation of the subfamily Cicadellinae is controversial (see Table 1.1). Traditionally, several authors included in this subfamily different combinations of the tribes Cicadellini, Proconiini, Mileewini,

Errhomenini, Evacanthini, and Makilingiini [110, 229, 262]. However, David A. Young, in his monographic taxonomic revision of this subfamily [392–394], only considered the nominate tribe and the Proconiini as belonging to the Cicadellinae. Young [391,392] explicitly transferred Mileewini to Typhlocybinae, and briefly indicated that the higher categories based on Makilingia, Errhomenelus, and Evacanthini itself should be treated as subfamilies of their own. However, the lack of a hypothesis concerning the phylogenetic position of the excluded groups was possibly the main reason why recent authors still treated those groupings as tribes of Cicadellinae [9,136,147,196,251,264]. These groups have been considered related by these authors based on few morphological traits, such as inflated frons and consequent displacement of the ocelli to a dorsal position, which are most probably correlated with their feeding mode, rather than phylogenetic proximity.

Inflation of the frons allows an increase in the area of attachment of the cibarial muscles to cope with negative tension and facilitate high feeding rates, necessary because of low nutrient contents of xylem

fluid [218, 256]. This mode of feeding is thought to be a plesiomorphic condition in the

(=Clypeorrhyncha sensu Campbel et al. [45]), found throughout the superfamilies Cicadoidea (cicadas) and

Cercopoidea (spittlebugs), and apparently retained by cicadelline leafhoppers [84]. This topic of xylem fluid specialization by sharpshooters and other cicadomorphans is discussed in CHAPTER 2, where ancestral feeding modes of the major lineages of Hemiptera are optimized onto a tree topology to study the effect of xylem feeding on body size increase in a phylogenetic framework.

Another complication of xylem specialization is the low nutrition contained in such a dilute food source.

Xylem fluid is more than 95% water, and is by far the most dilute food source encountered by herbivores [8].

Nitrogen concentrations in xylem are typically 10 times lower than in phloem sap [215] and organic profiles are usually unbalanced with a predominance of glutamine and asparagine [7], which are not essential for

3 Table 1.1: Previous classifications of the subfamily Cicadellinae (= Tettigellidae, Tettigellinae). Tribes (or nominate genus) listed are included in Cicadellinae when marked with a black square (), treated as a separate subfamily (lozenge ) or family (star F). Some taxa were included in subfamilies Aphrodinae (Aphr.) and Typhlocybinae (Typhl.). Taxa not treated in respective classification scheme are marked with a dash (-).

References Evans Oman Metcalf Young Linnavuori Kwon and Hamilton Oman, Nielson Anufriev & Knight & Knight Emel’yanov & Nielson Tribes included [109, 110] [262] [227–229] [391, 392] [195, 196] [9, 186] [147] [264] [251]

Cicadellini          (=Tettigellinae, -ini) Proconiini      -    (=Proconiinae) Mileewini  -  Typh.      (=Mileewanini) Errhomenini Aphr.  Aphr.       (=Errhomenellini) Evacanthini Aphr.  F      

Makilingiini  -    -   

Pagaroniini Aphr.  Aphr. - -     (= Euacantheliini) Phereurhinini  -  ---   

Anoterostemmini Aphr. - Aphr. - - - Aphr.  

Hylicini F - F ---   

Signoretiini Aphr. - Evacanthidae -  -   

insect growth and development. More limiting still is the mean concentration of organic carbon, which can range from 1-10 mol m−3 in xylem sap, while in phloem sap it is greater than 5 kmol m−3 [295]. One way to overcome this deficiency by phytophagous insects is through long-term hosting of symbiotic bacteria, which in turn can be an additional source of some essential amino acids and vitamins [237, 354, 355, 379]. Most of these associations are obligate, as hosts when deprived of the bacteria suffer sterility, slower growth and higher mortality rates [101, 131, 164]. At the same time, these bacteria apparently cannot survive outside their hosts (in culture medium) and their genomes are stripped down to a minimum set of genes necessary for the symbiotic lifestyle [134, 163, 379]. An extreme example is Carsonella ruddii, the hackberry psyllid endosymbiont, currently the smallest bacterial genome (approximately 160,000bp), with more than half of its genome devoted to the production of proteins and amino acids and considered to have reached organelle- like status [245]. Sharpshooters harbor two primary bacterial endosymbionts, Baumannia cicadellinicola and Sulcia muelleri, which complement eachother for the nutrition of their hosts [379]. The evolutionary histories of sharspshooters and the two bacterial endosymbionts are compared in CHAPTER 3.

Few sharpshooters are reported as causing direct damage to the plant (due to xylem sap withdrawal or physical damage to tissues), e.g. Tapajosa rubromarginata Signoret (tribe Proconiini) a pest on sugarcane, corn, oat and sorghum in Argentina [298], and only two are known as dubious or unimportant vectors of non-bacterial plant diseases, such as the chlorotic streak virus of sugarcane and the western-X strain of peach [137, 248]. On the other hand, all species of this subfamily are potential vectors of strains

4 of the xylem-borne bacterium Xyllela fastidiosa, which has been associated with causing diseases of many economically important plants: grapevine, alfalfa, peach, plum, almond, , sycamore, , maple, olean- der, mulberry, citrus, and coffee [160, 214, 278, 280, 296]. Some species of the genera Amphigonalia Young,

Xyphon Hamilton, Ball, Van Duzee (tribe Cicadellini), Cuerna Melichar,

Homalodisca St˚aland Oncometopia St˚al(tribe Proconiini) were identified as vectors of X. fastidiosa causing

Pierce’s disease of grape [1, 129, 130]. Species of the three last mentioned genera were also identified as vectors of the same bacterium causing phony peach disease [248,361,362]. In Southeastern Brazil, species of

Dilobopterus Signoret (tribe Cicadellini), Oncometopia and Acrogonia St˚al(tribe Proconiini) were identified as vectors of this bacterium to citric groves causing the variegated chlorosis of citrus [142,209–211,299], and these polyphagous leafhoppers are also probable vectors of X. fastidiosa causing coffee leaf scorch [203,214] in the same region. CHAPTER 4 is dedicated to a taxonomic study of the genus Homalodisca, which includes the discovery of a senior synonym for the glassy-winged sharpshooter, a major introduced vector of

Pierce’s disease in California; the exclusion from Homalodisca of the previous type-species, Cicada triquetra

(Fabricius); the description of the previously unknown male of H. ignota; and of a probable new vector of variegated chlorosis of citrus in Northeastern Brazil.

Recent phylogenetic evidence based on morphological and molecular datasets [83,88,91] has shown that the subfamily Cicadellinae sensu lato (as opposed to sensu Young [392–394]) is probably not a monophyletic unit (Figure 1.2). Based on these analyses, the Cicadellinae as delimited by Young seem to be closely related, but may be paraphyletic without the inclusion of the components of the subfamily Phereurhininae in this assemblage. Furthermore, the cosmopolitan tribe Cicadellini could be a paraphyletic taxon with respect to the New World restricted Proconiini.

There are very few available studies on phylogenetic relationships within the subfamily Cicadellinae.

Young [392, 393] organized the New World genera into diagrams of morphological resemblance, further dividing the more speciose Cicadellini into generic groupings. Although he even stated that “this graphic representation may have very little relationship to phylogeny”, these groupings seem to be very useful as a starting point to phylogenetic studies using the cladistic method [218] and were used by the following authors as a source for outgroup choices. Dietrich [82] studied the species relationships of the genus Draeculacephala and its sister group Hamilton, based on Young’s Latreille generic grouping. Cavichioli [49] studied the species relationships of the genus Parathona Melichar. In his M. Sc. and D. SC. dissertations,

Felix [120, 121] produced phylogenetic analyses among species of the wasp-mimicking genus Lissoscarta

St˚aland Apogonalia Evans, repectively. Takiya & Mejdalani [345] presented a phylogenetic analysis among species of the Eryngium (Apiaceae) specialist genus Balacha Melichar, included in the Erythrogonia Melichar

5 Figure 1.2: Relationships of Cicadellinae groups in previous parsimony analyses. A: Morphology-based analysis of Cicadellidae by Dietrich [83]. B: 28S rDNA-based analysis of Membracoidea with regions of length-polymorphism included by Diterich et al. [91]. Taxa in capitals and boldface refer to Cicadellinae sensu Young [392–394], other capitalized taxa were once included in Cicadellinae sensu lato. p: in part.

generic grouping. Only three works include phylogenetic relationships among genera of Cicadellinae. In their

doctoral theses, Cavichioli [48] analyzed the relationships among the components of the generic grouping

Paromenia Melichar, and Mejdalani [219] analyzed the relationships among 21 proconiine genera, which

have their posterior meron exposed when the forewings are in rest position. In this work, Mejdalani found

this group divided into two main lineages, one named the Abana Distant generic group including 7 genera,

which was the focus of the subsequent published analysis of Ceotto & Mejdalani [51]. In the present thesis,

a phylogenetic analysis of the whole tribe Proconiini is presented based on 183 characters of the external

morphology (CHAPTER 5) and based on the analysis of 4 gene regions (CHAPTER 6). More than

20 species of Cicadellinae sensu lato are included as outgroups to test the monophyly of Cicadellinae sensu

Young. Finally, CHAPTER 7 includes a combined analysis of morphological and DNA-sequence characters

to produce a robust phylogeny of the Proconiini to study the evolution of morphological traits associated

with an unique maternal care behavior called egg-powdering.

6 Chapter 2

All Xylem Specialists Great and Small: Revisiting Minimum Body Size Constraints

2.1 Introduction

Hemiptera, the largest order of non-holometabolous insects, currently comprises over 80,000 species placed

into four suborders [328]. Morphologically, they are clearly characterized by their mouthparts, which are

modified into a piercing-sucking proboscis. Prosorrhyncha currently includes two main lineages, the phy-

tophagous [57, 114] and the ancestrally predaceous (true bugs). Most represen-

tatives of the three remaining suborders, (, psyllids, scale-insects, and whiteflies),

Archaeorrhyncha (), and Clypeorrhyncha (cicadas, leafhoppers, treehoppers, and spittle- bugs), feed by a technique called “sheath feeding”, in which the insects typically seal their stylet tips into a vascular plant cell via a solid sheath made of saliva [17]. The majority feed preferentially on phloem sap, while only cicadas (Cicadoidea), spittlebugs (Cercopoidea), and a few leafhopper lineages feed preferentially on xylem sap. Exceptionally, typhlocybine leafhoppers do not feed on vascular sap, but through a technique called “cell rupture feeding”, in which a sheath is not made and stylets move continuously or intermittently, lacerating cells, secreting watery saliva, and ingesting the resulting slurry of cell contents, usually from the mesophyll [17, 59,144].

Several obstacles are faced by phytophagous insects to fulfill their nutritional needs and, as a probable consequence, shifts to this habit occurred only in 9 out of 30 extant insect orders [185]. Plants have acquired during their evolution a wide variety of physical (e.g., trichomes and spines) and chemical (e.g., toxic secondary compounds) impediments for protection against herbivores. On the nutritional side, plant tissues generally contain a much lower total concentration of nitrogen than do tissues [116] and very low amounts of specific amino acid and micronutrients that may limit insect herbivore growth [31]. Herbivores feeding on xylem sap, although they rarely encounter chemical plant protection (e.g., nicotine in Nicotiana),

are much more limited by the total amount of nitrogen and carbon available [7, 8, 295] than phloem and

cell-content feeders.

Xylem specialists adopted several behavioral solutions in an attempt to deal with the dilute content of

7 the food source. Species tend to be mainly polyphagous [255, 277] using host-plants with varied chemical profiles, e.g., spittlebugs seem to prefer nitrogen-fixing angiosperm hosts, which likely have higher organic nitrogen concentrations in their xylem [357]. In addition, sharpshooters adjust their feeding rates to cir- cadian fluctuations in xylem chemistry, showing higher feeding rates when amino acid concentrations are greater [8,41]. Xylem specialists also exhibit physiological specializations that help maximize the utilization of xylem sap [41]. Sharpshooters excrete ammonia as the primary waste product, which is very rare in ter- restrial insects and confers maximal caloric gain; they exhibit extremely high metabolic efficiency of organic compounds (over 90% of sugars, organic acids, and most amino acids); and very high feeding rates [8,41,232].

Very high feeding rates (up to approximately 300 times their own body weight per day) have also been no- ticed for other xylem feeders, such as the meadow spittlebug [206]. Because the widely accepted cohesion theory suggests that xylem sap flows at several megapascals below atmospheric pressure [316], this creates an interesting paradox between the zoological and botanical worlds [68, 232].

Biologists interested in this paradox have proposed that the presence of a large cibarial pump in clypeor- rhynchans, which functions as a mechanical water pump with high energetic cost, may be a sine qua non of xylem feeding [277,295]. The cibarial pump is surrounded by heavily sclerotized walls providing attachment for the very powerful dilator muscles (see schematic representation in Backus [14]), which when contracted create a pressure gradient that allows suction of fluids. Additionally, the length and radius of the food canal should influence the flow of fluid into the pharynx passing by the cibarial pump. Given that, Novotny &

Wilson [256] showed that the food canal measurements and the volume of the cibarial pump are approxi- mately isometrically related to body size in Auchenorrhyncha (Clypeorrhyncha + Archaeorrhyncha). This relationship supported their further suggestion that there should be a minimum adult body size threshold

(about 8 mm) for xylem feeders, above which the suction pressure necessary to overcome resistance, as well as its energetic cost, becomes negligible [256]. Based on measurements of Auchenorrhyncha body size taken from the literature, they showed that minimum body sizes in xylem-feeders tend to be larger than those of phloem and mesophyll feeders, but only one xylem feeding lineage (Cercopoidea + Cicadoidea) is significantly larger (ANOVA of mean body size) than its putative phloem feeder sister group.

Although Novotny & Wilson [256] recognized the importance of comparing xylem-feeding lineages with their respective non-xylem feeder sister lineages, since then several phylogenetic hypotheses have been pro- posed for auchenorrhynchan lineages [45, 69, 71, 91, 100, 107, 241, 328, 382, 386]. These recent phylogenetic analyses of the Hemiptera are more detailed, including more taxa and data, than the schemes Novotny &

Wilson utilized. At the same time, they are controversial with respect to the position of Archaeorrhyncha and relationships among Clypeorrhyncha lineages (Cicadoidea + Cercopoidea + Membracoidea). Therefore

8 depending on the phylogenetic scenario, xylem-feeding in Auchenorrhyncha may have evolved more than 2 times, which was the number assumed by Novotny & Wilson [256]. Moreover, making statistical comparisons among species is problematic as these analyses assume that data points are independent. Because species are linked by their shared ancestry, this phylogenetic effect has to be taken into account. The objectives of this study were to re-investigate the evolution of body size of Clypeorrhyncha using phylogenetic con- trasts [124] as a means to correct for the phylogenetic effect and test the hypothesis that shifts to xylem feeding were associated with an increase in body size. Body size data were compiled from the literature and composite phylogenetic trees were constructed combining previous published phylogenetic hypotheses. The influence of alternative hypotheses of higher level phylogenetic relationships of the Euhemiptera (Hemiptera except Sternorrhyncha) on the hypothesis of body size increase in xylem feeding lineages was investigated.

2.2 Materials and Methods

2.2.1 Classification

To avoid confusion created by the different higher level names used by different authors usually with in- consistent endings, Hemiptera subordinal names followed those used or coined by Sorensen et al. [328].

These groupings are: Euhemiptera Zrzavy [402] (=Hemelytrata Fallen [117]), Prosorrhyncha (=Het- eropteroidea Schlee [312], =Heteropterodea Zrzavy [403]), Archaeorrhyncha (=Neurohomoptera or Ful- goroides Crampton [67], =Fulgoromorpha [11, 37], =Fulgoroidea [125, 243]), and Clypeorrhyncha (=Eu- homoptera or Cicadoides Crampton [67], =Cicadomorpha [69,84], =Clypeata Shcherbakov [320]).

Heteroptera classification followed Schuh & Slater [318]. Cicadoidea species were placed in modern genera using Duffels & van der Laan [103], then generic placement into tribes followed Moulds [241]. Cercopoidea genera placement into tribes followed Metcalf [226]. Classification of leafhoppers followed Oman et al. [264], except of Myerslopiini (Ulopinae) which herein is treated as a family of its own following Hamilton [150].

Although the assumed phylogeny treats leafhoppers as a paraphyletic taxon in respect to treehoppers, the term leafhopper will still be used while referring to Cicadellidae+. Classification of New

World Membracidae followed Deitz [78], while that of Old World tribes followed Wallace [369]. Position of

Euwalkeria and Holdgatiella in Nicomiini follows phylogenetic results of Albertson & Dietrich [3].

2.2.2 Feeding Categories

Relative to the number of Euhemiptera species, there have been few studies demonstrating their feeding niches and most of these concern a handful of pest species. Feeding niches of Euhemiptera groups were

9 Table 2.1: Feeding niches and body-size measurements of Prosorrhyncha. Feeding niches (FN) scored for Coleorrhyncha family and Heteroptera infraorders. Taxa in bold were represented as terminals in phyloge- netic trees utilized. N refers to the number of Coleorrhyncha genera and of Heteroptera families measured in those references cited. Feeding niches are: (C) carnivores (predators and vertebrate blood-suckers); (H) herbivores (mycophagous and phytophagous, excluding vascular tissue specialists); (P), phloem sap feeders; and (X), xylem sap feeders.

Taxa FN Range of body size N References Mean ± SD COLEORRHYNCHA H 1.9-5.2 (3.1 ± 0.6) 14 [57, 114] HETEROPTERA Enicocephalomorpha C 2.0-15.0 (7.5 ± 1.4) 2 [318] C 0.8-3.0 (2.3 ± 0.6) 5 [318] C 1.0-36.0 (6.4 ± 6.0) 8 [318] C H 1.0-110.0 (13.9 ± 16.9) 11 [318] C 1.1-7.4 (3.1 ± 1.7) 4 [318] C H 1.0-40.0 (6.6 ± 5.1) 16 [318] C H P X 1.0-55.0 (10.8 ± 7.2) 29 [318]

classified as stated in Tables 2.1-2.6.

Treatment of peloridiid species as herbivores was based on accounts of moss feeding by China [57] and morphological evidence by Cobben [61], who suggests xylophagy, even though Popov & Shcherbakov [273] believe that extinct Coleorrhyncha most probably fed on phloem sap. Independent shifts to phytophagy in

Heteroptera occured within Nepomorpha, Cimicomorpha, and ancestrally in Pentatomorpha. Phytophagous true bugs may feed on algae, fungi, angiosperm inflorescences, pollen, endosperm, and other parts, some species can feed on both plant and animal tissues, and comparatively few were reported also to feed on vascular sap [213, 318].

Although nymphal stages of , , and apparently feed on fungi [378] and those of the delphacid Javesella opaca feed on mosses [374], Archaeorrhyncha were classified as primarily phloem feeders, because adults most probably do so like all other species. This behavior was recorded directly for some delphacids [174,327], but indirect records based on honeydew excretion and hymenopteran tending are more common and cover a wider taxon diversity [21,143,189,387]. Similarly, phloem feeding by leafhoppers and treehoppers was mostly documented indirectly via observations of mutualistic associations

[62,80,89,190,242,284], along with a few direct observations of phloem and cell-content feeding recorded using histological and EMIF methods [16,170,207,225,244]. Additionally, other indirect records of phloem feeding by leafhoppers and planthoppers rely on their efficient vectoring of phloem-restricted phytopathogens, such as few members of the Proteobacteria and many Mollicutes, such as Spiroplasma and Candidatus Phytoplasma species [38].

Xylem feeding has been recorded directly for cicada nymphs and adult spittlebugs [200, 364, 375], but

10 Table 2.2: Feeding niches and body-size measurements of Archaeorrhyncha. Feeding niches (FN) scored for Archaeorrhyncha families. References refer to source of measurements. Taxa in bold were represented as terminals in phylogenetic trees utilized. All Archaeorrhyncha are considered phloem sap specialists (P).

Taxa FN Range of body size References Mean ± SD P 7.9-8.5 (8.2) [125] Achilidae P 3.0-7.0 (4.5 ± 1.0) [95, 98, 125, 265] Cixiidae P 2.0-11.0 (5.7 ± 1.4) [95, 125, 183, 184, 265] P 1.2-7.5 (3.7 ± 1.4) [95, 125, 265] Derbidae P 2.0-14.0 (4.4 ± 2.1) [95, 125] P 6.0-20.0 (10.6 ± 1.8) [95, 125] P 6.0-23.0 (14.5) [95] P 4.5-22.0 (8.1 ± 2.9) [95, 98, 125] P 12.0-47.0 (27.2 ± 3.2) [95, 125] P 3.5-12.0 (6.2 ± 1.5) [95, 125] P 1.9-2.1 (2.0 ± 0.1) [125] P 4.0-14.5 (9.2) [95] P 5.5-15.0 (8.3 ± 2.2) [95, 125, 182] P 3.5-11.0 (7.2) [95] P 3.0-14.0 (8.7 ± 2.5) [95, 98, 125]

indirect evidence, such as very dilute excreta, high excretion rates, and the ability to lose water for ther- moregulation, has also been suggestive that xylem feeding is the general rule in Cicadoidea and Cer- copoidea [56, 206, 307]. Among leafhoppers, presently only species belonging to Mileewini [255], and to

Cicadellini and Proconiini, commonly called sharpshooters, have been recorded as feeding primarily on xylem sap. Sharpshooters are so named because while feeding they constantly shoot out watery droplets of excrement as a result of their high excretion rate. As added indirect evidence, many sharpshooter species are vectors of the xylem-borne proteobacterium , as are spittlebugs and some members of the leafhopper tribe Pagaroniini [296]. Despite the few feeding records, xylem feeding is also assumed herein for leafhopper representatives of Errhomenini, Evacanthini, Makilingiinae, Phereurhininae, Signoretiinae, and

Tinterominae due to the enlarged frontal sclerite of the face (=lining of the cibarial chamber) shared by these groups and all proven xylem specialists, which supports strong muscles for efficient inward water pumping needed by xylem-specialists. Following the same reasoning, Hamilton [150] proposed previously that mem- bers of the leafhopper family Myerslopiidae were also xylem-specialists, like extinct early membracoids [149].

However, although myerslopiids are herein treated as xylem-specialists, no studies have been published on their biology and they also have been hypothesized to be fungivores [339] or even carnivores [250].

2.2.3 Body Size Measurements

Fifty-four published taxonomic monographs and comprehensive journal articles were selected to compile 212 body size ranges for the Euhemiptera. Heteroptera and Archaeorrhyncha body size ranges were compiled

11 Table 2.3: Feeding niches and body-size measurements of Cercopoidea (Clypeorrhyncha). Feeding niches (FN) scored for Cercopoidea families and tribes. N refers to the number of Cercopoidea genera measured in those references cited. Taxa in bold were represented as terminals in phylogenetic trees utilized. All Cercopoidea are considered xylem sap specialists (X).

Taxa FN Range of body size N References Mean ± SD APHROPHORINAE Aphrophorini X 5.1-17.0 (9.3 ± 3.7) 10 [96, 98, 112, 146] Cloviini X 5.5-11.0 (8.2) 1 [96, 112] Lepyroniini X 5.0-8.5 (6.7 ± 0.6) 3 [96, 112, 146, 266] Philaeniini X 3.5-9.2 (6.5 ± 1.2) 5 [96, 146] Philagrini X 5.0-13.0 (7.8 ± 3.9) 2 [96] Ptyelini X 5.0-12.0 (8.1 ± 1.7) 3 [96, 146] CALLITEXTIINAE Callitextiini X 6.0-12.0 (8.1 ± 2.0) 4 [96] Considiini X 11.0-11.5 (11.2) 1 [98] CERCOPINAE Cercopini X 7.5-9.8 (8.6) 1 [146] Cosmoscartini X 5.5-22.0 (16.4 ± 4.4) 3 [96, 98] Eoscartini X 3.5-9.0 (7.1 ± 1.9) 2 [96, 98, 112] Locrisini X 5.5-6.0 (5.7) 1 [112] Rhinaulacini X 4.0-8.6 (6.4 ± 0.9) 1 [98, 112] Suracartini X 17.5-19.0 (18.2) 1 [112] Tomaspidini X 4.5-5.0 (4.7) 1 [98] X 2.7-5.3 (4.0) 1 [146] X 6.1 1 [151] HINDOLINAE Hindolini X 4.0-10.0 (6.6 ± 0.6) 3 [96, 98, 112] Hindoloidini X 3.5-8.0 (6.1 ± 2.6) 2 [98, 112] MACHAEROTINAE Machaerotini X 4.0-8.0 (6.0) 1 [96, 112]

from the literature at the familial level, Membracidae at the tribal level, and Cercopoidea, Cicadoidea,

Cicadellidae, and Coleorrhyncha at the generic level. These ranges and respective bibliographic references can be found in Tables 2.1-2.6. Emphasis was placed on Cicadellidae, for it is the largest Hemiptera family and has the most variation in both body size and sap-sucking niches.

Most body length measurements compiled included forewings at rest. Exceptions were all Cicadoidea measurements and those taken from Distant [95, 96, 98] for Cercopoidea, Achilidae, Cixiidae (in part), Der- bidae (in part), Dictyopharidae, Eurybrachidae, Flatidae (in part), Fulgoridae, Kinnaridae, Lophopidae (in part), Nogodinidae (in part), and Ricaniidae. In most of these groups, unlike other Hemiptera, the forewings at rest extend considerably beyond the tip of the abdomen; thus, including them would substantially inflate the body length measurement. Cicadoidea measurements from Moulds [240] were based on photographs given.

12 Table 2.4: Feeding niches and body-size measurements of Cicadoidea (Clypeorrhyncha). Feeding niches (FN) scored for Cicadoidea families and tribes. N refers to the number of Cicadoidea genera measured in those references cited. Taxa in bold were represented as terminals in phylogenetic trees utilized. All Cicadoidea are considered xylem sap specialists (X).

Taxa FN Range of body size N References Mean ± SD CICADETTINAE Chlorocystini X 11.0-41.0 (21.9 ± 7.7) 8 [94, 102, 240] Cicadettini X 9.4-34.0 (18.3 ± 4.6) 12 [75, 94, 95, 98, 102, 240, 266] Huechysini X 12.0-30.0 (22.3 ± 6.2) 3 [94] Parnisini X 12.5-19.0 (15.7 ± 3.0) 3 [94, 95, 98] Prasiini X 21.0-30.0 (25.5) 1 [240] Sinosenini X 27 1 [94, 95] Taphurini X 10.0-42.0 (18.6 ± 9.8) 7 [28, 94, 95] Burbungini X 13.8-17.7 (15.8) 1 [240] Cicadini X 19.0-32.0 (24.3 ± 1.6) 2 [94, 95, 240] Cryptotympanini X 13.0-51.0 (32.6 ± 7.5) 8 [28, 94, 95, 98, 102, 240] Cyclochilini X 25.0-37.0 (33 ± 3.5) 2 [240] Dundubiini X 12.0-62.0 (28.8 ± 7.2) 21 [94, 95, 98, 102, 240] Fidicinini X 8.0-45.0 (22.0 ± 19.8) 2 [94, 98] Gaeanini X 25.0-38.0 (29.0 ± 4.2) 2 [94, 95] Jassopsaltriini X 12.7 1 [240] Moganniini X 13.0-19.0 (16.0) 1 [94, 95] Oncotympanini X 17.0-30.0 (23.5) 1 [94, 95] Platypleurini X 15.0-32.0 (21.2 ± 2.8) 3 [94, 95] Polyneurini X 30.0-46.0 (37.5 ± 4.9) 3 [94, 95] Psithyristriini X 14.0-68.0 (31.5 ± 18.4) 2 [94, 95, 98] Tacuini X 47.0-57.0 (52.0) 1 [94] Talaingini X 23.0-26.0 (24.5) 1 [94, 95] Tamasini X 13.5 1 [240] Thophini X 24.4-35.6 (30.0) 1 [240] Zammarini X 26.2-31.3 (28.8) 1 [307] TETTIGADINAE Platypediini X 13.5-30.0 (19.8 ± 2.7) 2 [28, 75] Tettigadini X 18.0-23.0 (20.5) 1 M. Villet, pers. comm. Tibicinini X 21.0-35.0 (30.2 ± 4.1) 3 [28, 94, 95] X 27.0-33.0 (30.0) 1 [240]

2.2.4 Higher-Level Phylogenetic Hypotheses

Alternative incongruent phylogenetic hypotheses for major groupings of the Hemiptera have been proposed by different authors [69,107,111,113,382,386]. However, based on recent molecular evidence, there is general agreement that Sternorrhyncha is the sister group to Euhemiptera [45, 100, 328]. Because sternorrhynchans are primarily phloem feeders, they were not considered in the present analysis. Five scenarios concerning the phylogenetic relationships of Prosorrhyncha, Archaeorrhyncha, Cicadoidea, Cercopoidea, and Membracoidea were selected to check the influence of phylogeny on body size increase in euhemipterans (see Figure 2.1).

13 Table 2.5: Feeding niches and body-size measurements of treehoppers (Clypeorrhyncha). Feeding niches (FN) scored for treehopper families including Membracidae subfamilies and tribes. References refer to source of measurements. Taxa in bold were represented as terminals in phylogenetic trees utilized. All treehoppers are considered phloem sap specialists (P).

Taxa FN Range of body size References Mean P 3.0-30.0 (16.5) [79, 96] MELIZODERIDAE P 4.0-6.0 (5.0) [79] MEMBRACIDAE CENTROTINAE Beaufortianini P 4.8-9.0 (6.9) [369] Boccharini P 6.0-6.7 (6.4) [369] Boocerini P 2.0-7.0 (4.5) [78, 369] Centrocharesini P 3.7-4.5 (4.1) [369] Centrodontini P 2.0-4.7 (3.3) [369] Centrotini P 3.0-10.0 (6.5) [96, 266, 369] Centrotypini P 6.0-14.0 (10.0) [369] Choucentrini P 5.3-9.0 (7.1) [369] Ebhuloidesini P 3.3-5.5 (4.4) [369] Gargarini P 2.0-7.0 (4.5) [28, 96, 266, 369] Hypsauchenini P 4.5-8.0 (6.2) [96, 369] Leptobelini P 5.3-10.0 (7.6) [369] Leptocentrini P 4.0-10.0 (7.5) [96, 98, 369] Lobocentrini P 6.0-7.3 (6.6) [369] Maarbarini P 3.0-9.0 (6.0) [369] Micreunini P 7 [369] Monobelini P 2.0-4.8 (3.4) [369] Nessorhinini P 4.5-10.0 (7.2) [369] Oxyrhachini P 5.0-9.0 (7.0) [96, 369] Pieltainellini P 4.3-5.4 (4.8) [369] Platycentrini P 4.0-7.5 (5.7) [78, 369] Terentiini P 2.6-12.0 (7.3) [112, 369] Xiphopoeini P 4.7-6.3 (5.5) [369] DARNINAE Cymbomorphini P 6.0-9.0 (7.5) [78] Darnini P 4.0-10.0 (7.0) [78] Hemikypthini P 8.0-15.0 (11.5) [78] Hyphinoini P 8.0-12.0 (10.0) [78] Procyrtini P 3.5-4.0 (3.7) [78] ENDOIASTINAE P 3.0-4.0 (3.5) [79] HETERONOTINAE P 3.0-8.0 (5.5) [78] MEMBRACINAE Aconophorini P 4.0-6.0 (5.0) [78] Hoplophorionini P 3.0-14.0 (8.5) [28, 78] Hypsoprorini P 3.0-7.0 (5.0) [78] Membracini P 2.0-8.0 (5.0) [28, 78] Talipedini P 3.0-5.0 (4.0) [78] NICOMIINAE Nicomiini P 5.1-10.0 (7.6) [3] Tolaniini P 4.5-8.9 (6.7) [4] SMILIINAE Acutalini P 4.5-6.0 (5.2) [78] Amastrini P 3.0-5.5 (4.2) [78] Ceresini P 4.0-12.2 (8.1) [28, 78, 180] Micrutalini P 3.5-4.0 (3.7) [78] Polyglyptini P 3.0-6.0 (4.5) [28, 78] Quadrinareini P 3.0-3.5 (3.2) [78] Smiliini P 3.0-10.0 (6.5) [28, 78] Telamonini P 7.0-13.0 (10.0) [28] Thuridini P 4 [78] Tragopini P 2.5-5.0 (3.7) [78] STEGASPIDINAE Centronodini P 4.0-9.0 (6.5) [78] Microcentrini P 4.5-9.0 (6.75) [28, 78] Stegaspini P 4.0-9.0 (6.5) [78]

14 Figure 2.1: Alternative phylogenetic scenarios for higher-Level Euhemiptera. Scenarios numbered 1-5. Let- ters indicate relationships recovered in analyses of: a, Evans [111]; b, Evans [113]; c, Emel’yanov [107] and Yang [382]; d, Campbell et al. [45]; e, Sorensen et al. [328]; f, von Dohlen & Moran [100]; g, Yoshizawa & Saigusa [386]

2.2.5 Lower-Level Phylogenetic Hypotheses

Phylogenetic relationships within the abovementioned Euhemiptera suborders were compiled at different taxonomic levels depending on the availability of published phylogenies and diversity of feeding niches.

Prosorrhyncha (Coleorrhyncha and Heteroptera infraorders) relationships were based on Wheeler et al. [373]

(see Figure 2.2). Cercopoidea tribal relationships were inferred from Cryan’s [69] phylogeny based on com- bined 18S rRNA, 28S rRNA, and H3, for Cicadomorpha, which had the largest sample of Cercopoidea species

(see Figure 2.3). A phylogeny for Cicadoidea tribes was given by Moulds [241] (see Figure 2.4).

Multiple phylogenetic hypotheses were published for each of the remaining Hemiptera groupings. Thus, phylogenetic supertrees were produced using the MRP consensus method [24, 25, 285], which allows for the combination of multiple source hypotheses independent of their taxon sample, type of data, or the tree building algorithm used to construct them. Clades of these rooted source phylogenies without outgroups were used to construct a large consensus matrix by coding using Purvis’ [281] modification with the aid of the computer program RadCon 1.1.5 [358]. Polyphyletic taxa present in source phylogenies were divided into monophyletic units and numbered sequentially, e.g., Nogodinidae, Nogodinidae1, Nogodinidae2. . . , but keeping the same name for congeneric or contribal terminals across source phylogenies.

Source phylogenies for Archaeorrhyncha families were the morphological analyses of Asche [11], Bourgoin

[36] (as modified and depicted in [37]), Chen & Yang [54], Emel’yanov [108], and Muir [243], strict consensus of parsimony trees based on 18S rDNA of Bourgoin et al. [37], parsimony and mixed-model Bayesian trees based on combined 18S rDNA, 28S rDNA, H3, and wg of Urban & Cryan [363], and NJ and ME trees based on 16S rDNA modeled by T3P of Yeh et al. [385]. Achilidae, Issidae, and Nogodinidae were polyphyletic.

15 Table 2.6: Feeding niches and body-size measurements of leafhoppers (Clypeorrhyncha). Feeding niches (FN) scored for leafhopper families including Cicadellidae subfamilies and tribes. Numbers in parentheses are estimated number of genera/species included in respective lineage. N refers to the number of genera measured in those references cited. Taxa in bold were represented as terminals in phylogenetic trees utilized. Feeding niches are: (M) cell content feeders; (P), phloem sap feeders; and (X), xylem sap feeders.

Taxa FN Range of body size N References Mean ± SD MYERSLOPIIDAE (3/21) X 3.1-7.6 (5.5 ± 0.1) 2 [112, 178, 340] CICADELLIDAE ACOSTEMMINAE (7/20) P 10.0-14.5 (11.7 ± 2.5) 3 [96, 199] AGALLIINAE (37/580) P 2.2-6.0 (3.8 ± 0.6) 14 [81, 96, 98, 99, 112, 192, 198, 260, 261, 266] APHRODINAE (7/199) P 3.0-7.0 (4.8 ± 0.7) 5 [81, 96, 192, 266] AUSTROAGALLOIDINAE (1/8) P 5.0-10.0 (7.5) 1 [112] BYTHONIINAE (1/5) P 7.0-9.0 (8.0) 1 [194] CICADELLINAE Anoterostemmini (4/12) P 3.0-4.0 (3.5) 1 [99] Cicadellini (245/2150) X 3.4-25.1 (9.5 ± 3.0) 239 [81, 112, 192, 266, 393, 394] Errhomenini (10/70) X 7.0-8.0 (7.5) 1 [266] Evacanthini (14/100) X 4.5-8.0 (6.3 ± 0.8) 10 [81, 96, 99, 192, 266] Mileewini (4/90) X 3.5-6.5 (4.7± 1.0) 3 [96, 99, 192, 198, 391] Pagaroniini (4/60) X 6.7-10.9 (8.8) 1 [155] Proconiini (56/600) X 6.5-22.5 (13.8 ± 3.1) 56 [81, 392] COELIDIINAE Coelidiini (52/387) P 7.0-8.0 (7.5) 1 [192] Teruliini (50/300) P 5.5-8.0 (6.7) 1 [81] Tharrini (3/125) P 5.3-6.0 (5.6) 1 [112] Tinobregmini (5/15) P 4.0-6.5 (5.2) 1 [81] DELTOCEPHALINAE Acinopterini (2/50) P 4.0-6.5 (5.2) 1 [194] (300/2300) P 1.3-10.0 (5.4 ± 1.1) 111 [81, 96, 99, 112, 192, 194, 197, 267] Balcluthini (2/60) P 2.0-4.9 (3.3 ± 0.1) 2 [81, 96, 99, 194, 267] Chiasmini (1/25) P 2.5-5.0 (3.75) 1 [96, 99, 112] Cicadulini (5/50) P 3.2-7.0 (4.9 ± 0.3) 2 [81, 96, 99, 267] Coryphaelini (1/1) P 4.5-6.0 (5.2) 1 [267] Deltocephalini (140/1000) P 1.8-9.5 (3.9 ± 1.0) 41 [81, 96, 99, 112, 192, 194, 197, 267] Doraturini (7/300) P 2.5-6.0 (3.9 ± 0.5) 5 [81, 96, 99, 194, 267] Fieberiellini (10/50) P 7.0-7.2 (7.1) 1 [81] Goniagnathini (2/58) P 5.0-7.5 (6.2) 1 [96] Grypotini (3/10) P 4.0-5.0 (4.5) 1 [267] Hecalini (30/260) P 2.3-11.0 (6.1 ± 2.2) 8 [81, 96, 98, 99, 112, 194, 197] Luheriini (1/1) P 7 1 [194] (30/475) P 2.0-7.0 (4.0 ± 0.9) 10 [81, 112, 194, 267] (24/285) P 2.5-5.0 (3.6 ± 0.8) 6 [81, 96, 112, 192, 194, 267] Paralimnini (70/350) P 1.9-6.8 (3.9 ± 0.8) 25 [81, 96, 99, 112, 194, 197, 267] Platymetopiini (27/170) P 3.5-8.2 (5.6 ± 1.3) 8 [81, 112, 192] Scaphoideini (14/306) P 3.5-7.0 (5.4 ± 0.4) 3 [81, 96, 99, 112, 192] Scaphytopiini (9/260) P 3.5-6.0 (4.7 ± 0.2) 4 [81, 99, 194] Stenometopiini (10/100) P 2.0-7.0 (4.3 ± 0.7) 10 [81, 96, 99, 112, 194] EUACANTHELLINAE (1/2) P 4.5-8.0 (6.2) 1 [112] EUPELICINAE Dorycephalini (4/20) P 5.5-14 (4.2 ± 8.5) 2 [81] Eupelicini (1/10) P 5.3-7.2 (6.2) 1 [267] Paradorydiini (4/65) P 3-14 (7.8 ± 1.0) 2 [99, 112] EURYMELINAE (33/120) P 3.2-15.0 (6.3 ± 1.9) 29 [112] GYPONINAE (90/1300) P 7.0-12.0 (9.1 ± 0.8) 5 [81, 197] HYLICINAE (9/30) P 9.0-18.0 (12.9 ± 3.4) 7 [96, 99] IASSINAE Iassini (40/650) P 3.0-9.5 (6.2 ± 1.2) 8 [81, 96, 98, 112, 192, 199, 266] Krisnini (1/40) P 8.5-14.5 (11.5) 1 [96, 194, 199] Reuplemmelini (3/6) P 5.8-10.0 (8.1 ± 0.6) 2 [112] Trocnadini (2/10) P 4.5-10.0 (7.25) 1 [112] IDIOCERINAE (90/720) P 2.2-9.0 (4.8 ± 1.1) 16 [81, 96, 112, 266] KOEBELIINAE (1/5) P 4.8-9.2 (7.0) 1 [263]

16 Table 2.6: ...Continued.

Taxa FN Range of body size N References Mean ± SD CICADELLIDAE (cont.) LEDRINAE Ledrini (7/50) P 6.0-28.0 (13.2 ± 6.5) 8 [96, 112, 266] Petalocephalini (30/200) P 7.5-18.5 (12.5 ± 3.1) 9 [96, 98, 181, 192] Stenocotini (6/20) P 7.0-24 (12.7 ± 3.1) 6 [112] Thymbrini (16/90) P 4.6-16.0 (8.2 ± 2.5) 13 [112] Xerophloeini (3/30) P 4.5-9.5 (7.7 ± 1.4) 2 [81, 181] MACROPSINAE Macropsini (24/540) P 2.8-7.0 (4.8 ± 1.4) 9 [81, 98, 112, 148, 192, 266] Neopsini (2/10) P 3.5-5.3 (4.6 ± 0.3) 2 [148, 197] MAKILINGIINAE (1/23) X 6 1 [110] MEGOPHTHALMINAE (7/40) P 2.5-4.0 (3.2) 1 [266] MUKARIINAE (2/20) P 4.0-4.0 (4.0) 1 [96] NEOBALINAE (9/40) P 3.7-12.0 (5.6 ± 1.8) 7 [194, 197] NEOCOELIDIINAE (18/130) P 4.0-5.0 (4.5 ± 0.7) 2 [81] NIONIINAE (2/12) P 4.0-5.0 (4.5) 1 [81] NIRVANINAE Balbilini (2/20) P 7.0-7.0 (7.0) 1 [96] Macroceratogoniini (1/2) P 9.0-9.0 (9.0) 1 [112] Nirvanini (36/150) P 3.5-10.0 (5.9 ± 1.8) 14 [96, 99, 112, 192, 194] Occinirvanini (1/1) P 6.0-6.0 (6.0) 1 [112] PARABOLOPONINAE (20/68) P 7.0-7.3 (7.1 ± 0.2) 2 [112, 192] PENTHIMIINAE (46/245) P 2.8-9.0 (5.5 ± 1.4) 15 [81, 96, 99, 112, 194, 381] PHEREURHININAE (3/11) X 9.0-13.8 (11.0 ± 1.7) 3 [182] SELENOCEPHALINAE Bhatiini (7/13) P 4.8-7.0 (5.6 ± 1.2) 2 [96, 112] Drabescini (4/52) P 7.0-10.0 (8.5) 1 [96, 112, 192] Ianeirini (2/12) P 6.5-7.0 (6.7) 1 [96] Selenocephalini (19/127) P 4.0-8.0 (6.0 ± 0.0) 2 [96] SIGNORETIINAE (2/20) X 7.0-9.0 (8.0) 1 [96] TARTESSINAE (38/200) P 4.0-13.0 (9.1 ± 1.4) 4 [96, 112] TINTEROMINAE (1/2) X 5.0-5.6 (5.3) 1 [136] TYPHLOCYBINAE Alebrini (32/200) M 2.5-4.6 (3.4 ± 0.5) 27 [99, 105, 197, 266, 388] Dikraneurini (74/553) M 2.0-4.2 (3.4 ±0.5) 9 [99, 112, 266] Empoascini (83/1400) M 2.3-5.2 (3.6 ± 0.4) 16 [96, 99, 104–106, 112, 192, 197, 266] Erythroneurini (60/2000) M 1.9-6.0 (3.4 ± 0.7) 43 [99, 104–106, 112, 266] Helionini (1/2) M 3.0-4.0 (3.5) 1 [96] Typhlocybini (62/615) M 1.2-5.5 (3.8 ± 0.6) 19 [96, 99, 104, 105, 112, 192, 266] Zyginellini (29/136) M 2.6-5.1 (3.7 ± 0.8) 8 [104] ULOPINAE Cephalelini (6/50) P 3.5-15.0 (9.3 ± 0.1) 2 [112, 178] Ulopini (22/110) P 2.56-6.0 (3.9 ± 0.7) 11 [96, 98, 112, 178, 192, 266] XESTOCEPHALINAE Portanini (2/40) P 4.0-7.1 (5.5) 1 [194] Xestocephalini (5/160) P 2.0-5.7 (3.1 ± 0.9) 3 [81, 96, 99, 112, 194]

Source phylogenies for Membracoidea including treehopper families and Cicadellidae tribes were the mor- phological analyses of Membracoidea lineages of Dietrich & Deitz [79], leafhopper subfamilies of Dietrich [83],

Evacanthinae tribes of Dietrich [85], and Deltocephalinae tribes of Zahniser & Dietrich [397]; Membracoidea topology recovered by the parsimony analysis of combined 18S rDNA, 28S rDNA, and H3 of Cryan [69]; and mixed-model Bayesian best estimate based on combined morphology and 28S rDNA of Dietrich et al. [88].

Terminal subfamilies from Dietrich [83] were, whenever necessary, assumed to be represented by the typical

17 Figure 2.2: Phylogeny of Prosorrhyncha. According to Wheeler et al. [373]. Colored circles on nodes represent ancestral feeding niches optimized using parsimony. Green = herbivore; Red = carnivore; Blue = phloem specialist; and Black = xylem specialist.

tribe. The unplaced genus Paulianiana was excluded from source trees. Polyphyletic lineages were Coe-

lidiinae (Coelidiini+ Equeefini+ Macroceratogoniini+ Teruliini), Typhlocybinae (Alebrini+ Empoascini+

Erythroneurini+ Jorumini+ Typhlocybini), Neobalinae, and the tribes Athysanini, Hecalini, and Opsiini.

Source phylogenies for Membracidae tribes were the morphological analyses of Dietrich et al. [90] and

Wallace [369], and the parsimony analysis based on combined EF-1α and 28S rDNA of Cryan et al. [70],

and the parsimony analysis based on combined morphology, EF-1α, 28S rDNA, 18S rDNA, and wg of

Cryan et al. [71]. Unplaced genera Antillotolania, Ceraon, Deiroderes, Eufairmairia, Elaphiceps, Sextius,

Smilidarnis, and Tyrannotus were removed from source trees. Polyphyletic tribes were Amastrini, Boocerini,

Leptocentrini, Membracini, Polyglyptini, and Smiliini.

Heuristic most-parsimonious trees searches were run using the large consensus matrix under the parsi- mony criterion in PAUP* 4.0b10 [336], with characters treated as irreversible and enforcing constraints for the polyphyletic groupings mentioned above. Searches for the Archaeorrhyncha supertree were performed with 10,000 random addition replicates and TBR branch swapping. On the other hand, due to the high amount of missing data and the limited computing resources, maxtrees was set to 10,000 and 100 indepen- dent searches with TBR banch swapping for the Membracoidea and Membracidae supertrees were run with an initial step of 1,000 random addition replicates, saving only 20 optimal trees each replicate (nchuck=20); followed by a final swapping step on the optimal trees saved from the previous step. The last step would

18 Figure 2.3: Phylogeny of Cercopoidea. According to Cryan [69]. Gray branches were recovered with less than 80% bootstrap support. Colored circles on nodes represent ancestral feeding niche optimized using parsimony. Black = xylem specialist. invariably terminate prematurely due to reaching the maxtrees limit. Majority rule consensus of most- parsimonious supertrees obtained for Archaeorrhyncha, Membracoidea, and Membracidae were used as the assumed phylogenetic scenario.

2.2.6 Ancestral Character Optimizations

Ancestral states of the categorical feeding modes and continuous body size measurements were reconstructed using Mesquite 1.11 [205]. Categorical feeding niches were optimized using unordered parsimony, while continuous measurements of minimum, maximum, and mean body sizes were reconstructed using squared- change parsimony [204] assuming a rooted topology and simultaneous divergences in polytomous nodes.

2.2.7 Phylogenetic Independent Contrasts

Relationships between a shift to xylem feeding and body size increase in Euhemiptera were assessed using

Felsenstein’s [124] method of independent contrasts as a way to correct for non-independence of data points due to phylogenetic inertia [153]. The independent categorical variable for feeding mode was coded as 0:

19 Figure 2.4: Phylogeny of Cicadoidea. According to Moulds [241]. Body size measurements were not available for the taxon marked with an asterisk. Colored circles on nodes represent ancestral feeding niche optimized using parsimony. Black = xylem specialist. non-xylem specialist and 1: xylem specialist. The dependent continuous variables of minimum, maximum, and mean body sizes were log-transformed to approximate normality. Phylogenetic contrasts for body sizes were calculated with the aid of CAIC 2.6.9 [282] using the Brunch algorithm, which is the preferred algorithm when the predictor variable is categorical. Branch lengths of the phylogeny were assumed to be equal, following a punctuational model of change. Contrasts were tested for statistical significance against zero using a one-tailed Wilcoxon signed ranked test.

20 2.3 Results

2.3.1 Constructed Supertrees

The MRP consensus matrix of Archaeorrhyncha families included 27 terminal taxa and 16 characters and resulted in 387 most-parsimonious trees (L=257, CI=0.49, RI=0.80, RC=0.39), from which the majority-rule consensus is shown in Figure 2.5. The majority-rule consensus of 99,987 most-parsimonious trees (L=288,

CI=0.62, RI=0.93, RC=0.57) resulting from the analysis of the Membracoidea consensus matrix, which included 104 subfamilies and tribes and 177 characters, is shown in Figure 2.6. Likewise for Membracidae subfamilies and tribes, in Figure 2.8 is shown the majority-rule consensus of 94,152 most-parsimonious trees

(L=146, CI=0.68, RI=0.91, RC=0.62) derived from the analysis of the consensus matrix, which included 66 terminal taxa and 99 characters. Terminal taxa counts did not include the MRP ancestor.

2.3.2 Ancestral Feeding Niches

Optimization of feeding habits on the phylogenies utilized (Figures 2.2-2.8) suggests that, three or four shifts to xylem-feeding occurred in Euhemiptera. In scenarios supporting the monophyly of Clypeorrhyncha

(scenarios 1-4 in Figure 2.1), xylem-feeding appears to be the ancestral habit, which consequently indicates that the ambiguous Membracoidea ancestor was a xylem feeder. However in scenario 5, where Membracoidea is the sister-group to Archaeorrhyncha, the Membracoidea ancestor is recovered as a phloem feeder, and an independent origin of xylem-feeding in myerslopiids must be assumed. Additionally, two shifts to xylem- feeding occurred within Cicadellidae (see Figure 2.6): one in Errhomenini and another in a large lineage that includes the proven xylem feeders. From within this xylem-feeding clade, shifts to cell-content and back to phloem feeding occurred.

2.3.3 Ancestral Body Sizes and Phylogenetic Contrasts

Minimum, maximum, and mean ancestral body sizes calculated by squared-change parsimony are shown in Table 2.7 for xylem feeders and their sister clades. Six sister group comparisons were made in each phylogenetic scenario, five of them within Membracoidea and, therefore, shared by all scenarios. Although in all scenarios most phylogenetic contrasts were positive, suggesting an increase in body size of the xylem- feeding lineage, no scenario supported a statistically significant overall increase in body size of xylem-feeders

(p>0.05). This lack of significance was mainly due to the sole, but strong, negative contrast between putative xylem-feeding myerslopiids and the phloem-feeding ancestor of the remaining Membracoidea, found for all body size measurements and in every phylogenetic scenario (Table 2.7). In summary, no statistical evidence

21 Figure 2.5: Phylogeny of Archaeorrhyncha. Majority-rule consensus of supertrees generated based on Asche [11], Bourgoin [36], Bourgoin et al. [37], Chen & Yang [54], Emel’yanov [108], Muir [243], Urban & Cryan [363], and Yeh et al. [385]. Gray branches correspond to branches collapsed in strict consensus. Body size measurements were not available for taxa marked with an asterisk. Colored circles on nodes represent ancestral feeding niche optimized using parsimony. Blue = phloem specialist. was found to support an increase in body size related to a xylem-feeding habit in Clypeorrhyncha.

2.4 Discussion

After correcting for phylogenetic dependence and taking into consideration possible alternative higher-level phylogenetic scenarios, statistical analyses of euhemipteran body sizes did not show a significant increase in xylem feeding lineages, predicted by a previous study. Novotny & Wilson [256] suggested a tendency in xylem feeder minimum body sizes to be larger than minimum body sizes of taxa with other modes of feeding and, in particular, a statistically significant increase in body size of the xylem-feeding clade of cicadas+spittlebugs against other lineages, but not of xylem-feeding leafhoppers. The possible reasons why the present analysis yielded disparate results from the previous are: (1) incongruent body size datasets, (2) the correction for

22 Figure 2.6: Phylogeny of Membracoidea. Majority-rule consensus of supertrees generated based on Diet- rich [83, 85], Dietrich & Deitz [79], Dietrich et al. [88], Cryan [69], and Zahniser & Dietrich [397]. Gray branches correspond to branches collapsed in strict consensus. Non-Cicadellidae taxa in capitals. Body size measurements were not available for taxa marked with an asterisk. Detail of the Deltocephalinae clade shown in Figure 2.7. Colored circles on nodes represent ancestral feeding niche optimized using parsimony. Blue = phloem specialist; Pink = cell-content feeder; and Black = xylem specialist.

23 Figure 2.7: Phylogeny of Deltocephalinae. Expanded clade from the majority-rule consensus of supertrees shown in Figure 2.6. Gray branches correspond to branches collapsed in strict consensus. Body size mea- surements were not available for taxa marked with an asterisk. Colored circles on nodes represent ancestral feeding niche optimized using parsimony. Blue = phloem specialist.

24 Figure 2.8: Phylogeny of Membracidae. Majority-rule consensus of supertrees generated based on Dietrich et al. [90], Cryan et al. [70,71], and Wallace [369]. Gray branches correspond to branches collapsed in strict consensus. Body size measurements were not available for taxa marked with an asterisk. Colored circles on nodes represent ancestral feeding niche optimized using parsimony. Blue = phloem specialist.

25 Table 2.7: Body size ancestral reconstructions and phylogenetic constrasts of xylem and non-xylem feeding lineages. Ancestral minimum, maximum, and mean body sizes reconstructed by squared-change parsimony of xylem (in bold) and non-xylem feeder sister lineage. Phylogenetic contrasts of ln body sizes of corresponding sister group comparison are given in italics within parentheses. Ranges of body sizes in mm and constrasts are given to accommodate different results across higher-level phylogenetic scenarios. No statistically significant increase in body size of xylem-feeders was found in all scenarios (p>0.05).

Scenarios Sister group comparisons Minimum Maximum Mean

1 I Clypeorrhyncha 7.14 14 9.77 vs. (0.47 )(0.19 )(0.31 ) Archaeorrhyncha 3.7 11.31 6.06 2-4 II Clypeorrhyncha 7.44-11.56 14.23-19.07 10.34-14.53 vs. (0.50-0.63 )(0.21-0.31 )(0.34-0.45 ) Prosorrhyncha+Archaeorrhyncha 3.80-5.03 11.42-12.87 6.43-7.69 5 III Cicadoidea+Cercopoidea 10.9 18.22 13.90 vs. (0.46 )(0.19 )(0.31 ) Archaeorrhyncha+Membracoidea 5.33 12.27 8.05 1-5 IV Myerslopiidae 3.1 7.6 5.50 vs. (-0.36 )(-0.27 )(-0.28 ) remaining Membracoidea 6.50-7.78 13.10-14.24 9.59-10.70 1-5 V Errhomenini 7 8 7.50 vs. (0.23 )(0.02 )(0.12 ) Clade A 5.17-5.18 8.33-8.34 6.16-6.17 1-5 VI Cicadellinae clade 5.41 9.44 6.25 vs. (0.09 )(0.19 )(0.14 ) Coelidiinae+Neocoelidiinae 5.06 7 4.54 1-5 VI Evacanthini+Pagaroniini 5.54 9.28 7.21 vs. (0.04 )(0.04 )(0.06 ) Balbilini+Nirvanini 5.31 8.64 6.48 1-5 VIII Mileewini+Tinterominae 4.17 6.3 4.73 vs. (0.38 )(0.04 )(0.14 ) Typhlocybinae 2.38 5.88 2.94

phylogenetic dependence while assuming different phylogenetic scenarios, and/or (3) the addition of lineages assumed to be xylem-feeders not considered previously.

Although for the present study, body size data were compiled from an additional 47 bibliographic sources, these data seem to be congruent with those of the previous study [256]. The tendency for minimum body sizes of xylem feeders to be large is also seen in the present data (Figure 2.9). Moreover, without phylogenetic correction, the present data do suggest that the mean body size of xylem feeders is significantly larger, even of xylem-feeding leafhoppers (ANOVA, α=0.05). On the other hand, Novotny & Wilson [256] stipulated a threshold of 8.0 mm in body length, whereby xylem feeders would be large enough to overcome the negative tension and efficiently feed on xylem fluid (assuming xylem tension of 1-2 MPa). The present data do not support this conclusion because only in two out of the 30 sister taxa comparisons (across all scenarios) was the minimum size of the xylem feeder lineage more than 8 mm (Table 2.7). Comparisons that suggested an

26 Figure 2.9: Distribution of minimum ln body size of Auchenorrhyncha by feeding niche. Xylem feeders are further partitioned by higher-level groupings (A: cicadas+spittlebugs and B: leafhoppers). Vertical lines represent size ranges; box surrounds the interquartile range; plus sign (+) represents the median; and n=number of genera studied. increase in minimum body size over 8 mm were due to Cicadoidea (ancestral minimum size 21.51-21.79 mm) influencing drastically the optimization of minimum body size of the xylem feeder lineage.

This was the first attempt to study ecological traits of Auchenorrhyncha lineages using an explicit method of phylogenetic correction and estimation of ancestral states. Even though a large amount of phylogenetic information has been published in the last 20 years, many lineages are still understudied, and many published phylogenies are based on small datasets. Consequently, their results are not very robust. Due to the immense number of species contained in Euhemiptera, terminal taxa in this study had to be higher taxonomic categories (202 used herein). For taxa with multiple phylogenetic hypotheses proposed, supertrees were constructed using MRP even though theoretical problems with its algorithm have been suggested [132,272] and useful branch length information was lost. All the same, with the lack of robust phylogenetic hypotheses,

MRP is still a suitable method because of its ability to combine topologies derived from different kinds of datasets and its supertrees are fairly easy to construct and interpret [32].

Although statistical analyses on the contrasts of each alternative higher-level phylogenetic scenario sug- gested the same results, they must be viewed cautiously. Five out of the six contrasts in each scenario were essentially the same, as they represented comparisons between leafhopper lineages (Table 2.7). On the other hand, every contrast that differed among alternative higher-level scenarios (sister group comparisons I-III in

27 Table 2.7) was positive and of similar magnitude, suggesting that the present results are indeed independent of the phylogenetic uncertainty of higher-level lineages of the Hemiptera. In fact the major reason why the present results did not support a statistical increase in body size of xylem-feeders was because of the strong negative contrast of myerslopiids and the remaining Membracoidea ancestor.

Myerslopiidae was recently raised to familial level [150], being previously recognized as a lineage of leafhoppers related to Ulopinae [110, 178, 264]. Its members are rare, ground-dwelling bugs with a disjunct distribution in Chile and New Zealand. As mentioned previously, whether myerslopiids are truly xylem- feeders remains a mystery. In fact another limitation of the present study is the scarce record of feeding habits of leafhopper lineages, even though leafhoppers are major agricultural pests; over 21,000 species are described with estimates that in the tropics only 10% are actually known [93, 179, 248]. In the present study, leafhoppers showing a characteristic inflated cibarial chamber were considered xylem-feeders. If in the future myerslopiids are discovered to have a feeding habit other than xylem-feeding, it could still be argued that a contrast between phloem and xylem feeders exists at the base of Membracoidea based on the fossil record. Extinct and presumably basal lineages of Membracoidea from the Mesozoic, including the Upper

Jurassic Karajasidae, and the Lower Cretaceous Jascopidae and Ovojassini (extinct Myerslopiidae), were probably xylem feeders based on their inflated faces [149,150]. These insects were mostly larger than extant myerslopiids, ranging from 4.5-11.1 mm [149, 150, 319], but still not large enough to render this contrast positive.

The hypothesized need for a larger minimum body size in xylem specialists is based on the extreme paucity of nutrients in xylem sap, which in turn can be achieved only by an energetically costly muscular pumping system capable of sucking fluids out of xylem vessels under negative pressure [256,277,295]. Thus, one might expect that variation in the negative pressure in xylem vessels would be a strong predictor of feeding rate. Recent evidence, on the other hand, showed that feeding rates in sharpshooters are better predicted by the chemical profile in xylem sap, rather than xylem tension (as long as it is below a maximum threshold). For example, the glassy-winged sharpshooter (Homalodisca vitripennis) may, depending on host plant, feed at the same rate throughout the day when xylem tension is increasing steadily from approximately

0.1 to 1.0 MPa and even increase its feeding rate when xylem tension is very high [8]. Evidence from this sharpshooter can be viewed as one strong argument against the large negative xylem pressures advocated by the cohesion theory [400] and in favor of the compensating theory of ascent of sap [46,47]. The compensating theory advocates that xylem parenchyma and ray cells press the tracheary elements, thus compensating for the negative pressure caused by transpiration, and keeping the pressure within the conduits close to zero [46].

This theory may explain the numerous records of non-primarily xylem feeders occasionally ingesting xylem

28 sap (see Table 2.8, and additional records in Press & Whittaker [277]) as a means to compensate for either desiccation [275] or osmotic stress caused by ingestion of phloem sap [72]. If negative pressures were so common in xylem conduits, xylem ingestion by these herbivores that do not possess a strong cibarial pump, would presumably be very difficult.

In conclusion, although there seems to be a tendency for xylem-feeders to be larger than other phy- tophagous clypeorrhynchans, a tendency certainly accentuated by the extremely large body sizes of cicadas, the present study showed no statistical support for an overall increase of body size in xylem-feeding Clype- orrhyncha lineages, contrary to the conclusions of Novotny & Wilson [256]. The present results not only contradict Novotny & Wilson’s [256] contention that xylem feeders should be larger than 8 mm to efficiently overcome the xylem tension, but also lend support to the opponents of the cohesion theory of sap ascent, who believe that negative pressures in xylem conduits cannot be as high as proposed. However, until more robust phylogenies, especially of the large Membracoidea lineage, and more detailed studies on the feeding mode of many important leafhopper lineages become available, it is still advisable to view any such result with caution.

29 Table 2.8: Records of primary (*) or secondary (+) xylem sap feeding by “Homoptera”. Life stage (LS) studied: a=adults, ap=apterae forms, g=gynoparae forms, n=nymphs, and v=virginoparae (winged and wingless). Feed- ing niches: X=xylem; P=phloem; M=cell content. Feeding site detection methods are based on: MS=microscopic sections of leaf tissue and inserted stylets or salivary sheaths; OP=osmotic pressure of excreta; EPG= electri- cal penetration graph; ER=excretion rates; pH: pH of excreta; OCP= comparisons of the organic compounds (aminoacids, organic acids, and/or sugars) profile between excreta and xylem sap of host plant

Taxa LS X P M Detection Method Reference Archaeorrhyncha Nilaparvata lugens (Stl) a + * pH [30] Perkinsiella saccharicida Kirkaldy a + * + EPG [53] Sternorrhyncha Aphis fabae Scopoli v, g + * EPG [275, 360] Aphis fabae Scopoli ap * EPG [275] Brevicoryne brassicae (Linnaeus) ap + * EPG [63] Myzus persicae (Sulzer) ap + * EPG [63] Cercopoidea Chaetophyes compacta (Walker) n * MS [208] Cosmoscarta abdominalis Donovan n * MS [208] Machaerota coronata Maa n * MS [208] Philaenus spumarius (Linnaeus) a, n * + + MS, ER, OP [162, 206] Cicadoidea Cryptotympana mimica Walker a * MS, OP [56] Cyclochila australasiae Don a, n * MS, OP [56] Gaeana maculata Walker a * MS, OP [56] Magicicada spp. n * MS [375] Membracoidea Cicadellini (Linnaeus) a * MS [244] Draeculacephala floridana Ball a, n * OCP [305] Ball a, n * + + MS [165] Graphocephala atropunctata (Signoret) a, n * + + EPG, MS [15, 53, 165] sp. a * MS [165] Athysanini virescens (Distant) aa * + pH [58] Nephotettix virescens (Distant) a + * pH [58] Macrostelini (Naud) ab + * + MS [225] Cicadulina mbila (Naud) ac + + * MS [225] Cicadulina mbila (Naud) ad + * * MS [225] Proconiini Cuerna costalis (Fabricius) a * ER, OCP [41] Homalodisca vitripennis (Germar) a * ER, OCP [8, 41] Homalodisca insolita (Walker) a * ER, OCP [41]

afeeding on Tungro virus resistant rice (Oryza sativa) varieties. bfeeding on preferred hostplants: Digitaria sanguinalis and corn (Zea mays). cfeeding on rice. dfeeding on sugar cane (Saccharum officinarum).

30 Chapter 3

Codiversification of Sharpshooter Bacterial Endosymbionts

3.1 Introduction

One of the most prominent aspects of biological complexity is the occurrence of intimate symbiosis, link-

ing the ecology and evolution of phylogenetically distant lineages. Recent studies have shown that such

symbioses can persist for periods of hundreds of millions of years and that they can have major impact

on the evolution of interacting partners. Instances of such symbioses occur in insects, many of which rely

on mutualistic associations with bacteria to supplement amino acid or vitamin deficiencies in specialized,

nutritionally unbalanced diets [233]. Such symbionts typically live within host cells called bacteriocytes that

form aggregates called bacteriomes and are transmitted by infection of eggs within the mother [43, 246].

So far, molecular studies of insect symbioses have been focused on groups in which only a single bacterial

lineage is universally present in any given insect group, restricted to bacteriocytes, and cospeciating with

their hosts. Obligate symbionts, living within bacteriomes with a universal distribution in a clade of hosts,

are referred to as primary symbionts [27]. Both genomic and experimental studies indicate that primary

symbionts studied to date have a major role in provisioning their hosts with nutrients [27, 322]. This nutri-

tional role has been elucidated in the primary symbiont of aphids, Buchnera aphidicola, which provides its hosts with essential amino acids [26,27,101,238]. Although primary symbionts provide benefits to hosts, they also present constraints on host evolution, especially because they typically undergo degenerative evolution involving irreversible loss of genes and regulatory capacities [238, 349].

Multiple symbiont types often co-occur in one insect host (summarized in [27, 43]). In some cases, insects with a primary symbiont harbor additional symbionts with less restricted distributions among host tissues; these are called secondary or guest symbionts, and typically are not required for host development or reproduction [27,43,233]. Secondary symbionts tend to be phylogenetically diverse associates with relatively short histories in host lineages. Thus, closely related host species often differ in the presence of particular secondary symbionts, which can vary in presence among members of the same host species. A large number of phylogenetic studies indicate that secondary symbionts do not show phylogenetic congruence with hosts over

This chapter was mostly published in Takiya, Tran, Dietrich, & Moran (2006) [348]. long periods (e.g., [353] for psyllid hosts, [351] for whitefly hosts, review in [27]), although they may persist

within a closely related cluster of species such as members of a genus of (Pseudococcidae) [355]

or a subgenus of aphids [309]. In general, primary symbionts are also distinguished by their unusually large

cell size and shape, whereas secondary symbionts have more typical cell size and shape (overview in [27]).

Finally, recent studies have revealed major genomic differences between the two kinds of symbionts, with

primary ones having near-minimal genomes that do not take up new genes [77, 349] and secondary ones

featuring larger genomes that are dynamic and capable of gene acquisition and recombination [236, 359].

In numerous insects, more than one symbiont type appears to be an obligate associate, restricted to

bacteriomes and universally present within a species. Among the most prominent examples are the complex

assemblages of symbiotic microbes that occur in most members of the hemipteran suborder Auchenorrhyncha,

a diverse group that comprises approximately 45,000 species of leafhoppers, treehoppers, cicadas, planthop-

pers, and spittlebugs [43]. The most evident ecological explanation for the high frequency of symbionts in

Auchenorrhyncha is that they feed on plant fluids, which are notoriously unbalanced nutritionally with low

nitrogen and essential amino acid content [308]. Leafhoppers (Cicadellidae) include more than 20,000 de-

scribed species [91], among which are some of the most important vectors of plant diseases [156,279,280,296].

While members of most leafhopper lineages feed preferentially on phloem sap, those from the subfamily Ci-

cadellinae (commonly called sharpshooters) specialize on the very dilute sap of xylem. Xylem sap is among

the most nutritionally limited diets used by any , with a severe scarcity of organic carbon and nitrogen

concentrations that are 10 times less than those of phloem sap [7, 296].

According to earlier microscopy-based studies of symbiosis, performed before the availability of molecular

methods, auchenorrhynchan species may harbor up to six morphologically distinct symbiont types, but

the most common condition involves the presence of two obligate bacteriome-associated symbionts and

from zero to two secondary symbionts. Sharpshooters appear to exhibit the latter condition [43, 52, 173].

Sharpshooter bacteriomes are paired structures located near the anterior end of the abdomen [43, 173,

235] (See Figure 3.1). As is the case for all obligate symbiotic bacteria, the identities and evolutionary

relationships of organisms in these bacteriomes could not be ascertained before the advent of PCR and

DNA sequencing techniques. Recently, Moran et al. [235, 239] linked these two morphologically distinct organisms to respective 16S rDNA sequences, enabling their placement on the bacterial phylogenetic tree and correlating sequences with cells observed in light and electron microscopy. Candidatus Baumannia

cicadellinicola (hereafter called Baumannia), an irregularly spherical bacterium approximately 1.5-2.0 µm

in diameter, occurs in the bacteriomes of five sharpshooter species studied previously [235]. Although these

represented a very limited sample of species, phylogenetic analyses indicated that they formed a strongly

32 Figure 3.1: Sharphsooter bacteriomes. Cuerna sayi with basal abdominal tergites removed exposing the paired clusters of bacteria-housing cells. Sharpshooter bacteriomes are typically divided into reddish-orange and yellow portions supported clade within the γ-3 Proteobacteria and that their relationships were consistent with those of the insect hosts. However, this evidence for codiversification is inconclusive because so few species were included, representing only two genera. More intensive studies of Homalodisca vitripennis (formerly H. coagulata and also called the glassy-winged sharpshooter) revealed that a second organism, named Candidatus Sulcia muelleri (hereafter called Sulcia) in the phylum Bacteroidetes, is present in all sampled populations [235,

239]. Sulcia is widespread in Auchenorrhyncha and is documented in hosts from Fulgoroidea, Cicadoidea, and Cercopoidea, in addition to Membracoidea (which includes leafhoppers). Furthermore, phylogenetic relationships among Sulcia strains from diverse auchenorrhynchan hosts are congruent with current estimates of host phylogenies, suggesting that this symbiont descended from an ancestor that infected an ancient ancestor of Auchenorrhyncha, over 260 million years ago [239].

Sulcia and Baumannia are the dominant microbes residing in the bacteriomes, based on an intensive study on H. vitripennis; large clone libraries were obtained and few bacterial sequences were obtained that could not be definitively assigned to these organisms [379]. Furthermore, these two organisms possess complementary sets of pathways for the biosynthesis of nutrients needed by their insect hosts, with Baumannia able to provision a large set of cofactors (including most B-vitamins) and Sulcia encoding genes for the biosynthesis of essential amino acids [379]. This result along with fluorescent in situ hybridization studies of Baumannia and Sulcia [239, 379] verified earlier conclusions that two bacteriome-associated symbionts are consistently present in sharpshooters [43, 52, 173]. Although numerous symbioses involving an insect host group and a

33 primary symbiont have been analyzed, including symbioses of aphids [237], psyllids [354], mealybugs [355], whiteflies [352], cockroaches [201], carpenter ants [310], and tsetse flies [2], the sharpshooters are the first insect clade for which two apparently widespread bacteriome-restricted symbionts have been characterized using molecular data.

In addition to Baumannia and Sulcia, Wolbachia pipientis was the most common organism represented in the clone libraries of H. vitripennis, although much less abundantly than the other two bacteria and sometimes absent from some host individuals [235,379]. Wolbachia are found in all major insect orders [371], and, like Baumannia and Sulcia, are vertically transmitted through the egg cytoplasm. On the other hand, Wolbachia undergo extensive horizontal transmission between insect taxa [372], leading to completely incongruent histories with their hosts independent of gene or taxonomic-level studied [175,234, 324, 366].

In this chapter, the question of the distribution of Baumannia, Sulcia, and Wolbachia among sharp- shooters and related leafhoppers is addressed. Our central question is the extent to which these specialized associations have persisted throughout the diversification of the group and therefore might impose major constraints on host evolution. Thus, we focus on the evaluation of whether the two dominant bacteria have undergone long-term codiversification with their hosts. Prior to our study, sequences of sharpshooter sym- bionts were few and insufficient for rigorous testing of the extent of codiversification of these two symbionts with their hosts. Here, 24 additional leafhopper species, spanning six tribes with emphasis on sharpshoot- ers, were characterized for both Baumannia and Sulcia. Phylogenetic analyses were performed based on symbiont rDNA gene sequences and on four nuclear and mitochondrial insect gene sequences to generate independent hypotheses regarding the evolutionary histories of insects and symbionts. These datasets and phylogenies were then compared to assess the extent of support for a history of codiversification between host species and their two microbial associates.

3.2 Materials and Methods

3.2.1 Taxon Sampling

Included in the analyses were 29 species belonging to four leafhopper subfamilies, with emphasis on the

Cicadellinae and related leafhopper lineages [83, 91]. Table 3.1 lists the species names and collection data.

Leafhopper higher-level classification follows Dietrich [85], Hamilton & Zack [152], Oman et al. [264], and

Young [392, 393]. Specific names used herein for the glassy-winged sharpshooter and the Smoketree sharp- shooter (H. liturata) follow Takiya et al. [344] and Burks & Redak [44], respectively. Samples from Moran et al. [235] were used for Homalodisca vitripennis (=H. coagulata), H. liturata (=H. lacerta), Graphocephala

34 hieroglyphica, G. aurora, and G. cythura. Sequences of host and symbiont genes were obtained for the same

leafhopper species with the exception of the three previously studied species of Graphocephala, which were

not analyzed for host genes. In most cases, host and symbiont sequences were obtained from specimens

collected on the same day and locality (Table 3.1), including from the same insect specimen in eight of the

species studied.

Symbiont 16S rDNA sequences were newly determined in this study, except for five previously deposited

sequences for Baumannia (AF465793-7) and the single Sulcia (AY147399). Sequences of 16S rDNA for

symbiont outgroups were obtained from GenBank. Outgroups were chosen to represent several of the closest

BLAST hits for each of the symbiont groups, based on blastn searches of GenBank. These included both free-living bacteria and some other insect endosymbionts belonging to the same bacterial division. Insect symbionts included were those associated with aphids, mealybugs, psyllids, and ants for the phylogenetic analyses of Baumannia, which falls in the γ-Proteobacteria (NC002528, AF476100, AF476106, AF263556,

AJ250715, AF476102, AF263560, AF366378, and AE005632), and those with ladybird beetles, termites, and cockroaches for the analyses of Sulcia, which falls in the Bacteroidetes (M58789, M93152, AB071953,

Z35665, Z35666, AF363713, Y13889, and AJ009687).

3.2.2 DNA Preparation

Insect specimens were collected in the field directly into 95-100% ethanol and stored at 20◦ until processed.

For amplification of host genes, genomic DNA was extracted from a single hind leg and associated muscles using a modified ethanol precipitation/resuspension protocol [29] or the DNEasy tissue kit (QIAGEN Inc.).

All leafhopper vouchers were dried, pinned, and deposited at the INHS.

For amplification of bacterial genes, bacteriomes were isolated in the lab by immersing the freshly collected etherized insect in 0.85% saline solution under a dissecting microscope, slitting the cuticle and teasing out the structure using insect pins. Bacteriomes were separately placed into 95% ethanol and subjected to

DNA extraction using the DNEasy tissue kit (QIAGEN Inc.). An individual bacteriome was placed in a

1.5 ml microfuge tube, frozen by immersion in liquid nitrogen and crushed by grinding with a disposable pestle. Following addition of 180µl of the tissue homogenization buffer from the DNEasy kit, the tissue was homogenized further by grinding. Then 20µl of Proteinase K were added, followed by vortexing and incubation at 65◦C for 30 min. To remove RNA, 4µl of RNAse A (100mg/ml) were added, followed by 200µl

of the A1 buffer from the DNEasy kit, to enhance precipitation. This mixture was incubated at 70◦C for 10

min, followed by purification steps as specified in the kit. Each extraction was performed on one bacteriome

from a single individual, with one to ten individuals extracted for each species (see Table 3.1).

35 Table 3.1: Material examined with accession numbers for insect and endosymbionts Candidatus Baumannia cicadellinicola and Candidatus Sulcia muelleri genes. Symbiont DNA was extracted either from specimens from the same collection event as the specimen from which host DNA was extracted (same collection), which may include the same specimen, or from specimens from a different locality as indicated. Numbers in parentheses following symbiont locality are the number of host individuals which were tested positive for the presence of Wolbachia using wsp and ftsZ diagnostic primers / total number of individuals tested. The total number of individuals tested is the same as the number of individuals from which comparable sequences of 16S for Baumannia and Sulcia were recovered. Dash (-) indicates that no Wolbachia test was performed.

Taxon Locality GenBank Accession(s) Host Symbiont Host: COI, COII, 16S, H3 Baumannia 16S Sulcia 16S Coelidiinae: Teruliini Jikradia olitoria (Say) USA: Illinois same collection (0/1) — , —, AY869828, AY869758 — AY676913 Evacanthinae: Pagaroniini Pagaronia tredecimpunctata Ball USA: California same collection (0/2) AY869733, AY869785, AY869827, AY869755 — AY676911 Phereurhininae: Phereurhinini Clydacha catapulta Kramer PERU: Hu´anuco same collection (21/3) AY869743,AY869795,AY869804,AY869769 AY676878 AY676898 Cicadellinae: Bathysmatophorini Hylaius oregonensis (Baker) USA: Oregon same collection (0/2) AY869729, AY869784, AY869825, AY869753 — AY676905 Cicadellinae: Cicadellini Cicadella viridis (Linnaeus) KYRGYZSTAN: GERMANY: AY869735, AY869786, AY869826, AY869760 — AY676915 Dzhalal-Abad Brandenburg (0/2) Graphocephala aurora (Baker) — USA: Arizona (-/1) — AF4657973 4 (Forster) USA: Illinois USA: Illinois (11/4) AY869730, AY869789, AY869807, AY869763 AY676891 AY676916 Graphocephala cythura (Baker) — USA: Arizona (0/1) — AF4657953 AY676919 Graphocephala hieroglyphica (Say) — USA: Arizona (-/1) — AF4657963 4 Helochara communis Fitch USA: Arizona same collection (0/10) —, AY869783 ,AY869819, AY869752 AY676877 AY676897 Pamplona spatulata Young PERU: Pasco same collection (21/2) AY869744, AY869779, AY869821, AY869770 AY676887 AY676908 Paromenia isabellina (Fowler) COSTA RICA: San Jos´e same collection (0/32) AY869734, AY869782, AY869822, AY869759 AY676896 AY676914 36 Cicadellinae: Proconiini Acrogonia virescens (Metcalf) PERU: Jun´ın same collection (21/5) AY869746, AY869797, AY869809, AY869772 AY676883 AY676903 Cuerna costalis (Fabricius) USA: Florida USA: Illinois (0/7) AY869739, AY869791, AY869808, AY869765 AY676895 AY676918 Cuerna gladiola Oman & Beamer USA: California same collection (21/2) AY869741, AY869793, AY869818, AY869767 AY676892 AY676917 Cuerna sayi (Nielson) USA: Maryland USA: Wisconsin (0/5) AY869751, AY869802, AY869823, AY869777 AY676894 AY676921 Cuerna striata (Walker) USA: Michigan USA: Wisconsin (0/5) AY869750, AY869801, AY869824, AY869776 AY676893 AY676922 Cyrtodisca major (Signoret) MEXICO: Jalisco MEXICO: Jalisco (0/3) AY869749, AY869800, AY869810, AY869775 AY676886 AY676907 Diestostemma excisum Schmidt PERU: Pasco same collection (0/3) —, AY869778, AY869817, AY869756 AY676889 AY676910 Diestostemma stesilea Distant PERU: Pasco same collection (0/1) AY869732, AY869780, AY869813, AY869754 AY676884 AY676904 Homalodisca vitripennis (Germar) USA: Florida USA: California (11/1) AY869740, AY869792, AY869803, AY869766 AF4657933 AY1473993 Homalodisca elongata Ball USA: Arizona USA: Arizona (21/2) AY869747, AY869798, AY869806, AY869773 AY676881 AY676901 Homalodisca liturata Ball USA: California USA: Arizona (0/1) AY869738, AY869790, AY869820, AY869764 AF4657943 AY676920 Homoscarta irregularis (Signoret) PERU: Pasco PERU: Hu´anuco (0/2) AY869745, AY869796, AY869814, AY869771 AY676882 AY676902 Oncometopia orbona (Fabricius) USA: Illinois USA: Illinois (0/2) AY869736, AY869787, AY869811, AY869761 AY676879 AY676899 Paraulacizes irrorata (Fabricius) USA: Illinois USA: Illinois (21/3) AY869737, AY869788, AY869815, AY869762 AY676880 AY676900 Phera obtusifrons Fowler MEXICO: Puebla same collection (0/1) AY869748, AY869799, AY869805, AY869774 AY676888 AY676909 Proconosama alalia (Distant) PERU: Pasco same collection (0/1) AY869742, AY869794, AY869812, AY869768 AY676885 AY676906 Proconosama columbica (Signoret) PERU: Jun´ın same collection (31/4) AY869731, AY869781, AY869816, AY869757 AY676890 AY676912

1Accession numbers for Wolbachia wsp are DQ450148-DQ450164. 2Baumannia and Sulcia sequences recovered from only a single specimen. 3Sequences from [235]; all other sequences were obtained in current study. 4Lack of DNA for diagnostic PCR. 3.2.3 PCR and Sequencing of Host Genes

Modified primers based on [326] were used to amplify parts of the host mitochondrial genes COI, COII, and

16S rDNA (see Table 3.2). Nuclear H3 sequences were amplified using the primers HexAF and HexAR [259]

(see Table 3.2). Host templates and controls were amplified with Taq DNA polymerase (Promega Corp.)

added at 80◦C and after initial denaturation at 94◦C for 3 min, followed by 30 cycles of 94◦C for 1 min,

50◦C for 1 min, 72◦C for 2 min; and a final extension of 72◦C for 7 min. Double-stranded PCR amplification products were checked for yield and specificity on 1% agarose electrophoresis gels stained with ethidium bromide under UV light. Amplicons were purified using QIAquick PCR purification kit (QIAGEN Inc.) and both strands sequenced using ABI Prism BigDye terminator kit version 3 (PE Applied Biosystems).

Sequencing products were run on an ABI 3730 capillary sequencer or purified using Sephadex columns and run on an ABI 377 lane automated sequencer at the Biotechnology Center of the University of Illinois at

Urbana-Champaign.

3.2.4 PCR and Sequencing of Symbiont Genes

For all species except those of the sharpshooter genus Cuerna, PCR for Baumannia was conducted using the eubacterial primers 10F and 1507R, which together amplify most of the 16S rDNA sequence (≈500

bases) for most Proteobacteria. For Cuerna species, PCR was conducted using 10F and primer 35R, which together amplify most of the 16S, a small part of the 23S rDNA, plus the intergenic spacer. These reactions consistently yielded a single product, corresponding to the Baumannia gene. Products were purified (PCR purification kit, QIAGEN Inc.) and submitted for DNA sequencing using the PCR primers, as well as 320R,

559F, 650R, and 1128R (Table 3.2). All intergenic spacer sequence was removed prior to analyses, using

Escherichia coli as a reference. As previously noted [235], the 10F primer does not amplify Sulcia 16S

rDNA sequences, due to mispairing at the 3’ end. To obtain a portion of the Sulcia sequence, primer 10FF

was used instead, which binds in the same position as 10F but has a change in the 3’ end compatible with

sequences available in GenBank for most members of the Bacteroidetes phylum. Approximately 1,350bp of

16S rDNA of Sulcia was amplified using primer 10FF plus 1370R, binding near the 3’ end of the 16S rRNA.

Amplifications and sequencing protocols for rRNA genes of both Sulcia and Baumannia followed [235].

Sequences were obtained for both Sulcia and Baumannia with one to ten individuals of each host species.

Because multiple sequences from the same host species revealed almost no sequence differences, we obtained

polished sequences for only one individual per species, with at least two reads in each direction. Other

individuals were sequenced using only the sequencing primers, giving just one read over most of the sequence.

Wolbachia screenings were performed for 27 of the 29 species included in our analysis (DNA samples were

37 Table 3.2: Oligonucleotide primer sequences. Primers used in polymerase chain and sequencing reactions of COI, COII, H3, 16S, wsp, and ftsZ. Those indicated with asterisk (*) were only used in sequencing reactions.

Primer Organism Locus Sequence 5’ → 3’ C1-J-2195 Leafhopper COI TTG ATT TTT TGG TCA YCC WGA AGT TL2-N-3014 Leafhopper COI TTC ATT GCA CTA ATC TGC CAT ACT A TL2-J-3037 Leafhopper COII TAG TAT GGC AGA TTA GTG CAA TGA A C2-N-3661 Leafhopper COII CCR CAA ATT TCW GAR CAT TGA CCA HexAF Leafhopper H3 ATG GCT CGT ACC AAG CAG ACG GC HexAR Leafhopper H3 ATA TCC TTG GGC ATG ATG GTG AC LR-J-12887 Leafhopper 16S CCG GTY TGA ACT CAR ATC A LR-N-13398 Leafhopper 16S CRM CTG TTT AWC AAA AAC AT 10F Baumannia 16S AGT TTG ATC ATG GCT CAG ATT G 35R Baumannia 16S CCT TCA TCG CCT CTG ACT GC 320R* Baumannia 16S ACC AGC TAG AGA TCG TTG C 650R* Baumannia 16S CAC CGG TAC ATA TGA AAT TCT 1128R* Baumannia 16S GGG ACT TAA CCC AAC TTT CAC 1507R Baumannia 16S TAC CTT GTT ACG ACT TCA CCC CAG 10FF Sulcia 16S AGT TTG ATC ATG GCT CAG GAT AA 270F* Sulcia 16S TTA GTT GGT AAG GTA ATG GC 700R* Sulcia 16S ACA TTC CAG CTA CTC CAA ACT 1370R Sulcia 16S CGT ATT CAC CGG ATC ATG GC 559F* Baumannia and Sulcia 16S CGT GCC AGC AGC CGC GGT AAT AC WspF Wolbachia wsp TGG TCC AAT AAG TGA TGA AGA AAC TAG CTA WspR Wolbachia wsp AAA AAT TAA ACG CTA CTC CAG CTT CTG CAC FtsZF1 Wolbachia ftsZ GTT GTC GCA AAT ACC GAT GC FtsZR1 Wolbachia ftsZ CTT AAG TAA GCT GGT ATA TC

not sufficient for screening two of the species), using the PCR conditions listed above. The same individuals

screened for Sulcia and Baumannia were also screened for presence of Wolbachia using Wolbachia-specific

primers for the genes wsp [399] and ftsZ [372]. Reactions were performed using Taq DNA Polymerase

(Promega, Corp.). A touchdown PCR cycle was used, with denaturation step (94◦C for 2 min); followed by 10 cycles of 94◦C for 45 sec, 65◦C for 45 sec, 72◦C for 1 min; and 28 cycles of 94◦C for 45 sec, 55◦C for 45 sec, 72◦C for 1 min and a final extension of 72◦C for 5 min. Diagnostic PCR reactions consistently yielded a single product at ≈0.6 kb and ≈1 kb for Wolbachia wsp and Wolbachia ftsZ genes, respectively.

All wsp sequences and some ftsZ products were sequenced, using the PCR primers as sequencing primers

(Table 3.2).

Sequencing products were run on an ABI 377 sequencer at the University of Arizona Genomic Analysis and Technology Center.

38 3.2.5 Alignments

GenBank accession numbers for the sequences generated are listed in Table 3.1. Correction of chromatogram

sequences, reconciliation of complementary strands, and alignment of protein coding host genes across species

were facilitated by Sequencher 4.1.2 (Gene Codes Corp.). Alignments for the host ribosomal 16S rRNA

sequences were made using ClustalX 1.81 [356] with gap opening:extension costs 50:1 and IUB DNA

weight matrix. Alignments of the host genes resulted in 783bp of COI, 591bp of COII, 328bp of H3, and

481 bp of 16S rDNA (total of 2,183 bp, 804 parsimony-informative). Including outgroup taxa, alignments

for the 16S rDNA of Baumannia was 1,495bp (352 parsimony-informative) and of Sulcia was 1,489bp (372

parsimony-informative) in length.

Although sequences for 16S rDNA and histone H3 for the coelidiine Jikradia olitoria were sequenced and

deposited in GenBank, this species was not included in the host phylogenetic analyses. Preliminary results

for this species were highly inconsistent with the currently accepted classification and previous phylogenetic

analyses [83, 91, 264], probably due to the unavailability of COI and COII sequences for this species, which

accounted for 63% of character data in the combined host dataset.

3.2.6 Phylogenetic Analyses

All analyses were run using PAUP* 4.0b10 [336] unless otherwise stated. Combination of molecular datasets for the host analysis was supported by the lack of incongruence among matrices as suggested by the ILD test [119,230] run with 1,000 replicates and heuristic tree searches of 10 random addition replicates and TBR branch swapping. Constant and uninformative characters were excluded prior to the test [73].

Pairs of partitions tested were COI vs. COII (P=0.15); mitochondrial protein encoding (COI and COII) vs. ribosomal (16S) (P=0.27); and mitochondrial (COI, COII, and 16S) vs. nuclear (H3) (P=0.60). All gaps were treated as missing data.

Models of molecular evolution for use in maximum likelihood analyses were estimated by a hierarchical likelihood ratio test [122,167] using Modeltest 3.06 [274]. Models chosen were TVM+Γ+I for the combined host dataset were, GTR+Γ+I for Baumannia, and TrN+Γ+I for Sulcia. All models assume unequal base frequencies, unequal rates across sites modeled by a Γ distribution [383], and some estimated proportion of invariant sites. DNA substitution matrices assumed by each model include one transition rate in TVM and two different of such rates in TrN and GTR, as well as one transversion rate in TrN and four transversion rates in TVM and GTR. These model parameters were used when searching heuristically for the most likely trees with 10 random addition replicates and TBR branch swapping.

Heuristic parsimony tree searches were performed with 1,000 random addition replicates and TBR branch

39 swapping without setting a maximum limit on number of trees saved in memory, except for the Sulcia dataset.

Due to the immense numbers of trees that resulted from preliminary analyses of Sulcia sequences, it was necessary to set the maximum number of trees saved (maxtrees) to 100,000, and 100 independent replicates of heuristic parsimony searches were performed, each with 5 random addition replicates and SPR branch swapping.

Branch support was assessed by non-parametric character bootstrapping [123], using 100 replicates in likelihood (starting tree obtained by neighbor-joining, followed by NNI branch swapping) and 1,000 replicates in parsimony (5 random additions and SPR branch swapping) analyses. Once more, due to memory and time limitations, maxtrees was set to 100,000 for the Sulcia dataset. Bremer and partitioned Bremer decay indices [40] were calculated based on symbiont and host most likely trees, respectively, with the aid of

TreeRot 2.0 [330]. These indices are conservative estimates as heuristic parsimony searches were conducted with only 20 random replicates and TBR branch swapping (maxtrees set to 10,000 when analyzing the

Sulcia dataset).

3.2.7 Host-Symbiont Associations

To assess the evidence for codiversification of the two symbionts with their hosts, three statistical tests based on different assumptions and algorithms were conducted. A parsimony-based ILD test was conducted under the null hypothesis that the host combined dataset and each symbiont rDNA dataset are congruent, suggesting a history of cospeciation. Settings for ILD tests were as specified above. The advantage of the

ILD test is that it is not biased due to uncertainty regarding the topology of individual trees, as are the following two tests. Congruence of host and symbiont topologies was also assessed with a S-H likelihood- based test [138,323] run with 10,000 RELL bootstrap replicates. Considering the H0 that the -lnL score of a given host tree calculated using the host dataset and model of evolution is the same as the score calculated using the symbiont dataset and model of evolution (and vice-versa), a failure to reject H0 is suggestive of a perfect cospeciation scenario. For these tests, a new data matrix containing only the taxa represented in all three datasets (n=22) was constructed, and those taxa not included were pruned from most likely and parsimonious trees tested. All most likely and parsimonious trees were tested, except in the case of Sulcia, where only 10 randomly chosen most-parsimonious trees were tested (Topologies for host n=3; Baumannia n=6; Sulcia n=13). Finally, an event-based tree-fitting method, implemented in the program TreeFitter

1.0 [301, 302], was used to hypothesize parallel evolutionary events between host and symbiont trees. This method also tests whether cospeciation events hypothesized are more numerous than expected by chance, by comparing host trees with 1,000 randomly generated symbiont trees. Because TreeFitter does not allow

40 input of polytomous trees, the most likely tree for Baumannia, which contained one trifurcation, was resolved into the only two possible strictly bifurcating trees. The less resolved tree for Sulcia was resolved randomly into 20 different bifurcating topologies. These 22 symbiont topologies were compared to the most likely host tree and events calculated based on the following costs: 0 for codivergence and duplication, 1 for sorting, and 0-30 for horizontal transfer (=host switches).

3.2.8 Evolutionary Rates of Bacterial 16S rDNA

To test whether the 16S rDNA of Baumannia and Sulcia were evolving with a constant rate across different host-associated lineages, a likelihood ratio test was performed [122, 167]. After removing outgroups from the symbiont datasets, evolutionary models were chosen using Modeltest as described above. Maximum likelihood analyses were then run in PAUP*, with one analysis constrained to follow a molecular clock and another unconstrained. The likelihood ratio test statistic, which should follow a χ2 distribution, was calculated as twice the difference between the -lnLs of trees resulting from the unconstrained and constrained analyses, i.e. LRT = 2(−lnL non clock−lnL clock). The H0 that the rate of substitution is homogeneous among all branches in the topology was tested, with df = number of terminal taxa − 2, i.e., df=23 for

Baumannia and df=25 for Sulcia.

Because phylogenetic results were consistent with an origin of symbionts predating or simultaneous with the origin of the common ancestor to sharpshooters, we inferred that the symbionts had likely evolved in sharpshooters over the same time interval. To compare rates of 16S rDNA evolution between Baumannia and Sulcia, nodes with compatible groupings of taxa in both phylogenies, i.e., potentially cospeciating nodes, were identified. Branches leading from these nodes to their most recent ancestor in each phylogeny were recognized as copaths, i.e., homologous evolutionary branches [268]. Copath maximum likelihood lengths were compared using a Wilcoxon signed rank test (α=0.05, two-tailed). Failure to reject the H0 that members of a copath have undergone the same amount of evolution would imply that the two symbionts have the same substitution rate in their 16S rDNA. The slope of a reduced major axis regression [171, 217], i.e., the ratio of standard deviations of copath members, was calculated as a relative measure of the rate difference between the 16S rDNA of the two symbiont lineages.

41 3.3 Results

3.3.1 Distribution of Baumannia, Sulcia and Wolbachia among Hosts

Of the 29 species included in the study, Baumannia was present in 25, including all species of the cicadelline tribes Proconiini and Cicadellini except Cicadella viridis, and also in the single species of the subfamily

Phereurhininae. Baumannia was not detected in Hylaius oregonensis (Bathysmatophorini), Pagaronia tre- decimpunctata (subfamily Evacanthinae) or in Jikradia olitoria (subfamily Coelidiinae). To further test for presence of Baumannia in species not initially yielding a Baumannia product we used several other primer pairs expected to amplify Baumannia 16S rDNA and still obtained no positive reactions. All negative re- actions were repeated, and positive controls, run concurrently, did yield PCR products. Some species not yielding Baumannia sequences did produce 16S rDNA sequences from unrelated bacteria (not in the γ-

Proteobacteria), possibly corresponding to other symbionts or to contaminants that amplified in the absence of high copy numbers of the Baumannia chromosome.

All species considered, representing six tribes, possessed Sulcia, based on PCR amplification followed by DNA sequencing, blast searches, and phylogenetic analyses as described below. In every case, the first blastn hit against the non-redundant GenBank nucleotide database was the single sequence of this symbiont type from the sharpshooter host H. vitripennis, previously deposited in GenBank [235].

From one to ten individual insects from each host species were used for independent determinations of sequences from both symbionts (Table 3.1). In all cases, the sequences were identical or near-identical for different individuals of the same species, with no cases of more than three nucleotide differences between pairs of either Sulcia or Baumannia sequences within a species. The earlier study of Baumannia from H. vitripennis from Florida and California recovered identical 16S rDNA sequences from the two localities [235].

The most common additional sequences obtained corresponded to Wolbachia pipientis, a widespread symbiont of insects and other arthropods that is known to modify reproductive biology of some hosts. Using screens based on diagnostic primers for two protein coding genes (wsp and ftsZ ), we found Wolbachia in a total of nine of the 27 species screened. Sequences of wsp were obtained and deposited in GenBank

(Table 3.1). Because Wolbachia was absent from most species and often not universal in species in which it was found, it was not considered to be an obligate symbiont. Its distribution showed no evident pattern with respect to host phylogeny or classification. The sequences indicated the presence of several Wolbachia haplotypes among our samples, and no two were identical. Because recent findings indicate that Wolbachia in insects shows high rates of recombination among genes and among regions of the wsp locus [18], we did not use these sequences for phylogenetic reconstructions. The few other sequences obtained from 16S rDNA

42 amplifications were restricted to the few species not containing Baumannia. These were either contaminants or possibly additional obligate or facultative symbionts and are not reported since they were not confirmed.

3.3.2 Host Trees

The combined host dataset yielded a single most likely tree (-lnL=19,451.13), shown in Figure 3.2, and two most-parsimonious trees (L=4,025, CI=0.41, RI=0.42, RC=0.17). Clades that were present in the parsimony trees are indicated in bold, and clade support is shown in Figure 3.2. Host trees were largely consistent with generic and tribal taxonomic groupings, but some differences were observed (discussed below).

3.3.3 Symbiont Phylogenies and Divergences

Phylogenetic analyses of Baumannia 16S rDNA sequences yielded a single most likely tree (-lnL=9,175.09)

(Figure 3.3) and five most-parsimonious trees (L=1,558, CI=0.43, RI=0.6, RC=0.26). Sequence divergences within Baumannia ranged from 1% to 19% (Table 3.3). The analyses gave strong support for the mono- phyly of Baumannia within the clade of γ-3 Proteobacteria. Phylogenetic analyses of the Sulcia 16S rDNA sequences resulted in three most likely trees: the least resolved of these is shown in Figure 3. Parsimony analyses gave approximately 200,000 most-parsimonious trees (L=1,012, CI=0.73, RI=0.78, RC=0.56). The immense number of parsimonious trees reflects the low divergence among the sequences from different host species, with sequence divergence ranging from 0% to 7% (Table 3.3). Nonetheless, some nodes were strongly supported in both maximum likelihood and parsimony analyses, indicating that some phylogenetic informa- tion was present in the Sulcia dataset. Both likelihood and parsimony analyses gave strong support for the monophyly of Sulcia within the Bacteroidetes.

3.3.4 Host-Symbiont Associations

The maximum-likelihood topologies for hosts and symbionts were slightly different. However, these dif- ferences were not significant under the criterion of the maximum-likelihood-based S-H test. That is, the

Baumannia dataset and evolutionary model generated topologies not significantly less likely than those pro- duced from the host dataset under its respective evolutionary model (Table 3.4). This result is consistent with the hypothesis of perfect phylogenetic congruence of Baumannia and hosts. Results of the S-H test involving Sulcia were not self-consistent. Although host and Baumannia best topologies were not statisti- cally different from Sulcia best topologies when optimized with the Sulcia dataset and evolutionary model

(Table 3.4), Sulcia best topologies were found to be statistically different when optimized with the host or

Baumannia datasets and evolutionary model (Table 3.4).

43 Hylaius oregonensis BATHYSMATOPHORINI

Pagaronia tredecimpunctata PAGARONIINI

Homoscarta irregularis 30

25

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CICADELLINI 67/* Pamplona spatulata

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-5 -10 19 -15 0.05 substitutions/site Cuerna striata

Figure 3.2: Single maximum-likelihood phylogram for leafhopper hosts, based on the combined gene dataset (-lnL=19,451.13). Thicker branches represent clades also recovered by maximum parsimony analyses. Num- bers above branches are likelihood/parsimony bootstrap percentages (asterisks represent those below 50), and those below are Bremer decay indices. Node-associated graphs represent partitioned Bremer decay in- dices (y-axis scale ranging from -15 to 30) for the partitions (from left to right) histone H3, COI, COII, and 16S rDNA. Numbered nodes indicated by black circles are congruent with those indicated in both symbiont phylogenies.

44 Yersinia enterocolitica

60/* Escherichia coli Buchnera aphidicola of Acyrthosiphon pisum */99 S-endosymbiont of Cyphonococcus alpinus

61/65 S-endosymbiont of Calophya schini 86/* 2 Blochmannia of Camponotus herculeanus S-endosymbiont of Aphalaroida inermis S-endosymbiont of 98/94 Amonostherium lichtensioides 7 S-endosymbiont of Melanococcus albizziae

100/100 Diestostemma excisum 1 26 Diestostemma stesilea 100/100 3 14 Homoscarta irregularis 91/94 5 Paraulacizes irrorata 99/99 9 100/100 Proconosama columbica 2 11 90/68 4 Proconosama alalia Paromenia isabellina Helochara communis

1 Graphocephala coccinea 95/98 5 57/* 8 Graphocephala aurora 84/85 Candidatus Graphocephala cythura 69/* Baumannia

53/* Graphocephala hieroglyphica 6 cicadellinicola Pamplona spatulata Acrogonia virescens Clydacha catapulta

100/100 Homalodisca vitripennis 16 Homalodisca liturata

100/100 17 92/81 Homalodisca elongata 1 Phera obtusifrons 73/62 1 Oncometopia orbona 83/70 1 Cyrtodisca major 89/76 4 Cuerna gladiola

100/100 0.05 substitutions/site 9 Cuerna costalis

62/79 2 Cuerna sayi Cuerna striata

Figure 3.3: Single maximum-likelihood phylogram for Baumannia symbionts and relatives in the γ- Proteobacteria, based on 16S rDNA sequences (-lnL = 9,175.09). Thicker branches represent clades also recovered by maximum parsimony analyses. Numbers above branches are likelihood/parsimony bootstrap percentages (asterisks represent those below 50), and those below are Bremer decay indices. Numbered nodes indicated by black circles are congruent with both host and Sulcia phylogenies, while the one indicated by the black diamond is congruent only with the Sulcia phylogeny.

45 Flexibacter tractuosus

100/95 Weeksella virosa 13 Chryseobacterium miricola

100/100 Endoparasite of Coleomegilla maculata 29 Endoparasite of */64 Adonia variegata 4 Blattabacterium of Blaberus craniifer 100/100 19 Blattabacterium of Mastotermes darwiniensis of sp. 100/100 Blattabacterium punctulatus Cryptocercus 24 Jikradia olitoria Hylaius oregonensis 0.05 substitutions/site 100/100 Pagaronia tredecimpunctata 55 Homoscarta irregularis Paraulacizes irrorata Diestostemma excisum Diestostemma stesilea J. olitoria Proconosama alalia 80/62 H. oregonensis Proconosama columbica 2 P. tredecimpunctata H. irregularis Paromenia isabellina P. irrorata Helochara communis 82/* 59/* 3 54/* D. excisum 1 Cicadella viridis 2 D. stesilea Candidatus 51/77 P. alalia Graphocephala cythura 2 Sulcia 2 63/* P. columbica 4 Graphocephala coccinea P. isabellina muelleri H. communis Homalodisca vitripennis

93/* C. viridis Homalodisca liturata 5 1 G. cythura Phera obtusifrons G. coccinea Pamplona spatulata 6 H. vitripennis H. liturata Clydacha catapulta P. obtusifrons Homalodisca elongata P. spatulata Cyrtodisca major */62 C. catapulta 1 H. elongata Oncometopia orbona C. major Acrogonia virescens O. orbona Cuerna costalis 2 A. virescens C. costalis Cuerna gladiola 1 C. gladiola Cuerna sayi C. sayi C. striata Cuerna striata

Figure 3.4: Selected maximum-likelihood phylogram (one of three) for Sulcia symbionts and relatives in the Bacteroidetes phylum of Bacteria, based on 16S rDNA sequences (-lnL = 6,993.82). The smaller cladogram on the left shows the same topology as the phylogram, but with outgroups pruned for better visualization of clades and support. Thicker branches represent clades also recovered by maximum parsimony analyses. Numbers above branches are likelihood/parsimony bootstrap percentages (asterisks represent those below 50), and those below are Bremer decay indices. Numbered nodes indicated by black circles are congruent with both host and Sulcia phylogenies, while the one indicated the black diamond is congruent only with the Baumannia phylogeny.

46 Table 3.3: Maximum likelihood pairwise distances among 16S rDNA sequences of selected Baumannia (A) and Sulcia (B) endosymbionts and related bacteria. Host names are in parentheses. Values represent number of substitutions per 100 bases based on models of molecular evolution specified. (a) GTR+Γ+I Candidatus Baumannia cicadellinicola

2 3 4 5 6 7 8 9 10 11 12 13 14 15 1 Yersinia enterocolitica 7.64 23.79 18.69 17.76 19.69 23.09 19.05 25.53 21.08 20.53 22.96 22.86 22.21 22.49 2 Escherichia coli 17.68 19.46 18.72 18.19 24.81 19.82 26.07 22.53 23.46 24.29 23.28 25.03 25.22 3 Buchnera aphidicola 21.92 24.41 25.91 28.01 24.77 25.98 25.77 28.78 23.31 28.29 22.94 21.68 4 S-symb (A. lichtensioides) 14.22 15.86 14.23 13.44 13.65 14.3 14.6 14.87 16.02 15.7 13.86 5 S-symb (A. inermis) 16.86 16.69 13.97 15.72 14.09 15.49 16.6 15.3 18.37 16.71 6 Blochmannia (C. herculeanus) 17.22 16.14 18 17.67 19.39 19.75 17.63 21.17 18.38 7 (G. coccinea) 9.3 10.15 9.14 11.17 14.33 10.58 14.63 14.65 8 (P. isabellina) 8.56 6.53 9.38 12.23 8.56 13.62 12.47 9 (C. catapulta) 6.74 10.2 12.59 10.14 14.78 12.79 10 (A. virescens) 7.9 12.17 7.31 13.78 12.74 11 (C. costalis) 14.3 4.49 17.14 14.71 12 (D. excisum) 16.11 8.36 7.25 13 (H. vitripennis) 19.25 16.19 14 (H. irregularis) 5.8 47 15 (P. alalia) —

(b) TrN+Γ+I Candidatus Sulcia muelleri

2 3 4 5 6 7 8 9 10 11 12 13 14 15 1 Flexibacter tractuosus 23.27 25.3 27.54 30.24 29.91 34.85 39.04 30.91 30.83 31.01 31.42 29.37 29.32 29.44 2 Weeksella virosa 25.98 26.44 32.76 29.51 33.4 61.56 29.23 32.35 29.7 31.08 28.14 30.24 29.5 3 Endoparasite (A. variegata) 13.89 15.59 16.38 18.79 22.38 16.33 16.8 16.53 17.6 16.92 16.31 16.04 4 Blattabacterium (M. darwiniensis) 18.8 18.99 21.79 29.28 18.55 20.47 18.52 20.28 18.85 19.39 18.92 5 (J. olitoria) 2.12 4.13 3.13 2.58 2.73 2.58 3.12 3.11 2.21 2.13 6 (P. tredecimpunctata) 2.35 2.93 1.65 1.83 1.58 1.11 1.94 1.11 1.03 7 (G. coccinea) 4.87 1.15 1.84 0.98 1.93 0.74 2.28 1.7 8 (P. isabellina) 0.2 1.42 0.62 3.74 1.3 1.69 2.32 9 (C. catapulta) 0.4 0.49 1.15 0.83 1.23 1.06 10 (A. virescens) 0.08 2.09 0.57 1.26 1.27 11 (C. costalis) 1.08 0.66 1.16 1.16 12 (D. excisum) 1.43 0.71 0.08 13 (H. vitripennis) 1.52 1.51 14 (H. irregularis) 0.23 15 (P. alalia) — Table 3.4: Maximum likelihood (-lnL) scores for pruned most-parsimonious (MP) and most likely (ML) topologies. Scores were calculated based on sharpshooter, Baumannia and Sulcia datasets and respective model of molecular evolution. Best topologies (=most likely) are shown in bold. Scores that based on the Shimodaira-Hasegawa test are statistically different from the best topology are followed by an asterisk (*, α=0.05). MPsc refers to strict consensus of most-parsimonious trees and MP1-10 are 10 representatives chosen randomly from most-parsimonious Sulcia trees.

Host dataset Baumannia dataset Sulcia dataset TVM+Γ+I GTR+Γ+I TrN+Γ+I Host topologies ML 16473.94 5483.5 2757.38 MP1 16500.16 5472.09 2744.2 MP2 16493.8 5477.44 2757.37 Baumannia topologies ML 16559.83 5435.74 2740.55 MP1 16571.24 5441.88 2745.7 MP2 16572.49 5437.82 2740.55 MP3 16572.49 5437.82 2740.55 MP4 16570.96 5431.44 2740.39 MP5 16570.96 5431.44 2740.39 Sulcia topologies ML1 17033.01* 5834.06* 2699.87 ML2 17034.33* 5834.06* 2699.87 ML3 17033.01* 5836.22* 2699.87 MPSc 17806.23* 6514.06* 2933.14* MP1-10 17109.04-17527.79* 5860.02-6176.72* 2712.68-2731.88

In the event-based parsimony method of tree-fitting (Table 3.5), whenever one to three horizontal transfers

(host switching cost=10-15) were allowed, the maximum number of cospeciation events between hosts and

symbionts (12 for both symbionts) was found. This number was significantly higher than would be expected

to occur by chance (p<0.05 for all of Baumannia trees and for most Sulcia trees), supporting a significant

history of cospeciation of each symbiont with hosts. Whenever no (host switching cost=30) or a high number

of horizontal transfers (7-12, host switching cost=0-2) were allowed, estimates of codivergence decreased.

When host switching was not allowed, the null hypothesis of no switching could not be rejected for most

Sulcia trees. Also, we note that the higher number of transfer events is not biologically realistic for Sulcia,

which consists of huge cells confined to host bacteriocytes. The disparate result for Sulcia in the S-H test

and the failure to reject the H0 for some of its reconstructions in Treefitter is likely to be an artifact of the low phylogenetic information content of the 16S rDNA sequences of Sulcia, which yielded polytomous

topologies and low clade support statistics overall.

Finally, the ILD test did not reject the H0 that the gene sequences of hosts and each of the two symbionts share a common evolutionary history (P=0.46 for host and Baumannia and P=0.96 for host and Sulcia).

In summary, the results of all tests suggest that the diversification of both endosymbionts was largely or

entirely dependent on the phylogenetic history of their host leafhoppers.

48 3.3.5 Evolutionary Rates of Bacterial 16S rDNA

For both symbionts, the H0 of rate constancy among lineages was rejected (P<0.001). The differences between the -lnLs of analyses with unconstrained rates and rates constrained to be constant were 37.35 and

49.65 for Baumannia and Sulcia, respectively.

If Baumannia and Sulcia both cospeciated with their hosts, as supported by our analyses, then comparing corresponding sequence divergences for symbionts from the same host pairs will give an estimate of the relative substitution rates, since codiversification implies that these divergences accumulated over the same time intervals, for each pair. Inspection of the values in Table 3.3 suggests that Sulcia sequences evolve more slowly. However, many of these pairwise comparisons share overlapping branches of the tree, or correspond to parts of the tree in which codiversification was less strongly supported. In order to derive a more accurate estimate of the relative amount of sequence evolution in Baumannia and Sulcia, substitution rates were estimated by the lengths of branches in the topologies generated, and only on those branches leading to cospeciating nodes that are present in both symbiont trees (copaths). Based on the maximum likelihood topologies, six copaths between Baumannia and Sulcia were identified and five of these are congruent with the host topology (Figures 3.2, 3.3, 3.4). A paired statistical analysis showed that copath branch lengths from Baumannia are significantly different from those of Sulcia (W=10.5, P=0.03), which implies different

16S rDNA substitution rates for those two symbionts. In fact, the 16S rDNA of Baumannia appears to be evolving 4.88 times faster than that of Sulcia, as suggested by the slope of the major axis regression analysis

(SD of Baumannia=0.0156 and SD of Sulcia=0.0032).

Table 3.5: Results of TreeFitter analyses of coevolutionary events between leafhopper hosts and their two bacterial endosymbionts. Number of symbiont binary topologies tested in parentheses. α=0.05.

Symbiont Host switch Codivergence Duplication Sorting Host switch topologies cost events events events events Baumannia (n=2) 0 0-8a 0 0 13-21 2 11-12a 0-1 1-5 8-10 10-15 12a 8 27 1 30 11b 10 44 0 Sulcia (n=20) 0 0-9a 0 0 12-21 2 8-11b 0-2 1-11 7-13 10 9-12c 7-10 31-46 1-3 15 8-12b 7-12 32-58 1-3 30 7-10c 11-14 58-93 0

a All reconstructions rejected the H0. b Most reconstructions rejected the H0. c Most reconstructions failed to reject the H0.

49 3.4 Discussion

3.4.1 Codiversification in a Dual Symbiosis

A congruent evolutionary history of both Baumannia and Sulcia with their sharpshooter hosts is supported based on all (Baumannia) or most (Sulcia) statistical tests conducted here, suggesting a long-term association of these bacteria with their hosts. Prior to our study, molecular phylogenetic studies of hosts and symbionts have documented long-term cospeciation of only a single symbiont clade with its hosts. These examples of primary symbionts include B. aphidicola in aphids [27,237], Wigglesworthia in tsetse flies [55], Blochmannia in carpenter ants [310], Blattabacterium in cockroaches and termites [20,201], Carsonella in psyllids [331,354],

Tremblaya in mealybugs [355], and Portiera in whiteflies [352]. Each of these host groups is considered to have a single primary symbiont representing an ancient infection followed by codiversification.

We identified Sulcia and Baumannia in all but one of the sharpshooters tested (Baumannia was absent from C. viridis), and both have other features of typical primary symbionts such as large irregularly shaped cells, restriction to bacteriomes, and small genome sizes [238]. Neither Sulcia nor Baumannia can be considered a secondary symbiont, in view of their ancient associations with hosts, supported by our results, and their apparent nutritional contributions to hosts, supported by genomic results [379]. We suggest the term co-primary symbionts for this and other similar cases, in which two or more symbionts are obligate and ancient bacteriome-associates of a host group. Baumannia was found only in the sharpshooter tribes

Cicadellini and Proconiini and the subfamily Phereurhininae; whereas Sulcia was present in all leafhoppers tested (Table 3.1). Preliminary observations on members of other groups of Auchenorrhyncha suggest that

Baumannia is not found in groups outside of Cicadellidae, but that Sulcia is widely distributed among auchenorrhynchan lineages [239].

Our findings suggest that both Sulcia and Baumannia are ancient bacteriome-associates that reflect long-term codiversification of two bacteria clades with sharpshooters and relatives. These findings are in general agreement with Buchner’s views: he recognized leafhoppers and their relatives as having the most elaborate symbioses of any insects and hypothesized long-term coevolution of two or more bacterial associates with particular leafhopper subfamilies or tribes. Thus, in his proposed evolutionary scenario, based largely on the work of his student, H. J. M¨uller, one symbiotic association was ancestral to the

Auchenorrhyncha, with different host lineages acquiring and losing additional symbionts during the radiation of this large clade, resulting in a mosaic of different symbiont combinations across modern subfamilies and tribes. Buchner hypothesized that the sharpshooters retained the ancestral (“a”) symbiont plus a more recently acquired symbiont, designated the “t” symbiont. Under this view, the paired, bilateral bacteriomes

50 are homologous organs retained by most groups of Cicadomorpha (the lineage that includes leafhoppers, treehoppers, spittlebugs and cicadas), during their evolution from an ancestor containing an original symbiont type.

3.4.2 Nutritional Roles for Primary Symbionts and Implications for Host Diversification

The evidence for coinheritance of both Baumannia and Sulcia across millions of years strongly suggests that both symbionts are essential for the host insect. Other ancient bacteriome-associated symbionts are known to provide needed nutrients to host insects, a role inferred in part from genomic analyses of the symbionts

(e.g., [322]). The xylem sap diet of sharpshooters lacks many nutrients required by animals, including essential amino acids and cofactors [296]. Stable co-primary symbiosis might result if different symbiont types are providing different sets of required nutrients. Genomic analyses of Baumannia and Sulcia of the host H. vitripennis provide strong evidence that the two symbionts indeed serve complementary nutritional roles in the provisioning of amino acids and cofactors [379]. Despite its small genome size, Baumannia retains pathways for numerous cofactors (vitamins) needed by the host, with a total of about 12% of its genome devoted to these processes. Sulcia was partially sequenced and has genes for production of most essential amino acids.

The dependence on more than one obligate symbiont is potentially a constraining factor in the evolution and host plant range of sharpshooters and other auchenorrhynchans. Genome comparisons for primary symbionts of both aphids and carpenter ants indicate that these bacteria do not acquire foreign genes and do undergo continued erosion of the ancestral genome [77,349]. For example, Buchnera lineages continue to lose genes, and some of these appear to impose new nutritional requirements on hosts, such as a requirement for dietary sources of fixed sulfur, available in only some plants [349]. In H. vitripennis, Baumannia has a very small genome (686 kilobases, [238, 379]) and Sulcia also has a genome considerably under 1 megabase

(P. Tran and N. Moran unpublished data, [379]), as do all primary symbionts for which genome sizes are known. Indeed, an apparent distinction between primary and secondary symbionts is the extent to which their genomes continue to acquire novel genes and gene arrangements, and thus the extent to which they represent a source of novelty in host evolution. In both aphids and tsetse flies, recent genomic studies indicate that secondary symbionts have larger and more dynamic genomes, with phage, repetitive elements and evidence of recent gene acquisition, contrasting with the primary symbionts in the same hosts, which have near-minimal genome sizes [236,359].

Although the Baumannia-Sulcia-sharpshooter symbiosis is the first case in which multiple obligate and

51 apparently ancient microbial symbionts have been studied using molecular data, many more similar cases

of complex symbioses appear to exist, based on microscopy of other systems [43]. Hosts with multiple

primary symbionts are likely to be highly dependent on the gene sets of each endosymbiont, with possible

consequences for their own evolution. Alternatively, stable co-occurrence of more than one symbiont might

enable exchange of metabolites between them that enables even more extensive loss of genes in one or both

genomes. Further studies of symbiont genomes and interactions with hosts will be required to assess how

coevolution with multiple symbionts affects host biology and diversification.

3.4.3 Variable Rates of Evolution in Symbiont Sequences

Our estimates of divergences for 16S rDNA sequences of Baumannia, based on maximum-likelihood models,

are similar to the distance divergences estimated using a JC model for those five representatives studied

previously [235]. In their study, the maximum divergence of about 7% was found between lineages of

Graphocephala (Cicadellini) and Homalodisca (Proconiini). Furthermore, they suggested that Baumannia has an approximately equal rate of nucleotide base substitution for 16S rDNA to Buchnera aphidicola, the primary endosymbiont of aphids, based on the observation that Baumannia and Buchnera sequences show very similar divergences to outgroups such as Escherichia coli and Yersinia pestis (Table 3.3). Based on previous molecular studies, B. aphidicola shows a per lineage substitution rate of about 1-2% per 50 million years, corresponding to a divergence rate between paired lineages of 2-4% per 50 million years [237].

Accordingly, Moran et al. [235] hypothesized that the Baumannia common ancestor was associated with the sharpshooter common ancestor around 80-175 million years ago. However, the larger taxon sample of the present study facilitated our discovery that the minimum and maximum divergences between members of the most basal clade of Baumannia (associated with hosts of the Proconiini clade which includes Diestostemma) and other Baumannia representatives were 11% and 19%, respectively. If the 16S rDNA of Baumannia is evolving at the same rate as that of Buchnera, then these observed divergences would imply that the sharpshooter lineage is between 138 and 475 million years old. These dates are substantially greater than those suggested by the fossil record, suggesting that at least some lineages of Baumannia have a faster 16S rRNA substitution rate than do Buchnera. Although the earliest fossils of Cicadomorpha are 270 million years old, leafhoppers (Cicadellidae) do not appear in the fossil record until 130 million years later [321], and fossil leafhoppers definitely assignable to extant subfamilies do not appear until 50 million years ago (Baltic amber, [338]). Nevertheless, no true sharpshooter is known from this period (including Ambericarda skalskii

Szwedo and Gebicki 1998, which was incorrectly placed in Proconiini [341], J. Szwedo, pers. comm.). Only in Dominican amber (25-40 Mya) do true sharpshooters first appear as fossils [92]. This implies that at

52 least some lineages of Baumannia evolve at a faster rate than does Buchnera, and/or that the sharpshooter lineage is older than indicated by available fossil evidence. Previous studies have shown that bacterial groups can have very different rates of substitution in DNA sequences [258].

Indeed, a primary result of the present study is that Baumannia and Sulcia have dramatically different rates of 16S rDNA substitution, with Baumannia evolving approximately five times faster than Sulcia. The

basis for the much slower rate of evolution in Sulcia is not known. Because it is the first bacteriome-associate

outside of the Proteobacteria to be studied using molecular sequence data, its slow rate may reflect basic

differences in mutational processes between bacterial phyla. We note that Sulcia does show some other

genomic features that are characteristic of bacteriome-associated symbionts, including AT-biased genome

composition (about 24% G+C) and small genome size [379]. Finally, results of the likelihood-ratio test

suggest that both Baumannia and Sulcia 16S rDNA substitution rates are not constant among the different

lineages, which would introduce errors in age estimates based on the assumption of a constant molecular-

clock.

3.4.4 Wolbachia in leafhoppers

Our findings for Wolbachia are consistent with a previous study [176] showing that leafhoppers are fre-

quent hosts for this symbiont group and that it has an erratic distribution with respect to host phylogeny.

Noncongruent phylogenies are typically found between Wolbachia and their hemipteran [175] or other insect

hosts [34,187,324,372]. Results for Wolbachia are a striking constrast to those for Baumannia and Sulcia and

weigh against a role for Wolbachia as an obligate mutualist of leafhoppers. Indeed its most well-documented

effect on insects hosts are modifications of the reproductive biology, such as cytoplasmic incompatibility and

son-killing [372]. We do not know what phenotypes Wolbachia confers upon the leafhopper hosts that we examined. Wolbachia is often variably present within host species. Since most of our study species were represented by one or few collections, more extensive sampling might reveal Wolbachia infections in some of

the species for which we found none.

3.4.5 Implications for Leafhopper Phylogenetics and Classification

Results of the present analysis of sharpshooter hosts agree with recent higher-level phylogenetic studies

of leafhoppers suggesting that the subfamily Cicadellinae sensu Oman et al. [264] is not a monophyletic unit [83,85,91]. These previous studies, based on analyses of morphology and 28S rDNA sequences, support

Young’s [392, 393] inclusion of only two tribes, Cicadellini and Proconiini, in the subfamily, but also sug- gest that Phereurhininae is derived from the same lineage. This delimitation of Cicadellinae is consistent

53 not only with our present phylogenetic analyses of host nuclear and mitochondrial, but also of symbiont

genes, suggesting the possibility of using these endosymbiont genes for inferring host phylogenetic histories.

Moreover, Baumannia appears to be found only in this leafhopper lineage, and its presence could itself be

considered a highly informative character for phylogenetic reconstruction.

3.4.6 Conclusions

Initial molecular studies on bacterial-eukaryote symbioses focused on relatively simple systems, involving

a single bacterial lineage or a single primary symbiont combined with opportunistic secondary symbionts

[27]. However, complex symbioses involving multiple partners are common in eukaryotes. For example,

Buchner [43] describes numerous systems with several co-inherited bacterial and/or fungal symbionts in

animal hosts. Our results support the hypothesis that the sharpshooter dual symbiosis has been stably

inherited during the evolution of this insect group and raise the likelihood that bacterial symbiosis has

been a major element governing the ecological diversification of these insects. In the case of the single

primary symbiont Buchnera in aphids, the symbiosis benefits hosts by providing a source of needed nutrients

[27,322]. But, because Buchnera lineages undergo irreversible gene loss over time, this symbiosis can also be a constraining factor, and current evidence from genomics suggests that gene losses and genome degradation in Buchnera may limit ecological capabilities of aphids, confining them to certain host plant groups [349].

In the case of multiple bacterial partners, these constraints are likely to be more complex, Baumannia and

Sulcia resemble Buchnera is having reduced genomes and limited metabolic capabilities [379]. The evolution

of each symbiont is likely to have had major consequences for the other as well as for hosts. For the first

time, a combination of phylogenetic and genomic approaches is enabling us to recognize these intertwined

histories and to understand their consequences for host ecology and evolution.

54 Chapter 4

Taxonomy of the genus Homalodisca St˚al

4.1 Introduction

Sharpshooters are members of the cosmopolitan leafhopper subfamily Cicadellinae, which contains over 2,600

species among 372 genera. Fifty-eight of these genera, and over 400 species, belong to the New World tribe

Proconiini [50,135,293,389]. Among the most economically important proconiine genera is Homalodisca St˚al,

1869, which was not properly redescribed until Young’s [392]revision of Proconiini. Young [392] included 19

valid species in Homalodisca, making four new synonymies and describing three new species. In addition,

he noted that Homalodisca was closely related to Phera St˚al,1864, Dichrophleps St˚al,1869, Oncometopia

St˚al,1869, and Propetes Walker, 1851. Since Young’s [392] revision, only a single published work included

taxonomic changes to this economically important genus. Burks & Redak [44] transferred Phera lacerta

Fowler, 1899 back to its original genus and reinstated Young’s junior synonym Homalodisca liturata Ball,

1901, as the valid name for the species occurring in Mexico and the U.S.A. (California and Arizona) commonly referred to as the smoketree sharpshooter.

Most species included in Homalodisca occur in Central and North America, but the genus is widely distributed throughout the Americas. Furthermore, the glassy-winged sharpshooter, native to southeastern

North America, has been recently introduced in some Pacific islands (reported from Tahiti in 1999, Moorea in

2002, and Hawaii in 2004 ; [158,329]). Four Homalodisca species occur in the southern United States and three of these are known vectors of the bacterium Xylella fastidiosa, which causes millions of dollars of damage in a number of fruit and horticultural crops [248,296]. The glassy-winged sharpshooter transmits the bacterium to grapevines causing the lethal Pierce’s Disease, virtually precluding viticulture in the southeastern United

States and northeastern Mexico [5,296]. In addition, this species can transmit this bacterium to peach, plum, and nectarine trees, and to the ornamental Nerium oleander (L.) [66, 280, 296, 362]. Among other North

American Homalodisca, H. insolita (Walker, 1858) is a vector of phony peach disease and the smoketree sharpshooter is a vector of Pierce’s Disease and oleander leaf scorch, the latter especially in drier areas of southern California where this sharpshooter is most abundant [33, 130, 362]. Of the 17 valid Homalodisca

This chapter was published in: Takiya, McKamey, & Cavichioli (2006) [344] and Takiya, Cavichioli, & McKamey (2006) [343] species, only three described species are known to occur in Brazil: H. ignorata Melichar, 1924; H. ignota

Melichar, 1924; and H. lucernaria (Linnaeus, 1758). A fourth species formerly in Homalodisca, H. triquetra

Fabricius, is herein referred to Propetes Walker, while a fifth species described from Brazil, H. vitripennis, had been mislabeled as to its origin. In Southeastern Brazil, H. ignorata transmits X. fastidiosa to citrus [380] and coffee, causing coffee leaf scorch [212]. On the other hand, not only can X. fastidiosa infect many other plant species, but sharpshooter vectors tend to be polyphagous, e.g., the glassy-winged sharpshooter has been associated with over 100 host species [159,362], increasing the potential economic impact of the genus

Homalodisca.

Considering its great economic importance, the identity of the genus Homalodisca is of primary signifi- cance and it is necessarily tied to its type-species. Here evidence is provided that the previously designated type-species of Homalodisca was erroneously identified by St˚al(1869), and to maintain stability of nomen- clature, we fix the identity of the type of the genus and establish the valid name of the economically important glassy-winged sharpshooter based on the lectotype specimen. This new fixation facilitates reten- tion of Homalodisca as a valid genus, despite the transfer of the previously designated type-species, Cicada triquetra Fabricius, 1803, to the genus Propetes.

Additionally, the rediscovery of the allegedly “lost” Hemiptera collection of Ernst F. Germar at the Ivan

Franko National University (Lviv, Ukraine) permitted the designation of a lectotype for Tettigonia vitripen- nis Germar, 1821. Homalodisca vitripennis is a senior synonym of Tettigonia coagulata Say, 1832, thus becoming the valid name for the glassy-winged sharpshooter. With the spread of Pierce’s Disease in Califor- nia vineyards, the glassy-winged sharpshooter and other Homalodisca species are receiving an unprecedented level of research. Despite the grave nature of changing the scientific name of such an economically important species, it is necessary from a nomenclatural standpoint and such changes should be made sooner rather than later.

Study of specimens from citrus orchards in Bahia and Sergipe states (Brazil) resulted in the discovery of a species of Homalodisca that differs in its external and genitalic morphology from all Homalodisca species described. This new species, Homalodisca spottii sp. nov., is herein described and illustrated and may be associated with the transmission of the bacterium Xylella fastidiosa strain that causes the citrus variegated chlorosis (CVC) in Northeastern Brazil.

After studying the female lectotype of Homalodisca ignota, the only previously known specimen, conspe- cific male and female specimens were found at the collection of the Museu Nacional (Rio de Janeiro, Brazil).

H. ignota is redescribed herein, to include information on the previously unknown male and an illustration of the female sternite VII, which was inaccurately provided previously by Young [392]. Both species de-

56 scribed here have a well developed and sclerotized (at least partially) internal sternite VIII in females. They

represent the first record of this structure in Homalodisca. Nielson [247] stated that Homalodisca does not

have this structure sclerotized, but did not specify which species he studied. Sclerotization of this structure

occurs in fairly closely related genera, such as Propetes Walker [346] and Dichrophleps St˚al[220].

Finally, notes and new locality records for other Homalodisca species occurring in Brazil are given, as

well as a taxonomic key.

4.2 Materials and Methods

In quoting label data of type material, a bar (|) separates lines on a label. Morphological terminology follows mainly Young [392, 393] , except for the head, which follows Hamilton [145], leg chaetotaxy which follows

Rakitov [287], and genitalia which follows Nielson [247]. Techniques for preparation of genital structures are those of Oman [262]. The dissected parts are stored in microvials with glycerin. Acronyms for depositories of material studied are given in the list of specimen depositories in the beginning of this thesis.

Drawings of genitalia structures of H. vitripennis were based on photographs and pencil sketches made during the visit to IFNU.

4.3 Taxonomy

4.3.1 Homalodisca St˚al

Homalodisca St˚al, 1869: 63 [332].

Type-species: Cicada triangularis Fabricius, 1803: 63. New designation. C. triangularis was misidentified by St˚al[332] as C. triquetra Fabricius, 1803: 63 [115]. C. triangularis (preoccupied) was replaced by Homalodisca fabricii Metcalf, 1965: 501 [229], which was considered by Young [392] to be a junior subjective synonym for H. lucernaria (Linnaeus, 1758): 434 [193].

St˚al[332] erected the genus Homalodisca including Cicada triquetra and C. triangularis F., 1803, which he redescribed based on Fabricius’ type-specimens. In a footnote in the same paper, St˚alalso included Proco- nia admittens Walker, 1858 and Tettigonia coagulata in Homalodisca. Distant [97] subsequently designated

C. triquetra as the type-species, meeting the requirements of the current International Code of Zoological

Nomenclature (ICZN [401]: Articles 67.2, 67.4, and 69.1). Unfortunately, St˚al[332] apparently interchanged the descriptions of C. triquetra and C. triangularis in his manuscript. Fabricius’s [115] description, although short, states clearly that C. triquetra is black and has fuscous hyaline forewings without markings (Figure

57 4.3), while St˚al[332] described the species as mostly brown mottled with yellow with forewings hyaline with

a dark costal macula (as in Figure 4.1). The latter description fits C. triangularis (now correctly referred

to as H. lucernaria), the other Fabrician species upon which St˚al’sgenus description was based, not only

in the external characters and color pattern, but also in the shape of the female sternite VII, which was

not mentioned previously by Fabricius [115]. At the same time, St˚al’s redescription of C. triangularis ap- proximated the original description of C. triquetra and, perhaps most importantly, emphasized the basally constricted abdomen, a character shared by C. triquetra with other Propetes species (Figure 4.3, see notes below), but not present in Homalodisca. Furthermore, although Fabricius [115] did not mention the gender of the specimens studied, St˚al’s [332] descriptions indicated that he studied one or more males of “C. trian- gularis” and more than one female of “C. triquetra” from the Fabricius collection. Females of C. triquetra

are not known (see below) and according to Young [390] there is only one male specimen of C. triquetra

in the Fabricius collection (designated as the lectotype). Taken together, the aforementioned comments are

consistent with only one conclusion: that St˚al’sdescription of “C. triquetra” in fact refers to C. triangularis.

Nevertheless, it warrants mention that Young [390] stated that out of four specimens of C. triangularis in

the Fabricius collection, at least one is a male, a point not noted by St˚al[332], who may have mistaken males

for females, a possibility corroborated by the unusually large body size range St˚algave (“Long. 11.5-14”) in

his “C. triquetra” description.

An early entomologist in possession of St˚al’s [332] work, of which the generic key was translated into

English by Dallas [74], but not familiar with the original species descriptions by Fabricius [115] would almost

certainly identify a glassy-winged sharpshooter or any similar Homalodisca as H. triquetra. Erroneous use

of the name H. triquetra as applied to North American species dates back to Weed [370], who recorded this

species from Mayersville (MS, U.S.A.). Weed [370] mentioned being in contact with Dr. Van Duzee, who

most probably played a role in identifying his specimens as C. triquetra. Two years later, Van Duzee [365]

in his catalogue of North American jassoids, recorded C. triquetra from Mexico and U.S.A. and ignored the

fact that this species was originally described from South America. These previous accounts consisted only

of taxonomic lists, until Ball [19] redescribed and illustrated what appear to be glassy-winged sharpshooter

specimens, and perhaps its close relative H. ichthyocephala, under the name H. triquetra. Following Ball [19],

the name H. triquetra was widely accepted by authors erroneously for the glassy-winged sharpshooter (e.g.,

[81,188,229,262]), including Distant [97], who designated C. triquetra as the type-species for Homalodisca. It

was only after Schr¨oder[317] redescribed and illustrated the genitalia of C. triquetra based on its lectotype

(Figure 4.3) that the identity of this species was clarified (see below). Young [389] reviewed the species of

Homalodisca in the United States, widely publicizing Schr¨oder’s (1957) discovery and reinstating H. coagulata

58 Figure 4.1: The glassy-winged sharpshooter, Homalodisca vitripennis (Germar). (A) Dorsal habitus, female, USNM. (B) Lateral habitus, female, USNM. (C) Aedeagus, lateral view, lectotype, IFNU. (D) Aedeagus, ventral view, lectotype, IFNU. (E) Abdominal sternite VII in situ, ventral view, female paralectotype, IFNU. A, aedeagal atrium; AP, atrial processes; S, aedeagal shaft. as the valid name for the glassy-winged sharpshooter. This name was accepted and has been in use by virtually all of the many researchers on the glassy-winged sharpshooter, but must now be synonymized under H. vitripennis (see below).

By interchanging the descriptions of the two Fabrician species upon which he based his original description of Homalodisca, St˚al[332], in effect, misidentified both species, one of which, C. triquetra, was later designated by Distant [97] as the type species of the genus. In the case of misidentification of a type-species, the

ICZN [401] allows an author to select and fix as type-species either the “nominal species previously cited as type-species” (Article 70.3.1) or the “taxonomic species actually involved in the misidentification” (Article

70.3.2). In this case, stability and universality of nomenclature are best served by using the second option, because the first option would require sinking Homalodisca as a junior synonym under Propetes and the proposal of a new genus to receive the glassy-winged sharpshooter and all other species currently placed in Homalodisca. Thus, we hereby fix C. triangularis F. (=C. triquetra sensu St˚al[332] as type-species of

Homalodisca under Art. 70.3.2 of the Code [401]. Cicada triangularis is a junior synonym of H. lucernaria.

59 4.3.2 Homalodisca vitripennis Germar

Tettigonia vitripennis Germar, 1821: 61 [133].

Tettigonia coagulata Say, 1832: 13 [311]. New synonymy.

Type-locality. Unknown. The species is distributed in the southeastern U.S.A. and northern Mexico; introduced to California, Hawaii, and French Polynesia. The lectotype was erroneously labeled as being from S˜aoPaulo, Brazil.

Length. Lectotype |, including forewings in repose: 12 mm. Previous attempts to locate the Hemiptera collection of Ernst F. Germar, which housed specimens eligible to be primary types of H. vitripennis, were unsuccessful [317, 392]. Schr¨oder [317] described and illustrated the common Central and North American Homalodisca species, H. ichthyocephala (Signoret, 1854), and reluctantly considered it to be conspecific with H. vitripennis based on the short original description [133].

Later, Young [392] rejected this proposal based on the fact that no species of the genus has a range that includes both Brazil and Central America, which left the identity of H. vitripennis in doubt. The Germar

Hemiptera collection, however, has not been lost and, as correctly indicated previously [161, 317], it was kept in “Lemberg,” now known as Lviv in Ukraine. This collection has been recently made accessible at the Ivan Franko National University of Lviv. A recent visit by DMT to this collection made possible the study of a male and a female identified as H. vitripennis by Germar (Figure 4.2). The handwriting on these specimens’ labels is Germar’s (compare to [161]: Plate 37, Fig. 36) and the specific epithet is followed by “m.,” an abbreviation of mihi (= I, me), referring to himself as the author of the species. There is no reason to believe that these specimens of H. vitripennis were mislabeled after Germar’s study, because the external morphology of both specimens and the type-locality on the label (S˜aoPaulo) agree with the original description ( [133]: 61). The original description of H. vitripennis, however, was based only on external morphological characters, and there are no other sharpshooters in that collection that would agree with it (DMT, personal observation). Furthermore, that another male and female of H. vitripennis from

“Carolina” were added to the collection, most probably after Germar’s [133] work (labeled “vitripennis Gr”), is evidence that whoever worked on that collection tried to keep the original disposition intact. Therefore, the two specimens erroneously labeled as being from Brazil are viewed as syntypes and the male specimen is herein designated as the lectotype for Tettigonia vitripennis Germar, 1821, the female specimen consequently becomes a paralectotype ( [401]: Article 74.1.3). The taxonomic purpose of designating a lectotype for T. vitripennis is to assure a correct and consistent application of this name, associated with a specimen, in order to promote nomenclatural stability. Other sharpshooter specimens found in this collection will be designated as primary types individually for other species in a future paper (DMT, in preparation).

60 Figure 4.2: Lectotype of Homalodisca vitripennis. (A) Dorsal habitus, male lectotype, IFNU. (B) Lateral habitus, male lectotype, IFNU. (C) Original green label handwritten by E. F. Germar associated to lectotype, IFNU.

Based on the study of external and male genitalic characteristics, no Brazilian (or South American) species closely resemble H. vitripennis, which is, in fact, conspecific with H. coagulata. According to the principle of priority ( [401]: Article 23), H. vitripennis is the oldest valid name and therefore has priority over

H. coagulata This precedence may be reversed only if two conditions are met ( [401]: Article 23.9.1): (1) if the junior synonym has been used as a valid name in at least 25 works, published by at least 10 authors, and encompassing a span of not less than 10 years in the last 50 years; and (2) if the senior synonym has not been used as a valid name after 1899. In the case of H. vitripennis vs. H. coagulata, the first condition is met but not the second. Homalodisca coagulata has been used as the valid name for the glassy-winged sharpshooter since Young’s [389] revision of North American Homalodisca and, just in the last 25 years, at least 53 journal articles with 33 different primary authors were published using the junior synonym (Biological Abstracts search for “coagulata”). Nevertheless, H. vitripennis has been used as a valid name after 1899. Homalodisca vitripennis was listed in Metcalf’s [229] catalogue of the Homoptera, but not as valid name, because it was erroneously considered by many American authors as a junior synonym to Cicada triquetra. On the other hand, Schr¨oder [317] did consider H. vitripennis a valid name, although he misdetermined it, and Young [394] included H. vitripennis as a valid species in Homalodisca, even though he did not see the type-specimen or have any idea of its true specific identity.

61 An older name may be suppressed even when both aforementioned conditions are not met by referring the

matter to the International Commission of Zoological Nomenclature ( [401]: Article 23.9.3). The authors

are not taking this course of action because the usage of the older synonym is not considered a threat

to universality or stability. The scientific community working on economically important sharpshooters is

very cohesive and in less than a year embraced the revalidation of H. liturata as the valid name for the smoketree sharpshooter [44]. More importantly, accepting H. vitripennis as the valid name for the glassy- winged sharpshooter is a major step toward stability because it is represented by a primary type specimen, whereas the type of H. coagulata has been lost. Apart from 770 non-cicadellid specimens currently deposited at the Museum of Comparative Zoology (Harvard University), the Thomas Say Entomological Collection is assumed to have been destroyed [216] and with it specimens of Tettigonia coagulata eligible to become primary types. So far, no neotype has been designated for this species.

The erroneous labeling of locality, as happened with the lectotype of H. vitripennis, is a problem fairly

common in old collections (for more cases in leafhopper collections, see [222, 342, 345]). The glassy-winged

sharpshooter is native to the southeastern U.S.A. and northern Mexico. Say [311] described the junior

synonym, Tettigonia coagulata, from Louisiana and the species has been recorded subsequently from an

unknown locality in Mexico and the U.S.A. states of Alabama, Arkansas, Florida, Georgia, Mississippi,

Missouri, North Carolina, South Carolina, Texas, and Wisconsin [362,389]. It is herein newly recorded from

San Luis Potos´ıState in Mexico. Recently, the glassy-winged sharpshooter expanded its range to California

in 1989, Tahiti and Moorea in 1999, and Hawaii in 2004 [158, 329].

Taxonomic notes. Homalodisca vitripennis can be easily distinguished from other Homalodisca species

by its (1) brown dorsum mottled with yellow (Figure 4.1), (2) mesoscutellum concolorous with mesoscutum

(Figure 4.1), (3) forewings mostly membranous (Figure 4.1), (4) female hindtibiae with less than 5 modified

apical setae in anteroventral row ( [291]: Fig. 11L), (5) first and second visible abdominal tergites with tan

to yellow lateral maculae much larger than those on remaining posterior tergites (Figure 4.1), (6) aedeagus

without conspicuous concavity between atrium and shaft (Figure 4.1), and (7) aedeagal atrium with two

pairs of processes (Figure 4.1).

Homalodisca vitripennis has the aedeagal atrium processes (Figure 4.1, AP) variable in their shape and

especially in orientation (see Young [389]: plate I), the latter due to having a weakly sclerotized base. The

lectotype has both pairs of processes (Figure 4.1, AP) directed posteriorly, in lateral view at an approximately

right angle to the shaft (Figure 4.1, S), but other males have the dorsal pair of processes directed dorsally

(in lateral view parallel to the shaft). Both forms, and intermediates, are often found coexisting at the same

locality studied herein from various localities in the U.S.A. Intraspecific variation in the shape and length

62 of aedeagal atrium processes is common in related proconiine genera, such as Molomea China, Oncometopia

St˚al,and Pseudophera Melichar.

Type Material. LECTOTYPE: |, green label on drawer “vitripennis m. |St. Paulo”, label on pin “ET p477”, IFNU. ˜ Material Examined. BRAZIL (erroneous record): SAO PAULO: 1 ~paralectotype, same data as ´ lectotype, IFNU. MEXICO: SAN LUIS POTOSI: 1 |, 122 m, Tamazunchale, Nov 1954, N.L.H. Krauss,

USNM; 3 ~~, 2 ||, same as preceding except, 15 Jul 1963, Duckworth & Davis, 3 ~~, 3 ||, same as preceding except, 2 Aug 1963, Duckworth & Davis, USNM. U.S.A.: ALABAMA: 1 |, Montgomery

County, Montgomery, Picket Springs, 05-06-VIII-16, AMNH; 1 |, Dallas County, Hazen, 28-I-21, L. B.

Woodruff, AMNH; CALIFORNIA: 3 ||, Ventura County, Fillmore, 06-V-98, R. Rakitov, on Ulmus sp.,

INHS; FLORIDA: 2 ||, Fort Myers, one at 25- and other at 27-VII-57, J. C. Denmark, INHS; 2 ||,

Leesburg, 30-XI-55, L. H. Stover, at Vitis, INHS; 2 ||, Gainesville, 27-VI-64, F. W. Mead, light, INHS;

GEORGIA, 1 |, Wayne County, Jesup, 3 Nov 1968, Davidson, USNM; LOUISIANA: 1 |and 1 ~, Iberville

Parish, Bayou Paul, 21-VII-2003, C. R. Bartlett, UDCC; 3 ||, East Baton Rouge Parish, Baton Rouge, nr. LSU Campus, 20-VII-2003, Hg vapor light, UDCC; 1 |, East Baton Rouge Parish, Baton Rouge, 01-

IX-95, M. G. Karge, UDCC; 1 |, Tangipahoa Parish, Arcola, 07-IX-2002, Mumma, sweeping, UDCC; 1 |,

Bossier Parish, Benton hunting lease, 04-VII-2003, K. E. Landry, sweeping, UDCC; 1 |, Franklin County,

12 Jun 1963, D.R. Whitehead, USNM; MISSISSIPPI: 3 ||, Yazoo County, 14-IX-72, J. M. McWilliams,

INHS; TEXAS: 2 ||, Brownsville, 24-VI-08, light, INHS; 1 |, Brownsville, 09-XII-10, sweeping, INHS;

1 |, Brownsville, 8 May 1967, A. Blanchard; USNM; 2 ||and 2 ~~, Brazos County, nr. Millican, 07-

VI-80, S. J. Merritt, on Helianthus sp., TAMU; 1 |, Brazos County, Highway 30 9.1 miles east of junction with Highway 6, 07-08-VI-80, S. J. Merritt, on Helianthus sp., TAMU; STATE UNKNOWN: 1 |and 1 ~, Carolina, IFNU.

4.3.3 Propetes Walker

Propetes Walker, 1851: 797 [368].

Type-species: P. compressa Walker, 1851, by monotypy.

Young [392] included two valid Brazilian species in the rare genus Propetes: the type-species P. compressa

Walker, 1851, recorded from Amazonas, Mato Grosso, and Par´astates; and P. schmidti Melichar, 1925, recorded from Mato Grosso do Sul and S˜aoPaulo states. Young [392] also studied specimens of a possibly undescribed species from Guyana. Species of Propetes can be easily distinguished from other proconiines by the following characteristics [346]: (1) head with median longitudinal carina on transition crown-frons

63 Figure 4.3: Dorsal habitus of type-specimens of Propetes Walker species. (A) Male lectotype (abdomen detached) of P. triquetra (Fabricius), comb. nov., University of Copenhagen. (B) Female holotype of P. compressa Walker, BMNH.

(Fig. 9); (2) pronotum with transverse sulcus anteriorly (Figure 4.3); (3) mesoscutellum swollen (Figure

4.3); (4) forewings hyaline (Figure 4.3); and (5) abdomen strongly constricted basally (Figure 4.3). The last three morphological characteristics, along with the color pattern consisting of black and yellow, have been associated in P. schmidti with mimicry of epiponine wasps [346].

4.3.4 Propetes triquetra (Fabricius) comb. nov.

Cicada triquetra Fabricius, 1803: 63 [115]. [nec Cicada triquetra sensu St˚al,1869]

Type-locality. South America.

The study of a photograph of the lectotype deposited at the University of Copenhagen (Figure 4.3,

Copenhagen, Denmark) and two additional males of Cicada triquetra confirms that this species, having all aforementioned genus characteristics, pertains to Propetes. Previously, Young [392] realized that this species probably belonged to Propetes, but because C. triquetra was designated as the type-species of Homalodisca and he thought that proper placement would lead to a generic synonymy, he chose not to take action at that time. Having newly fixed the identity of the type-species of Homalodisca as C. triangularis, we herein transfer C. triquetra to Propetes without any change in status of the genera involved. Propetes triquetra, comb. nov., was described from an unknown locality in South America [115] based on material collected

64 by Smidt. According to St˚al( [332]: 3), Smidt’s material was most probably collected in Guyana (Essequibo

and Demerara), therefore, P. triquetra is herein newly recorded from Brazil (Mato Grosso and Par´astates).

Taxonomic notes. The male lectotype of P. triquetra (Figure 4.3) was redescribed and illustrated by

Schr¨oder[317] and Young [392]. Males of this species can be easily distinguished from males of the only other

Propetes with known males, P. schmidti, by their (1) crown and pronotum completely black without yellow markings (Figure 4.3), (2) membranous connection between the aedeagal shaft and atrium ( [392]: Fig.

180f), and (3) three pairs of atrial processes ( [392]: Fig. 180f). Females of this species are still unknown.

Similarly, P. schmidti was only known from males until Takiya et al. [346] discovered that females have a strikingly different color pattern than males, making the genders difficult to associate. Although sexual dimorphism in the color pattern of sharpshooters is uncommon, it is possible that females of P. triquetra,

when discovered, will differ from the mostly black males.

Material examined. BRAZIL (New Country Record), MATO GROSSO: 1 |, Barra do Tapirap´e, ´ 01-I-63, B. Malkin, CAS (compared with type by D. A. Young); PARA: 1 |, Serra Norte, Caldeir˜ao,27-X-84, M. Zanuto, MPEG.

4.3.5 Notes on and New Records of Brazilian Homalodisca

Homalodisca ignorata was described from Paraguay and recorded as associated with Citrus orchards in South-

ern and Southeastern Brazil (S˜aoPaulo and Rio Grande do Sul states) [12, 211]. Surprisingly, this species

was not found associated with Citrus sinensis (L.) Osb. in northeastern Argentina (Misiones Province) [297].

This species is newly recorded from Minas Gerais, Paran´a,Rio de Janeiro, and Santa Catarina states.

Homalodisca lucernaria is the most common species in the Brazilian Amazonia. It is widely distributed

in northern South America (Colombia, French Guyana, Guyana, Surinam, and Venezuela) including Brazil

[392]. Here this species is newly recorded from Tobago island and the Brazilian states Par´aand Roraima.

4.3.6 Homalodisca ignota Melichar

Type locality. Brazil.

Length. |, 13.5 mm; ~~, 14.0-16.0 mm. External morphology. Crown (Figure 4.4) with median length approximately nine-tenths interocular

width and half transocular width; anterior margin rounded in dorsal aspect; transition crown-face slightly

angulate and with median longitudinal blunt elevation; disc flattened; median fovea incomplete, becoming

broader and shallower anteriorly; pubescence dense; posterior margin with M-shaped elevation. Frontogenal

sutures extending onto crown and attaining ocelli. Ocelli (Figure 4.4) located on imaginary line between

65 anterior eye angles; each equidistant from adjacent eye angle and median line of crown. Antennal ledges protuberant in dorsal aspect; dorsally carinate and with anterior margin slightly oblique in lateral view. Frons

flattened medially and depressed; median area mostly smooth or slightly striated; muscle impressions distinct pubescent. Epistomal suture incomplete for short median distance. Clypeus (Figure 4.4) continuing profile of frons; apical margin convex; pubescent. Prothorax with dorsopleural carinae complete. Pronotum narrower than transocular width; lateral margins parallel to slightly divergent anteriorly; median length approximately seven-tenths transhumeral width; disc rugose, punctate, and pubescent; posterior margin broadly concave.

Mesothorax with katepisternum enlarged and inflated; scutellum not striate. Forewings (Figure 4.4) hyaline, except for large sclerotized area on costal region covering entirely outer discal, base of inner discal, and apices of all anteapical cells; veins distinct and elevated; anteapical cells closed; with four apical cells, base of third more apical than those of second and fourth; without supernumerary crossveins; claval veins fused at mid-length for distance equal or longer than longer separated brancH. Hind wings extending almost as far posteriorly as forewings; vein R2+3 incomplete. Hind legs apical femoral setal formula 2:0:0; tibial anteroventral (AD) setal row without intercalary macrosetae; tibial setal row AV in females with over thirty elongate and hook-shaped modified setae throughout most of its length; first tarsomere slightly shorter or subequal than combined length of two more distal tarsomeres; with two parallel rows of short setae on plantar surface.

Male genitalia. Pygofer (Figure 4.5) moderately elongate; posterior margin broadly round; ventral process well developed, spiniform; microsetae numerous on posterior half; few macrosetae on dorsal portion. Valve

(Figure 4.5) linear, transverse. Subgenital plates (Figure 4.5) subtriangular, extending posteriorly as far as mid-length of pygofer; several microsetae distributed throughout disc; apex narrowly rounded. Connective

(Figure 4.5) approximately U-shaped, arms widely separated; with short dorsal keel. Styles (Figure 4.5) short, extending posteriorly slightly beyond apex of connective; with distinct pre-apical lobe; microsetae present; apex narrowly rounded. Aedeagus (Figure 4.5) symmetrical; preatrium (Figure 4.5) elongate, articulated basally with connective; atrium with basolateral flange and single pair of short spiniform processes directed posterolaterally (Figure 4.5: AP); apex of shaft with pair of short spiniform processes directed laterally (Figure 4.5: SP).

Female genitalia. Abdominal sternite VII (Figure 4.5) with microsetae distributed throughout disc; posterior margin with shallow median concavity. First valvifers longer than tall; few microsetae on pos- teroventral margin. First valvulae of ovipositor (Figure 4.5) with bases broadly round. Internal abdominal sternite VIII (Figure 4.5) mostly membranous, forming two main lobes; ventral lobe in lectotype (Figure

4.5), in dorsal view, more anteriorly produced than in other specimens, in which it is positioned just be-

66 Figure 4.4: Dorsal and lateral habitus of Homalodisca. (A-B) Homalodisca ignota Melichar, female lectotype, MMBC. (C-D) Homalodisca spottii sp. nov., male holotype, DZUP. Scale bars in mm. low dorsal fold; dorsal fold with pair of conspicuous sclerotized lateral regions over bases of first valvifers.

Pygofer (Figure 4.5) in lateral view moderately produced; microsetae distributed throughout disc; posterior margin narrowly round. Second valvulae of ovipositor (Figure 4.5) regularly broadened beyond basal cur- vature throughout apical four-fifths; blade bearing approximately 31 continuous teeth; each tooth (Figure

4.5) subtriangular, declivous posteriorly, with denticles throughout entire dorsal margin; preapical ventral prominence present; apical portion with denticles on dorsal and ventral margins; apex narrowly obliquely truncate.

Coloration. Head, thorax, and legs tan (lectotype) to dark-brown; mesokatepisternum dark-brown to black (Figure 4.4). Forewings (Figure 4.4) sclerotized areas and venation purplish-red (lectotype) or dark brown. Abdomen tan to dark brown; dorsal median region black; sternites with dark brown to black transverse band basally.

Material examined. LECTOTYPE, ~, “Br´esil”, “Collectio |Dr. L. Melichar |Moravsk´emuseum Brno”, “ignotus M. |det. Melichar”, “type”, “Lectotype |Homalodisca |ignota |Melichar |Young & Lauterer”,

67 Figure 4.5: Homalodisca ignota Melichar, genital structures. (A-E) Male genitalia: (A) pygofer, valve, and subgenital plate, lateral view; (B) valve and subgenital plate, ventral view; (C) connective and style, dorsal view; (D) aedeagus and anal tube, lateral view; (E) aedeagus, caudal view. (F-K) Female genitalia: (F) sternite VII, ventral view; (G) base of first valvula of ovipositor, ventral view; (H base of sternite VII and internal sternite VIII, dorsal view; (I) sternite VII, gonoplac, and pygofer, lateral view; (J) second valvula of ovipositor, lateral view; (K) tooth of median portion of second valvula of ovipositor, lateral view. Scale bars in mm. AP=aedeagal atrium process, PA=aedeagal preatrium, and SP=aedeagal shaft process.

68 “Invent |2992/Ent. |Mor. Museum, Brno”, MMBC. Additional material: BRAZIL, RIO DE JANEIRO:

|, Petr´opolis, XI.1940, Parko, MNRJ; ~, Rio de Janeiro, Jacarepagu´a,12.XI.1952, N. Santos, MNRJ; ~, ˜ Rio de Janeiro, Parque Nacional da Tijuca, III.1951, C. Seabra, MNRJ; SAO PAULO: ~, Ubatuba, Parque Estadual da Serra do mar, N´ucleoPicinguaba, Equipe Laborat´oriode Entomologia da UFRJ, 4-7.XII.2002,

MNRJ.

Taxonomic notes. The lectotype of H. ignota was designated by Young & Lauterer [396] and its female sternite VII illustrated in Young [392]. Although the unprepared female sternite VII of this specimen is slightly distorted, it was indicated to be strongly distorted by Young ( [392]: Fig. 189i). After preparation using KOH, the female sternite became completely symmetrical as is herein illustrated (Figure 4.5).

Homalodisca ignota may be mistakenly keyed out to either Dichrophleps or Pseudophera Melichar in

Young’s [392] key to Proconiini genera. This is because its claval veins are fused for a much longer distance than in most other Homalodisca and that females can measure up to 16 mm. One female specimen deposited at MNRJ was previously misidentified as Pseudophera sp. by leafhopper specialist W. J. Knight (The Natural

History Museum, London).

Homalodisca ignota is apparently more closely related to H. ignorata, H. apicalis Schmidt, and H. nitida

(Signoret). Although specimens of the latter species were not studied here, this species is possibly con- specific with H. apicalis [392]. These species share with related genera (e.g. Dichrophleps, Propetes, and

Pseudophera) the plesiomorphic hind leg setal row AV with modified elongate and hook-shaped setae along most of apical half of tibiae and the ventral pygofer processes spiniform and elongate (Figure 4.5). In most other Homalodisca species (e.g. H. spottii sp. nov.), row AV is only modified apically (up to about 6 setae) and the ventral pygofer process is reduced to a dentiform projection (Figure 4.6). H. ignota can be easily separated from other Homalodisca species by its aedeagal morphology, being the only Homalodisca species with processes on the apical portion of the shaft (Figure 4.5: SP). This species was described from Brazil without further specification of its distribution; herein it is first recorded from Rio de Janeiro and S˜aoPaulo states, in areas dominated by Atlantic Rainforest.

69 4.3.7 Homalodisca spottii Takiya, Cavichioli et McKamey, sp. nov.

Type-locality. Rio Real, Bahia State, Brazil.

Length. ||, 10.8–11.4 mm; ~~, 12.0–12.3 mm. External morphology. Crown (Figure 4.4) with median length approximately equal to interocular width and half transocular width; anterior margin rounded in dorsal aspect; transition crown-face slightly angulate and with median longitudinal blunt elevation; disc flattened; median fovea incomplete, becoming broader and shallower anteriorly; pubescence scarce; posterior margin with M-shaped elevation. Frontogenal sutures extending onto crown and attaining ocelli. Ocelli located on imaginary line between anterior eye angles; each equidistant from adjacent eye angle and median line of crown. Antennal ledges protuberant in dorsal aspect; dorsally carinate and with anterior margin slightly oblique in lateral view. Frons flattened me- dially and depressed; median area mostly smooth or slightly striated; muscle impressions distinct; pubescent.

Epistomal suture incomplete for short median distance. Clypeus continuing profile of frons; apical margin convex; pubescent. Prothorax with dorsopleural carinae complete. Pronotum narrower than transocular width; lateral margins parallel to slightly divergent anteriorly; disc rugose, punctate, and pubescent; poste- rior margin broadly concave. Mesothorax with katepisternum enlarged and inflated; scutellum not striate.

Forewings (Figure 4.4) hyaline, except for small sclerotized area on costal region covering apex of outer discal and base of outer anteapical cells; veins distinct and elevated; anteapical cells closed; with four apical cells, base of third more apical than those of second or fourth; without supernumerary crossveins; claval veins fused at mid-length for distance shorter than longest free branch. Hind wings extending almost as far posteriorly as forewings; vein R2+3 incomplete. Hind legs apical femoral setal formula 2:0:0; tibial setal row

AD without intercalary macrosetae; tibial setal row AV in females with only five elongate and curved setae apically; first tarsomere slightly shorter or subequal than combined length of two more distal tarsomeres and with two parallel rows of short setae on plantar surface.

Male genitalia. Pygofer (Figure 4.6) short; posterior margin obliquely truncate, slightly concave dor- sally and convex ventrally; ventral process poorly developed; microsetae numerous along posterior and ventral margins. Valve (Figure 4.6) linear, transverse. Subgenital plates (Figure 4.6) subtriangular, extend- ing posteriorly almost as far as pygofer apex; microsetae distributed throughout disc; apex narrowly round.

Connective (Figure 4.6) T-shaped, dorsal keel present. Styles (Figure 4.6) elongate, extending posteriorly beyond apex of connective; preapical lobe not strongly developed; apex narrowly round. Aedeagus (Figure

4.6) symmetrical; preatrium (Figure 4.6: PA) elongate, articulating basally with connective; atrium with dorsal and ventral pair of elongate spiniform processes directed posterolaterally (Figure 4.6: AP), dorsal pair longer than ventral one; apex of shaft without processes.

70 Figure 4.6: Homalodisca spottii sp. nov., genital structures. (A-E) Male genitalia: (A) pygofer, valve, sub- genital plate, style, and anal tube, lateral view; (B) valve and subgenital plate, ventral view; (C) connective and style, dorsal view; (D) aedeagus, lateral view; (E) aedeagus, caudal view. (F-L) Female genitalia: (F) sternite VII (setae not illustrated) and bases of first valvulae of ovipositor, ventral view; (G) first valvifer and base of first valvulae of ovipositor, lateral view; (H) sternite VII and internal sternite VIII, dorsal view; (I) sternite VII, pygofer, and gonoplac, lateral view; (J) second valvula of ovipositor, lateral view; (K) tooth of median portion of second valvula of ovipositor, lateral view; (L) apex of second valvula of ovipositor, lateral view. Scale bars in mm. AP=aedeagal atrium processes and PA=aedeagal preatrium.

71 Female genitalia. Abdominal sternite VII (Figure 4.6) with microsetae distributed throughout disc; posterior margin with broad median concavity. First valvifers (Figure 4.6) taller than long; microsetae along posterior margin. First valvulae of ovipositor (Figure 4.6) with bases broadly round. Internal abdominal ster- nite VIII (Figure 4.6) well developed and mostly sclerotized. Pygofer (Figure 4.6) in lateral view moderately produced; microsetae distributed along posteroventral margin; posterior margin round. Second valvulae of ovipositor (Figure 4.6) regularly broadened beyond basal curvature throughout apical three-fourths; blade bearing approximately 50 individual teeth; each tooth (Figure 4.6) subrectangular with denticles on anterior and posterior margins; preapical ventral prominence present (Figure 4.6); apical portion (Figure 4.6) with denticles on dorsal and ventral margins; apex (Figure 4.6) broadly round.

Coloration. Crown, pronotum and mesoscutum dark-brown, mottled with yellow spots (Figure 4.4).

Face yellow; frons with two pairs of small maculae ventrally and transverse complete or incomplete band over epistomal suture, black. Mesoscutellum yellow. Forewings (Figure 4.4) hyaline; sclerotized areas and venation purplish-red or dark brown (holotype). Thoracic pleura and sterna mostly yellow with several irregular dark-brown to black markings. Abdomen yellow with irregular dark brown markings; dorsal median region black; sternites with dark brown median transverse band basally.

Material examined. HOLOTYPE: |, “Rio Real, Bahia |Brasil 19/III/2003 |Citros |Miranda, M. P. leg”, DZUP. PARATYPES: 3 ||and 3 ~~, same data as holotype, DZUP; 1 |and 1 ~, same data as holotype, USNM; |and ~, “Sergipe |Brasil 14/II/2000 |Citros |Miranda, M. P. leg”, DZUP. Etymology. This species is named after Dr. Jo˜aoR. Spotti Lopes (Escola Superior de Agricultura

“Luiz de Queiroz”, Universidade de S˜aoPaulo, Brazil) for his contributions on Brazilian leafhopper ecology, vector biology, and pest management.

Taxonomic notes. Homalodisca spottii sp. nov. appears most closely related to H. lucernaria, sharing with the latter the mottled crown and contrasting yellow mesoscutellum (Figure 4.4, also present in other

South and Central American species) and the broadly round concavity of the posterior margin of the female sternite VII (Figure 4.6). The new species can be distinguished from H. lucernaria and other species of the genus by the combination of characters mentioned above and by the aedeagus atrium not expanded dorsally (i.e., not forming a lobe in between dorsal processes) and having two pairs of straight spiniform atrial processes.

72 4.3.8 Taxonomic Key to Brazilian Homalodisca species

Acronyms in parentheses refer to Brazilian states from which species are recorded.

1. Crown and pronotum tan to dark-brown not mottled; mesoscutellum concolorous with mesoscutum;

female hind tibiae with anteroventral (AV) setal row with over 25 modified elongated and curved setae

throughout apical 2/3 ( [291]: Fig. 11K); male pygofer processes long, almost attaining or extending

posteriorly beyond apex of pygofer (Figure 4.5); aedeagus with single pair of atrial processes (Figure 4.5:

AP); female sternite VII posterior margin not broadly concave (Figure 4.5)

2. Smaller, males 10.5-11.0 mm, females 12.0-13.0 mm; frons with large contrasting black macula ventrally

( [211]: Fig. 2B); clavus mostly sclerotized between outer vein and suture; aedeagus without pair of

apical processes on shaft ( [392]: Figs 182f, g) (MG, PR, RJ, RS, SC, SP) ...... H. ignorata

2. Larger, male 13.5 mm, females 14.0-16.0 mm; frons without large contrasting black macula ventrally

(some specimens with frons becoming darker ventrally) (Figure 4.4); clavus membranous between outer

vein and suture (Figure 4.4); aedeagus with pair of laterally directed apical processes on shaft (Figure

4.5: SP) (RJ, SP) ...... H. ignota

1. Crown and pronotum dark-brown mottled with yellow; mesoscutellum bright yellow contrasting with

darker mesoscutum; female hind tibiae with AV setal row with approximately 5 modified elongated and

curved setae restricted to apex ( [291]: Fig. 11L); male pygofer processes very short modified into a

dentiform projection (Figure 4.6); aedeagus with two pairs of atrial processes (Figure 4.6); female sternite

VII with posterior margin broadly concave (Figure 4.6)

3. Aedeagus with atrium expanded dorsally, forming lobe between pair of dorsal atrial processes ( [392]:

Figs 188f, g); ventral pair of atrial processes as long as dorsal pair (except on specimens from Trinidad)

( [392]: Fig. 188f) (PA, RR) ...... H. lucernaria

3. Aedeagus with atrium not expanded dorsally (Figure 4.6); ventral pair of atrial processes shorter than

dorsal pair (Figure 4.6) (BA, SE) ...... H. spottii sp. nov.

4.3.9 Additional Material Studied

´ Homalodisca apicalis. COSTA RICA, SAN JOSE: 2 ||and 2 ~~, San Jos´e,08.VIII.1932, C. H. Ballou, NCSU.

Homalodisca ignorata. LECTOTYPE: 1 ~, “Museum Paris |Paraguay |Paraguay Jaguaron |Santa- Clara |Cosset 1900”, “Collectio |Dr. L. Melichar |Moravsk´emuseum Brno”, “ignorata; det. Melichar”,

“Typus”, “Lectotypus |Homalodisca |ignorata |Melichar |Young & Lauterer”, “Invent. c. |2993/Ent. |Mor.

73 Museum, Brno”, MMBC. Additional material: BRAZIL, MINAS GERAIS: |and ~, Cafelˆandia, IV.1997, ´ Fundecitrus, DZUP; ~, Comendador Gomes, III.1997, Fundecitrus, DZUP; PARANA: ~, Fˆenix, Reserva

Estadual de Vila Rica, 04.X.1986, Profaupar, DZUP; RIO DE JANEIRO: |, Niter´oi, 19.IV.1987, A. L. ˜ Carvalho, MNRJ; |, Rio de Janeiro, Botafogo, 13.VI.1994, A. C. J. Carvalho, MNRJ; SAO PAULO: 2

||and 2 ~~, Bebedouro, 14.V.1998. S. Roberto, INHS (donated by R. C. Marucci); |, Colina, II.1998,

A. H. Purcell, INHS (donated by A. H. Purcell); 4 ~~, Olimpia, unknown date or collector, DZUP; |and 5

~~, Paulo Faria. 10.VII.1997, P. Yamamoto; |, Araraquara, unknown date, Fundecitrus, DZUP; SANTA

CATARINA: |, Chapec´o,18.XI.2003, Z. Meneguzzi, DZUP. ´ Homalodisca lucernaria. BRAZIL, PARA: ~, Bel´em,23.I.1984, O. Silveira, MPEG; ~, Bel´em,Campus

do MPEG, 29.III.1990, J. M. Rocha, MPEG; ~, Bel´em, Campus do MPEG, 08.II.1989, R. M. Valente,

MPEG; 2 ||and 2 ~~, Santo Antonio do Tau˜a,15-23.X.1979, M. Boulard, MHNP; ~, S˜aoJo˜aode

Pirabas, 18.XII.1996, J. Dias, MPEG; RORAIMA: |, Rio Uraricoera, Ilha de Marac´a,23-24.XI.1987, N. O.

Aguiar, mixed light trap, DCMB. TRINIDAD AND TOBAGO: |, Tobago, 13-15.VII.1962, J. Maldonado C., USNM.

Propetes compressa. HOLOTYPE: 1 ~, “Type”, “I. Propetes compressa”, “Para”, “294”, BMNH.

Propetes trimaculata (= jun. syn. of P. compressa). LECTOTYPE: 1 ~, “Cuyab´a |Matto [sic!] Grosso”, “Typus”, “Propetes |trimaculata |Schmidt |Edm. Schmidt |determ. 1928”, “Mus. Zool. Polonicum

|Warszawa |12/45”, “Lecto- |typus”, MZPW.

4.4 Conclusions

Name changes of well-known species are never comfortable to adopt at first. They require a change in vocab-

ulary by those who need taxonomy to communicate about a handful of species with which they personally

deal. It is only in recognition of the vast number of species needing classification that the application of a

consistent set of international rules can be fully appreciated and respected despite its inconveniences. For

a useful perspective on name changes by non-taxonomists, it is worth reiterating that until Schr¨oder’s[317]

and Young’s [389] work, the glassy-winged sharpshooter was comfortably being referred to as H. triquetra.

Due to the discovery of a type specimen, the scientific name has necessarily changed again, this time from

H. coagulata to H. vitripennis; the common name remains the same for day-to-day communication. We

believe the name established in this paper now provides a stable, well-justified new classification.

74 Chapter 5

Morphological Analysis of the Proconiini

5.1 Introduction

The tribe Proconiini Haupt, 1924 is restricted to the New World, except for recent introductions of the glassy-

winged sharpshooter, Homalodisca vitripennis, to several Pacific islands [158, 329]. Currently, it comprises approximately 355 species divided into 55 genera included in the last revision [392] and three genera described more recently: Cleusiana Cavichioli et Sakakibara [50], Brevimetopia Godoy [135], and Paraquichira Rakitov et Godoy [293]. Of these 58, 12 genera (21%) are monotypic, i.e., include a single species. External morphological differences among genera of Proconiini are usually conspicuous and few genitalia characters are used for generic diagnoses. This pattern contrasts to the one observed in the putative sister tribe

Cicadellini, as previously noted by D. A. Young: “[Cicadellini] morphology suggests rapid radiation and often shows small discontinuities compared with those found in many of the Proconiiini.”

The present chapter includes a phylogenetic analysis of the generic relationships of the tribe Proconiini based on morphological characters. This analysis included a large sampling of outgroup taxa to test its monophyly and investigate its relationships with some other Cicadellinae sensu lato tribes. The present results suggest the inclusion of Phereurhinini (previously treated as a separate subfamily) in Cicadellinae and division of Proconiini as previously defined into two tribes, the nominate tribe and Oncometopiiini, trib. nov.. Henceforth, for easier readability the term Proconiini will refer to the previous delimitation [392], and

Proconiini sensu stricto to the newly proposed circumscription.

5.2 Material and Methods

5.2.1 Taxon Sampling

Species exemplars of all 58 Proconiini genera were selected to estimate the genus-level phylogeny of pro- coniines [276]. Type-species were selected when available, otherwise preference was given to species recently collected in ethanol for the molecular analyses (CHAPTER 6). Additional species were included for larger

75 genera, especially those belonging to the Oncometopiini, which are generally harder to characterize morpho- logically and some are apparently para- or polyphyletic, based on unpublished preliminary analyses [219,347].

Outgroup taxa included were selected in order to test the monophyly of the Proconiini and provide us insight on the monophyly of Cicadellini and of Cicadellinae as delimited by Young [392–394]. Most of these outgroups are currently placed in the extremely speciose tribe Cicadellini (36 taxa, approximately 70% from the New World), but the sample also included three unplaced Cicadellinae species, three represen- tatives of the Phereurhininae (Phereurhinus, Clydacha, and Dayoungia), two Mileewinae ( Amahuaka and

Archeguina), one Evacanthinae (Pagaronia), one Signoretiinae (Signoretia), one Bathysmatophorini (Hy- laius), one Aphrodinae (Aphrodes), and one Acostemminae (Acostemma). Except for the two last, these higher groupings have been suggested to be closely related to Cicadellinae sensu Young [392, 392, 393] in previous phylogenetic or phenetic analyses [83, 91, 136].

Acronyms of the institutions that loaned the specimens are listed in the beginning of this thesis. A list of material studied is provided in Table 5.1.

5.2.2 Notes on Specimen Identifications

Aulacizes sp.: In the absence of a male specimen, most characters of the male genitalia were scored based on the male specimen of A. quadripunctata.

Bothrogonia cf. addita: The female DNA voucher keys to this species in Young [394], but no male specimens from the same locality were studied to confirm this identification. In the absence of a male specimen, characters of the male genitalia were scored using male specimens from Indonesia, of similar color pattern, but probably not conspecific.

Bhoria sp. nov.: The male genitalia are similar to B. princeps, but the aedeagus shaft does not have ventral teeth, in lateral view, and the apex is curved anteriorly.

Diestostemma excisum: Identification was based on the original description [314] and illustration of the female sternite VII by Young [392]. Males of this species were previously unknown, and although the genital structures seem to be distinct from any other described Diestostemma illustrated by Young [392], the male specimen studied seems to be malformed.

Homalodisca elongata: One male specimen from OSU has an extra pair of atrial aedeagal processes not present in typical form [389]. It is herein considered intraspecific variation.

Oncometopia alpha: Male specimens were determined based on the illustrations of the aedeagus given by Young [392]. However, Young explicitly states that O. alpha has the R2+3 of the hindwing (char. 87) complete, while the specimens studied have it incomplete (character which often varies among specimens),

76 and this species is treated under the subgenus Similitopia, whose members possess distinct enlarged lobes

of the anal tube segment X, which was not seen in these specimens.

Table 5.1: Species studied for the phylogenetic analysis of the Proconiini. Underlined localities refer to where DNA voucher specimens were collected. Depository acronyms follow the list of acronyms in beginning of thesis.

Taxa Localities Depositories Acostemminae Acostemma sp. MADAGASCAR: Toliara INHS Aphrodinae Aphrodes bicinctus USA: Illinois INHS Bathysmatophorini Hylaius oregonensis USA: Oregon INHS Evacanthinae Pagaronia tredecimpunctata USA: California INHS Signoretiinae Signoretia sp. VIETNAM ROM Mileewinae Amahuaka sp. nov. USA: Queretaro INHS Archeguina disparata PAPUA NEW GUINEA: Eastern Highlands BMNH, BPBM Phereurhininae Clydacha catapulta PERU: Hu´anuco AMNH, INHS Dayoungia magister PARAGUAY AMNH, DZUP Phereurhinus sosanion PERU: Hu´anuco INHS Cicadellinae unplaced Cicadellinae gen. nov. PERU: Pasco, Jun´ın INHS Ectypus coriaceus ”New Granada” NMW Homalogoniella pubescens BRAZIL: Rio de Janeiro MTD Jilijapa armata LAOS: Luang Prabang, VIETNAM BMNH, MNHN Cicadellini Atkinsoniella alternata TAIWAN: Chiai, Nantou INHS, TARI Backhofella pulchra PERU: Jun´ın NCSU Bhandara semiclara THAILAND INHS Bhooria sp. nov. SOUTH AFRICA: Western Cape INHS addita THAILAND INHS Cicadella viridis KYRGYZSTAN: Dzhalal-Abad, Talas INHS Cicadellini gen. nov. PERU: Pasco INHS, MUSM spectra TAIWAN: Kaohsiung; THAILAND; INHS, ROM, TARI VIETNAM: Tuyen Quang Conogonia sp. INDONESIA; PAPUA NEW GUINEA: Eastern Highlands INHS, USNM Diedrocephala bimaculata BRAZIL: Mias Gerais; MEXICO: Chiapas INHS Dilobopterus costalimai BRAZIL: S˜aoPaulo, Minas Gerais INHS Emadiana conformis MADAGASCAR: Fianaranboo, Fianarantsoa INHS Erythrogonia areolata MEXICO: Chiapas, Jalisco INHS Graphocephala coccinea USA: Illinois INHS Helochara communis USA: Illinois INHS Kolla sp. TAIWAN: Nantou INHS Latiguina oribata PAPUA NEW GUINEA: Chimbu, Eastern Higlands, INHS Sandaun Macugonalia leucomelas BRAZIL: Minas Gerais; Rio de Janeiro MNRJ, INHS Madaura superba MADAGASCAR: Fianarantsoa INHS Mucrometopia caudata BRAZIL: Par´a;PERU: Cuzco, Hu´anuco, Pasco AMNH, INHS, USNM Oragua obscura MEXICO: Chiapas INHS Pamplona feralis COLOMBIA: Meta; ECUADOR: Los Rios INHS, USNM Pamplona sp. n. spatulata PERU: Pasco INHS Pamplonoidea yalea BRAZIL: Rio de Janeiro; COSTA RICA: Heredia; MNRJ, INHS, UDCC, ECUADOR: Orellana; FRENCH GUYANA USNM Paracatua rubrolimbata COLOMBIA: Cundinamarca INHS, USNM Parathona cayennensis VENEZUELA: Barinas INHS Paromenia isabellina COSTA RICA: San Jos´e INHS Plesiommata tripunctata USA: Illinois INHS Sonesimia dimidiata BRAZIL: Rio Grande do Sul MNRJ Stehlikiana halticula PERU: Pasco INHS Subrasaca sp. BRAZIL: Rio de Janeiro MNRJ Tettigoniella cosmopolita ZAMBIA: Copperbelt INHS Tettisama quinquemaculata BRAZIL: Rio de Janeiro MNRJ Torresabela fairmairei BRAZIL: Rio Grande do Sul MNRJ Versigonalia ruficauda BRAZIL: Minas Gerais, Rio de Janeiro MNRJ, INHS Vidanoana sp. nov. CHILE: Tarapac´a INHS

77 Table 5.1: ...Continued

Taxa Localities Depositories Proconiini Abana gigas COSTA RICA INHS, NCSU Abana horvathi ECUADOR: Napo, Orellana; PERU: Cuzco, INHS, NCSU, UKL, Hu´anuco, Madre de Dios UNAM Acrobelus rakitovi ECUADOR: Orellana INHS, USNM Acrobelus reflexus COSTA RICA: Guanacaste, Lim´on, Turrialba INHS, USNM Acrocampsa integra BRAZIL MNRJ Acrocampsa pallipes FRENCH GUYANA ; GUYANA NCSU, UKL Acrogonia citrina BRAZIL: S˜aoPaulo INHS Acrogonia terminalis ECUADOR: Orellana; PERU: Amazonas INHS Acrogonia virescens BRAZIL: Amazonas, S˜aoPaulo; PERU: Jun´ın AMNH, INHS Amblydisca rubriventris MEXICO: Chiapas, Tamaulipas,; NICARAGUA INHS, TAMU, USNM Anacrocampsa wagneri BRAZIL: Rio de Janeiro MNRJ Anacuerna centrolinea CHILE: Tarapac´a, PERU: Cuzco INHS, NCSU Aulacizes quadripunctata BRAZIL: Santa Catarina NCSU Aulacizes sp. BRAZIL: S˜ao Paulo MNRJ Brevimetopia hermosa COSTA RICA: San Jos´e FSCA, INHS Catorthorrhinus resimus COSTA RICA: Puntarenas, BMNH, FSCA, NMW, PANAMA: Chiriqui INHS Cicciana latreillei BRAZIL: Esp´ırito Santo, Santa Catarina, S˜aoPaulo DZUP, NCSU, DZUP Cicciana obliqua BRAZIL: Paran´a MNRJ Ciccus adspersus FRENCH GUYANA NCSU Ciccus sp. nov. n. adspersus ECUADOR: Orellana EPNC, INHS, USNM Ciccus sp. nov. n. viridivitta ECUADOR: Orellana EPNC, INHS, USNM Cleusiana hyalinata BRAZIL: Mato Grosso DZUP Cuerna costalis USA: Florida, Illinois, Texas INHS, TAMU Cuerna gladiola USA: California, Oregon INHS, USNM Cuerna obesa MEXICO: Zacatecas; USA: Texas INHS, TAMU Cuerna sayi USA: Maryland, Montana INHS Cuerna striata USA: Michigan, Wisconsin INHS Cuerna yuccae USA: Utah OSAC Cyrtodisca major MEXICO: Jalisco INHS Dechacona missionum BOLIVIA: Santa Cruz; BRAZIL: Mato Grosso; PERU: Piura INHS, NCSU Depanana bugabensis COSTA RICA: Puntarenas; PANAMA: Chiriqui INHS, USNM Depanisca sulcata COLOMBIA: Magdalena; VENEZUELA: Lara FMNH, INHS, USNM Desamera intersecta BOLIVIA BRAZIL: Mato Grosso; PERU: Ancash BMNH, FMNH, USNM Deselvana excavata BRAZIL: Santa Catarina NCSU Deselvana sp. nov. ECUADOR: Orellana INHS Dichrophleps boliviana ECUADOR: Orellana INHS Dichrophleps despecta BRAZIL: Par´a MNHN Dictyodisca salvini COSTA RICA: Lim´on,Puntarenas; PANAMA: Chiriqui INHS, LACMm USNM Diestostemma albipenne GUYANA NCSU Diestostemma excisum PERU: Pasco INHS Diestostemma reticulatum PERU: Hu´anuco INHS Diestostemma stesilea PERU: Pasco Egidemia anceps GUATEMALA: Izabal; MEXICO: Chiapas CAS, NCSU Egidemia paranceps COSTA RICA INHS Egidemia inflata BELIZE; COSTA RICA: Puntarenas; EMEC, NCSU, INHS, MEXICO: Chiapas, Oaxaca OSU Egidemia sp. nov. COSTA RICA: Puntarenas INBIO, INHS, USNM Egidemia speculifera BRAZIL: Paran´a, Santa Catarina NCSU Homalodisca apicalis COSTA RICA NCSU Homalodisca elongata MEXICO: Durango, Michoac´an; USA: Arizona FMNH, INHS, OSU Homalodisca ichthyocephala MEXICO: Colima, Oaxaca, Zacatecas INHS Homalodisca ignorata BRAZIL: Paran´a, S˜aoPaulo INHS Homalodisca ignota BRAZIL: Rio de Janeiro, S˜ao Paulo MNRJ Homalodisca insolita GUATEMALA: Sacatep´equez; MEXICO: Chiapas, Jalisco; AMNH, CMNH, MZPW, USA: Florida Texas NCSU, INHS, OSU Homalodisca liturata USA: Arizona, California INHS, SEMC Homalodisca lucernaria BRAZIL: Par´a,Roraima; VENEZUELA: Aragua DCMB, MNHN, INHS Homalodisca sp. n. apicalis PERU: Pasco INHS Homalodisca vitripennis USA: California, Florida, Louisiana, Texas INHS, UDCC, TAMU Homoscarta boliviana BOLIVIA NCSU Homoscarta irregularis PERU: Chanchamayo, Pasco INHS, USNM Hyogonia batesi COLOMBIA; ECUADOR: Napo, Orellana FSCA, INHS, UKL Hyogonia youngi PERU: Jun´ın, Napo INHS, USNM

78 Table 5.1: ...Continued

Taxa Localities Depositories Proconiini Ichthyobelus bellicosus ECUADOR: Orellana INHS Ichthyobelus regularis PERU: Pasco INHS Lojata ohausi ECUADOR: Loja MZPW Mareba curuna FRENCH GUYANA NCSU, INHS Molomea alternata BRAZIL: Mato Grosso, Minas Gerais, Goi´as DZUP, MPEG, MNRJ, Par´a INHS Molomea exaltata PERU: Hu´anuco INHS, USNM Molomea fatalis BRAZIL: Par´a,Rondˆonia; ECUADOR: Orellana FSCA, INHS, MPEG Molomea personata BRAZIL: Santa Catarina, Paran´a, AMNH, DZUP, NCSU, Rio Grande do Sul NMW Molomea virescens COLOMBIA; ECUADOR: Napo, Orellana INHS, NCSU, USNM Ochrostacta physocephala BRAZIL: Rio Grande do Sul MNRJ, INHS Omagua fitchi ECUADOR: Orellana, PERU: Madre de Dios INHS Oncometopia alpha USA: Arizona AMNH, FMNH, INHS Oncometopia facialis BRAZIL: Minas Gerais, S˜aoPaulo INHS Oncometopia herpes MEXICO: Chiapas, Veracruz; PANAMA: Chiriqui AMNH, INHS, NCSU Oncometopia nigerrima MEXICO: Yucat´an MTD, NCSU Oncometopia orbona USA: Illinois, Virginia AMNH, INHS Oncometopia venosula PERU: Jun´ın INHS Paracrocampsa amida ECUADOR: Suc´umbios AMNH, BMNH Paracrocampsa discreta ECUADOR: Guayas, Los Rios, Pichincha FMNH, NCSU, USNM, Paracrocampsa laboulbeni VENEZUELA: Lara USNM Paraquichira costaricensis COSTA RICA: Cartago INBIO, INHS Paraulacizes figurata MEXICO: Jalisco INHS Paraulacizes irrorata USA: Illinois INHS Peltocheirus sp. nov. ECUADOR: Orellana EPNC, INHS, USNM Phera lacerta MEXICO: Chiapas, Jalisco, Puebla INHS Phera lanei MEXICO: Chiapas, Jalisco, Oaxaca INHS Phera luciola MEXICO: Guerero, Oaxaca INHS, OSU Phera obtusifrons COSTA RICA, GUATEMALA, UNAM, NCSU, INHS, MEXICO: Chiapas, Puebla OSU Procama fluctuosa PANAMA NCSU, USNM Procandea ochracea PERU: Hu´anuco INHS Procandea sp. nov. ECUADOR: Orellana EPNC, INHS, USNM Proconia cf. esmeraldae GUYANA NCSU Proconia cf. marmorata BRAZIL: Par´a MNRJ Proconia sp. ECUADOR: Orellana INHS Proconiini gen. nov. COLOMBIA: Narino; ECUADOR: Orellana CMNH, EPNC, UMO Proconobola callidula BOLIVIA: La Paz MTD, USNM Proconopera pullula PERU: Chanchamayo, Pasco NCSU, INHS, USNM Proconopera sp. n. cumingi COLOMBIA MTD Proconosama alalia PERU: Pasco INHS, UNSM Proconosama columbica PERU: Jun´ın, Pasco INHS Propetes schmidti BRAZIL: MIna Gerais, S˜aoPaulo INHS Propetes triquetra BRAZIL : Par´a MPEG Pseudometopia phalaesia ECUADOR: Orellana; PERU: Pasco INHS Pseudophera contraria GUATEMALA; PANAMA: Bocas del TOro INHS, NCSU Pseudophera heveli COSTA RICA: Puntarenas FMNH, SEMC Quichira tegminis COSTA RICA: Cartago, Lim´on, San Jos´e INBIO, INHS Raphirhinus phosphoreus BOLIVIA: Cochabamba; ECUADOR: Napo, Orellana INHS Splonia acutalis ECUADOR: Napo MZPW, USNM Splonia sp. nov. PERU: Hu´anuco MUSM Stictoscarta sulcicollis BRAZIL: Rio de Janeiro, Santa Catarina MNRJ, NCSU Tapajosa doeringi ARGENTINA: C´ordoba NCSU Tapajosa fulvopunctata BRAZIL NCSU Tapajosa rubromarginata ARGENTINA: Tucum´an INHS, NCSU Teletusa limpida BRAZIL: Minas Gerais, ECUADOR: Orellana INHS Tretogonia bergi BRAZIL:Rio Grande do Sul MNRJ Tretogonia cribrata BRAZIL:Rio Grande do Sul MNRJ Yotala boliviana PERU: Madre de Dios, Loreto AMNH, FSCA, INHS Yunga cartwrighti PANAMA: Panam´a USNM Yunga coriacea MEXICO: Veracruz BMNH, FMNH haenschi ECUADOR: Bol´ıvar NCSU Zyzzogeton viridipennis ECUADOR: Pichincha LACM, USNM

79 Phera lacerta: Females were identified based on comparisons of photographs of the lectotype in Burks

& Redak [44]. However, these authors associated the Mexican female lectotype with a male from southern

Brazil, which we do not believe to be conspecific. The illustration of the male given by Burks & Redak agrees with P. carbonaria, a southern South American species, which resembles P. lacerta externally. Herein, the male genitalia of Phera lacerta is coded based on specimens collected in Mexico from the same localities as the females studied. The male genitalia of this species resemble closely that of another Central American species, P. obtusifrons.

Proconia cf. esmeraldae: Apices of styles different from those illustrated by Young [392].

Proconopera cf. cumingi: Determined based on the color pattern [313]. The pygofer process is bifurcate, as illustrated in Young [392], but the aedeagal shaft bears no dorsal spines.

Proconopera pullula: The DNA voucher specimen keys to P. cumingi, because its pygofer process is bifurcate and the aedeagal shaft bears few teeth on dorsal margin.

Versigonalia ruficauda: Specimens from Minas Gerais State are quite different than those from Rio de

Janeiro State in the length of the male pygofer processes and paraphyses, and in the concavity of the female sternite VII. Nevertheless, they do not differ in the characters studied.

Subrasaca sp.: In the absences of a male specimen, characters of the male genitalia were scored using illustrations of S. flavoornata given by Young [393].

Sonesimia dimidiata: In the absence of a male specimen, characters of the male genitalia were scored using illustrations given by Young [393].

Vidanoana sp. nov.: Specimens are externally similar to V. flavomaculata, but the male pygofer has few macrosetae on the dorsal margin, the subgenital plates are only slightly longer than pygofer, and the aedegal shaft is shorter than in the latter species.

5.2.3 Terminology

Morphological terminology follows mainly Young [392, 393] and Dietrich [86]. Terminology of head sclerites follows Hamilton [145] (see Figure 5.1) as suggested by Mejdalani [218], of wing venation Comstock &

Needham [64, 65] (see Figure 5.2), of leg chaetotaxy Rakitov [287] (see Figure 5.3), and of genitalia Nielson

[247] (see Figure 5.4). Techniques for preparation of genital structures were those of Oman [262]. The dissected parts are stored in microvials with glycerin. Acronyms for depositories of the studied material are given in the beginning of this thesis.

80 Figure 5.1: Morphology of the head and thorax. (A) Antenna of Cicciana latreillei showing basal flagel- lomeres extremely elongate (char. 21, state 1), a generic apomorphy. Line drawing by G. Mejdalani. (B) Head and thorax, dorsal view, of Hyogonia brasiliensis showing antennal ledges prominent (char. 19, state 1), ocelli on crown (char. 16, state 1) and on imaginary line between anterior eye angles (char. 17, state 1), pronotum width smaller than transocular width (char. 32, state 0), and with deep punctures (char. 40, state 1). (C) Head and thorax, lateral view, of Acrogonia virescens showing crown acclivent anteriorly (char. 7, state 2), mesokatespisternum enlarged and inflated (char. 38, state 1), and metepisternum ventrally not closely associated with trochantin and basisternum (char. 50, state 1). Line drawing by M. Felix and G. Mejdalani. (D) Head and thorax, lateral view, of Phera obtusifrons showing crown declivent anteriorly (char. 7, state 1), clypeus profile continuing profile of frons (char. 30, state 0), lateral lobe of the pronotum with ventral margin strongly depressed, forming a long straight ridge (char. 46, state 1), forewing base straight exposing metameron (char. 52, state 1), and metepimeron with shelf-like projection (char. 51, state 1). Line drawing by M. Felix and G. Mejdalani.

81 Figure 5.2: Morphology of the wings. (A) Forewing of Homalodisca vitripennis showing venation not retic- ulate (char. 64, state 0), vein R1 present (char. 68, state 0), M segment between r-m1 and m-cu2 forming an acute angle with R4+5 (char. 72, state 1), crossvein rs present (char. 73, state 0), m-cu1 basad of R+M fork (char. 75, state 1), and claval veins confluent (char. 83, state 1). (B) Hindwing of H. showing R2+3 incomplete (char. 87, state 1) and ambient vein widely separated from margin (char. 88, state 0). (C) Forewing of Amahuaka sp. showing r-m1 absent (char. 70, state 1), crossvein rs and m-cu1 absent (char. 73, state 1 and char. 74, state 1), claval veins parallel (char. 83, state 0), and appendix of inner margin extremely broad (char. 85, state 2).

5.2.4 Notes on Characters Used in Previous Studies

The following characters previously thought to be important in Proconiini taxonomy were either not coded

or were reinterpreted for the reasons given below. Most intergrade gradually among taxa, which makes

difficult the process of objectively delimiting character states.

The thick carina on the vertex-face transition, which is distinctly present in Yotala, Peltocheirus, and

Acrocampsa, is developed to a variable degree in other genera, e.g., Dictyodisca, Ectypus, Proconia. This carina seems to be the result of the simultaneous presence of distinct depressions at the apex of the crown

(char. 8, state 1) and at the superior portion of the frons (char. 28, state 1).

The distinct dorsal carina on the antennal ledges in lateral aspect mentioned by Young [392] was observed in Dichrophleps and Propetes, but it is developed to a lesser degree in some Oncometopia and Homalodisca,

and is completely absent in Egidemia. This character was not coded.

Apical head processes are present in multiple unrelated genera of Cicadellinae and their presence/absence

was coded in character 15, while their aspects in character 16. However, two exceptions were made. Although

Clydacha has a produced head, its structure seems to have a different origin because the frons is limited to

the underside of the process; therefore it was not scored as state (1) in character 14. Also, the head process

of Raphirhinus phosphoreus seems to originate on the superior portion from the frons rather than from the

82 Figure 5.3: Morphology of the legs. (A) Profemur, anterior view, of Cicadella viridis showing uniseriate row IC (char. 79, state 0), AV row restricted to AV1 only (char. 94, state 2), and AM row restricted to AM1 only (char. 92, state 1). (B) Profemur, anterior view, of Proconia sp. showing uniseriate AV (char. 95, state 0) reduced to 2-6 apical setae (char. 94, state 1). (C) Proleg, posterior view, of Deselvana sp. showing inferior portion of PD edge expanded (char. 96, state 1). Illustration by G. Mejdalani and L. Costa. (D) Hindleg, anterodorsal view, of male Quichira tegminis showing metatibial row AD without intercalaries (char. 101, state 1), AD setae similar to those in PD (char. 103, state 0), and shorter than tibial width (char. 102, state 1). Illustrated by R. Rakitov. (E) Hindleg, anterodorsal view, of female Q. tegminis showing metatibial PD row extending from base (char. 104, state 0), AV row with distinctly longer setae in females (char.107, state 1) distributed throughout most of tibia (char. 108, state 0), and metatarsomere I with scattered setae (char. 112, state 2). Illustrated by R. Rakitov.

83 Figure 5.4: Morphology of the abdomen. (A) Dorsal habitus of Teletusa limpida showing forewings with costal supranumerary veins (char. 66, state 1), and abdomen short and wide (char. 3, state 2). Illustrated by L. Costa, G. Mejdalani, and C. Souza. (B) Males sternum I of Acrobelus rakitovi with elongate lateral projections (char. 114, state 0). (C) Male sternum I of Oncometopia venosula showing no elongate projec- tions (char. 114, state 1). (D) Genital chamber, lateral view, of male Hyogonia brasiliensis showing pygofer with ventrobasal elongate and spiniform process (char. 130, state 0), lateral margins of valve articulated with pygofer (char. 136, state 1), and anal tube segment X with posteroventral margin produced posteriorly (char. 181, state 1). (E) Aedeagus, lateral view, of krameri showing sclerotized ejaculatory reservoir apex (char. 162, state 1) approximately as long as wide (char. 163, state 0) and conspicuous dorsal apodemes (char. 158, state 1). (F) Aedeagus, lateral view, of Oncometopia facialis showing elongate preatrium (char. 164, state 1) and asymmetrical ventral atrial processes (char. 176, state 4). (G) Valve and subgenital plate, ventral view, of O. krameri showing valve articulated posteriorly with subgenital plates (char. 135, state 0) and plate uniseriate macrosetae (char. 148, state 1). (H) Genital chamber, ventral view, of male Acrobelus rakitovi showing valve fused laterally to pygofer (char. 136, state 0) and plate setae multiseriate (char. 148, state 0). Illustrated by P. Ceotto. (I) Abdomen, ventral view, of female Propetes schmidti showing basal abdominal constriction (char. 3, state 1) and gonoplac apices enclosed within pygofer (char. 183, state 0).

84 vertex, and appears to be homologous to the scars observed in this position on the adults of some other

genera (vestiges of nymphal apical head processes). Therefore, the R. phosphoreus apical process was scored

as a separate state (2) under character 27.

Pronotum with distinct median lateral depressions (char. 36, state 1), previously recovered as a synapo-

morphy of Molomea [219], was found to be more widespread within the Oncometopiini and Proconiini sensu stricto.

Young [392] treated most of the Proconiini male pygofer and subgenital plates setae, as “microsetae” in contrast to “macrosetae” on Cicadellini structures. Herein we refer to most of Young’s “microsetae” as “fine and elongate macrosetae” (chars. 119 and 147), because in most instances these fine and elongate setae have also distinctly thicker bases like in the “macrosetae”, although not as conspicuous.

Accessory male genital structures (paraphyses) are structures between the base of the aedeagus and the apex of the connective. As suggested by Young [392–394], they probably arose independently multiple times within Cicadellinae. For the lack of a better hypothesis, herein paraphyses in the outgroup and some ingroup taxa were treated as primarily homologous structures and coded under the same character (presence in char.

156, state 3, aspect in char. 157). However, the presence of “paraphyses” in some Proconiini was coded under char. 176 state 5, because they are thought to be derived from the basal atrial aedeagal processes, present in congeners, but still fused to the base of the aedeagus, e.g., Diestostemma reticulatum and Splonia spp.

Furthermore, “paraphyses” described by Young [392] in Homoscarta and Yunga are herein thought to be homologous with processes of the conjunctiva IX-X (char. 137), which are well developed in Desamera and

Ciccus. Interestingly, Young refers to Depanana as having “paired, large conspicuous sclerites in conjunctiva

IX-X”; however, these are thought herein to be the ventral arms of the dorsal connective (char. 161, state

1), which are bizarrely enlarged and inflated in D. bugabensis.

5.2.5 Character Sampling and Assumptions

Some morphological characters available from previous analyses concerning Proconiini and Cicadellidae relationships [51, 85, 219] were recoded based on the specimens available and many additional characters were defined. Characters were identified based on their topographical identity before proposing hypotheses of primary homology by defining the states in the data matrix [42,271]. Characters were assigned equal weights and reductively coded (sensu Wilkinson [377]), particularly when both presence/absence of a structure and its aspect were studied [127, 154]. When two states of the same character were found to be present in one terminal taxon, both states were coded in the matrix and the character was treated as polymorphic in optimal tree searches [337]. Multistate characters were treated as unordered under the Fitch parsimony

85 [126]. Parsimony-uninformative autapomorphic characters were included in the data matrix following the suggestion of Yeates [384].

5.2.6 Phylogenetic Analysis

Analyses of the morphological dataset were conducted using the cladistic method [6, 157, 376], considered as the current paradigm for systematic research based on morphological characters [271]. Unrooted a priori diagrams [254] were estimated using the maximum parsimony criterion. Heuristic parsimony tree searches were performed in two steps: (1) 1,000 random addition sequence replicates and TBR branch swapping, but only keeping 100 optimal trees each replicate (nchuck=100), then (2) a full TBR branch swapping starting with the best trees saved in the previous step, but with maxtrees set to 100,000 due to computational limitations. Analyses were also conducted using the parsimony ratchet [253], which for large datasets has been proven to be very effective in finding optimal trees in short amounts of time [139]. For each iteration, the Ratchet uses instead of random, starting trees that retain information from previous optimal islands found; additionally it overcomes getting stuck in a large suboptimal tree island by perturbing a randomly chosen subset of characters (either by changing their weighting scheme or by jacknifing). Batch files for analyses in PAUP* 4.0 [336] were modified from those output from PAUPRat 1.0b [325] to conduct 100 independent runs of 200 iterations, perturbing the dataset by adding +1 weight to 15% of randomly selected characters. Finally, all the distinct optimal trees found in these 100 runs were used as starting point for a full TBR branch swapping with 10,000 maxtrees set.

Branch support was evaluated based on clade posterior probabilities by 4 independent analyses of the morphological dataset (datatype=standard), each using 4 Markov chains accommodating for rate variation across characters (lset rates=gamma) and flat priors using a Bayesian algorithm implemented by Mr. Bayes

3.1 [168,169,303]. The burnin was calculated based on the plot of sampled likelihoods. Additional support was calculated using parsimony character bootstrap [123] run with 1,000 pseudoreplicates, each with 10 random addition replicates and TBR branch swapping.

5.3 Results

5.3.1 Morphological Matrix

A morphological character matrix (Table 5.2) including 50 outgroup taxa and 121 ingroup species represent- ing all 58 described genera of the Proconiini was coded based on 183 delimited morphological characters. Of these morphological characters, 12 characters were autapomorphies and 42 were multistate.

86 Table 5.2: Matrix of morphological characters for the phylogenetic analysis of the Proconiini. Innaplicable charaters coded as “-”, missing data as

“?”, and polymorphic taxa as “&”. 87 Table 5.2: ...Continued. 88 Table 5.2: ...Continued. 89 Table 5.2: ...Continued. 90 Table 5.2: ...Continued. 91 Table 5.2: ...Continued. 92 Table 5.2: ...Continued. 93 Table 5.2: ...Continued. 94 Table 5.2: ...Continued. 95 5.3.2 Morphological Characters and States

1. Body: (0) not depressed, (1) depressed dorsoventrally.

2. Head and thorax, dorsal view: (0) without callosities, (1) with nodular callosities.

3. Abdomen shape: (0) uniformly narrowing distally, (1) with distinct basal constriction (Figure 5.4: A),

(2) short and wide (Figure 5.4: I).

4. Crown texture, region between ocelli: (0) unsculptured, (1) rugose.

5. Crown sculpturing: (0) without punctures, (1) with punctures.

6. Crown pubescence: (0) absent, (1) conspicuously pubescent.

7. Crown, lateral view: (0) approximately parallel to dorsum, (1) declivent anteriorly continuing prono-

tum slope (Figure 5.1: D), (2) acclivent anteriorly (Figure 5.1: C).

8. Crown anterior portion: (0) not depressed, (1) with distinct depression.

9. Crown median region: (0) not distinctly foveate, (1) with elongate longitudinal fovea.

10. Crown apical half: (0) without distinct carina, (1) with longitudinal carina.

11. Crown median region, color pattern: (0) without contrasting dark markings, (1) with an A-shaped

marking, (2) with a dark triangle.

12. Crown laterad of ocelli: (0) flattend or slightly concave, (1) with distinct depression.

13. Crown laterad of ocelli: (0) flattened or concave, (1) with short longitudinal carinae.

14. Crown apex: (0) not produced into process, (1) produced into process.

15. Crown apex process aspect: (0) elongate and grooved, (1) elongate and ventrally bifurcate, (2) broad

and concave, (3) tapering or almost cylindrical.

16. Ocelli position: (0) on crown-face transition, (1) on crown (Figure 5.1: B).

Although Ectypus coriaceus appears not to have ocelli, dark maculae on the crown near the posterior

end of the frontogenal sutures mark their supposed location.

17. Ocelli position: (0) anterad of imaginary line between anterior eye angles, (1) on or posterad of this

imaginary line (Figure 5.1: B).

96 18. Ocelli in females: (0) present, (1) absent.

19. Antennal ledges: (0) not prominent, (1) prominent (Figure 5.1: B).

20. Antennal ledges anterior end: (0) round or truncate, (1) with concavity, (2) produced laterad farther

than dorsal margin of crown.

21. Antennal flagellum, basal 5 segments length: (0) subequal to width, (1) elongated, more than thrice

as long as wide (Figure 5.1: A).

22. Vertex-frons transition: (0) with distinct transverse carina, (1) without carina.

23. Vertex-frons transition angle, lateral view: (0) acute, (1) right or obtuse.

24. Genae lateral margins: (0) produced laterally, covering proepimeron, (1) not produced laterally,

proepimeron exposed.

25. Frontogenal sutures: (0) not extending onto crown, (1) almost extending to ocelli.

26. Frons texture: (0) unsculptured, (1) striated, granulose, or rugose.

27. Frons superior portion: (0) without scar or process, (1) with round scar remnant of nymphal process,

(2) with elongate cylindrical process directed dorsally.

28. Frons dorsomedian area: (0) evenly convex or flat, (1) with distinct median depression.

29. Clypeus pubescence: (0) present, (1) absent.

30. Clypeus ventral portion profile, lateral view: (0) continuing profile of frons (Figure 5.1: C, D), (1)

angulate with frons.

31. Pronotum length: (0) not posteriorly produced, (1) produced posteriorly, uncovered scutum shorter

than scutellum.

32. Pronotum width: (0) smaller than transocular width (Figure 5.1: B), (1) subequal to transocular

width, (2) larger than transocular width.

33. Pronotum lateral margins: (0) parallel, (1) convergent anteriorly, (2) divergent anteriorly.

34. Pronotum dorsal profile, lateral view: (0) evenly convex, (1) anterior portion at lower plane than

posterior portion.

35. Pronotum posterior two-thirds: (0) not dorsally produced, (1) produced in median dorsal keel.

97 36. Pronotum midlength: (0) flattened or convex, (1) with lateral depressions.

37. Pronotum posterior portion: (0) continuousy convex, (1) produced dorsally into paired projections.

38. Pronotum posterior portion paired projections aspect: (0) round bumps directed dorsally, (1) acute

processes directed dorsolaterally.

39. Pronotum posterior third texture: (0) striated or rugose, (1) unsculptured.

40. Pronotum posterior third sculpturing: (0) without punctures, (1) with deep punctures (Figure 5.1: B).

In some species, e.g., in Ciccus, Depanisca, Procama, and Pseudometopia, round dark areas are present

underneath the integument surface that bear hairs, but these were not in pits and therefore not coded

at state (1).

41. Pronotum posterior margin shape: (0) straight, concave, or convex, (1) strongly bilobed.

42. Pronotum dorsopleural carina: (0) present, (1) absent.

43. Pronotum dorsopleural carina aspect: (0) not strongly elevated, (1) strongly flared.

44. Pronotum dorsopleural carina anterior end alignment: (0) with posterior corner of eyes, (1) below

posterior corner of eyes.

45. Pronotum lateral lobe posterior margin: (0) not projected posteriorly, (1) with digitate projection.

46. Pronotum lateral lobe ventral margin: (0) not depressed or slightly depressed, (1) with strong depres-

sion forming a long longitudinal ridge (Figure 5.1: D).

47. Mesoscutellum: (0) flattened or slightly convex, (1) inflated.

48. Mesokatepisternum: (0) not enlarged, (1) enlarged and usually inflated (Figure 5.1: C, D).

Whenever the mesokatepisternum is enlarged and/or inflated, the rostrum never attains its posterior

margin.

49. Mesokatespisternum anterodorsal region: (0) without fold, (1) with folded projection(s).

In most species bearing a folded projection they appear as a simple produced fold along the sclerite

margin. However in Mareba and Proconobola there can be more than one fold forming conspicuous

finger-like processes.

50. Metepisternum: (0) closely associated with trochantin and basisternum, (1) ventrally projected later-

ally, not closely associated with trochantin and basisternum (Figure 5.1: C).

98 51. Metepimeron: (0) without projection, (1) with shelf-like projection (Figure 5.1: C, D).

52. Forewing costal base: (0) round, concealing meron, (1) approximately straight, exposing meron (Figure

5.1: C. D), (2) straight, concealing meron.

53. Forewings development: (0) females and males macropterous, (1) females submacropterous and males

macropterous, (2) females and males brachypterous, (3) females macropterous and males submacropter-

ous, (4) females brachypterous and males submacropterous.

54. Forewings texture: (0) coriaceous or opaque, (1) membranous.

55. Forewings clavus base sclerotization: (0) as sclerotized as base of corium, (1) distinctly more sclerotized

than base of corium.

56. Forewings apical membrane: (0) not distinctly membranous, (1) with distinct apical membrane.

57. Forewings apical membrane extension: (0) including apical and some part of anteapical cells, (1)

including apical, some part of anteapical, and brachial cells, (2) completely including anteapical cells.

58. Forewing membranous region: (0) hyaline or fuscous, (1) darkly pigmented (brown or black).

59. Forewings sculpturing: (0) not sculptured, (1) with deep punctures.

60. Forewings punctures distribution: (0) punctures throughout, (1) punctures restricted to clavus.

61. Forewings chaetotaxy: (0) not distinctly pubescent, (1) pubescent.

62. Forewings pubescence: (0) distributed throughout, (1) mostly restricted to venation.

63. Forewings costal setae in females: (0) uniformly distributed, (1) distinctly concentrated.

64. Forewings venation: (0) not reticulate (Figure ??), (1) reticulate.

65. Forewings reticulate venation extension: (0) corium apical half, (1) corium apical third, (2) entire

wing, (3) entire corium, (4) basal two-thirds corium and clavus.

State (4) is a synapomorphy of the genus Hyogonia, however some specimens do not have reticulations

on the corium part and may have supranumerary crossveins in anteapical cells.

66. Forewings supranumerary apical costal crossveins: (0) absent, (1) more than 2 present (Figure 5.4: A).

67. Forewings supranumerary apical costal crossveins extension: (0) apical third, (1) apical half.

68. Forewings vein R1: (0) present (Figure 5.2: A), (1) absent.

99 69. Forewings vein R1 location: (0) distad of R fork, (1) basad of R fork.

Whenever species have supranumerary costal veins, the basalmost vein was considered R1.

70. Forewings crossvein r-m1: (0) present, (1) absent.

71. Forewings crossvein r-m1 R connection: (0) R4+5, (1) R or Rs.

72. Forewings segment of M between connections with r-m1 and m-cu2: (0) forming acute angle with

R4+5 (Figure 5.2: A), (1) parallel with R4+5.

73. Forewings crossvein rs: (0) present (Figure 5.2: A), (1) absent (Figure 5.2: C).

74. Forewings crossvein m-cu1: (0) present, (1) absent (Figure 5.2: C).

75. Forewings crossvein m-cu1 location: (0) distad of R+M fork, (1) basad of R+M fork (Figure 5.2: A).

This character is related to the size of the outer discal cell, of which the R+M fork forms the base.

When the outer discal cell is long, m-cu1 is located distad of the fork, while when this cell is short,

m-cu1 is basad of the fork. However, exceptionally in Pamplona and Pamplonoidea, even though the

outer discal cell seems short, m-cu1 is distad of the fork because its relative position seems to be more

distal than in other oncometopiines.

76. Forewings distance between basal m-cu1 to R+M fork: (0) shorter than outer discal cell length, (1)

much longer than outer discal cell length.

77. Forewings crossvein m-cu2: (0) present, (1) absent.

78. Forewings crossvein m-cu2 position: (0) distad of r-m1, (1) aligned with r-m1, (2) basad of r-m1.

79. Forewings brachial cell anterior end: (0) not abruptly narrowed, (1) abruptly narrowed.

80. Forewings inner apical cell aspect: (0) acuminate apically, M3+4 curving to inner margin of wing, (1)

parallel-sided, M3+4 reaching apical margin of wing.

81. Forewings inner apical cell width: (0) approximately as wide as other apical cells, (1) narrower than

half width of other apical cells, (2) twice as wide as other apical cells.

82. Forewings outer claval vein distal end: (0) unbranched, (1) branched.

83. Forewings claval veins: (0) parallel (Figure 5.2: C), (1) median area converging, may touch for short

distance, (2) confluent for long distance (Figure 5.2: A).

100 84. Forewings clavus crossveins: (0) absent, (1) present.

This character was coded as inapplicable (-) for species that have reticulate venation in clavus.

85. Forewings appendix of inner margin largest width: (0) as wide as of apical margin, (1) two times

broader than that of apical margin, (2) more than three times broader than that of apical margin.

86. Forewings apex aspect: (0) convex, (1) truncate, (2) concave, (3) trilobed.

87. Hindwings vein R2+3: (0) complete, (1) incomplete (Figure 5.2: B).

88. Hindwings ambient vein: (0) separated from wing margin by distance distinctly longer than its width

(Figure 5.2: B), (1) close to wing margin by distance smaller or approximately the same as its width.

89. Profemora setal row IC chaetotaxy: (0) uniseriate (Figure 5.3: A, B), (1) multiseriate.

90. Profemora multiseriate setal row IC: (0) distinct of AV, (1) indistinct of AV.

In proconiines both rows IC and AV can be multiseriate, e.g., see Ciccus for multiseriate AV and

Depanana for multiseriate IC, so in cases where there are scattered setae in between both rows, it is

not possible to distinguish which row is mutiseriate. In those cases an arbitrary deecision was made

and they were charaterized as multiseriate IC indistinct of AV and coded as state (1).

91. Profemora setal row IC setae: (0) subequal, (1) basal 3-7 setae enlarged.

92. Profemora setal row AM: (0) with additional AM setae, (1) reduced to AM1 only (Figure 5.3: A).

93. Profemora setal row AV: (0) uniseriate (Figure 5.3: B), (1) multiseriate.

94. Profemora setal row AV extension: (0) continuous from base or midfemur, (1) reduced to 2-6 apical

setae, (2) reduced to AV1 only.

95. Profemora setal row AV setae: (0) subequal, (1) AV1 well developed, (2) larger setae intercalated with

smaller setae.

96. Protibiae PD edge: (0) not expanded, (1) inferior portion flattened and expanded (Figure 5.3: C), (2)

completely flattened and expanded.

97. Metatibiae: (0) compressed laterally, (1) approximately square in cross section.

98. Metafemora length: (0) reaching lateral lobes of pronotum, (1) not reaching lateral lobes of pronotum.

99. Metafemora apical setal formula: (0) 2:2:1 or 2:2:1:1, (1) 2:2:2:2, (2) 2:1:1 or 2:1:1:1, (3) 2:1:0, (4)

2:0:0, (5) 3:1:1.

101 100. Metafemora apical setae penultimate pair: (0) close together, (1) widley separated.

101. Metatibiae setal row AD: (0) with intercalary macrosetae, (1) without intercalary macrosetae (Figure

5.3: D, E).

102. Metatibiae setal row AD setae: (0) much longer than tibial dorsal width, (1) shorter than or subequal

to tibial dorsal width (Figure 5.3: D, E).

103. Metatibiae setal row AD setae: (0) similar to PD setae (Figure 5.3: D, E), (1) more robust than PD

setae.

104. Metatibiae setal row PD extension: (0) extends from base (Figure 5.3: D, E), (1) restricted to apical

half, (2) absent.

105. Metatibiae setal row PD setae length: (0) subequal to or longer than distance between setae, (1)

shorter than half distance between setae.

106. Metatibiae setal row AV extension: (0) extending from base, (1) restricted to apical half.

107. Metatibiae setal row AV distal setae length: (0) subequal to remaining setae, (1) with distinctly longer

setae in females (Figure 5.3: E), (2) with distinctly longer setae in males and females.

108. Metatibiae long distal AV setae in females: (0) throughout apical half to most of tibiae (Figure 5.3:

E), (1) restricted to apices of tibiae (approximately 5 setae).

109. Metatibiae setal row PV: (0) uniseriate, (1) multiseriate.

110. Metatibiae setal row PV setae: (0) subequal, (1) alternating: 1 long and 1 short, (2) alternating: 1

long and 2-4 short, (3) addtional 3 longer intercalated setae on apical fourth.

111. Metatibiae setal row PV basal setae length: (0) subequal to remaining setae, (1) elongate and hair-like.

112. Metatarsomeres I plantar chaetotaxy: (0) two uniseriate rows, (1) uni- or biseriate basally, multiseriated

apically, (2) scattered (Figure 5.3: D), (3) single row.

113. Metatarsomeres I plantar setae aspect: (0) simple, (1) some peg-like.

114. Abdominal sternum I, lateral margins: (0) with elongate projections (Figure 5.4: B), (1) without

elongate projections (Figure 5.4: C).

Some species, e.g., Paracrocampsa amida, Conogonia sp., and Backhofella pulchra, may have tiny

tooth-like projections in the same position as the elongate projections. These were not coded as state

(0).

102 115. Abdominal sternum II: (0) not projected posteriorly, (1) with conspicuous apodemes directed posteri-

orly.

116. Abdominal sternum II apodemes shape: (0) round lobes, (1) narrow and elongate with expanded apex.

117. Pygofer apical half ventral margin, lateral view: (0) mostly convex, (1) narrowed by strong concavity.

118. Pygofer disc macrosetae: (0) with macrosetae, (1) without macrosetae.

119. Pygofer disc macrosetae aspect: (0) robust, (1) elongate and fine, (2) both fine and robust.

120. Pygofer apical half chaetotaxy: (0) ventral margin as setose as remaining of disk, (1) with distinct

bare strip along ventral margin.

121. Pygofer dorsal and ventral margins: (0) not projected, (1) both projected as similar processes.

122. Pygofer dorsal and ventral margins processes aspect: (0) broad directed mesally, (1) tufts of elongate

fine processes.

123. Pygofer base of dorsal margin, lateral view: (0) continuously straight or concave, (1) with deep round

emargination.

124. Pygofer dorsal margins: (0) not projected, (1) projected as processes.

125. Pygofer dorsal margins processes aspect: (0) spiniform directed posteriorly, (1) spiniform directed

inwardly, (2) slender and elongate directed ventrally, (3) robust and inflated directed ventrally.

126. Pygofer apex dorsal margin, lateral view: (0) continuous, (1) with deep emargination forming sub-

quadrate lobe.

127. Pygofer inner margins, caudal view: (0) concave, (1) with sclerotized lobes, (2) with elongate processes

directed posteriorly.

128. Pygofer ventral margins: (0) not projected, (1) projected as processes.

129. Pygofer ventral processes origin: (0) basal, (1) apical.

130. Pygofer ventrobasal processes, aspect: (0) elongate and spiniform directed posteriorly (Figure ??), (1)

small and dentiform.

131. Pygofer ventrobasal elongate processes articulation: (0) base not separated from pygofer, (1) distinctly

articulated with remaining pygofer lobe.

103 132. Pygofer ventroapical processes aspect: (0) apical, elongate spiniform processes directed inward- or

posteriorly, (1) apical, short acute projections directed dorsally.

133. Pygofer ventral margins preapical area: (0) convex, (1) with ventrally directed lobes.

134. Pygofer apices, lateral view: (0) convex, (1) truncate, (2) produced into spiniform processes.

135. Valve posterior margin: (0) articulated with subgenital plates (Figure 5.4: D, H), (1) fused to subgenital

plates.

136. Valve lateral margins: (0) fused to pygofer (Figure 5.4: H), (1) articulated with pygofer (Figure 5.4:

D).

137. Conjunctiva IX-X: (0) membranous, (1) with sclerotized plates, (2) with elongate processes forming

paired paraphysis.

138. Subgenital plates shape, ventral view: (0) subtriangular with acute apex, (1) short and lobiform, (2)

filiform, (3) ligulate.

139. Subgenital plates shape, lateral view: (0) continuously straight or upturned, (1) abruptly upturned.

140. Subgenital plates length: (0) approximately half or subequal to pygofer length, (1) distinctly longer

than pygofer length, (2) shorter than half pygofer length.

141. Subgenital plates fusion: (0) connected by membrane or fused by basal short distance, (1) fused along

basal half, (2) fused by most of their lengths.

142. Subgenital plates inner margin: (0) straight, contiguous, (1) concave, aedeagus base exposed.

143. Subgenital plates outer margin of basal half, ventral view: (0) without concentrated setae, (1) with

close-set setae.

144. Subgenital plates dorsal suface apical half texture: (0) smooth, (1) sclerotized and serrate.

145. Subgenital plates dorsal surface: (0) slightly concave, (1) with dentiform processes associated with

styles apices.

146. Subgenital plates dorsal surface preapical area: (0) slightly concave, (1) with dorsally directed lobe.

147. Subgenital plates, ventral surface: (0) with macrosetae, (1) without macrosetae.

148. Subgenital plates macrosetae, ventral surface: (0) multiseriate (Figure 5.4: H), (1) uniseriate (Figure

5.4: G).

104 149. Subgenital plates apex: (0) not projected, (1) projected into process.

150. Subgenital plates apex process aspect: (0) short and dentiform, (1) elongate and spiniform.

151. Styles preapical region texture: (0) sclerotized and smooth, (1) weakly sclerotized and wrinkled, (2)

sclerotized and serrate.

152. Connective anterior arms direction: (0) strongly divergent, (1) converging anteriorly or when parallel,

distance between arms is smaller than arm width.

153. Connective posteromedian region: (0) produced as a stem, (1) not produced posteriorly.

154. Connective dorsal surface median area: (0) without distinct depression, (1) with distinct central pit.

155. Connnective dorsal surface posterior half: (0) not projected, (1) produced into a dorsal longitudinal

keel.

156. Connnective apex or subapical region: (0) articulated with aedeagus, (1) fused with aedeagus, (2)

linked by long membrane to aedeagus, (3) articulated with additional sclerite (paraphyses).

157. Paraphyses aspect: (0) paired symmetrical processes, (1) single process or small sclerite, (2) single

or paired asymmetrical processes, (3) trifurcate, (4) single process modified into sheath surrounding

shaft.

158. Connection of aedeagus to base of anal tube: (0) membranous, (1) through conspicuous aedeagal base

dorsal apodemes (Figure 5.4: E), (2) through additional sclerite(s) forming dorsal connective.

159. Dorsal connective aspect: (0) pair of dorsal arms, (1) U-shaped sclerite, (2) H-shaped sclerite, (3) long

subquadrate sclerite.

In some case, the U-shaped dorsal connective may be interrupted mesally, e.g., Abana, Omagua,

Raphirhinus, and Teletusa.

160. Dorsal connective dorsal arms length: (0) approximately the same or shorter than twice distance in

between, (1) longer than thrice distance in between.

161. Dorsal connective transverse bar and ventral arms aspect: (0) approximately same width as dorsal

arms, (1) robust and inflated.

162. Ejaculatory reservoir apex: (0) membranous, (1) sclerotized (Figure 5.4: E).

105 163. Ejaculatory reservoir sclerotized apex length: (0) approximately as long as wide (Figure 5.4: E), (1)

approximately thrice longer than wide.

164. Aedeagus preatrium: (0) short, (1) elongate (Figure 5.4: F).

In all oncometopiines with elongate preatrium, the preatrium base articulates with the ventral surface

of the connective, which is not the case with Old World cicadellines. In both species of Dichrophleps

the preatrium was scored as long and articulated ventrally to the connective. However, it is also

articulated to the shaft, therefore having the appearance of a basal triangular plate. It has been

described as and interpreted as a separate sclerite by Mejdalani & Emmrich [220] in D. nielsoni and

as a paraphyses shaped as basal sclerite in D. truncata by Young [392]. This articulated preatrium is

present in many other Dichrophleps species, including the type species, and may bear processes, as is

the case of D. despecta, herein considered homologous to ventral atrial processes (char. 175, state 1).

Acostemma sp. has a very bizarre elongate preatrium, which curves ventrally and attaches dorsally to

the connective.

165. Aedeagus shaft aspect: (0) with thick cuticular wall around gonoduct, (1) with very thin cuticular

wall around gonoduct.

166. Aedeagus shaft at midlength dorsal margin: (0) not projected, (1) with paired elongate processes

directed dorsally.

167. Aedeagus shaft at midlength lateral margins, caudal view: (0) not projected, (1) with pair of short

dentiform or lobed processes.

168. Aedeagus shaft apex dorsal margin: (0) not projected, (1) projected as dorsal flanges or process(es).

169. Aedeagus shaft apex dorsal margin projections aspect: (0) paired and elongate processes, (1) sin-

gle projection continuing shaft, may be branched or bear processes, (2) paired and short dentiform

projections, (3) flattened flanges extending basally, (4) 2 pairs of elongate processes.

170. Aedeagus shaft apex lateral margins: (0) not projected, (1) projected as lateral flanges or processes.

171. Aedeagus shaft apex lateral margins projections aspect: (0) paired elongate processes directed pos-

terodorsally or laterally, (1) paired flattened flanges extending basally, (2) paired dentiform projections

directed laterally.

172. Aedeagus atrium: (0) not distinctly expanded, (1) expanded posteriorly, (2) expanded posteriorly and

laterallly.

106 173. Aedeagus posterior atrial lobe, lateral view: (0) continuous with shaft, (1) separated by deep concavity

from shaft.

174. Aedeagus atrium lateral margin: (0) not projected, (1) with pair of flattened expansisons.

175. Aedeagus atrium ventral margin: (0) not projected, (1) projected as ventral process(es).

176. Aedeagus atrium ventral margin processes aspect: (0) single and spiniform, (1) single and scoop-shaped,

(2) trifurcate, (3) paired and elongate (may be slightly asymmetrical), (4) paired and drastically

asymmetrical processes (Figure 5.4: F), (5) paired, but modified into paraphyses, (6) paired and

elongate, but arising dorsally to the shaft.

Due to overall resemblance, the aedeagal processes in Anacuerna, Dechacona, and T. doeringi are

thought to be homologous to the ventral atrial processes of other Oncometopiini, even though they

seem to arise in a dorsal position to the shaft (state 6).

177. Aedeagus atrial ventral processes apices sculpturing: (0) smooth, (1) with long spiniform cuticular

processes.

Young [392] mentions that the ventral processes in D. missionum are “finely hairy”. However, this in

not the case as these processes bear small cuticular processes rather than hairs.

178. Gonoduct apex: (0) internal to sclerotized shaft, (1) distinctly extending beyong sclerotized portion

of shaft.

179. Anal tube segment X base lateral margins: (0) without processes, (1) with finger-like process.

180. Anal tube segment X base ventral margin: (0) without lobed processes, (1) with paired lobed processes

close to midline.

181. Anal tube segment X posteroventral margin: (0) not produced, (1) produced posteriorly (Figure 5.4).

182. Anal tube segment X lateral margins: (0) not expanded, (1) expanded ventrally.

183. Ovipositor gonoplacs apices: (0) mostly enclosed within pygofer, (1) largely exposed, produced poste-

riorly past pygofer apex by more than their height.

5.3.3 Phylogenetic Analysis

The heuristic parsimony search ran for approximately 20 processor hrs on a Power Mac G5 (dual PPC

2.7 GHz) and saved 100,000 suboptimal trees (L=1409). However, the 100 independent runs of Parsimony

107 Ratchet on a Power Mac G4 (733 Mhz) took a combined approximately 7 processor hrs and optimal trees were found in 21 of these runs (non-distinct optimal trees found in 4 of these replicates). The strict consensus of the 10,000 most parsimonious trees (L=1408, CI=0.25, RI=0.74, RC= 0.19) found by the full TBR step is shown in Figures 5.5 and 5.6. One randomly selected tree is shown with non-ambiguous characters optimized in Figures 5.7-5.9.

Bayesian posterior probabilities were calculated by computing a majority-rule consensus of the 36,000 trees sampled post-burnin (calculated at 500,000 generations based on Figure 5.10). These probabilities are shown in Figures 5.11 and 5.12 along with parsimony bootstrap percentages.

5.4 Discussion

5.4.1 Changes in the Higher-Level Classification

The present study is the first higher level phylogenetic study including multiple genera and a wide sampling of species of the subfamily Cicadellinae. Although not focused on elucidating tribal composition or testing the monophyly of Cicadellinae sensu lato, the present results corroborates restriction of Cicadellinae to the tribes Cicadellini and Proconiini as previously proposed by Young [392–394], but additionally suggests the inclusion of members of the subfamily Phereurhininae, treated herein as a tribe of Cicadellinae. Cicadellinae as delimited herein was recovered as a monophyletic clade in the most parsimonious trees, with the exception of the single representative of Signoretiinae grouping with members of Cicadellini. This position was not supported statistically and it is believed to be an artifact of missing data, because the male genitalia could not be scored for this particular species.

Furthermore, this study suggests that the tribe Proconiini sensu Young [392] is not monophyletic, but comprises two main lineages, roughly corresponding to the two groups Young [392] delimited based on the exposure of the posterior meron by resting the forewings. The clade including the type-genus Proconia was recovered with high clade support and is given priority to keep its nominate status. Proconiini Haupt,

1924 sensu stricto retains two previously recognized subjective junior synonyms, Ciccini Baker, 1915 and

Ciccianini Metcalf, 1952. However, the second clade, most members of which were considered previously related and informally referred to as the Oncometopia group by Rakitov [288] and the Cleusiana group by

Mejdalani [219], has never been formally recognized as a separate higher taxon. This clade is herein elevated to tribal status, Oncometopiini, trib. nov. (type-genus: Oncometopia).

108 Figure 5.5: Morphological analysis: Strict consensus, part 1. Consensus of 10,000 most parsimonious trees (L=1408, CI=0.25, RI=0.74, RC=0.19). Clades are colored accordingly to proposed classification. The colored names refer to the taxa affected by the present classification proposal.

109 Figure 5.6: Morphological analysis: Strict consensus, part 2. Consensus of 10,000 most parsimonious trees (L=1408, CI=0.25, RI=0.74, RC=0.19). Clades are colored accordingly to proposed classification. The colored names refer to the taxa affected by the present classification proposal.

110 Figure 5.7: Morphological analysis: Optimized tree, part 1. One of 10,000 most parsimonious trees (L=1,408, CI=0.25, RI=0.74, RC= 0.19) with non-ambiguous characters optimized.

111 Figure 5.8: Morphological analysis: Optimized tree, part 2. One of 10,000 most parsimonious trees (L=1,408, CI=0.25, RI=0.74, RC= 0.19) with non-ambiguous characters optimized.

112 Figure 5.9: Morphological analysis: Optimized tree, part 3. One of 10,000 most parsimonious trees (L=1,408, CI=0.25, RI=0.74, RC= 0.19) with non-ambiguous characters optimized.

113 Figure 5.10: Morphological analysis: Posterior likelihoods plot. Plot of posterior likelihoods of the trees sampled every 500 generations in the 4 independent runs.

Finally, the speciose tribe Cicadellini appears to be a polyphyletic taxon. However, the character and taxon sampling of the present study are insufficient to justify proposing any changes in the classification of this tribe.

5.4.2 Changes in the Composition of Tribes

Two genera included in the present analysis were previously not explicitly placed within Cicadellinae. Young

[393] never studied specimens of Ectypus coriaceus Signoret and, although, based on the illustration in the original description, he thought it might be a Proconiini, he kept this monotypic genus unplaced. Oman et al. [264] dubiously placed it in Proconiini following Young [393]. The present analysis furthermore supports the inclusion of Ectypus in Proconiini sensu stricto based on high clade support. Its basal position in the recovered trees, however, should be viewed cautiously, because it is probably an artifact of missing characters

–the single known specimen is a brachypterous female, which made it impossible to code characters from the wings and male genitalia for this species. Another monobasic genus, Homalogoniella, was studied by

Young [393] who thought it to be “ more like ledrine leafhoppers than like Cicadellinae in appearance.” The genus was catalogued dubiously under Cicadellini by Oman et al. [264] but it here confidently transferred to

Phereurhinini, bringing the total number of genera included in this tribe to four.

114 Figure 5.11: Morphological analysis: Clade support, part 1. Support based on Bayesian posterior likelihoods (BPL) and parsimony boostrap (PB). Percentages are given above branches as BPL or BPL/PB. Clades are colored accordingly to proposed classification. The colored names refer to the taxa affected by the present classification proposal.

115 Figure 5.12: Morphological analysis: Clade support, part 2. Support based on Bayesian posterior likelihoods (BPL) and parsimony boostrap (PB). Percentages are given above branches as BPL or BPL/PB. Clades are colored accordingly to proposed classification. The colored names refer to the taxa affected by the present classification proposal.

Even though Proconiini was delimited as a tribe strictly confined to the New World, the Oriental mono- typic genus Jilijapa Melichar was catalogued under Proconiini by Metcalf [229] and this classification followed by Oman et. al. [264]. Additionally, its close relative Namsangia was placed in Ciccini by the former and Ci- cadellinae without further tribal reference by the latter author. Previous authors most probably assigned this genus to Proconiini based on their short hindfemora, which do not attain the lateral lobes of the pronotum at rest position, prominent antennal ledges, and elongate head process. The first two characters were thought to be characteristic of Proconiini; however, the present study highlights that many Old World Cicadellini also have these traits. Elongate head processes appear to arise independently in many instances in the Pro- coniini sensu stricto, but never in Cicadellini. In the present analysis, Jilijapa grouped with other members of the Old World Cicadellini and should be transferred, together with Namsangia to this tribe. In addition to these genera, the New Guinean endemic Archeguina is also transferred to the Cicadellini. The original description of Archeguina and its five included species [395] was published posthumously for D. A. Young by

116 L. Deitz and the generic placement based on Young’s manuscript note “I think this should be placed in the

Mileewanini [sic!] or very near - not in Cicadellini. There are only 2 anteapical cells.” Archeguina indeed

lack crossvein rs (char. 73, state 1), which closes the outer anteapical cell, and this characteristic has been

used to separate Cicadellinae sensu stricto from other members of Cicadellinae sensu lato. However, Young

had previously placed other genera with only two anteapical cells in Cicadellini, e.g., Ciminius and Hadria,

and those other members can also show variation in this trait, e.g., Evacanthinae [85]. The present analysis

showed that, despite many autapomorphies, Archeguina shares many characters of the male genitalia with members of the Cicadellini, which are very different from Mileewini-related groups. In fact, based on the male genitalia and wing characters, the genus Vidanoana is apparently related to Mileewinae rather than to members of Cicadellini, where the genus is presently placed. Vidanoana is a Chilean endemic and is herein transferred provisionally to Mileewinae. Mileewinae is thought to be related to Typhlocybinae [391] and

Tinterominae [136]. Its members are morphologically very conservative and mostly distinguished by their small size and large appendix of the inner margin of the forewings (char. 85, state 2). Placement of Vi- danoana in Mileewinae would require the morphological concept of this subfamily to be broadened. However a formal delimitation is not proposed herein, because there are several other undescribed (and described) species are related to members of this clade, but were not included in this study.

The genera Pamplona and Pamplonoidea were previously included in Cicadellini, but Young [393] noted that Pamplona shared many characters with members of the Proconiini. Herein this highly supported clade was recovered as a sister group to the remaining Oncometopiini except of Acrogonia in the most

parsimonious trees, while in the Bayesian analysis it grouped with that genus with high posterior probability.

Their present placement in the Oncometopiini is based on their elongate aedeagal preatrium and ventrally

projected metepisternum, the latter shared by all members studied (in addition to the cicadelline genus

Mucrometopia).

Finally, the genus Ochrostacta, previously included in Proconiini, was never recovered herein associated with neither Proconiini sensu stricto or Oncometopiini. Therefore, it is excluded from Proconiini and

transferred to Cicadellini. The two species included in Ochrostacta are dorsoventrally flattened and have

inflated heads, which probably misled other authors on the proper tribal placement of this genus [229,392].

Although the present analysis failed to elucidate relationships of this genus within Cicadellini, it is thought

to be related to the cicadelline genus Exogonia (not included) based on male genitalic characters, such as the aspect of the pygofer processes and the unique two-rami stalked paraphyses.

117 5.5 Conclusions

This was the first attempt focused on recovering the relationships of sharpshooters including a broad taxon sampling. The resulting topologies based on the morphological characters were not extremely well resolved and generally poorly supported, especially with regards at relationships between genera. Many characters underwent a high degree of convergence, which is not surprising for such a species-rich group. However, most of the proposed classification changes are also supported by preliminary analyses of DNA sequences of

4 gene regions. These analyses are presented in CHAPTER 6, with a short discussion on the topological agreement of the morphological and molecular results.

The proposed classification changes are made in order to circumscribe natural higher-level groupings. As in most insect groups, applied comparative studies are not based on phylogenies as relationship hypotheses, but rather on classification schemes, such as studying congeners or members of the same tribes. Therefore, it is important that higher-level classifications reflect the evolutionary history of sharpshooters.

118 Chapter 6

Molecular Analyses of the Proconiini

6.1 Introduction

The present chapter summarizes the sequence data gathered for the phylogenetic analyses of the Proconiini.

In addition to a combined molecular analysis, the phylogenetic information content of each gene region is evaluated.

6.2 Materials and Methods

6.2.1 DNA Sequence Data Gathering

Protocols for DNA extraction, PCR amplification, and sequencing mostly follow those described in CHAP-

TER 3. Additionally, some amplicons were purified using the GENECLEAN III purification kit (QBiogene) and some were sequenced using an Applied Biosytems 3730XL High-throughput DNA capillary sequencer at the Biotechnology Center of the University of Illinois at Urbana-Champaign.

Some regions were not sequenced for all species because no PCR product could be obtained in multiple trials using the primers mentioned. For a few other species, one of the DNA strands was not sequenced because trials yielded bad chromatograms, possibly due to mispairing of the respective primer. These exceptions are noted in Table 6.1. Except for sequences obtained for the study in CHAPTER 2, these sequences are not yet deposited in GenBank.

6.2.2 Alignment

Sequences were assembled and COI, COII, and H3 regions aligned using Sequencher 4.5 (Gene Codes Corp.).

These protein encoding genes were partitioned by codon position. The ribosomal 16S rDNA was initially aligned using ClustalX 1.81 [356] with gap opening:extension costs 50:1 and IUB DNA weight matrix and subsequently corrected by hand. This alignment is shown in Table 6.2. Hypervariable regions, which were not confidently aligned (69 characters), were excluded from subsequent analyses.

119 Table 6.1: DNA sequences of Proconiini obtained. X refer to the number of DNA strands sequenced. Origin of voucher specimens are given in Table 5.1.

TAXA COI COII 16S H3 TAXA COI COII 16S H3 Bathysmatophorini Proconiini cont. Hylaius oregonensis XX XX XX XX Deselvana sp. nov. – XX – XX Pagaroniini Dichrophleps boliviana XX XX XX X Pagaronia tredecimpunctata XX XX XX XX Dictyodisca salvini XX XX XX XX Phereurhinini Diestostemma excisum XX XX XX XX Clydacha catapulta XX XX XX XX Diestostemma stesilea – XX XX XX Phereurhinus sosanion XX XX XX X Egidemia paranceps XX XX XX XX Cicadellini Egidemia sp. nov. XX–– Bhandara semiclara XX XX XX X Homalodisca vitripennis XX XX XX XX Cicadella viridis XX XX XX XX Homalodisca elongata XX XX XX XX Cicadellini gen. sp. XX – XX – Homalodisca ichthyocephala XX XX XX XX Diedrocephala bimaculata XX XX XX – Homalodisca ignorata – XX XX XX Dilobopterus costalimai XX – XX XX Homalodisca insolita XX XX XX XX Erythrogonia areolata XX XX XX – Homalodisca liturata XX XX – XX Graphocephala coccinea XX XX XX XX Homalodisca lucernaria XX–– Helochara communis – XX XX XX Homalodisca n. apicalis XX XX XX XX Kolla sp. XX X XX X Homoscarta irregularis XX XX XX XX Latiguina oribata XX X – X Hyogonia batesi XX XX XX X Macugonalia leucomelas XX XX XX – Hyogonia youngi XX – XX XX Madaura superba XX X X XX Mareba curuna XX XX X XX Mucrometopia caudata XX XX XX X Molomea alternata XX XX XX XX Oragua obscura XX XX XX XX Molomea exaltata XX XX XX XX Pamplona spatulata XX XX XX XX Molomea fatalis XX eX XX XX Pamplonoidea yalea XX XX XX – Molomea virescens – XX XX XX Paromenia isabellina XX XX XX XX Ochrostacta physocephala XX XX XX XX Plesiommata tripunctata XX X XX XX Omagua fitchi XX XX XX XX Sonesimia dimidiata XX XX – XX Oncometopia alpha XX XX XX XX Subrasaca sp. – XX X XX Oncometopia facialis XX XX XX XX Tettisama quinquemaculata XX XX – – Oncometopia herpes XX XX XX X Torresabela fairmairei XX XX XX XX Oncometopia orbona XX XX XX XX Vidanoana sp. nov. XX X – – Oncometopia venosula XX XX XX XX Proconiini Paraquichira costaricensis – XX XX XX Abana horvathi XX X X XX Paraulacizes figurata XX X XX X Acrobelus rakitovi XX XX XX XX Paraulacizes irrorata XX XX XX XX Acrocampsa integra XX XX X XX Peltocheirus sp. nov. XX XX XX X Acrogonia citrina – X XX XX Phera lanei XX – – XX Acrogonia terminalis XX XX – X Phera luciola XX XX XX XX Acrogonia virescens XX XX XX XX Phera obtusifrons XX XX – XX Amblydisca rubriventris XX X – – Procandea sp. nov. XX XX XX XX Anacuerna centrolinea XX XX – X Proconia cf. marmorata XX XX X XX Aulacizes sp. XX X – XX Proconopera cumingi XX XX XX – Brevimetopia hermosa XX XX X XX Proconosama alalia XX XX XX XX Catorthorrhinus resimus – XX XX XX Proconosama columbica XX XX XX XX Cicciana obliqua XX–X Propetes schmidti XX XX – XX Ciccus sp. nov. n. adspersus – XX X X Pseudometopia phalaesia XX X XX XX Ciccus sp. nov. n. viridivitta XX – X X Pseudophera contraria XX – XX XX Cuerna costalis XX XX XX XX Quichira tegminis XX XX XX XX Cuerna gladiola XX XX XX XX Raphirhinus phosphoreus eX XX XX XX Cuerna sayi XX XX XX XX Splonia sp. nov. XX XX XX XX Cuerna striata XX XX XX XX Stictoscarta sulcicollis XX XX – XX Cyrtodisca major XX XX XX XX Tapajosa rubromarginata XX XX – XX Dechacona missionum – X – XX Teletusa limpida XX XX XX XX Depanana bugabensis XX XX XX XX Tretogonia bergi XX X XX XX Depanisca sulcata XX–– Tretogonia cribrata XX XX XX XX

120 Table 6.2: Alignment of 16S rDNA of Proconiini. Hypervariable regions are positions 38-45, 247-286, 312-317, 342-344, 355-356, and 363-372. 121 Table 6.2: ...Continued. 122 Table 6.3: Descriptive statistics on sequence data. Models of molecular evolution were selected by hLRT for each molecule and codon position in protein encoding genes. The 16S rDNA dataset was analyzed with hypervariable positions excluded. Percent pairwise divergences (%PD) in between Cicadellinae taxa are given. Skewness (g1) was calculated based on 1,000 random trees. Percentages of clades with parsimony bootstrap support >50% is based on strict consensus trees (%BS).

Gene Model Length # #Variable #Informative %A %C %G %T %PD g1 %BS Selected (bp) Taxa sites (%) sites (%) COI GTR+I+Γ 783 91 409(52) 375(48) 33 14 14 39 0-25 -0.22 21 COI pos1 GTR+I+Γ 261 COI pos2 GTR+I+Γ 261 COI pos3 GTR+I+Γ 261 COII GTR+I+Γ 591 95 381(64) 321(54) 37 14 11 38 1-27 -0.3 43 COII pos1 GTR+I+Γ 197 COII pos2 GTR+I+Γ 197 COII pos3 GTR+I+Γ 197 H3 SYM+I+Γ 328 90 313(95) 101(31) 16 28 33 23 1-16 -0.2 93 H3 pos1 GTR+Γ 110 H3 pos2 JC 109 H3 pos3 GTR+I+Γ 109 16S F81+I+Γ 415 80 226(54) 177(43) 16 28 33 23 0-21 -0.33 28 Combined 2,117 102 1132(53) 974(46) 31 15 17 37 1-23 -0.35 48

6.2.3 Phylogenetic Content

As measures of phylogenetic content, the g1 (skewness) statistics of 1,000 random trees [166] and the per- centage of clades supported by >50% parsimony bootstrap among all resolved clades in the strict consensus trees were calculated.

Additionally, likelihood mapping [333] was conducted with TREE PUZZLE 5.3 [315] by examining

10,000 quartets using an approximate likelihood function based on the selected model parameters for each molecule. Models of molecular evolution were selected by hLRTs conducted by MrModeltest 2.2 [257] and are given in Table 6.3 for each molecule and codon position.

6.2.4 Phylogenetic Analyses

Phylogenetic analyses were performed on each gene separately and on a combined molecular dataset. Com- bination of the molecular datasets was supported by 1,000 replicate ILD tests (each with 10 random addition replicates saving only 100 trees, with uninformative and constant characters excluded) of the partitions: COI vs. COII (p=0.31), CO vs. 16S (p=0.81), and mitochondrial vs. nuclear (p=0.95). This dataset included representatives of 46 out of the 58 genera previously included in the Proconiini [50, 135,293].

Heuristic parsimony tree searches were performed with 1,000 random addition replicates and TBR branch swapping, except for the H3 dataset. Due to the immense numbers of trees that resulted from preliminary analyses of the H3 and the limits of available computing resources, searches were conducted by firstly keeping

100 optimal trees from each of the 1,000 random addition replicate (nchuck=100), then performing full TBR branch swapping starting with the best trees saved in the previous step, but with maxtrees set to 100,000.

123 Figure 6.1: Molecules: Likelihood mapping diagrams. Diagrams for each gene region based on 10,000 quartets. Each point represents a four-taxon statement, with those quartets that are well resolved towards a certain topology falling in the three corners (high phylogenetic signal), while those in the central triangle are those that do not strongly support any of the 3 possible relationship statements (low phylogenetic signal).

Bayesian analyses were conducted for each gene using Mr. Bayes 3.1 [168, 169,303]. Protein encoding genes were partitioned by codon position and a mixed model approach was conducted by applying different models of molecular evolution to each partition (see Table 6.3). Each analysis consisted of 2 independent runs of 1,000,0000 generations, each with 4 chains, and sampling topologies from every 500 generations.

Clade support was calculated using Bayesian clade probabilities, as recovered from the majority-rule consensus of the 4,600 post-burnin sampled trees and parsimony character boostrap [123]. Boostrap searches were conducted with 1,000 pseudoreplicates each with 10 random addition replicates and TBR branch swapping, except for only 100 pseudoreplicates each with 5 addition replicates and SPR branch swapping in the case of the H3 dataset.

6.3 Results

6.3.1 Phylogenetic Content of Datasets

Descriptive information on the molecules studied was summarized in Table 6.3. Likelihood mapping diagrams for each molecule are given in Figure 6.1.

124 Figure 6.2: Molecules: Posterior likelihoods plots. Posterior likelihoods of the trees sampled per 500 gener- ations in the 2 independent runs for each molecule.

6.3.2 Phylogenetic Analyses

Bayesian analysis runs appeared to converge after 100,000 generations (Figure 6.2) and the topologies sam- pled before this arbitrary cut-off were discarded (200 trees per run), except for the combined molecular dataset in which 600 trees were discarded (plot not shown). The remaining topologies were used to produce a 50% majority-rule consensus phylogram with mean branch lengths shown in Figures 6.3-6.7, along with their clade posterior probabilities and parsimony bootstrap support.

Heuristic parsimony searches resulted in: 4 most parsimonious trees for COI (L=5501, CI=0.14, RI=0.34,

RC=0.05), 25 for COII (L=4205, CI=0.17, RI=0.41, RC=0.07), 6 for 16S (L=1835, CI=0.22, RI=0.49,

RC=0.11), 10,000 for H3 (L=817, CI=0.25, RI=0.64, RC=0.16), and 2 for the combined molecular dataset

(L=12737, CI=0.17, RI=0.40, RC=0.07). These most parsimonious trees were summarized into strict con- sensus and clades compatible with the Bayesian trees were highlighted in Figures 6.3-6.7.

125 Figure 6.3: COI: Bayesian consensus phylogram. Mixed-model Bayesian analysis of the COI dataset (3 codon partitions: each GTR+I+Γ). Thicker clades refer to those also found by maximum parsimony. Clade support indices based on posterior probabilities >80% (BPL) and parsimony boostrap >50% (PB) are shown as percentages associated to their respective clades either as BPL or BPL/PB. Clades are colored accordingly to proposed classification. 126 Figure 6.4: COII: Bayesian consensus phylogram. Mixed-model Bayesian analysis of the COII dataset (3 codon partitions: each GTR+I+Γ). Thicker clades refer to those also found by maximum parsimony. Clade support indices based on posterior probabilities >80% (BPL) and parsimony boostrap >50% (PB) are shown as percentages associated to their respective clades either as BPL or BPL/PB. Clades are colored accordingly to proposed classification.

127 Figure 6.5: 16S: Bayesian consensus phylogram. Bayesian analysis of the 16S dataset (GTR+I+Γ). Thicker clades refer to those also found by maximum parsimony. Clade support indices based on posterior prob- abilities >80% (BPL) and parsimony boostrap >50% (PB) are shown as percentages associated to their respective clades either as BPL or BPL/PB. Clades are colored accordingly to proposed classification.

128 Figure 6.6: H3: Bayesian consensus phylogram. Mixed-model Bayesian analysis of the H3 dataset (3 codon partitions: pos1=GTR+Γ, pos2=JC, pos3=GTR+I+Γ). Thicker clades refer to those also found by maxi- mum parsimony. Clade support indices based on posterior probabilities >80% (BPL) and parsimony boos- trap >50% (PB) are shown as percentages associated to their respective clades either as BPL or BPL/PB. Clades are colored accordingly to proposed classification.

129 Figure 6.7: Molecular: Bayesian consensus phylogram. Mixed-model Bayesian analysis of the combined molecular dataset (6 partitions: H3 pos2=JC, 16S=F81+I+Γ, H3 pos1=GTR+Γ, COI, COII, and H3 pos 3=GTR+I+Γ). Thicker clades refer to those also found by maximum parsimony. Clade support indices based on posterior probabilities >80% (BPL) and parsimony boostrap >50% (PB) are shown as percentages associated to their respective clades either as BPL or BPL/PB. Clades are colored accordingly to proposed classification.

130 6.4 Discussion

6.4.1 Topological Agreement with Morphological Analysis: Support for Classification Changes

The present phylogenetic analyses based on 4 gene regions provide further support for the classification

changes proposed in CHAPTER 5. Bayesian analysis of the combined molecular dataset recovered the

monophyly of the Phereurhinini stat. nov., Proconiini sensu stricto, and Oncometopiini trib. nov., the first

two with high levels of clade support. Additionally, it supports the transfer of Pamplona and Pamplonoidea

to the Oncometopiini.

As in the morphological study, the gene regions selected herein did not provide any further insight on

the relationships of the polyphyletic Cicadellini, with few noteworthy exceptions. The analysis recovered a

highly supported clade containing Ochrostacta, all included members of the Erythrogonia generic group and

Subrasaca, further supporting the exclusion of the former from Proconiini and transfer to Cicadellini. Also,

it divided the included Cicadellini into two major groups: a basal Old World clade, which includes tropical

Asian groups and the New Guinea endemic, and a possibly paraphyletic or polyphyletic group that contains

the New World genera in addition to Cicadella and a Madagascar endemic. The latter assemblage groups with high support with the Phereurhinini and Oncometopiini.

Finally, the only major disagreement between morphology and molecules found was on the position of

Vidanoana was transfered from Cicadellini to Mileewinae based on wing and male genitalia morphology.

The present analyses recover this genus (without great clade support) within of what is herein treated as the Cicadellinae, although in a basal position and not grouping with the remaining New World Cicadellini.

This could be an artifact of missing data, as only sequences from COI and COII were included so far.

131 Chapter 7

Evolution of Egg-Powdering Behavior

7.1 Introduction

Sharpshooter species exhibit several types of the oviposition behavior. Most commonly females simply insert

their eggs into plant tissues, like the majority of leafhopper species ( [60,334]. Others can either (1) lay their

eggs in the soil (only in Sonesimia Young, [224]), (2) lay their eggs into plant tissues and cover the scars with specialized brochosomes [252,335], or (3) lay their eggs exposed and cover them with specialized brochosomes

(only in Acrogonia St˚al,[270]). Brochosomes are proteinaceous particles, typically 0.2-2.0 µm in diameter

that are produced by glandular segments of the Malpighian tubules by all leafhoppers [76, 286]. Freshly

molted leafhoppers cover their integument with these particles through specialized behaviors of anointing

and grooming [289]. Their most probable function is providing protection against excessive humidity and

sticking of honeydew to the body [286, 290].

Sharpshooter females that cover their egg batches with brochosomes release droplets containing brocho-

somes and transfer them to their forewings with their hindlegs (Figure 7.1), in a behavior indistinguishable

from some anointing behaviors [291]. After these droplets dry, they become hard pellets that stay in place

while the females search for an oviposition site. After oviposition, females scrape brochosomes off these

pellets, onto their egg nests (Figure 7.1). This powdering behavior has been viewed by Rakitov [291] as a

possible modification of a regular grooming behavior, which is observed in females of Cicadella viridis just

after oviposition [10].

The egg-powdering behavior is the only known use of brochosomes other than covering of the integument

[290]. In addition, egg-powdering females display three kinds of structural modifications that are related to

this behavior: (1) large and elongated egg-brochosomes, distinct from those used for body covering, which

are produced only by gravid females [290]; (2) forewings with areas of differentiated setae that facilitate the

storing of brochosomes before oviposition (Figure 7.2); and (3) hindlegs with modified setae for scraping the

brochosomes off the wings and onto the egg-nest (Figure 7.3). A survey of museum collections found that

these traits are present in 15 genera of the Oncometopiini and one Phereurhinini (Dayoungia), but not in

132 Figure 7.1: Egg-Powdering Behavior. Sketches of Oncometopia orbona behaviors: (A) transfer of anal droplets to forewings prior to oviposition, and (B) powdering of egg nests by scraping brochosomes off the pellets with hindlegs. Illustrations by R. Rakitov. other leafhoppers [291]. Among these species, each of the structural traits involved exhibits gradient-like variation from unspecialized to highly specialized conditions. However, in a particular species, different traits do not always display the same degree of specialization, indicating that they did not necessarily evolve in concert.

Sharpshooter egg-powdering behavior may be viewed as an example of a novel complex of traits evolved in association with a new biological function. Its origin apparently involved the acquisition of a new function by an old trait: brochosomes, ancestrally used as a body coat only, began to be used as a coating to eggs.

Subsequently, the altered selection on various behavioral, structural, and physiological traits of females resulted in the evolution of modifications summarized above. In the present study a phylogenetic analysis based on morphological and molecular data of sharpshooters is used as a framework to elucidate the evolution of egg-powdering by reconstructing ancestral character states.

133 Figure 7.2: Egg-Powdering Behavior: Forewing setation dimorphism. Male and female forewings of a non- powdering (A, Homalodisca elongata) and a powdering species (B, H. vitripennis). Red dots represent setae. Illustrations by R. Rakitov.

7.2 Materials and Methods

7.2.1 Phylogenetic Analysis

Analyses of the morphological and molecular datasets presented in CHAPTERS 5 and 6 were conducted to obtain a robust hypothesis of relationships. Two of the three morphological characters associated with egg- powdering used in subsequent optimization analyses are included in the morphological dataset (chars. 63 and

107) used to reconstruct the combined evidence tree. The inclusion of these characters in the reconstruction process is justified because all heritable characters should be included to add to the robustness of the results [140] and also because they constitute only a small proportion of the 2,369 characters included and therefore should not lead to circular reasoning [283].

Mixed-model Bayesian analyses were conducted with 4 independent runs each with 4 chains for 5,000,000 generations assuming flat priors. The dataset was divided into 7 partitions: morphology, 16S, COI, COII, and three codon positions of H3. These analyses were conducted on the Tungsten Linux cluster (1,450 systems of 3.2 GHz) of the National Center for Supercomputing Applications (Urbana, IL). Additionally, heuristic maximum parsimony tree searches were performed in PAUP* 4.0b10 [336] with 1,000 random addition replicates and TBR branch swapping.

Clade support was calculated based on Bayesian clade probabilities, as recovered from the majority- rule consensus of the 36,000 post-burnin sampled trees and parsimony character boostrap [123]. Boostrap searches were conducted with 1,000 pseudoreplicates each with 5 random addition replicates and SPR branch swapping. Partitioned Bremer decay indices [40] were calculated based on one of the most parsimonious trees with the aid of TreeRot 2.0 [330]. The initial unconstrained and each constrained search were conducted with 20 random addition replicates and TBR branch swapping.

134 Figure 7.3: Egg-Powdering Behavior: Hindleg AV row setation dimorphism. Male and female hindtibiae of a non-powdering (A, Cuerna obesa) and a powdering species (B, Acrogonia sp.). Blue setae represent the AV row. Illustrations by R. Rakitov.

7.2.2 Character Optimization

Although the oviposition behavior has been studied in few sharpshooter species ( [202] and see [291] for a summary), the presence of the behavior may be inferred by the presence of egg brochosomes (museum female specimens occasionally carry brochosome pellets on their forewings). Data on the presence and absence of egg-brochosomes were obtained from Rakitov [291], and those species he did not study were coded as unknown. It should be noted that in the species coded as lacking egg powdering because no museum specimens with pellets were found, this character state cannot be considered as proven because the chances to collect these females with pellets are generally low. Data on forewings and hindlegs were also scored as in Rakitov [291]. This scoring in some species can be ambiguous, because of the range of variation in the expression of these traits.

The three egg-powdering related morphological traits were optimized on tree topologies using maximum parsimony and likelihood approaches. To avoid polytomous topologies, optimizations were conducted on the tree with the highest posterior likelihood (posterior -lnL=56,787.83) using branch length information. All optimizations were conducted in Mesquite 1.12 [205]. Likelihood optimization [269] was done with a MK1 model, which assumes that any particular change is equally probable.

Although outgroup taxa are not shown in the truncated Oncometopiini tree with optimized characters

(Figure 7.8), all optimizations were conducted with outgroups included [141]. In >50% of the Bayesian trees, the sister taxon to the Oncometopiini is Diedrocephala bimaculata, but in the estimate of the most likely tree used for optimizations, the sister taxon is the clade Dilobopterus+Mucrometopia.

135 Figure 7.4: Combined: Posterior likelihood plots. Posterior likelihoods of the trees sampled every 500 generations in the 4 independent runs for the analysis of the combined dataset.

7.3 Results

7.3.1 Phylogenetic Analyses

Bayesian analysis runs appeared to converge after 500,000 generations (Figure 7.4 and the topologies sampled before this arbitrary cut-off were discarded (1,000 trees per run). The remaining topologies were used to produce a 50% majority-rule consensus phylogram with mean branch lengths shown in Figures 7.5-7.7.

Parsimony analyses recovered 7 most parsimonious trees (L=13850, CI=0.17, RI=0.42, RC=0.73) and clades present in the strict consensus compatible with the Bayesian consensus were highlighted in Figures

7.5-7.7.

Clade support indices based on posterior probabilities >80%, parsimony bootstrap >50%, and partitioned

Bremer decay are also shown in Figures 7.5-7.7. Interestingly, for most well-supported clades, COI is the only partition disagreeing with the remaining partitions by showing negative values of decay indices.

7.3.2 Character Optimization

All the species included in the present study that show traits associated with the egg-powdering behavior fall into the Oncometopiini clade. Optimizations of these traits are summarized in Figure 7.8.

Based on the presence of egg-brochosome pellets, the parsimony optimization shows uncertainty at the base of the Oncometopiini clade, with either a single origin of egg-powdering and 8 subsequent losses (+ one unambiguous secondary origin in C. sayi) of this behavior in ACCTRAN or 8 independent origins (+ one

136 Figure 7.5: Combined: Bayesian consensus phylogram, part 1. Mixed-model Bayesian analysis of the com- bined morphological and molecular dataset (7 partitions: Morphology, H3 pos2=JC, 16S=F81+I+Γ, H3 pos1=GTR+Γ, COI, COII, and H3 pos 3=GTR+I+Γ). Thicker clades refer to those also found by maxi- mum parsimony. Clade support based on clade posterior probabilities >80% (BPL) and parsimony bootstrap >50% (PB) are shown as percentages over its respective clade either as BPL or BPL/PB. Partitioned Bre- mer indices shown as graphs below respective clades, with x-axis representing partitions: Morphology, COI, COII, H3, and 16S, and y-axis support ranging from -20 to 20. Clades are colored accordingly to proposed classification.

137 Figure 7.6: Combined: Bayesian consensus phylogram, part 2. Mixed-model Bayesian analysis of the com- bined morphological and molecular dataset (7 partitions: Morphology, H3 pos2=JC, 16S=F81+I+Γ, H3 pos1=GTR+Γ, COI, COII, and H3 pos 3=GTR+I+Γ). Thicker clades refer to those also found by maxi- mum parsimony. Clade support based on clade posterior probabilities >80% (BPL) and parsimony bootstrap >50% (PB) are shown as percentages over its respective clade either as BPL or BPL/PB. Partitioned Bre- mer indices shown as graphs below respective clades, with x-axis representing partitions: Morphology, COI, COII, H3, and 16S, and y-axis support ranging from -20 to 20. Clades are colored accordingly to proposed classification.

138 Figure 7.7: Combined: Bayesian consensus phylogram, part 3. Mixed-model Bayesian analysis of the com- bined morphological and molecular dataset (7 partitions: Morphology, H3 pos2=JC, 16S=F81+I+Γ, H3 pos1=GTR+Γ, COI, COII, and H3 pos 3=GTR+I+Γ). Thicker clades refer to those also found by maxi- mum parsimony. Clade support based on clade posterior probabilities >80% (BPL) and parsimony bootstrap >50% (PB) are shown as percentages over its respective clade either as BPL or BPL/PB. Partitioned Bre- mer indices shown as graphs below respective clades, with x-axis representing partitions: Morphology, COI, COII, H3, and 16S, and y-axis support ranging from -20 to 20. Clades are colored accordingly to proposed classification. 139 Figure 7.8: Optimizations of Egg-Powdering Behavior and related morphological characteristics. Parsimony optimization of the presence of egg-brochosomes (black clades) as an indicator of powdering behavior in Oncometopiini, ambiguous clades in black and white. Likelihood of ancestral states given as pie charts by each node referring to the presence of modified forewings (in red) and hindlegs (in blue), uncertain probabilities in grey. Species for which observations on the oviposition behavior were made [291] are marked with a plus (+) or minus (-) sign to indicate presence or absence of egg-powdering. Clades which were not highly supported are marked with asterisks (*). unambiguous loss in Brevimetopia and one ambiguous loss in the ancestor of H. elongata-P. obtusifrons) in

DELTRAN. Furthermore, likelihood optimization shows a 63% chance that egg-powdering was present at the base of the Oncometopiini clade. Likewise, there is a strong probability (83%) that hindlegs were already modified at the base of the Oncometopiini. However, the forewing modifications appear to have originated independently 4 times within the Oncometopiini (in the Oncometopia-Homalodisca clade, in Acrogonia, in

Pamplona, and within Molomea) with two subsequent losses. There is only a 1% chance that this trait was present in the ancestor of the Oncometopiini.

140 7.4 Discussion

7.4.1 Origin of the Egg-Powdering Behavior

The present analysis gives evidence for a single instead of multiple origins of the egg-powdering behavior.

This finding is not surprising because the egg-powdering behavior seems to be a rather complex trait,

involving physiological and behavioral changes in females, which are not very likely to evolve multiple times.

However, it is possible that the behavior was acquired previous to the diversification of the Oncometopiini.

Aside from members of the Oncometopiini, traces of egg-brochosomes were found on the forewings of the

phereurhinine Dayoungia virescens [291], and later also in the Malpighian tubules of another specimen [292].

This strong evidence should not be taken lightly and supports a sister-taxon relationship between the tribes

Oncometopiini and Phereurhinini. Unfortunately, the combined Bayesian analysis presented here did not

recover this relationship, although it did not disagree with it, as it showed no strong support for a putative

sister group to the Oncometopiini. Furthermore, the parsimony analysis of both the morphological dataset

(CHAPTER 5) and the combined dataset did recover (without clade support) the Phereurhinini as a

sister to the Oncometopiini. The rare tribe Phereurhinini is currently composed of four genera and less than

10 described species, and females are known for only the two described species of Dayoungia. Although in

Dayoungia there is no sexual dimorphism in the hindleg structure, the setae of the AV row are unusually long

in both males and females. It seems plausible that the egg-powdering behavior originated in the ancestor

of Phereurhinini and Oncometopiini. This behavior and the differentiation of the associated morphological

traits found in the Oncometopiini, especially the modification in the hindlegs, may have conferred a greater

selective advantage for the oncometopiine ancestor, contributing to the radiation of this clade. For example,

species of Acrogonia definitely occupy a whole new oviposition niche (exophytic) in the entire leafhopper

family.

Even though the egg-brochosomes represent solid evidence for the position of Phereurhiniini as a sister

taxon to the Oncometopiini, it is worthwhile to discuss the recovery of the clade Dilobopterus + Mucrome- topia in a small percentage of Bayesian trees found in the combined analysis as a putative sister to the

Oncometopiini. Although this relationship was not recovered in the morphological analysis, the monotypic genus Mucrometopia resembles oncometopiines, as previously noticed by Young [393], and shares with them many synapomorphies. Unfortunately, no females of this species are known. Interestingly, males of Mucrome- topia and the phereurhinines Clydacha and Phereurhinus have been collected together in large numbers in ephemeral aggregations along river banks in Peru, where they are found sucking water from the sand in a mud-puddling behavior [294]. Except in very few species, females apparently do not engage in this behavior.

141 Rakitov [291] discussed, based on the similarity of the stroke movements, the hypothetical transformation of post-ovipositional grooming into powdering of the eggs. This transformation would presumably occur through incidental scraping of the integument brochosomes off onto the egg-nests providing some selective advantage. This scenario seems particularly probable if conducted by species that normally have a thick coat of integument brochosomes, such as those found in the genera Diestostemma, Proconia, and Tretogonia. The present study, did not recover any of these genera as a possible sister group to the powdering clade. While the first two genera were recovered in the Proconiini sensu stricto (Figure 7.5), Tretogonia was nested within

Oncometopiini, so its radiation apparently occurred subsequent to the postulated origin of the egg-powdering behavior (Figure 7.8).

7.4.2 Gains, Losses, and Vestiges of Egg-Powdering Related Traits

The fact that some extant powdering species show modifications only in the hindlegs, others only of the forewings, and others of both traits simultaneously, suggests a hypothetical evolutionary scenario of asyn- chronous and possibly stepwise gains of these modifications following the origin of the behavior itself. The present results, on the contrary, indicate that both egg-powdering behavior and hindleg modification at the base of the Oncometopiini. The variation in the presence and absence of egg-powdering related traits, instead of being due to stepwise gains, is due to independent origins of forewing modifications aggravated by several episodes of secondary losses of both hindleg and forewing modifications (Figure 7.8). Interestingly, some powdering species showing only hindleg modifications, e.g., T. rubromarginata, C. striata, Q. tegminis, and P. lanei, were associated with clades that lost the behavior completely. This suggests that the partial reduction of structural traits involved in the behavior may actually precede and possibly facilitate loss of the behavior. Exceptions are the Molomea ancestor, whose sister taxon is ambiguous, and C. sayi, who most probably secondarily regained this behavior.

It is interesting to note that among the seven known instances of losses of egg-powdering in oncometopi- ines, in the case of B. hermosa modifications of the hindlegs are still present. This observation highlights the danger of predicting a species’ behavior based on related morphological characteristics, e.g. bioluminescence based on non-functional photic organs in fireflies (examples of non-functional organs given in [39]), complex staminode (oviposition site) transportation behavior based on related female frontal hook in Staminodeus weevils (extrapolation of oviposition observation of a single species to a generic character by [128]), or male sacrifice behavior based on male genitalia prone to mutilation (correlation of these characters by [231]).

142 Egg-brochosomes show a great morphological variability among species (Figure 7.9), which contrasts

immensely with the conservative shape of brochosomes for the integument throughout the whole family

Cicadellidae, based on the available ultrastructural data [13, 290–292]. Egg-brochosomes are usually large

(mostly >4 µm), elongate, and rod-like, while integument brochosomes are small (0.2-2.0 µm) and spher- ical [286]. However, in several cases, some species produce spherical egg-brochosomes, closely resembling integument ones, although usually larger [291]. The present analysis supports that spherical egg-brochosomes were not an ancestral condition in the Oncometopiini (Figure 7.9), but that these “reversals” to a spherical shape have occurred independently within Cuerna, Oncometopia, Homalodisca, and possibly ancestrally in

the Molomea-Tapajosa clade. More specifically, spherical brochosomes are produced by all Tapajosa, but only in M. alternata of all Molomea species studied so far [292].

Finally, the large variation in the degree of specialization of egg-powdering related traits among extant oncometopiine species does not appear to represent the actual stages in the acquisition of the suite. In such a hypothetical temporal succession scenario, basal oncometopiines would show structural features less modified than more derived clades. However, in the present analyses, the genus Acrogonia, which demonstrates

extreme degrees of modification in the forewing setation, hindleg setation, and egg-brochosome elongation,

was consistently recovered as sister to the clade comprising the rest of the egg-powdering group.

7.4.3 Multiple Losses of an Adaptive Behavior

Approximately 220 described species are presently included in Oncometopiini and many new species remain

still undescribed especially from speciose genera such as Cuerna, Molomea, and Oncometopia, which show

significant variation in egg-powdering traits. Although eight secondary losses of egg-powdering suggested by

our phylogeny do not seem too many for such a diverse clade, the question arises why a supposedly adaptive

behavior should have been lost multiple times.

The exact function of brochosomes as a coat of the egg-nests is not known. Rakitov [290, 291] hy-

pothesized that brochosomes protect the eggs against pathogens, parasitoids, and perhaps create improved

conditions for egg development. Only one of these possible roles has so far been tested. Velema et al. [367]

observed oviposition behaviors of females the mymarid parasitoid Gonatocerus ashmeadi on egg masses of

the glassy-winged sharpshooter with or without brochosome coats in the laboratory. Their results suggest

that brochosomes may hinder parasitoidism rates because when fairyflies oviposit on brochosome-coated

eggs, these particles adhere to themselves and trigger frequent bouts of intensive grooming, which lead to

a decreased tendency to drill into eggs. In a large evolutionary scale, it is hard to imagine that losses of

egg-powdering are due to the absence or lower selective pressure of egg-parasitoids. However, although not

143 144

Figure 7.9: Egg brochosome diversity. Scanning electron micrographs shown as terminals in the Oncometopiini phylogeny. Black clades represent species that produce egg-brochosomes, grey ones do not. Dotted clades are ambiguous. All micrographs by R. Rakitov, except of O. facialis by W. Azevedo-Filho. tested experimentally, it is possible that some egg parasitoids use egg-brochosomes as a cue to locate host eggs. If so, losing egg brochosome coats may provide an escape from the pressure of especialized egg- parasitoids.

7.4.4 Conclusions

This study suggests a single origin of the egg-powdering behavior, possibly in the ancestor of Phereurhinini and Oncometopiini. Modifications of the female hindlegs for scraping the brochosomes off onto the egg nests were also acquired once in the ancestor of the Oncometopiini, while modifications on the female forewing setation for better anchoring of brochosome pellets, seem to have been acquired multiple times. Multiple losses of the behavior and its related associated traits occurred in various oncometopiine lineages. Finally, there is a great need for more oviposition observations, and detailed studies in the selective advantage of egg-brochosomes, especially in the variety of habitats oncometopiines inhabit from Canada to Chile and

Argentina, to further elucidate the evolution of this complex of behavioral, physiological, and morphological traits.

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168 Curriculum Vitae

Personal Data

Name • Daniela Maeda Takiya Parents’ Name • Sim˜aoMikami Takiya and Christina Maeda Takiya Date of Birth • November 23 1976 Place of Birth • S˜aoPaulo, SP Nationality • Brazilian

Education MDegrees 2001-2007 • Ph.D. candidate in Entomology, University of Illinois at Urbana-Champaign, expected. 1999-2001 • M.Sc. in Zoology, Universidade Federal do Rio de Janeiro. 1995-1998 • B.Sc. in Zoology, Universidade Federal do Rio de Janeiro.

MCourses and Workshops May 22-31 2005 • Bayesian and Likelihood Inference of Phylogeny: Organisms to Genomes. Fredrik Ronquist, John Huelsenbeck, Dave Swofford, and Jeff Thorne. University of Illinois at Urbana-Champaign, IL, USA. May 5-10 2002 • Workshop in Applied Phylogenetics. Michael Sanderson, H. Bradley Schaf- fer and Peter Wainwright. Bodega Bay Marine Laboratory, University of Cali- fornia at Davis, CA, USA. Mar. 5-9 2001 • Phylogenetic Comparative Methods. Jos´eA. F. Diniz-Filho. Departa- mento de Ecologia, Universidade Estadual de Campinas, SP, Brazil. July 16-Aug. 17 2000 • Field Course: Ecology of the Amazon Forest. Biological Dynamics in Forest Fragments Project, Instituto Nacional de Pesquisa Amazˆonicas, Organi- zation for Tropical Studies and Universidade Estadual de Campinas, Manaus environs, AM, Brazil. Nov. 13-Dec. 8 2000 • Field Course: Zoological Inventories. P´os-Gradua¸c˜aoem Zoologia, Museu Paraense Em´ılio Goeldi, Esta¸c˜aoCientfica Ferreira Penna, Floresta Nacional de Caxiuan˜a,Melgao, PA, Brazil. Sep. 13-17 1999 • Molecular Systematics and Evolution of Microorganisms. James McIn- erney, Robert Hirt, Martin Embley and Mark Wilkinson. The Natural History Museum in London and Instituto Oswaldo Cruz, Rio de Janeiro, RJ, Brazil. Feb. 9-13 1998 • Insects: Main groups and Evolution. Rodney Cavichioli. XXII Congresso Brasileiro de Zoologia, Universidade Federal de Pernambuco, Recife, PE, Brazil. May 12-23 1997 • Logic and Comparative Biology. Nelson Papavero. Departamento de Biolo- gia Animal, Universidade Federal Rural do Rio de Janeiro, RJ, Brazil. May 9 1997 • Living and Learning with Nature Workshop. Pedro Menezes. Museu Nacional, Universidade Federal do Rio de Janeiro, RJ,Brazil. Jan. 11-12 1997 • Systematics and Ecology of Ants. Jos´eHenrique Schoereder. Semana da Biologia, Universidade Federal de Vi¸cosa,MG, Brazil. Nov. 4-6 1996 • Protozoology. XII Reuni˜aoAnual da Sociedade Brasileira de Protozoologia, Hotel Gl´oria, Caxamb´u,MG, Brazil.

169 Education (continued)

Sep. 23-25 1996 • Celular Differentiation. Vivaldo Moura-Neto, Radovan Borojevick and Rafael Linden. II Semana de Microbiologia e Imunologia, Instituto de Microbi- ologia Prof. Paulo de G´oes, Universidade Federal do Rio de Janeiro, RJ, Brazil. Nov. 5-7 1995 • Protozoology. XI Reuni˜ao Anual da Sociedade Brasileira de Protozoologia, Hotel Gl´oria, Caxamb´u,MG, Brazil.

Fellowships, Grants and Awards MFellowships Aug. 2001-Aug. 2005 • Doctoral Research Abroad Fellowship. Conselho Nacional de Desenvolvi- mento Cient´ıficoe Tecnol´ogico, Brazil (CNPq). Research Project: Phylogenetic analysis of the subfamily Cicadellinae (Hemiptera: Cicadellidae) with emphasis on the tribe Proconiini. July 1999-Mar. 2001 • M.Sc. Research Fellowship. Coordena¸c˜aode Aperfeicionamento de Pessoal de N´ıvel Superior, Brazil (CAPES). Research Project: Phylogenetic analysis of the genus Balacha Melichar (Hemiptera: Cicadellidae). Feb. 1997-Aug. 1998 • Scientific Initiation Fellowship for Undergraduate Research. CNPq- PIBIC. Research project: Taxonomy, morphology and biology of the Brazilian Cicadellidae (Homoptera), with emphasis on the Atlantic Forest fauna from Rio de Janeiro State. Aug. 1995-Nov. 1996 • Scientific Initiation Fellowship for Undergraduate Research. CNPq- PIBIC. Research project: Study of the benthonic ciliate (Protozoa: Ciliophora) fauna of Guanabara Bay, Rio de Janeiro State.

MGrants and Awards 2007 • J. H. Comstock Award for Outstanding Graduate Student Achieve- ment. North Central Branch, Entomological Society of America. US$ 100.00 and all-expense paid trip to the Annual Entomological Society of America Meet- ing. 2006 • Runner-up for the Ph.D. Student Competition for the President’s Prize. North Central Branch Meeting of the Entomological Society of America (Sections A, B & Ce). US$ 120.00. 2004 • Francis M. and Harlie M. Clark Research Support Grant. School of Integrative Biology, University of Illinois at Urbana-Champaign. US$ 1,200.00 for conducting field research in Brazil. 2005 • Herbert H. Ross Memorial Fund Award. Center for Biodiversity of Illinois Natural History Survey and Department of Entomology of University of Illinois at Urbana-Champaign. Proposed research: The phylogenetic status of the Sub- family Cicadellinae (Hemiptera: Cicadellidae): searching for nuclear markers for resolving deeper divergences of sharpshooters. US$ 1,200.00 for field work and travel to conduct research at Dr. Wiegmann’s lab in North Carolina State University. 2004 • SIB Enhancement Fund. School of Integrative Biology, University of Illinois at Urbana-Champaign, US$ 723.00 for travel expenses to XXIII Willi Hennig Society Meeting (Paris, France) and the Ivan Franko National University (Lviv, Ukraine). 2004 • Marie Stopes Student Travel Award for the XXIII Willi Hennig So- ciety Meeting. Willi Hennig Society, US$ 500.00 for travel expenses to the Meeting.

170 Fellowships, Grants and Awards (continued)

2004 • Francis M. and Harlie M. Clark Research Support Grant. US$ 500.00 for lodging, food, and travel within Australia to visit the Australian National Insect Collection (Canberra) and the NSW Agricultural Scientific collections Unit (Orange). 2004 • Entomological Society of America and National Science Foundation travel grant for the XXII International Congress of Entomology. US$ 2,300.00 for travel expenses and Congress registration fee. 2004 • Best Poster Presentation. XXV Brazilian Congress of Zoology (section En- tomology). 2003 • Herbert H. Ross Memorial Fund Award. Center for Biodiversity of Illi- nois Natural History Survey and Department of Entomology of University of Illi- nois at Urbana-Champaign. Proposed research: On taxonomy and phylogeny of sharpshooters (Insecta: Hemiptera: Cicadellidae: Cicadellinae). US$ 1,000.00. 2003 • Tinker Grant for Summer Field Research. Center for Latin American and Iberian Studies, University of Illinois at Urbana-Champaign. Proposed research: Taxonomic studies on Brazilian sharpshooters: from discovery to naming and describing (Insecta: Cicadellidae). US$ 1,526.00 for conducting field research in Brazil.

Professional Meetings MInvited Oral Presentations 2005 • Takiya, D. M. & Dietrich, C. H. Revisiting body size constraints in Auchenorrhyncha: the influ- ence of alternative phylogenetic scenarios and multiple origins of xylem-feeding. 12th Interna- tional Auchenorrhyncha Congress. Berkeley, USA. 2005 • Dmitriev, D. A. & Takiya, D. M. 3I, a new program for creating Internet-accessible interactive keys. 12th International Auchenorrhyncha Congress. Berkeley, USA. 2005 • Dietrich, C. H., Dmitriev, D. A., Rakitov, R. A., Takiya, D. M., & Zahniser, J. N. Phylogeny of Cicadellidae (Cicadomorpha: Membracoidea) Based on Combined Morphological and 28S rDNA Sequence Data. 12th International Auchenorrhyncha Congress. Berkeley, USA. 2003 • Takiya, D. M. Leafhoppers of Amazonia: Taxonomic Challenge and Habitat Diversity. Tinker Workshop on Pre-Dissertation Field Research. Champaign, USA.

MOral Presentations 2006 • Takiya, D. M., Rakitov, R. A., Dietrich, C. H., & Mejdalani, G. Phylogeny of the sharpshooter tribe Proconiini (Hemiptera: Cicadellidae) and its implication to classification. North Central Branch Meeting of Entomological Society of America. Bloomington, USA. 2005 • Takiya, D. M., Tran, P., Dietrich, C. H., & Moran, N. Coevolution of sharpshooters (Hemiptera: Cicadellidae) and their two bacterial endosymbionts. Entomological Society of America Annual Meeting. Fort Lauderdale, USA. 2005 • Takiya, D. M., Tran, P., & Moran, N. Coevolutionary patterns in the dual symbiosis of sharp- shooters (Hemiptera: Cicadellidae). 7th Annual Graduate Student Symposium of the Program in Ecology and Evolutionary Biology. University of Illinois at Urbana- Champaign, Champaign, USA. 2004 • Takiya, D. M., Rakitov, R. A., & Dietrich, C. H. Phylogeny of proconiine sharpshooters (Hemiptera: Cicadellidae) and the evolution of egg-powdering behavior. XXII International Congress of Entomology. Brisbane, Australia.

171 Professional Meetings (continued)

2004 • Takiya, D. M., Rakitov, R. A., & Dietrich, C. H. Evolution of egg powdering behavior in proconi- ine sharpshooters (Insecta: Hemiptera: Cicadellidae). XXIII Willi Hennig Society Meeting. Paris, France. 2003 • Takiya, D. M., Rakitov, R. A., & Dietrich, C. H. Phylogeny of Proconiine Leafhoppers and the Evolution of Egg-Powdering Behavior (Homoptera: Cicadellidae). Entomological Society of America Annual Meeting. Cincinnati, USA. 2003 • Takiya, D. M., Rakitov, R. A., & Dietrich, C. H. Phylogeny of proconiine sharpshooters and the evolution of egg-powdering behavior. 5th Annual Graduate Student Symposium of the Program in Ecology and Evolutionary Biology. University of Illinois at Urbana- Champaign, Champaign, USA. 2002 • Takiya, D. M., Rakitov, R. A., & Dietrich, C. H. Phylogeny of Proconiine Leafhoppers and the Evolution of Egg-Powdering Behavior (Cicadellidae: Cicadellinae: Proconiini). 11th Auchen- orrhyncha Congress. Potsdam, Germany. 1999 • Mejdalani, G., Takiya, D. M., & Felix, M. F. Preliminary notes on the phylogeny of the Proconiini with special reference to the genera with the exposed posterior meron (Hemiptera: Cicadellidae: Cicadellinae). Cardiff, Wales.

MPoster Presentations 2005 • Takiya, D. M., Dietrich, C. H., Rakitov, R. A., & Mejdalani, G. Phylogeny of the sharpshooter tribe Proconiini based on morphology and DNA sequences (Membracoidea: Cicadellidae: Cicadel- linae). 12th International Auchenorrhyncha Congress. Berkeley, USA. 2005 • Takiya, D. M., Tran, P., Dietrich, C. H., & Moran, N. Coevolution of sharpshooters (Hemiptera: Cicadellidae) and their two bacterial endosymbionts. 12th International Auchenorrhyncha Congress. Berkeley, USA. 2004 • Takiya, D. M. & Cavichioli, R. R. The Neotropical sharpshooter genus Onega (Hemiptera: Ci- cadellidae: Cicadellini): synonyms and new species. XX Brazilian Congress of Entomology. Gramado, Brazil. 2004 • Takiya, D. M., Dietrich, C. H., Rakitov, R. A., & Mejdalani, G. Morphology and DNA-sequences in the phylogeny of the tribe Proconiini (Hemiptera: Cicadellidae). Fifth Biennial Conference of the Program in Partnerships for Enhancing Expertise in Taxonomy. Urbana- Champaign, USA. 2004 • Takiya, D. M., Dietrich, C. H., Rakitov, R. A., & Mejdalani, G. Morphology and DNA-sequences in the phylogeny of the tribe Proconiini (Hemiptera: Cicadellidae). XXV Brazilian Congress of Zoology. Bras´ılia, Brazil. 2004 • Takiya, D. M. & Cavichioli, R. R. Three species of the new South American sharpshooter genus Lanceoscarta gen. nov. (Insecta: Cicadellidae: Cicadellinae). XXV Brazilian Congress of Zoology. Bras´ılia, Brazil. 2004 • Takiya, D. M. & Cavichioli, R. R. Notes on Brazilian Homalodisca St˚al,1869 (Hemiptera, Ci- cadellidae) with a description of a new species from Bahia. XXV Brazilian Congress of Zoology. Bras´ılia, Brazil. 2002 • Takiya, D. M. & Mejdalani, G. Phylogenetic analysis, host-plant shift, and distribution of the genus Balacha Melichar (Cicadellidae: Cicadellini). Entomological Society of America Annual Meeting. Ft. Lauderdale, USA. 2002 • Takiya, D. M. & Mejdalani, G. Phylogenetic analysis, host-plant shift, and distribution of the genus Balacha Melichar (Cicadellidae: Cicadellini). 11th Auchenorrhyncha Congress. Pots- dam, Germany. 2002 • Ceotto, P. C., Mejdalani, G., & Takiya, D. M. Taxonomy and phylogeny of the leafhopper genus Acrobelus (Cicadellidae: Cicadellinae: Proconiini) including two new South American species. 11th Auchenorrhyncha Congress. Potsdam, Germany.

172 Professional Meetings (continued)

2002 • Takiya, D. M., Rakitov, R. A., & Dietrich, C. H. A Case Study in the Evolution of a Novel Suite of Traits: Egg-powdering in Sharpshooters (Hemiptera: Cicadellidae). Evolution Meeting. University of Illinois at Urbana-Champaign, Champaign, USA. 2002 • Takiya, D. M., Mejdalani, G., & Webb, M. D. On the Amazonian Hyogonia China, 1927 (Hemiptera: Cicadellidae). 19th Brazilian Congress of Entomology. Manaus, Brazil. 2002 • Takiya, D. M. & Mejdalani, G. Notes on Tacora Melichar, 1926 (Hemiptera: Cicadellidae). 19th Brazilian Congress of Entomology. Manaus, Brazil. 2002 • Nessimian, J. L., da Silva, L. F., Lustosa, M. G. L., Takiya, D. M., Magalh˜aes, C., de Marco-Jr., P., Venticinque, E. M., & Zuanon, J. Riqueza de macroinvertebrados em igarap´esde pequena ordem em ´areas com diferentes estados de integridade da mata rip´ariana AmazˆoniaCentral. XXIV Brazilian Congress of Zoology. UNIVALI, Itaja´ı,Brazil. 2002 • Nessimian, J. L., da Silva, L. F., Lustosa, M. G. L., Takiya, D. M., Magalh˜aes, C., de Marco-Jr., P., Venticinque, E. M., & Zuanon, J. Distribui¸c˜aoespacial de macroinvertebrados em igarap´es de pequena ordem na AmazˆoniaCentral. XXIV Brazilian Congress of Zoology. UNIVALI, Itaja´ı,Brazil. 2000 • Takiya, D. M., Mejdalani, G., & Felix, M. A new genus of Cicadellini (Hemiptera: Cicadellidae) from Southeastern Brazil. XXI International Congress of Entomology / XVIII Brazilian Congress of Entomology. Foz do Igua¸cu,Brazil. 2000 • Takiya, D. M., Mejdalani, G., & Felix, M. Chave para as esp´ecies e notas sobre o gˆenero Balacha Melichar (Hemiptera, Cicadellidae). XXIII Brazilian Congress of Zoology. Universidade Federal do Mato Grosso, Cuiab, Brazil. 2000 • Felix, M., Mejdalani, G., & Takiya, D. M. Notas taxonˆomicas sobre o gˆenero Amblyscartidia (Hemiptera, Cicadellidae, Cicadellinae), incluindo uma nova esp´eciedo Brasil. XXIII Brazil- ian Congress of Zoology. Universidade Federal do Mato Grosso, Cuiab´a,Brazil. 1999 • Takiya, D. M., Mejdalani, G., & Felix, M. Batesian mimicry in the leafhopper subfamily Ci- cadellinae (Hemiptera: Cicadellidae). 10th Auchenorrhyncha Congress. Cardiff University, Cardiff, Wales. 1998 • Mejdalani, G., Takiya, D. M., & Felix, M. Cladistic analysis of the genera of Proconiini with the posterior meron exposed (Hemiptera: Cicadellidae: Cicadellinae). 17th Willi Hennig Society Meeting. Universidade de S˜aoPaulo, S˜aoPaulo, Brazil. 1998 • Felix, M., Mejdalani, G., & Takiya, D. M. Cladistic analysis of the genus Lissoscarta (Hemiptera: Cicadellidae: Cicadellinae). 17th Willi Hennig Society Meeting. Universidade de S˜aoPaulo, S˜aoPaulo, Brazil. 1998 • Mejdalani, G., Takiya, D. M., & Felix, M. Uma an´aliseclad´ıstica dos gˆeneros de Proconi- ini (Hemiptera, Cicadellidae, Cicadellinae) com proje¸c˜aono metep´ımero. XXII Brazilian Congress of Zoology. Universidade Federal de Pernambuco, Recife, Brazil. 1998 • Takiya, D. M., Mejdalani, G., & Felix, M. Propetes schmidti, um Proconiini (Hemiptera, Ci- cadellidae, Cicadellinae) que mimetiza vespas (Hymenoptera, Vespidae). XXII Brazilian Congress of Zoology. Universidade Federal de Pernambuco, Recife, Brazil. 1998 • Felix, M., Takiya, D. M., & Mejdalani, G. Descri¸c˜ao do macho de Deltolidia magnifica (Hemiptera, Cicadellidae, Coelidiinae). XXII Brazilian Congress of Zoology. Universi- dade Federal de Pernambuco, Recife, Brazil. 1996 • Takiya, D. M., Dias, C. T. M., & Silva-Neto, I. D. Structural and ultrastructural aspects of Tracheloraphis sp. (Ciliophora: Karyorelictida) a psammophilic ciliate from the Guanabara Bay, RJ. XII Annual Meeting of the Brazilian Protozoology Society. Caxamb´u,Brazil. 1996 • Silva-Neto, I. D., Dias, C. T. M., & Takiya, D. M. Study of the benthonic ciliated Protozoa at Guanabara Bay, RJ. XII Annual Meeting of the Brazilian Protozoology Society. Caxamb´u,Brazil.

173 Professional Meetings (continued) MElectronic Poster Presentations 2004 • Takiya, D. M. & Dmitriev, D. A. An interactive key to sharpshooter genera of the tribe Proconi- ini (Hemiptera: Cicadellidae). XXII International Congress of Entomology. Brisbane, Australia.

MParticipation 2006 • Entomological Collections Network. Indianapolis,USA. 2003 • Entomological Collections Network. Cincinnati,USA. 2002 • Entomological Collections Network. Fort Lauderdale,USA. 1998 • XVII Brazilian Congress of Entomology. Rio de Janeiro, Brazil. 1997 • 16o Brazilian Congress of Entomology. Salvador, Brazil. 1996 • XXI Brazilian Congress of Zoology. Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil.

Publications MPeer-Reviewed Journals 2006 • Cicadellinae (Hemiptera, Auchenorrhyncha, Cicadellidae) described by Leopold Melichar in the Hungarian Natural History Museum. Annales historico-naturales Musei nationalis hungarici, submitted. 2006 • Takiya, D. M., Tran, P., Moran, N. A., & Dietrich, C. H. Co-cladogenesis spanning three phyla: leafhoppers (Insecta: Hemiptera: Cicadellidae) and their dual bacterial symbionts. Molecular Ecology 15: 4175-4191. 2006 • Takiya, D. M., McKamey, S. H., & Cavichioli, R. R. Validity of Homalodisca and of H. vitripennis (Germar) as the name for Glassy-Winged sharpshooter (Hemiptera: Cicadellidae: Cicadellinae). Annals of the Entomological Society of America 99: 648-655. 2006 • Takiya, D. M., Cavichioli, R. R. & McKamey, S. H. Brazilian sharpshooters of the genus Homalodisca St˚al,1869 (Hemiptera, Cicadellidae): notes, new records, key to species, first description of the male of H. ignota Melichar, 1924, and a new Northeastern species. Zootaxa 1249: 23-36. 2006 • Mejdalani, G., Takiya, D. M., & Carvalho, R. A. Notes on Neotropical Proconiini (Hemiptera: Cicadellidae: Cicadellinae), IV: lectotype designations of Aulacizes Amyot & Audinet-Serville species described by Germar and revalidation of A. erythrocephala (Germar, 1821) Arthropod Systematics & Phylogeny 64: 105-111. 2005 • Takiya, D. M. & Cavichioli, R. R. The new South American sharpshooter genus Lanceoscarta gen. nov. (Hemiptera: Cicadellidae: Cicadellinae) with descriptions of three new species. Ento- mologische Abhandlungen 62: 175-183. 2004 • Takiya, D. M. & Mejdalani, G. Taxonomic revision and phylogenetic analysis of the sharp- shooter genus Balacha Melichar (Hemiptera: Cicadellidae: Cicadellini). Systematic Entomology 29: 69-99. 2004 • Takiya D. M. & Cavichioli, R. R. A Review of the Neotropical sharpshooter genus Onega Distant. Zootaxa 718: 1-19. 2004 • Ceotto, P. C., Mejdalani, G. & Takiya, D. M. Two new South American species of Acrobelus St˚al(Hemiptera: Cicadellidae: Cicadellinae) with a key to the species of the genus. Journal of Natural History 38: 2073-2983.

Underlined are names of the actual presenter of the paper.

174 Publications (continued)

2003 • Takiya, D. M., Mejdalani, G. & Webb, M. D. Notes on the Amazonian genus Hyogonia China (Hemiptera: Cicadellidae: Proconiini) with a description of a new species. Journal of Natural History 37: 2863-2869. 2003 • Takiya, D. M., Cavichioli, R. R. & Mejdalani, G. Caragonia, a new genus of Cicadellini (Hemiptera: Cicadellidae) from Southeastern Brazil. Zootaxa 335: 1-10. 2002 • Takiya, D. M. & Mejdalani, G. On the Central and Western Amazonian genus Tacora Melichar, 1926 (Hemiptera: Cicadellidae: Cicadellinae): key to species and descriptions of three new taxa. Amazoniana 17: 227-242. 2002 • Mejdalani, G., Takiya, D.M., Felix, M., Ceotto, P. & Yanega, D. Teletusa limpida (Signoret): a Neotropical proconiine leafhopper that mimics megachilid bees (Hymenoptera: Apoidea), with notes on Batesian mimicry in the subfamily Cicadellinae (Hemiptera: Cicadelli- dae). Denisia 4: 215-224. 2002 • Felix, M., Mejdalani, G., Nielson, M. & Takiya, D. M. The Neotropical leafhopper genus Deltolidia Nielson (Hemiptera: Cicadellidae: Coelidiinae): polymorphism and taxonomic notes. Studies on Neotropical Fauna and Environment 37: 161-167. 2000 • Takiya, D. M., Mejdalani, G. & Felix, M. A new genus and species of Cicadellini (Hemiptera Cicadellidae Cicadellinae) from Southeastern Brazil. Tropical Zoology 14: 175-183. 2000 • Mejdalani, G., Felix, M. & Takiya, D. M. Description of a new species of Amblyscartidia Young from Southeastern Brazil (Hemiptera: Cicadellidae: Cicadellinae). Bollettino di Museo Regionale di Scienze Naturali Turin 17: 131-140. 1999 • Takiya, D. M., Mejdalani, G. & Felix, M. Dual-mimicry of wasps by the Neotropical leafhopper Propetes schmidti Melichar with a description of its female (Hemiptera: Cicadellidae: Cicadellinae). Proceedings of the Entomological Society of Washington 101: 722-728.

MOnline Publications 2004 • Takiya, D. M. & Dmitriev, D. A. An interactive key to genera of the tribe Proconiini. http://ctap.inhs.uiuc.edu/takiya 2003 • Takiya, D. M. & Ceotto, P. C. (2003) Os 15 minutos de fama das cigarrinhas. Electronic Publication @ Bioletim: Revista de divulga¸c˜aocient´ıficados estudantes de Biologia da UFRJ, n´umero 3. http://www.bioletim.hpg.ig.com.br/III-3/Artigos/takyia%20e%20ceotto.htm

Didactic Experience MTeaching Assistantships Animal Biology • University of Illinois at Urbana-Champaign, USA. Fall 2005. Entomology I • Universidade Federal do Rio de Janeiro, Brazil. 2001/1, 2000/1, 1998/1. Zoology III • Universidade Federal do Rio de Janeiro, Brazil. 2001/1, 2000/1. 1998/1, 1997/1, 1996/2. Zoology II • Universidade Federal do Rio de Janeiro, Brazil. 1996/1. Zoologia IA • Universidade Federal do Rio de Janeiro, Brazil. 1995/2

MInvited Lectures Entomology I • Universidade Federal do Rio de Janeiro, Brazil. Lecture on “Social Insects”. 2001/1, 2000/1, 1999/1. Entomology I • Universidade Federal do Rio de Janeiro, Brazil. Lecture on “Homologies”. 1998/1. Hemipterology • Universidade Federal do Rio de Janeiro, Brazil. Lecture on “Auchenorrhyncha”. 1999/1.

175 Professional Service MEditorial Board Member 2005- • Systematic Entomology, Royal Entomological Society.

MJournal Reviews • African Entomology. • Entomological News. • Proceedings of the Entomological Society of Washington. • Revista Brasileira de Entomologia.

MMembership in Professional Societies • Entomological Society of America. • Sociedade Brasileira de Entomologia. • Sociedade Brasileira de Zoologia.

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