Integrating Vibrational Signals, Mitochondrial DNA and Morphology for Species Determination in the Genus Aphrodes (Hemiptera: Cicadellidae) JOANNA K

Systematic Entomology (2014), 39, 304–324

Integrating vibrational signals, mitochondrial DNA and morphology for species determination in the genus Aphrodes (Hemiptera: Cicadellidae) JOANNA K. BLUEMEL1, MAJA DERLINK2, PETRA PAVLOVCIˇ Cˇ 2, ISA-RITA M. RUSSO1, ROBERT ANDREW KING1,*, EMMA CORBETT1, ELEANOR SHERRARD-SMITH1, ANDREJ BLEJEC1, MICHAEL R. WILSON3, ALAN J. A. STEWART4, WILLIAM O. C. SYMONDSON1 andMETA VIRANT-DOBERLET2

1Cardiff School of Biosciences, Cardiff University, Cardiff, U.K., 2Department of Entomology, National Institute of Biology, Ljubljana, Slovenia, 3Department of Biodiversity & Systematic Biology, National Museum of Wales, Cardiff, U.K. and 4School of Life Sciences, University of Sussex, Brighton, U.K.

Abstract. Reliable delimitation and identification of species is central not only to systematics, but also to studies of biodiversity, ecology and pest management. In the era of Internet-based biodiversity databases misidentifications are rapidly disseminated and may have far-reaching consequences. Leafhoppers from the genus Aphrodes (Hemiptera, Cicadellidae) are common and abundant, but, nevertheless, they are still a taxonomically challenging group whose members are often assessed in ecological studies and are also potential vectors of plant diseases. Previous study has shown that the syntype series for A. aestuarina (Edwards) includes also specimens of A. makarovi Zachvatkin and has suggested that misidentifications may be widespread in museum collections. We studied Aphrodes individuals collected from the U.K. and Slovenia in order to provide a more comprehensive analysis of this genus using multiple criteria. Combined work using male and female vibrational signals emitted during courtship, and a 600-bp fragment within the barcoding region of the COI mtDNA gene, provided validated specimens that we also used for morphometric study. Analyses confirmed A. aestuarina, A. bicincta, A. diminuta and A. makarovi as behaviourally, genetically and morphologically distinct species. Although any of these approaches could be used alone to distinguish between species, combining morphological and molecular approaches will help to improve reliability, especially when identifying females. Morphological investigation of validated individuals from the U.K. and Slovenia also revealed geographic differences within species. By combining several body and aedeagus morphological characters males can be reliably identified, however, morphological differences between species are, nevertheless, relatively small. By contrast, observed genetic distances between Aphrodes species are relatively large (4.2–7.0%). At about half of our collecting sites more than one Aphrodes species was found and A. makarovi was collected together with every other species, including A. aestuarina on tidal saltmarshes. Due to low morphological variation between syntopic congeners it is likely that many museum specimens of Aphrodes have been assigned to the wrong species and species identification in ecological and vector studies may also be questionable.

Correspondence: Meta Virant-Doberlet, Department of Entomology, National Institute of Biology, Vecnaˇ pot 111, SI-1000 Ljubljana, Slovenia. E-mail: [email protected]

*Present address: College of Life and Environmental Sciences, University of Exeter, Exeter, U.K. Authors have no conﬂict of interest.

Introduction Aphrodes type specimens either to verify fresh material or to validate the type material in the light of updated knowledge. In recent years the concept of integrative taxonomy has Recently, however, molecular analysis of archived specimens received a lot of attention (Dayrat, 2005; Will et al., 2005; of Aphrodes revealed that the majority of analysed museum Valdecasas et al., 2008; Goldstein & DeSalle, 2010; Padial specimens had been assigned to the wrong species and that et al., 2010; Schlick-Steiner et al., 2010; Yeates et al., 2011). the syntype series for A. aestuarina (Edwards) includes also Integrating several sources of taxonomic information (mor- specimens of A. makarovi Zachvatkin (Bluemel et al., 2011). phological, molecular, behavioural and ecological data) into Confusion in the taxonomy of this genus is exemplified species descriptions improves the quality of taxonomic data by the fact that in the Fauna Europea database there are and such an approach is generally considered the most reliable 12 species listed under the genus Aphrodes, while Species method for delimiting species. In order to document biological 2000 & ITIS Catalogue of Life includes 28 names. The diversity before natural ecosystems are altered or destroyed, most frequently documented European species are A. bicincta a reliable delimitation and identification of species is cen- (Schrank), A. aestuarina, A. makarovi and A. diminuta Ribaut tral to biodiversity studies (Wheeler, 2004; Guralnick et al., (Tishechkin, 1998; Nickel & Remane, 2002). However, the 2007). Biodiversity data are now commonly shared via Inter- former database does not include A. diminuta (or its synonym net databases (Guralnick et al., 2007; Patterson et al., 2010; A. centrorossica Zachvatkin) while the latter one does not list Deans et al., 2012; Fontaine et al., 2012; Gibson et al., 2012; any of the most common species. Although older synonyms Jetz et al., 2012) and therefore misidentifications, which are undoubtedly exist for A. makarovi and A. diminuta, recent widespread even in museum collections (Meier & Dikow, authors prefer to use these names, because high variability and 2003), may be rapidly disseminated. Furthermore, inaccurate overlapping morphological characters make reliable synonymy species identifications can result in error cascades that can hard to establish. affect the conclusions of ecological studies which, in turn, In an attempt to resolve species status in the genus Aphrodes, impact on ecosystem management and conservation regula- Tishechkin (1998) recorded species-specific vibrational signals tions (Bortolus, 2008; Dexter et al., 2010). Taxonomic errors of two, three and ten males of A. makarovi, A. diminuta and in identifying pests and vectors can also have direct socioeco- A. bicincta, respectively, and afterwards examined the mor- nomic consequences (Van Bortel et al., 2001). phology of these individuals. Tishechkin (1998) also included Leafhoppers of the genus Aphrodes Curtis (Hemiptera: two archived specimens of male A. aestuarina in his study. Cicadellidae), are abundant, widely distributed over the However, regardless of the fact that he noticed that sometimes Palearctic and also, as neozoa, in North America (Hamilton, two Aphrodes species can be found together, he also included 1983; Tishechkin, 1998; Nickel & Remane, 2002). They are in his morphological examinations additional individuals of important species in grassland leafhopper communities (Nickel both sexes that he collected together with acoustically iden- & Achtziger, 2005) and they are also vectors of phytoplasmas tified males of each species. Although only a small fraction that cause plant diseases (Lee et al., 1998; Weintraub & of the leafhoppers examined morphologically was simulta- Beanland, 2006). The genus Aphrodes is considered a taxo- neously identified by acoustic characters, Tishechkin (1998) nomically challenging group and even trained experts often nevertheless concluded that none of the morphological char- designate these leafhoppers merely to a nominal Aphrodes acters is entirely reliable for distinguishing between species. bicincta s.l. species group. Species in this genus could even be Nevertheless, oscillograms of male vibrational signals provide considered cryptic (Bickford et al., 2007) because, due to their a reliable character to which further studies can be compared morphological similarities, they have in the past been classified and Tishechkin’s work forms the basis for the most recent as ecotypes of a single species (Le Quesne, 1965; Nast, 1972). morphological identification key for the genus Aphrodes (Bie- Identification of nymphs and females based on morphological dermann & Niedringhaus, 2004). Even a brief comparison of characters is currently not possible, while characters to distin- aedegal morphology of the four Aphrodes species as deter- guish males are unreliable. The colouration, size and aedeagal mined by Tishechkin (1998) with descriptions in earlier taxo- form are highly variable and there is considerable overlap in nomic work (Zakhvatkin, 1948; Ribaut, 1952; Duffield, 1963; the morphological characters used to separate males of dif- Le Quesne, 1965; Nast, 1976; Emmrich, 1980; Ossiannilsson, ferent species (Hamilton, 1975; Le Quesne, 1988; Tishechkin, 1981; Hamilton, 1983) shows significant differences in inter- 1998) and consequently interpretation of such characters often pretation of morphological characters between authors. relies on intuition. The problem is compounded by the fact The objective of the present paper is to provide a more com- that, due to both inconsistencies in identifying the species prehensive analysis of Aphrodes using multiple criteria (the and several taxonomic revisions, there are many unresolved iterative approach sensu Yeates et al., 2011) in order to pro- synonyms (Nast, 1972; Hamilton, 1983; Tishechkin, 1998). vide the information needed for reliable species identification, Until the early 20th Century aedeagus morphology was which is clearly needed. Besides being included in molecular not routinely used as a taxonomic character in leafhopper and morphological phylogenetic studies (Dietrich et al., 2001; systematics and species were often described, and individuals Zahniser & Dietrich, 2008, 2010), Aphrodes species are identified, on the basis of colouration, size, body shape and often collected in ecological studies due to their widespread ecology. Furthermore, many archived specimens are females distribution and abundance (Brown et al., 1992; Hollier et al., or nymphs. It is therefore often not possible to use archived 1994; Jobin et al., 1996; Huusela-Veistola & Vasarainen, 2000;

Eyre et al., 2001, 2005; Eyre, 2005; Fisher Barham & Stewart, a smaller subset of individuals from which we also obtained 2005; Morris et al., 2005; Nickel & Achtziger, 2005; Strauss complete morphometric data (Tables 1, S1). Leafhoppers & Biedermann, 2006, 2008; Kattwinkel et al., 2009, 2011; were collected using either a sweep net or a motor-driven Keathley & Potter, 2012; Kor¨ osi¨ et al., 2012; Schuch et al., suction sampler (McCulloch, BVM 250; Electrolux) using 2012) and some of them are even considered to be indicator an 11.5-cm diameter sampling cylinder. In the laboratory species (Maczey et al., 2005; Trivellone et al., 2012). They males and females were kept separately and according to ◦ have also been included as potential vectors in phytoplasma the locality, in plastic boxes (38 × 26 × 27cm) at 23–28 C, and mycoplasma transmission studies (Denes & Sinha, 1992; 50–70% humidity and photoperiod 16:8 h (L:D) until their Carraro et al., 2004; Bressan et al., 2006; Riedle-Bauer et al., species identity was determined by their species-specific 2008). Taking into account the obvious inconsistencies in calling signals (see below). Leafhoppers were fed with taxonomy of this genus, species identification in most of the alfalfa (Medicago sativa), red clover (Trifolium pratense)and above mentioned studies is questionable. knapweed (Centaurea jacea) placed in vials of water. We collected Aphrodes leafhoppers at various locations in the U.K. and Slovenia, thus including in the study specimens from geographically distant locations. As in other leafhoppers, Recording and analysis of vibrational signals mate recognition and location in Aphrodes leafhoppers is mediated exclusively via substrate-borne vibrational signals The species identity of the majority of individuals was (Claridge, 1985; Virant-Doberlet & Cokl,ˇ 2004; de Groot initially determined by recording their species-specific vibra- et al., 2011). Because behavioural characters are the most tional signals from a plant using a laser vibrometer (PDV 100, accurate ones to delimit species (Schlick-Steiner et al., 2010), Polytec GmbH, Waldbronn, Germany) as described previously we initially identified 10–15 individuals of both sexes of (de Groot et al., 2012). Males emitted calling signals spon- each species by recording species-specific vibrational signals. taneously, while emission of female vibrational signals was We followed the species determination of Tishechkin (1998), induced by stimulation with pre-recorded male calling signals except for A. aestuarina for which vibrational signals had (de Groot et al., 2012). Females were stimulated with a ran- not been previously recorded (see below). For the same dom sequence of four randomly selected male calling signals, individuals we also obtained a 600-bp sequence within the one from each Aphrodes species. Within the sequence, each barcoding region of the mitochondrial COI (cytochrome signal was repeated five times. The amplitude of stimulation oxidase subunit I) gene. After establishing that each acoustic was adjusted to that of naturally emitted male calling signals profile (signal type and female preferences) corresponded to a at the point of recording. Males of A. bicincta, A. makarovi distinct COI lineage, species identity of individuals included and A. diminuta were determined by comparing recorded sig- in the morphometric study could be determined by the acoustic nals to oscillograms obtained by Tishechkin (1998, 2000). and/or molecular approach. While sequences of the barcoding Vibrational signals of A. aestuarina had not been previously region of the COI gene are available from the Genbank, we recorded. Identification of this species was based on live speci- also provide audio files of representative vibrational signals and mens from the site where syntype specimens had been collected raw morphological data as Supporting Information. This should by Edwards (Wells, Norfolk, U.K.), on the original species enable other researchers to compare their own material with description (Edwards, 1908), on its specific ecology (habitat validated individuals. For each species ten individuals (five daily inundated during high tide), on vibrational signals that males and five females) included in the present study have also differ from the ones previously described for other species and been deposited as voucher specimens in the collection at the on the distinct COI lineage (see Results). The species iden- Department of Biodiversity and Systematic Biology at National tity of females was determined by their selective response to Museum Wales in Cardiff (U.K.). A taxonomic revision of conspecific male calls. Because only virgin females reply to Aphrodes leafhoppers was not the objective of the present male calls, species identity of silent females was established paper. However, in the Discussion we offer some insights into by molecular methods (see below). After their vibrational signals were recorded, leafhoppers were stored in 100% ethanol how the taxonomy of this group might be resolved. ◦ at −20 C until DNA extraction. Recorded vibrational signals were stored in a computer and Materials and methods analysed using Raven v1.3 (Charif et al., 2008) and Sound Forge (Sonic Foundry, Madison, WI, U.S.A.). Male calls Insect sampling are composed of highly species-specific elements and differ greatly among species [see Results and Audio files (Audio Adult Aphrodes leafhoppers were sampled from the end of S1–S8) included in the Supporting Information]. In the anal- May until mid-August during 2005–2011 at numerous sites in yses we included ten males of each species (five from U.K. the U.K. and Slovenia. Altogether we collected and identified, and five from Slovenia, except for A. aestuarina) and from using an acoustic and/or molecular approach (see below), from each male we analysed five calls in detail. The male calling both countries combined, 922 A. makarovi, 279 A. bicincta signals can be divided into sections which are composed from and 97 A. diminuta, as well as 120 A. aestuarina from the structurally different elements (see Results) (Fig. 2) and, where U.K. In the present study, however, we have included only appropriate, we measured the following parameters: total

Table 1. Collection details of Aphrodes individuals included in the study.

Altitude No. of Collection site Collection date Habitat type/dominant plant GPS coordinates (m) individuals ◦ ◦ Holme-next-the-sea 22 July 2008 Tidal saltmarsh/Atriplex N52 58.064 E00 31.696 04M ◦ ◦ Overy Marsh 22 July 2008 Tidal saltmarsh/Atriplex N52 57.792 E00 44.239 04M ◦ ◦ Stiffkey Marsh 21 July 2008 Tidal saltmarsh/Atriplex N52 57.621 E00 55.389 02M ◦ ◦ Morston Quay 21 July 2008 Tidal saltmarsh/Atriplex N52 57.543 E00 59.060 02M ◦ ◦ Wells East Quay 22 July 2008 Tidal saltmarsh/Atriplex N52 57.417 E00 51.851 05M ◦ ◦ Warham Marsh 21 July 2008 Tidal saltmarsh/Atriplex, Urtica N52 57.367 E00 54.505 0-5 3 M, 5 F ◦ ◦ Blakney Quay 21 July 2008 Tidal saltmarsh/Atriplex N52 57.343 E01 00.827 01M ◦ ◦ Horsey Island 31 July 2009 Tidal saltmarsh/Atriplex N51 51.552 E01 14.649 02F ◦ ◦ Canvey Bridge 6 July 2008; 31 July 2009 Tidal saltmarsh/Atriplex N51 32.551 E00 33.834 02M,3F ◦ ◦ Canvey Island 6 July 2008 Tidal saltmarsh/Atriplex N51 31.343 E00 37.025 01M ◦ ◦ Pegwell Bay 7 July 2008 Tidal saltmarsh/Atriplex N51 18.954 E01 21.590 02M,2F ◦ ◦ Rye Harbour 21 August 2007 Tidal saltmarsh/Atriplex N50 56.223 E00 45.822 08F ◦ ◦ Shoreham 13 July 2006 Tidal saltmarsh/Atriplex N50 50.456 W00 17.387 06M,3F ◦ ◦ Pagham Harbour 22 August 2007 Tidal saltmarsh/Atriplex N50 46.197 W00 45.354 01F ◦ ◦ Castle Hill 1 13 July 2006 Path border/Urtica N50 50.473 W00 04.400 166 6 M, 3 F ◦ ◦ Castle Hill 2 12 July 2006 Chalk grassland meadow N 50 50.868 W00 03.313 126 5 M, 3 F ◦ ◦ Mountain Ash 18 July 2006 Brown ﬁeld site/Fabaceae N 51 41.616 W03 24.406 114 18 M, 6 F ◦ ◦ Pen-clawdd 1 July 2008; 3 July 2009 Grassland/Urtica N51 38.609 W04 05.742 10 1 M, 5 F ◦ ◦ Lisvane 28 July 2007; 15 July 2008 Field border/Urtica N51 32.160 W03 10.173 52 6 M, 5 F ◦ ◦ Mackovciˇ 15 July 2010 Pannonian grassland/Urtica N46 47.325 E16 09.732 306 1 M ◦ ◦ Ledavsko jezero 15 July 2010 Pannonian grassland/Fabaceae N 46 45.663 E16 02.942 224 1 M ◦ ◦ Pod Poncami 8 August 2010 Alpine meadow/Fabaceae N 46 29.156 E13 43.595 885 3 M ◦ ◦ Belca 8 August 2010 Alpine grassland/Fabaceae N 46 28.407 E13 54.271 678 2 M, 3 F ◦ ◦ Dolinka 27 July 2011 Alpine meadow/Fabaceae N 46 27.645 E13 57.627 636 1 F ◦ ◦ Plavskiˇ Rovt 8 August 2010 Alpine meadow/Fabaceae N 46 27.052 E14 02.062 863 3 M, 2 F ◦ ◦ Kocnaˇ 8 August 2010 Alpine meadow/Fabaceae N 46 24.769 E14 04.688 668 1 M, 1 F ◦ ◦ Zgornje Jezersko 12 August 2010; 27 July 2011 Alpine meadow/Fabaceae N 46 23.722 E14 30.195 883 1 M, 2 F ◦ ◦ Potoceˇ 27 July 2011 Alpine grassland/Fabaceae N 46 17.982 E14 27.659 493 2 F ◦ ◦ Podbela 17 July 2010 Alpine grassland/Fabaceae N 46 15.287 E13 28.073 463 1 M, 1 F ◦ ◦ Ljubljana 20 June 2006; 23 July 2009 Alpine grassland/Urtica N46 03.259 E14 27.719 303 3 M ◦ ◦ Neblo 31 July 2010 Mediterranean grassland/ Fabaceae N 46 00.157 E13 30.175 83 1 M ◦ ◦ Ozeljan 15 June 2009 Mediterranean grassland/Fabaceae N 45 56.467 E13 43.517 84 1 F ◦ ◦ Stara Gora 7 July 2009 Mediterranean meadow N 45 56.071 E13 40.374 118 1 F ◦ ◦ Sempasˇ 17 June 2010 Mediterranean grassland N 45 55.681 E13 43.263 65 1 M, 4 F ◦ ◦ Malovseˇ 15 June 2009 Mediterranean grassland N 45 54.086 E13 47.367 158 2 M ◦ ◦ Lokavec 15 June 2009; 18 June 2010 Mediterranean grassland N 45 53.762 E13 53.247 134 2 M, 2 F ◦ ◦ Podnanos 15 June 2009 Mediterranean karst grassland/Fabaceae N 45 48.020 E13 58.086 163 1 F ◦ ◦ Selo pri Stjakuˇ 7 July 2009 Mediterranean karst grassland N 45 47.825 E13 53.911 509 1 M ◦ ◦ Gabrje Podgorje 31 July 2010 dinaric grassland/Fabaceae N 45 46.822 E15 16.632 431 2 M ◦ ◦ Gospodicnaˇ 31 July 2010 Dinaric grassland/Fabaceae N 45 46.136 E15 18.005 826 1 M ◦ ◦ Krajna vas 1 July 2010 Mediterranean karst grassland/Fabaceae N 45 45.979 E13 48.207 263 2 M, 1 F ◦ ◦ Krizˇ 1 July 2010 Mediterranean karst grassland/Fabaceae N 45 44.507 E13 51.896 318 1 F ◦ ◦ Zirjeˇ 1 July 2010 Mediterranean karst meadow N 45 42.293 E13 54.949 388 1 M, 3 F ◦ ◦ Goriceˇ 12 July 2011 Mediterranean karst meadow/Fabaceae - Anthyllis N45 40.595 E14 00.384 423 1 F ◦ ◦ Vrepolje Vremsˇcicaˇ 1 July 2010 Mediterranean grassland N 45 40.589 E14 05.924 585 1 F ◦ ◦ Gorice pri Famljah 1 July 2010 Mediterranean karst meadow/Fabaceae N 45 40.351 E14 00.883 439 3 M, 4 F ◦ ◦ Prelozeˇ Lokev 8 June 2009 Mediterranean karst grassland/Fabaceae N 45 39.487 E13 56.136 461 1 M, 1 F ◦ ◦ Lokev 1 July 2010 Mediterranean karst grassland/Fabaceae N 45 39.685 E13 55.365 444 2 M ◦ ◦ Osp 2 8 June 2009 Mediterranean karst meadow/Fabaceae N 45 34.379 E13 50.845 25 2 F ◦ ◦ Hoticnaˇ 12 July 2011 Mediterranean karst meadow/Fabaceae N 45 34.186 E14 01.150 550 1 M ◦ ◦ Dragonja 4 14 June 2006; 25 May 2009 Mediterranean grassland/Fabaceae - Medicago sativa N45 27.300 E13 42.078 31 3M,10F ◦ ◦ Dragonja 2 25 May 2009 Mediterranean grassland/Fabaceae - Medicago sativa N45 27.026 E13 41.252 26 1 M, 1 F

duration of the call, duration of each section within the call, duration (Broughton, 1963). Chirp was defined as a sound number of units in the section, pulse or chirp repetition time consisting of a group of pulses that is identifiable to the human and dominant frequency of each section. In A. makarovi pulse ear as a unitary event (Hunt et al., 1992; de Groot et al., 2012). repetition time within the call was measured from ten consecu- Female signals of all species consist of a series of single pulses tive pulses in the middle part of the last section. In A. bicincta, and we measured the call duration, pulse repetition time and chirp repetition time was measured from ten consecutive dominant frequency. In A. diminuta and A. makarovi, females chirps in the beginning of the species-specific section. Pulse start emitting their calling signals before the male call is com- was defined as a unitary homogenous parcel of sound of finite pleted and therefore pulse repetition time was measured from

© 2014 The Royal Entomological Society, Systematic Entomology, 39, 304–324 308 J. K. Bluemel et al. ten consecutive pulses following immediately after the end of (Ronquist et al., 2012) All trees were rooted using the closely the male call (de Groot et al., 2012). Due to marked changes related aphrodine species Anoscopus limicola (Edwards, 1908) in pulse repetition time during a female call in A. aestuarina, (GeneBank Accession numbers FR729924 and HE587046) we included in the analyses only the first ten pulses in a call. and Planaphrodes trifasciata (Geoffr., 1785) (GeneBank Accession numbers KF378763-KF378765) (Hemiptera: Cicadellidae: Aphrodinae). DNA extraction, amplification, sequencing and molecular Pairwise Kimura-2-parameter (K2P) distances (Kimura, analysis 1980) between haplotypes were used to carry out NJ analyses with 1000 bootstrap replicates to estimate nodal support. All six legs of ethanol-preserved specimens were removed A likelihood ratio test as implemented in jmodeltest under a stereo-microscope (Olympus SZX7 or Leica MZ95). v2.1.2 (Guindon & Gascuel, 2003; Darriba et al., 2012) was DNA was extracted from legs using the DNeasy Blood used to statistically select the best-fit model of nucleotide and Tissue kit (Qiagen, Hilden, Germany) following the substitution. ML analysis was conducted using the heuristic manufacturer’s protocol. search option with ten random addition replicates and the tree For leafhoppers collected in the U.K. a barcoding 710- bisection-reconnection (TBR) branch-swapping algorithm. bp region of the mitochondrial cytochrome oxidase subunit I Nodal support was estimated using 1000 bootstrap replicates (COI ) gene was amplified with the universal primers LCO1490 (Felsenstein, 1985). For the BI four chains were run for and HCO2198 (Folmer et al., 1994). PCR amplifications and 5 × 106 generations using random starting trees and flat the sequencing of the mtDNA COI gene were carried out as priors. Trees and parameters were recorded every 100th described previously (Virant-Doberlet et al., 2011). generation. Two runs were performed simultaneously and split For leafhoppers collected in Slovenia a 1524-bp COI frag- frequencies were compared every 100th generation to ensure ment was amplified with the primers LCO1490 (Folmer et al., convergence of the runs. Both runs used the default heating 1994) and TL2-N-3014 (Simon et al., 1994) using GeneAmp and swap frequency parameters. The first 5000 generations 9700 thermal cycler (Applied Biosystems, Carlsbad, CA, were excluded as the burn-in. Percentage sequence divergence U.S.A.) and the following PCR conditions: a 3-min denatura- estimates within and among species was also calculated based ◦ ◦ tion step at 94 C, followed by 5 cycles of denaturation at 94 C on pairwise K2P distances in PAUP. ◦ ◦ for 30 s, annealing at 45 C for 30 s and elongation at 72 Cfor 2 min. Thirty additional cycles were carried out as above with ◦ the annealing temperature increased to 48 C. Final extension Morphometric measurements and analyses ◦ was carried out at 72 C for 10 min. All reactions were carried out in a total volume of 50 μL consisting of 1 × PCR buffer, Species identity of all leafhoppers analysed morphometri- 2mm magnesium chloride, 0.2 mm dNTP, 0.25 μM each of the cally had been previously determined by the acoustic and/or forward and reverse primer, 1.25 U TopTaq DNA polymerase molecular approach. Only individuals that were not damaged (Qiagen) and 2 μL DNA. In each PCR run negative controls during leg dissection were included in the morphometric analy- of tissue culture water were included to detect possible ses. We analysed 21 males and 15 females of A. aestuarina,34 contamination. The PCR products were first visualized on males and 27 females of A. bicincta, 41 males and 44 females an ethidium bromide-stained 1.4% agarose gel. The PCR of A. makarovi, and 15 males and 7 females of A. diminuta. products were sequenced in both directions using a 3730XL For the latter species, all females included in the analyses were DNA sequencer (Macrogen, Amsterdam, The Netherlands). collected in Slovenia. Photographs were taken using AUTO- Sequences were edited in SEQUENCHER v4.9 (Gene MONTAGE PRO v5.0 image software (Synoptics, Cambridge, Codes, Ann Arbor, MI, U.S.A.) and a consensus sequence U.K.) and a JVC KY F70 3CCD digital camera mounted on was generated with each of the forward and reverse sequences a Leica M28 stereo-microscope. For illumination a cold light (U.K. samples) or in MEGA5 (Tamura et al., 2011) (Slovenian source (Schott KL 1500) was used. samples). Secondary alignment was carried out in CODON- Whole leafhoppers (without legs) were placed in a groove CODE ALIGNER v4.0.4 (CodonCode Co., Centerville, MA, made in a white-tack (UHU) block inside an excavated glass U.S.A.) using default settings. block and submerged in 100% ethanol. They were pho- For the final analysis we used the 600-bp overlapping tographed with a Planapo chromatic 1× lens at magnifications region between the two primer sets. Unique haplotype 1×, 1.6×, 2.0× or 2.5× depending on the size of the speci- sequences were identified and deposited in GenBank under mens, saving a corresponding scale bar with each image. the Accession numbers: FR727167, FR727169–FR727176, After imaging the whole specimen the last abdominal seg- FR727178, HE587025, HE587026, HE587031, HE587032, ments of males were removed with forceps under an Olympus HE587037, HE587042, HE587043, KF321746–KF321772. SZX7 stereo-microscope. The soft tissue was dissolved by Neighbour-joining (NJ) and Maximum Likelihood (ML) immersion for 20 min in hot 10% KOH and the remaining scle- analyses were used to determine phylogenetic relationships rotized parts were removed to obtain the aedeagus, which was between haplotypes for the four Aphrodes species using stored in glycerol. For imaging, each aedeagus was placed on a PAUP v4.0 beta (Swofford, 2001) or MEGA5 (Tamura et al., cavity slide filled with glycerol and glass beads, which helped 2011) and Bayesian Inference (BI) using mrbayes v3.2.1 to keep the aedeagus in the correct orientation. Each aedegaus

Table 2. Description of morphometric characters measured on the body and aedeagus of Aphrodes leafhoppers as shown in Fig. 1 and calculated ratios included in the ﬁnal linear discriminant analyses (LDA).

Variable Description/taxonomic character interpretation Body a Body length (from top of vertex to the tip of the wings) b Pronotum length c Head length d Head width e Eye width g* Eye head length – length between posterior tip of the eye and top of vertex (M) f Pronotum width Fig. 1. Morphometric characters measured using IMAGE J 1.40e on w* Wing length (M) the body and aeadegus of Aphrodes leafhoppers. a × f* Body area (M, F) a/f* Overall body length–width ratio (M, F) was photographed from the side and front view. Aedeagus f/b* Pronotum width–length ratio (F) images were taken at 5× magnification either with a Planapo f/c* Pronotum width–head length ratio (F) chromatic 1× lens or with a 1.5× Apo chromatic lens. A cor- f/d* Pronotum–head width ratio (M, F) a/c* Proportion of the body taken by head (M) responding scale bar was saved with each image. g/c* Ratio between eye head length and head length (F) Morphometric measurements were taken from photographs a/(c + b)* Proportion of the body taken by head and in jpg format imported into IMAGE J v1.40e (Rasband, 2006). pronotum (M) We measured various body and aedeagus size characters and a/w* Wing to body length ratio (F) from these calculated several ratios (Fig. 1, Table 2). After Aedeagus a1 Shaft length preliminary inspection we included in the final linear discrim- b1 Length between the top of the shaft to the tip of inant analysis (LDA) only those characters that showed the the lower pair of spines least overlap between species. Morphometric measurements b2 Length between the top of the shaft to the top of were analysed by LDA in MINITAB v15 (Minitab Inc, State the lower pair of spines College, U.S.A.), KYPLOT v5.0 (KyensLab Inc., Tokyo) c1 Length between the top of the shaft to the tip of and R (R Development Core Team, 2010). Individuals were the upper pair of spines c2 Length between the top of the shaft to the top of assigned to groups according to the acoustic profile and/or the upper pair of spines COI lineage. To avoid bias in the analysis due to correlations Z * Distance between tips of upper and lower aedeagus between variables, we additionally eliminated one of each pair pair of spines of variables exhibiting high correlation coefficients (r ≥ 0.80). d1 Shaft base width LDA with backward stepwise selection was used to determine d2 Shaft base height h ‘Hook’ length whether any variables could be removed from the analysis. a1 × d1* shaft area The ranges of obtained values for the informative characters, a1/c2* Relative position of the upper pair of spines on the as well for those that were ultimately excluded, are shown in shaft Figures S2–S7. c2/Z* Ratio between the position of the upper pair of spines and distance between tips of upper and lower pair of spines Results c1/b2* Relative position of upper and lower pair of spines a/a1* Body–aedeagus length ratio × × Vibrational signals a f/a1 d1* Body–aedeagus shaft area ratio Italics indicate measured variables; asterisks indicate 14 variables The male vibrational signals of A. bicincta, A. diminuta and included in initial LDA analyses of males and seven parameters used A. makarovi have been described by Tishechkin (1998, 2000) for females; bold indicate eight remaining characters for males after and vibrational signals, as well as the duetting behaviour of LDA backward stepwise selection. M, F, indicate whether the body A. makarovi, have been studied in detail by de Groot et al. parameter was used in LDA for males and/or females. (2012). The male calling signals of all four species had a species-specific stereotyped structure (Fig. 2) and differed The female calling signals of all four species were a series of greatly among species. In all species a nonspecific first regularly repeated single pulses (Fig. 3) and species-specificity section of the male call was followed by a species-specific of female calls was found primarily in call duration and also section composed of different elements. Tishechkin (2000) in pulse repetition time (Table 4). Male–female duets also had provided brief descriptions of female signals of A. bicincta a species-specific structure (Figure S8). Audio files of repre- and A. makarovi and de Groot et al. (2012) included detailed sentative male calls and Male–female duets of each species analyses of female calls of A. makarovi in their study. are provided in Supporting Information (Audio S1–S8).

Fig. 2. Male calls of four Aphrodes species. Sonogram (above) and oscillogram (below) of representative signals are shown. A1–A2, B1–B2, D1–D3, M1–M4 indicate sections of A. aestuarina (A), A. bicincta (C), A. diminuta (B) and A. makarovi (D) male calls, respectively. Note differences in scale.

Aphrodes aestuarina. In A. aestuarina themalecallwas characteristics of a chirp emitted by A. bicincta males were short (Table 3) and the species-specific section (A2) included similar to those of a chirp found in A. aestuarina.The only one chirp with broadband frequency characteristics female call of A. bicincta was shorter than female signal of (Fig. 2A). The female call of A. aestuarina differed from the A. aestuarina (Table 4) and in a duet a female reply was female call of A. bicincta only in duration (Table 4). In a duet inserted between continously emitted male chirps (Figure S8). the female reply followed after the male chirp (Figure S8). Aphrodes diminuta. The male call of A. diminuta was also Aphrodes bicincta. In A. bicincta the species-specific section short (Table 3), but it included two species-specific elements: a (B2) was formed by continuously repeated chirps and pulses frequency modulated ‘siren’ (section D2) followed by a series which had high and low dominant frequency, respectively of regularly repeated single pulses increasing in amplitude (Fig. 2C, Table 3). The duration of this section was highly (section D3) (Fig. 2B). The siren had a clear harmonic variable and the shortest and the longest observed calls lasted structure with relatively high dominant frequency (Table 3). 5 s and 4 min, respectively. In some calls the nonspecific The duration of female reply was substantially longer than in starting section was missing. The temporal and spectral A. aestuarina and A. bicincta and the female signal also had

A B

Fig. 3. Female calls of four Aphrodes species (A–D). Sonogram (above) and oscillogram (below) of representative signals are shown. Note differences in scale. a longer pulse repetition time (Table 4). In A. diminuta the signals this section was missing. The females of A. makarovi female reply followed after the ‘siren’ and was overlapped by started emitting vibrational signals during the male call and male pulses (Figure S8). the female reply always continued after the end of male call (Figure S8). In comparison with other Aphrodes species, the female call of A. makarovi was the longest and had the longest Aphrodes makarovi. The duetting behaviour and male and pulse repetition time (Table 4). The shortest and the longest female calling signals of A. makarovi have been described measured female calls lasted 5 and 61 s, respectively. in detail (de Groot et al., 2012). In this species chirps and regularly repeated pulses formed a complex signal with stereotyped overall structure (Fig. 2D). The male call is Molecular analysis relatively long, but its duration was less variable than in A. bicincta (Table 3). The shortest and the longest observed In total, we obtained from individuals included in the study calls lasted 11 and 24 s, respectively. The third section of ﬁve, eight, nine and 14 COI haplotypes for A. aestuarina, the call consisted of up to 5 units, but in a few recorded A. bicincta, A. diminuta and A. makarovi, respectively. Within

Table 3. Temporal and spectral properties of the male calling signals of the four Aphrodes species.

Species Call section Duration (s) Dominant frequency (Hz) Pulse repetition time (ms)

A. aestuarina Complete call 1.35 ± 0.33 – – A1 1.16 ± 0.33 176 (100–1061) – A2 0.095 ± 0.022 1260 (608–1965) – A. bicincta Complete call 33.79 ± 28.41 – – B1 3.199 ± 0.619 231 (111–458) – B2 30.586 ± 28.31 – 327 ± 45 SB2-chirp 0.085 ± 0.027 1328 (1037–2149) – SB2-pulse 0.105 ± 0.036 273 (194–351) – A. diminuta Complete call 1.66 ± 0.53 – – D1 1.17 ± 0.49 266 (75–1541) – D2 0.148 ± 0.10 1350 (208–2819) – D3 0.339 ± 0.81 1331 (264–2819) 22 ± 2.6 A. makarovi Complete call 16.90 ± 3.10 – – M1 5.0 ± 1.22 117 (97–1459) – M2 0.65 ± 0.095 178 (70–1104) – M3 2.48 ± 0.787 178 (73–1255) – M4 8.19 ± 3.09 164 (94–1255) 49 ± 2.5

For duration and pulse repetition time (PRT) means with standard deviations are shown while for dominant frequency medians, minimum and maximum measured values (in brackets) are given. For A. bicincta temporal and spectral parameters of a chirp and a pulse within the section 2 are also shown and repetition time of chirps in this section is given. For details about call sections see Fig. 2.

Table 4. Temporal and spectral properties of the female calling and Anoscopus) were much higher and ranged between 15.13 signals of the four Aphrodes species. and 17.54%. Phylogenetic analyses of mtDNA COI sequences showed that all four Aphrodes species form a monophyletic Pulse Dominant group with four well-supported lineages (Fig. 4). NJ, ML and Call repetition frequency BI analyses resulted in phylogenies with very similar topolo- Species Nn duration (s) time (ms) (Hz) gies. Each lineage corresponded to species identity determined A. aestuarina 14 96 0.414 ± 0.136 24.3 ± 3.8 560 (354–1378) by acoustic characters. Aphrodes bicincta and A. makarovi A. bicincta 26 126 0.140 ± 0.035 24.9 ± 3.2 563 (101–1284) were revealed as sister species (Fig. 4). Although in the pre- ± ± A. diminuta 7702.512.28 34.3 3.2 804 (559–967) vious study in which a shorter fragment and only the U.K. A. makarovi 15 92 16.47 ± 5.1 52.1 ± 5.7 547 (118–1166) haplotypes were used A. diminuta was positioned as the basal Means with standard deviations are shown while for dominant species within the genus (Bluemel et al., 2011), according frequency medians, minimum and maximum measured values (in to the phylogeny obtained in the present study A. diminuta brackets) are given. N , number of individuals analysed; n, total number and A. aestuarina may be sister species, albeit with weak of signals analysed. support. species there was only one shared haplotype (of A. diminuta) Morphometric analyses between the countries. Within-species sequence divergence ranged from 0.40 to 1.01%, whereas within-genus divergences The ranges of obtained values of morphological characters ranged from 4.21 to 7.0% (Table 5). Sequence diver- for each species are shown in Tables 6, 7 and Figures S2–S4. gences between aphrodine genera (Aphrodes, Planaphrodes Raw morphological measurements for each individual are

Table 5. Average percentage sequence divergence within Aphrodes species (on diagonal) and between Aphrodes and two other aphrodine species Anoscopus limicola and Planaphrodes trifasciata (below diagonal) based on Kimura-2-parameter distance measure.

Sequence divergence (%) – Kimura-2-parameter A. aestuarina A. bicincta A. diminuta A. makarovi A. limicola P. trifasciata

A. aestuarina 0.40 A. bicincta 6.86 0.52 A. diminuta 7.00 6.38 1.01 A. makarovi 6.71 4.21 6.84 0.67 A. limicola 17.10 16.41 16.14 16.76 0.17 P. trifasciata 17.18 16.64 17.54 15.85 15.13 1.58

Fig. 4. Neighbour joining phylogeny using Kimura 2-parameter distances (Kimura, 1980) for 600 bp of cytochrome oxidase subunit I gene for 36 haplotypes from four Aphrodes species, A. aestuarina, A. bicincta, A. diminuta and A. makarovi. Bootstrap support values greater than 70% are shown above the branches for neighbour-joining, maximum likelihood analysis and posterior probability support values higher than 0.7 are shown for Bayesian analysis, respectively. The phylogram is rooted with two aphrodine species, Anoscopus limicola and Planaphrodes trifasciata.The scale bar represents 0.006 substitutions per site. H and SH indicate haplotypes found in individuals collected in the U.K. and Slovenia, respectively. given in Tables S2, S3. Leafhoppers of both sexes collected had the largest aedeagus relative to their body size and the from Slovenia tended to be larger than U.K. individuals of the values of a/a1 ratio were always below 6 (Fig. 6, Figure S3). same species and for all measured characters values overlapped The second most informative character was the position of between at least two species. the spines on the aedeagus shaft (ratio a1/c2). In A. makarovi LDA based on 14 body and aedeagus characters grouped the upper spines were generally positioned lower on the shaft males of each species into four distinct, well-deﬁned clus- than in the other species (Fig. 6) and ratios a1/c2 and c2/Z ters that corresponded with species assignment based on included in LDA, as well as measured characters c1, c2, vibrational signals and/or COI sequences (Fig. 5). The most together with aedeagus length distinguished this species from informative character was the ratio between body and the A. bicincta (Fig. 6, Figure S3). aedeagus shaft length that clearly distinguished males of In comparison with A. makarovi, males of A. aestuarina A. diminuta from the other three species. Males of this species had overall narrower head (d) and body shape (ratio a/f),

Table 6. The ranges (means, with minimum and maximum measured Table 7. The ranges (means, with minimum and maximum measured values in brackets) of obtained values for male body and aedeagus values in brackets) of obtained values for female body characters characters shown in Table 2. Raw morphological measurements for shown in Table 2. Raw morphological measurements for each each individual are given in Table S2. individual are given in Table S3.

Morphometric Morphometric character A. aestuarina A. bicincta A. diminuta A. makarovi character A. aestuarina A. bicincta A. diminuta A. makarovi a(mm) 5.75 5.51 4.70 5.93 a(mm) 6.99 6.67 5.91 7.27 (5.40–6.10) (4.80–6.12) (4.44–5.09) (5.34–6.99) (6.62–7.66) (6.12–720) (5.72–6.20) (6.63–7.99) b(mm) 0.68 0.72 0.66 0.77 b(mm) 0.81 0.87 0.77 0.94 (0.62–0.81) (0.62–0.88) (0.59–0.69) (0.61–0.89) (0.73–0.87) (0.78–0.97) (0.74–0.81) (0.78–1.08) c(mm) 0.63 0.69 0.63 0.70 c(mm) 0.86 0.93 0.84 0.98 (0.54–0.72) (0.56–0.82) (0.56–0.70) (0.63–0.85) (0.80–0.96) (0.82–1.10) (0.75–0.89) (0.85–1.13) d(mm) 1.43 1.47 1.37 1.58 d(mm) 1.72 1.8 1.63 1.93 (1.33–1.54) (1.30–1.61) (1.24–1.45) (1.42–1.82) (1.62–1.82) (1.67–1.98) (1.54–1.72) (1.64–2.18) e(mm) 1.24 1.27 1.19 1.40 e(mm) 1.54 1.58 1.42 1.70 (1.16–1.48) (1.11–1.40) (1.08–1.23) (1.26–1.61) (1.42–1.67) (1.43–1.75) (1.35–1.48) (1.31–1.93) g(mm) 1.37 1.47 1.30 1.51 g(mm) 1.70 1.83 1.59 1.92 (1.27–1.48) (1.27–1.62) (1.20–1.41) (1.36–1.75) (1.61–1.85) (1.69–1.98) (1.5–1.68) (1.69–2.16) f(mm) 1.83 1.82 1.66 1.99 f(mm) 2.09 2.11 1.91 2.30 (1.71–1.96) (1.59–1.95) (1.57–1.75) (1.84–2.31) (1.99–2.23) (1.96–2.33) (1.87–1.98) (1.93–2.58) w(mm) 4.44 4.11 3.39 4.48 w(mm) 5.31 4.87 4.3 5.35 (4.09–4.74) (3.60–4.63) (3.19–3.73) (4.06–5.27) (5.00–5.84) (4.38–5.30) (4.17–4.59) (4.94–5.85) a × f 10.66 10.05 7.76 11.82 a × f 14.62 14.08 11.28 16.80 (9.22–11.73) (7.62–11.85) (6.97–8.70) (9.82–16.14) (13.20–17.08) (12.28–16.46) (10.67–12.25) (13.19–20.62) a/f 3.13 3.07 2.82 2.99 a/f 3.35 3.17 3.10 3.16 (3.00–3.33) (2.95–3.28) (2.74–3.00) (2.74–3.16) (3.17–3.44) (3.02–3.34) (3.06–3.14) (2.98–3.55) f/d 1.29 1.25 1.22 1.27 f/b 2.57 2.42 2.48 2.47 (1.18–1.33) (1.17–1.30) (1.17–1.27) (1.22–1.33) (2.48–2.72) (2.22–2.58) (2.39–2.56) (2.25–2.75) a/c 9.05 8.23 7.47 8.58 f/c 2.42 2.27 2.29 2.35 (8.21–10.67) (7.26–9.33) (6.95–8.58) (7.57–9.55) (2.25–2.57) (1.98–2.44) (2.11–2.57) (2.17–2.55) a/(c + b) 4.35 3.93 3.68 4.10 f/d 1.22 1.17 1.18 1.19 (4.06–4.61) (3.70–4.20) (3.48–3.92) (3.83–4.40) (1.19–1.26) (1.12–1.2) (1.09–1.24) (1.14–1.25) a1(mm) 0.79 0.79 0.88 0.90 g/c 1.98 1.96 1.9 1.95 (0.72–0.85) (0.72–0.87) (0.77–0.95) (0.83–1.02) (1.87–2.06) (1.8–2.08) (1.81–1.04) (1.80–2.12) b1(mm) 0.54 0.56 0.63 0.66 a/w 1.32 1.37 1.37 1.36 (0.49–0.59) (0.48–0.60) (0.55–0.72) (0.56–0.77) (1.30–1.34) (1.34–1.41) (1.37–1.39) (1.33–1.40) b2(mm) 0.40 0.40 0.49 0.51 (0.36–0.45) (0.34–0.44) (0.41–0.57) (0.41–0.61) c1(mm) 0.47 0.44 0.49 0.60 (0.41–0.53) (0.39–0.48) (0.38–0.56) (0.51–0.72) c2(mm) 0.34 0.30 0.34 0.43 aedeagus shaft and distance between the tips of aedeagus spines (0.29–0.38) (0.26–0.33) (0.26–0.41) (0.34–0.55) (Fig. 6, Figures S2, S3). Z(mm) 0.07 0.11 0.16 0.07 (0.05– 0.11) (0.08–0.14) (0.13–0.18) (0.03–0.10) Backward stepwise selection indicated that only eight d1(mm) 0.08 0.09 0.10 0.10 variables (g, a/f, a/(c + b), w, Z, a/a1, a1/c2, a1 × d1) could (0.06–0.09) (0.08–0.12) (0.08–0.13) (0.07–0.11) reliably separate the four species (Figure S9). It should be d2(mm) 0.12 0.12 0.10 0.15 (0.10–0.13) (0.11–0.16) (0.09–0.12) (0.12–0.17) emphasized that backward stepwise selection removed the h(mm) 0.48 0.46 0.49 0.52 variable c1/b2 which indicates the relative position of the (0.45–0.50) (0.42–0.50) (0.46–0.53) (0.46–0.55) a1 × d1 0.06 0.07 0.09 0.09 two pairs of adeagus spines to each other. Values of this ratio (0.05–0.07) (0.06–0.10) (0.07–0.12) (0.06–0.11) higher than 1 are associated with overlapping spines, while a1/c2 2.34 2.65 2.64 2.17 values below 1 indicate that the base of the lower pair is (2.17–2.89) (2.43–3.08) (2.32–3.01) (1.76–2.60) c2/Z 4.84 2.61 2.24 6.45 positioned at the tip of the upper pair or lower. This character (2.79–6.93) (2.07–4.26) (1.52–2.86) (4.26–15.22) has often been used as a character to distinguish between c1/b2 1.19 1.12 0.98 1.20 species (Hamilton, 1983; Tishechkin, 1998; Biedermann & (1.10–1.27) (0.99–1.26) (0.94–1.09) (1.07–1.31) a/a1 7.14 7.11 5.38 6.61 Niedringhaus, 2004), but in the present study it turned out to (6.61–8.01) (6.16–7.72) (4.98–5.90) (5.94–7.19) be highly variable, and for A. bicincta and A. diminuta the vari- a × f/a1 × d1 176.1 139.6 84.01 136.6 ability was also associated with the country of origin (Figure (134.6–220.8) (98.6–176.0) (69.7–116.8) (117.8–182.2) S3). Importantly, in several A. diminuta males collected from Slovenia, the lower and upper pair were overlapping. In general, A. diminuta did have the largest distance between two proportionally shorter head with pronotum and longer wings pairs of aedeagus spines of the four species (Fig. 6); however, (ratios: a/(c + b), a/w) and smaller aedeagus (Fig. 6, Figures the values for this character found in Slovenian specimens S2, S3). The values for all characters obtained for A. aestuarina overlap with values determined for A. bicincta (Figure S3). overlapped with values found in A. bicincta. The least overlap Similarly, A. makarovi had, in general, the shortest distance was observed in the body ratios a/(c + b), a/w, aedeagus shaft between the spines (Fig. 6), but the observed values overlap base width, the relative position of the upper spines on the with values for A. aestuarina and largely also with A. bicincta.

Fig. 6. Body and frontal of view of the aedeagus of males of four Aphrodes species.

head and wings in males were absent (Figs 6, 8). However, individuals of light colour and males with weakly expressed or absent broad white transverse bands on the head and white oblique bands on the forewings were observed also in other species, most frequently in A. makarovi.

Fig. 5. Linear discriminant analysis of 14 morphometric characters for males of four species of Aphrodes leafhoppers. Aphrodes aestuarina, open triangles; A. bicincta, grey circles; A. diminuta, white Ecological observations circles; A. makarovi, black circles. Views from two angles are shown. Altogether we collected and identified using the acoustic and/or molecular approaches Aphrodes specimens from 119 For females most of the measured lengths and calculated sites. At 53 of them we simultaneously found more than ratios were highly correlated. LDA based on seven characters one Aphrodes species. Although A. makarovi was the only showed overlap between species (Fig. 7) and 8.7% of females species collected on stinging nettles (Urtica dioica), we were assigned to the wrong species. Misidentified individuals also found it syntopically with every other species in very were mainly A. makarovi females identified as A. aestuarina different habitats. This species was present on sea purslane and A. bicincta. The size (body area) was the variable that (Atriplex portulacoides) at tidal saltmarshes together with contributed most to the analysis, followed by the wing to A. aestuarina, in alfalfa fields together with A. bicincta,aswell body length ratio. Aphrodes diminuta females were the smallest as in alpine meadows at altitudes above 800 m a.s.l. together while, in general, A. aestuarina females had the narrowest body with A. diminuta. Aphrodes bicincta was collected from a shape (a/f ratio) and proportionally the longest wings (ratio range of habitats, usually dominated by fabaceous plants, a/w) (Fig. 8, Figure S4). from brown field sites to alpine meadows above 800 m a.s.l. In both males and females, body colour and colouration In Slovenia, A. diminuta was found in alpine meadows and pattern were very variable. Pigmentation of A. aestuarina was Dinaric grassland dominated by fabaceous plants at altitudes in general lighter than in other species and white bands on the higher than 600 m a.s.l., while in the U.K. we collected this

Fig. 8. Bodies of females of four Aphrodes species.

acoustic and/or molecular methods allowed us to determine reliable morphological characters to distinguish species. The study also confirmed the presence of A. diminuta in the U.K. and the high adaptability of A. makarovi to different habitats. It also confirmed analyses of archived Aphrodes specimens (Bluemel et al., 2011) that suggested that both A. aestuarina and A. makarovi inhabit tidal saltmarshes. A situation usually observed in Auchenorrhyncha vibrational communication is that in closely related species male calls are variations of the same general pattern (e.g. den Bieman, 1986; Gillham, 1992; Cocroft et al., 2010). In contrast, male vibrational calls of Aphrodes leafhoppers are highly divergent and differ qualitatively. While male calls of A. aestuarina and A. bicincta are both composed of chirps, the overall structure of the call is different (one chirp as opposed to a series of chirps) and the species-specific duration of female replies reflects the difference in conspecific male call structure. In A. bicincta the female reply is shorter, because it has to be interpolated Fig. 7. Linear discriminant analysis of five morphometric characters between continuously repeated chirps. In all species, pair for females of four species of Aphrodes leafhoppers. Aphrodes formation begins with emission of a male advertisement call aestuarina, open triangles; A. bicincta, grey circles; A. diminuta, white and partners exchange signals in a stereotyped temporal pattern circles; A. makarovi, black circles. Views from two angles are shown. characteristic of a proper duet (Tishechkin, 2000; de Groot et al., 2012). As in other leafhoppers, Aphrodes males use a species from chalk grassland and heath-land. Our observations ‘fly/jump-call-walk’ strategy (Hunt & Nault, 1991; Eriksson are, in general, in agreement with previously described et al., 2011) to increase their signalling space by moving ecological differences among Aphrodes species (Nickel, 2003); from plant to plant. A reply from a sexually receptive virgin however, they also suggest that the ecological preferences of female triggers searching for the female on the plant and a the different species may be less fixed. precisely coordinated duet is crucial for successful location of the female. In A. makarovi it has been shown that the male–female duet is a dynamic interaction during which both Discussion partners modify their signals according to their partner’s reply and males fail to locate the source of a female reply if its A comprehensive analysis of leafhoppers of the genus temporal parameters are outside the species-specific values (de Aphrodes using multiple criteria confirmed A. aestuarina, Groot et al., 2011, 2012). A. bicincta, A. diminuta and A. makarovi as behaviourally, Our analyses of validated specimens revealed consistent dif- genetically and morphologically distinct species. Furthermore, ferences in aedeagal morphology among species; however, morphological studies of individuals from the U.K. and aedeagal characters alone were not enough to clearly distin- Slovenia also revealed geographic differences within species guish between species using LDA (results not shown). Fur- and morphometric analyses of individuals validated by the thermore, differences in male genital morphology seem not to

© 2014 The Royal Entomological Society, Systematic Entomology, 39, 304–324 Species determination in Aphrodes 317 prevent inter-specific matings (J. Bluemel and M. Derlink, per- be expected. Highly divergent male vibrational calls in the sonal observation). Given that the male genitalia evolve more absence of divergence in male genitalia have been described rapidly and divergently than other morphological characters also in the leafhopper genus Macropsis (Tishechkin, 2002). (Eberhard, 1985; Arnqvist, 1997; Hosken & Stockley, 2004), Similarly, a high level of divergence in vibrational songs found it seems surprising that the divergence in aedeagus morphol- in sympatric lacewings species of the Chrysoperla carnea ogy among Aphrodes species is low, especially as they live group is accompanied by very similar male genital morphology syntopically. Differences in genital morphology are consid- across the complex (reviewed in Henry et al., 2013). ered by taxonomists as the most reliable characters to distin- Taking into account the low morphological differentiation guish closely related and otherwise morphologically similar between Aphrodes species it has been generally assumed species (Eberhard, 1985, 2010) and aedeagus morphology has that they may have diverged only recently. However, the also been a prominent taxonomic character used in leafhopper observed average sequence divergences in the COI gene systematics since early 20th century (e.g. Ribaut 1936; Wag- between the four species were in the range of 4.2–7.0% which ner, 1937). Although the mechanisms driving rapid evolution corresponds to interspecies divergence levels found in other of male genitalia are still much debated (Shapiro & Porter, insect taxa (e.g. Hebert et al., 2003; Cywinska et al., 2006; 1989; Arnqvist, 1998; Eberhard, 2004, 2010; Hosken & Stock- Foottit et al., 2008; Seabra et al., 2010). While the level of ley, 2004; Wojcieszek & Simmons, 2013), the most widely genetic divergence of mtDNA is used to estimate the relative supported hypotheses link variation in genital morphology to time of divergence between species (e.g. Avise et al., 1987; sexual selection, either via cryptic female choice or sexual con- Brower, 1994; Papadopoulou et al., 2010), it has been shown flict. However, both hypotheses predict that if females typically that rates of molecular evolution in insects, even at the same mate only once and males cannot coerce females to copulate, mitochondrial gene region, differ substantially even between the rate of divergent evolution of male genitalia would be low closely related taxa (Zakharov et al., 2004; Whinnett et al., and, therefore, genital characters should show little species- 2005). While it is not possible to estimate whether Aphrodes specificity (Eberhard, 1985; Arnqvist, 1998). The mating sys- leafhoppers show a fast rate of evolution of the COI gene with tem generally observed in leafhoppers is that females, unlike the current data, the observed sequence divergence between males, most likely mate only once (but see Hayashi & Kimura, congeners is intermediate between those reported for two other 2002). Laboratory studies showed that in contrast to males leafhopper genera [Homalodisca (3.4%), de Leon´ et al., 2006; which start calling (i.e. searching for a mate) immediately after and Graminella (9 and 14%), Ballman et al., 2011]. copulation, mated leafhopper females stop replying and there- Although not comprehensive, the current molecular analyses fore males cannot locate them (Hunt & Nault, 1991; Mazzoni clearly showed that, within the Aphrodinae, Aphrodes species et al., 2009). Some females, however, may become responsive form a single group with four well-supported lineages; again after a post-mating refractory period, the length of which however, the phylogenetic relationships between them are less depends upon the size of the ejaculate (i.e. whether the male clear. While the similarity of male and female vibrational had repeatedly mated several times immediately prior to cop- signals and the overlap in morphological characters suggest ulation) (Bailey & Nuhardiyati, 2005). Although Chiykowski that A. aestuarina and A. bicincta may be the most closely (1970) suggested that in A. bicincta a single mating may not related species or may share the same direct ancestor, be sufficient to fertilize a female for life, our observations phylogeny based on the COI fragment does not support such showed that mated Aphrodes females did not reply to male an evolutionary relationship. As shown previously the sister calls even a month after observed copulation (M. Derlink, per- species of A. bicincta seems to be A. makarovi (Bluemel et al., sonal observation). While females live 2–4 months, even in 2011). While the nodal support for this relationship was high, laboratory conditions male mortality increases after 3–4 weeks the placement of A. aestuarina as a sister species of A. diminuta (Chiykowski, 1970; M. Derlink, personal observation). Taking is not well supported by bootstrap and posterior probability into account also protandry and that males also face greater values. However, in the present study the main aim of the predation risk (Virant-Doberlet et al., 2011) it seems likely molecular analyses was to establish a match between species- that under natural conditions females usually mate only once specific vibrational signals and a distinct COI lineage and not in their lifetime, as was also shown for female planthoppers to unravel phylogenetic relationships between species. For the (Heady, 1993) and treehoppers (Wood & Guttman, 1983). latter, nuclear genes should also be included (Nichols, 2001; Because the mating system is likely to have an important, if Ballard & Whitlock, 2004). not crucial, impact on male genital morphology, its implica- tions should also be considered in taxonomic studies when choosing the most informative and reliable characters. Fur- Species discrimination thermore, pre-zygotic reproductive isolation among species in the genus Aphrodes is maintained primarily by highly diver- According to our results, any approach used in the present gent species-specific male vibrational signals and precisely study could be used as a stand-alone method to distin- coordinated male–female duets. In such situations character guish between Aphrodes species, although combining the displacement in male genital morphology as an adaptation to morphological approach with molecular techniques should prevent hybridization between syntopic, closely related species improve the reliability of species identification, especially (Shapiro & Porter, 1989; Eberhard, 2010) may not necessarily when dealing with females. While including individuals from

© 2014 The Royal Entomological Society, Systematic Entomology, 39, 304–324 318 J. K. Bluemel et al. different countries provided the necessary robustness to our 3(2). Ratio of body length to combined length of head morphometric results and conclusions, the observed geographic and pronotum (a/(b + c)) > 4.06, w > 4.09 mm, Z < 0.11 mm, differences within species indicate that it is conceivable that aedeagus shaft base width (d1) < 0.09 mm, pale body colour individuals from other geographic areas, and different habitats, without white bands and stripes, habitat tidal saltmarshes and may reduce the observed species resolution. Our results show estuaries ...... A. aestuarina that aedeagus shaft length is an important character; however, – Ratio of body length to combined length of head and prono- the most recent morphological identification key for the genus tum (a/(b + c)) < 4.2, w < 4.63 mm, Z > 0.08 mm, aedeagus Aphrodes (Biedermann & Niedringhaus, 2004) does not incor- shaft base width (d1) > 0.08 mm, distance to the tip of the porate information on aedeagus size. We wish to emphasize upper pair of aedeagus spines (c1) < 0.48 mm, head and prono- that when identifying larger numbers of Aphrodes individuals tum with white band, wings with white stripes, habitat not collected in ecological or plant pathogen transmission studies, saltmarsh ...... A. bicincta all specimens should be properly identified, because more than one species is very likely to be collected at a given site. Even Aphrodes diminuta is morphologically the most distinct with available photographic equipment, morphological identi- species. Males of this species have always been considered fication of individuals may be time consuming, because for smaller than other Aphrodes species (Ribaut, 1952; Tishechkin, males both body and aedeagal characters are needed to cor- 1998). Our results showed that this is probably also true rectly identify the species. Ideally, LDA analysis that includes for females; however, the number of analysed females was validated individuals should also be performed. On the other low and they were all collected from Slovenia. Body size hand, for molecular identification sequencing may not always is generally not a reliable taxonomic character as it can be be necessary. Species-specific primers, at least for A. makarovi, influenced by many abiotic and biotic factors (see also below). have been published (Virant-Doberlet et al., 2011). Further- However, in the case of A. diminuta body size seems to be a more, with a number of Aphrodes COI sequences now avail- good predictor of species identity. Our results indicate that able in Genebank, a restriction fragment length polymorphism in contrast to A. bicincta and A. makarovi, there seems to (RFLP) analysis may provide a faster and cheaper solution for be very little variability in the body length of A. diminuta reliable species identification. However, when live individu- males associated with the country of origin, although this may als are needed, such as in plant pathogen transmission studies, also be a result of the relatively low number of individuals acoustic study seems to be the only available approach to iden- analysed. Interestingly, a greater variability between the U.K. tify them prior to use in experimental trials. and Slovenian A. diminuta male specimens was observed in aedeagus length; nevertheless, the ratio between body and aedeagus length proved to be the most reliable character Morphological key to male Aphrodes to identify males of this species. Besides its small size, A. diminuta has usually been distinguished from other species The key to male Aphrodes, based on the most reliable and primarily by the large distance between aedeagus spines. Our distinctive morphological characters obtained from validated results, however, suggest that the relative position of the two individuals used in the present study, is provided below. For pairs of aedeagus spines to each other shows a high degree of detailed description of morphological characters a, b, c, f, a/f, overlap between species, so it should not be used as taxonomic + a/(c b), a1, a/a1, c1 d1, Z , used in the key see Table 1, for character on its own. An additional distinguishing character graphic illustrations see Figs 1, 6; Figures S1, S10 and for that we did not include in the morphometric study, because it detailed distribution of the measured values see Table 6 and could not be reliably measured, is that the aedeagus shaft is Figures S2, S3. For individuals with measured values close to straight in the lateral view (Figure S10). the extreme species values we recommend performing LDA Aphrodes makarovi is considered as the largest in the analysis on a combined dataset which includes unknown and genus (Emmrich, 1980; Tishechkin, 1998; Biedermann & validated individuals used in the present study for which raw Niedringhaus, 2004). However, while our results are, in morphological data are given in Tables S2, S3. general, in agreement with this, we also observed great 1. Body length/aedeagus length ratio (a/a1) ≤ 5.9, variability in body size (body length and width) associated w < 3.73 mm, Z > 0.13 mm, aedeagus shaft straight in with the country of origin. Both males and females collected lateralview ...... A. diminuta from Slovenia were larger than specimens from the U.K. This – Body length/aedeagus length ratio (a/a1) > 5.9, was particularly evident in males, in which the size of the w > 3.60 mm, Z < 0.14 mm, aedeagus shaft bent in lateral U.K. males completely overlapped with the size of A. bicincta view ...... 2 from Slovenia. However, taking into account only the U.K. 2(1). Body length/pronotum width ratio (a/f) > 2.95, aedeagus individuals, only extremely small A. makarovi were of approx- length (a1) < 0.87 mm, distance from aedeagus tip to tip of the imately the same size as the largest A. bicincta. Aphrodes upper pair of aedeagus spines (c1) < 0.53 mm ...... 3 makarovi found in the U.K. were also not different in size – Body length/pronotum width ratio (a/f) < 3.16, aedeagus from A. aestuarina. Furthermore, individuals of A. makarovi length (a1) > 0.83 mm, distance from aedeagus tip to the of found on tidal saltmarshes have pale body colour and often the upper pair of aedeagus spines (c1) > 0.51 mm ...... lack white bands and stripes. The morphological characters ...... A. makarovi that include body size and aedeagus length, and position

© 2014 The Royal Entomological Society, Systematic Entomology, 39, 304–324 Species determination in Aphrodes 319 of the spines on the aedeagus shaft (but not the relative ecotypes (subspecies) of a single species A. bicincta (e.g. position of two spine pairs to each other), are the most reliable Ribaut, 1952; Le Quesne, 1965), we refer to them by species characters to distinguish A. makarovi from the other species. names used in the present study. We primarily relied on figures As noted by others (Zakhvatkin, 1948; Tishechkin, 1998) the and/or descriptions that included body and aedeagus length. aedeagus shaft was distinctly bent in lateral view; however, However, while it is usually possible to attribute the aedeagus this character does not reliably distinguish A. makarovi from shown in the figures to one of the species described in our A. aestuarina (Figure S10). study, the descriptions suggest that previous authors were Aphrodes aestuarina was traditionally identified primarily usually dealing with a mixture of A. bicincta and A. diminuta by its specific saltmarsh habitat and pale body colouration or A. makarovi and A. bicincta or A. aestuarina. without white bands and stripes (Edwards, 1908), while aedeagal characters were considered to be same as those of Aphrodes bicincta. The drawings of the aedeagus of A. makarovi (Biedermann & Niedringhaus, 2004). Our results A. bicincta in Ribaut (1952: 335, figs 882, 883) correspond are in broad agreement with these observations; however, in to the characters found in the present study (aedeagus shaft eastern England, A. aestuarina is found on tidal saltmarshes length shorter than in A. diminuta and slightly bent at the tip syntopically with A. makarovi and the syntype series of in the lateral view). Most previous authors, however, pro- A. aestuarina collected by Edwards in 1908 contains a mixture vided male genitalia drawings for a species that most likely of these two species (Bluemel et al., 2011). Our observations belongs to A. diminuta. Wagner (1937) showed on p. 66, fig. suggest that on saltmarshes colour is not a reliable character to 4a, b an aedeagus attributed to A. bicincta which shows char- distinguish A. aestuarina from A. makarovi; however, the for- acters that correspond to A. diminuta found in the present mer is morphologically characterized by narrower body shape, study and as described originally by Ribaut (1952) (large proportionally longer wings and males also have a smaller size - approx. 9 mm, large distance between tips of upper aedeagus (shorter and with a narrower base). While LDA and lower spine pair, aedeagus shaft straight in the lateral based on several morphological characters separates these two view). Duffield (1963) and Nast (1976) followed Wagner’s species, additional molecular identification may be needed to interpretation and their drawings of the aedeagus characters of identify saltmarsh individuals correctly. The reported presence A. bicincta on p. 157 and p. 159, respectively, correspond to of A. aestuarina in The Netherlands (den Bieman et al., 2011), A. diminuta. Similarly, Ossiannilsson (1981) described aedea- Germany (Nickel & Remane, 2002), Poland (Nast, 1987), gus characters for A. bicincta that are attributable to A. diminuta Italy (d’Urso, 1995) Ireland (Helden, 2005) and Denmark (p. 365, figs 1182, 1183). While aedeagus associated with (Endrestøl, 2013) should be re-examined and confirmed. A. bicincta by Hamilton (1983: 484, fig. 51) corresponds to the Aphrodes bicincta is probably morphologically the least findings of the present study, it seems likely that the aedea- distinct species and as in A. makarovi, individuals collected gus attributed to the progeny of A. bicincta (p. 484, fig. 52) from Slovenia were larger than individuals found in the U.K. belongs to A. diminuta. In contrast, individuals interpreted as The most reliable characters to distinguish males of A. bicincta A. bicincta by Le Quesne (1965: 124–125) show characters from the other two species with which it is found syntopically that correspond to A. makarovi found in the present study (large (A. makarovi, A. diminuta) are those that include body size and body size and large aeadeagus). Emmrich (1980) included Le aedeagus length, as well as the position of the spines on the Quesne’s data on A. diminuta (see below) into his interpreta- aedeagus shaft (c1 and ratio a1/c2). The relative position of the tion of A. bicincta. Zakhvatkin (1953) described the aedeagus two spine pairs to each other was too variable to be considered of A. bicincta as slightly bent near the tip in lateral view and a reliable character. As noted by others (Zakhvatkin, 1953; with the tips of the upper pair of aedegal spines reaching the Tishechkin, 1998) the aedeagus shaft in lateral view was only top third of the length of the lower pair. slightly bent near the tip (Figure S10). Aphrodes diminuta. Ribaut (1952) associated A. diminuta with a small body and a large aedeagus with two pairs of Interpretation of previously published taxonomic data on the aedeagal spines positioned far apart (p. 335, figs 884, 885) and genus Aphrodes Le Quesne (1965) accordingly attributed smaller individuals with large aedeagus to A. diminuta. The illustration in Duffield The published taxonomic work on Aphrodes leafhoppers (1963) shows for this species an aedeagus (p. 157, fig. 2) that reveals that authors interpreted morphological characters very most probably belongs to A. makarovi. The drawings of the differently. In addition, we believe that problems also arise aedeagus of A. striata (F.) in Zakhvatkin (1948: 188, figs 10, from the fact that taxonomists have not taken into account 11) correspond to the characters of A. diminuta found in the the fact that two or three Aphrodes species can be found present study (aedeagus shaft straight in lateral view, large syntopically at the same collecting site. While a taxonomic distance between the two pairs of spines). revision of Aphrodes leafhoppers is not the objective of the present study, we provide below some information on Aphrodes makarovi. Zakhvatkin (1948) described the aedea- how some authors have interpreted species according to the gus of A. makarovi as very similar to the aedeagus of currently established names. Although authors often treated A. aestuarina, with two pairs of aedegal spines mostly overlap- individuals of different species included in their studies as ping (p. 188, figs 2, 3). The aedeagal characters of A. makarovi

© 2014 The Royal Entomological Society, Systematic Entomology, 39, 304–324 320 J. K. Bluemel et al. found in the present study correspond to the drawings of vari- Such background work is crucial, not only for solving difficult ation of aedeagus morphology in this species provided by Nast cases in systematics, but also to provide reliable information (1976: 160). The aedeagus of A. makarovi shownbyOssian- for further more detailed investigations on evolutionary pro- nilsson (1981: 364, figs 1176, 1177) most probably belongs to cesses and speciation. A. bicincta, because this author attributed a smaller aedeagus to the larger species. Hamilton (1983) synonymized A. makarovi with A. costatus and his drawing for the latter species also Supporting Information corresponds to the aedeagus of A. makarovi. Additional Supporting Information may be found in the online version of this article under the DOI reference: Aphrodes aestuarina. It is likely that authors describing 10.1111/syen.12056 Aphrodes collected from tidal saltmarshes mostly dealt either with material that contained a mixture of A. makarovi and Figure S1. Morphometric characters measured on the body A. aestuarina or only A. makarovi. The fact that the distance and aedeagus of Aphrodes leafhoppers. between the tips of two pairs of aedeagal spines does not Figure S2. The ranges of obtained values for male body differ between these two species and that in both species characters shown in Table 2. Data are arranged according aedeagus shaft is bent is most probably the reason why the to species (A. a., A. aestuarina;A.b.,A. bicincta;A.d., existence of two species in this habitat was not recognized A. diminuta;A.m.,A. makarovi) and country of origin previously. The drawings of the aedeagus of A. aestuarina in Zakhvatkin (1948: 188, figs 7, 8) correspond to the characters (U.K., Slovenia). Boxplots with medians, quartiles, standard found in the present study. deviation and outliers are shown. See Figure S1 and Table 1 for detailed information. Figure S3. The ranges of obtained values for male aedeagus Conclusion characters shown in Table 2. Data are arranged according to species (A. a., A. aestuarina;A.b.,A. bicincta;A.d., While the results of the present study confirmed previous A. diminuta;A.m.,A. makarovi) and country of origin observations on the high variability of morphological characters in the leafhopper genus Aphrodes (Wagner, 1937; (U.K., Slovenia). Boxplots with medians, quartiles, standard Zakhvatkin, 1953; Le Quesne, 1965; Emmrich, 1980; Hamil- deviation and outliers are shown. See Figure S1 and Table 1 ton, 1983), they also suggest that at least some intraspecific for detailed information. variability encountered in the previous studies may be a result Figure S4. The ranges of obtained values for female body of grouping together specimens of different species. Not only characters shown in Table 2. Data are arranged according are individuals of different species sympatric and found in to species (A. a., A. aestuarina;A.b.,A. bicincta;A.d., the same type of habitat over a larger geographical area, but A. diminuta;A.m.,A. makarovi) and country of origin they also live syntopically at the same site. Even on coastal (U.K., Slovenia). Boxplots with medians, quartiles, standard tidal saltmarshes, which were always regarded as habitats deviation and outliers are shown. See Figure S1 and Table 1 exclusively inhabited by A. aestuarina, this species is found together with A. makarovi. Although males of A. aestuarina, for detailed information. A. bicincta, A. diminuta and A. makarovi can be reliably iden- Figure S5. The ranges of male body ratios excluded from tified by combining several body and aedeagus morphological the LDA analysis. Data are arranged according to species characters, morphological differentiation between species is, (A. a., A. aestuarina;A.b.,A. bicincta;A.d.,A. diminuta; nevertheless, still relatively small. Observed genetic distances A. m., A. makarovi) and country of origin (U.K., Slovenia). between Aphrodes species are relatively large in comparison Boxplots with medians, quartiles, standard deviation and with low morphological variation. Strong pre-zygotic repro- outliers are shown. ductive isolation based on highly divergent species-specific male vibrational signals and a mating system in which females Figure S6. The ranges of male aedeagus ratios excluded typically mate only once in their lifetime are the most likely from the LDA analysis. Data are arranged according to reasons for low divergence in aedeagus morphology among species (A. a., A. aestuarina;A.b.,A. bicincta;A.d., species. Taken together with previous work on archived A. diminuta;A.m.,A. makarovi) and country of origin Aphrodes specimens (Bluemel et al., 2011), our study sug- (U.K., Slovenia). Boxplots with medians, quartiles, standard gests that it is likely that many museum specimens have been deviation and outliers are shown. assigned to the wrong species and that species identification in previous ecological and vector studies is also questionable. Figure S7. The ranges of female body ratios excluded from This work showed that when dealing with genera such as the LDA analysis. Data are arranged according to species Aphrodes (low morphological differentiation between syntopic (A. a., A. aestuarina;A.b.,A. bicincta;A.d.,A. diminuta; congeners), integration of behavioural and molecular data to A. m., A. makarovi) and country of origin (U.K., Slovenia). obtain validated specimens enables the clarification of mor- Boxplots with medians, quartiles, standard deviation and phological characters and, ultimately, of species differentiation. outliers are shown.

Figure S8. Sonagrams and oscillograms of the duets of four Bailey, W.J. & Nuhardiyati, M. (2005) Copulation, the dynamics of Aphrodes species. sperm transfer and female refractoriness in the leafhopper Balclutha insica (Hemiptera: Cicadellidae: Deltoocephalinae). Physiological Figure S9. Linear discriminant analysis of eight morpho- Entomology, 30, 343–352. metric characters for males of four Aphrodes species. Views Ballard, J.W.O. & Whitlock, M.C. (2004) The incomplete natural from two angles are shown. history of mitochondria. Molecular Ecology, 13, 729–744. Ballman, E.S., Rugman-Jones, P.F., Southamer, R. & Hoddle, Figure S10. Side view of aedeagus of four Aphrodes M.S. (2011) Genetic structure of Graphocephala atropunctata species. (Hemiptera: Cicadellidae) populations across its natural range in California reveals isolation by distance. Journal of Economic Table S1. Summary information on individuals used in Entomology, 104, 279–287. morphometric analyses. Bickford, K., Lohman, D.J., Sodhi, N.S. et al. (2007) Cryptic species Table S2. Raw morphological data for males of eight body as a window on diversity and conservation. Trends in Ecology & Evolution 22 and eight aedeagus characters measured as shown in Fig. 1. , , 148–155. Biedermann, R. & Niedringhaus, R. (2004) Die Zikaden Deutschlands. Table S3. Raw morphological data of eight body characters Wissenschaftlich Akademischer Buchvertieb-Frund,¨ Scheeßel. measured in females as shown in Fig. 1. den Bieman, C.F.M. (1986) Acoustic differentiation and variation in planthoppers of the genus Ribautodelphax (Homoptera, Delphaci- Audio S1. Male call of A. aestuarina. dae). Netherlands Journal of Zoology, 36, 461–480. Audio S2. Male call of A. bicincta. den Bieman, K., Biedermann, R., Nickel, H. & Niedrighaus, R. (2011) The Planthoppers and Leafhoppers of Benelux. Wissenschaftlich Audio S3. Male call of A. diminuta. Akademischer Buchvertieb-Frund,¨ Scheeßel. Bluemel, J.K., King, R.R., Virant-Doberlet, M. & Symondson, W.O.C. Audio S4. Male call of A. makarovi. (2011) Primers for identification of type and other archived Audio S5. Male–female duet of A. aestuarina as shown in specimens of Aphrodes leafhoppers (Hemiptera, Cicadellidae). Figure S8. Molecular Ecology Resources, 11, 770–774. Bortolus, A. (2008) Error cascades in the biological sciences: the Audio S6. Male–female duet of A. bicincta as shown in unwanted consequences of using bad taxonomy in ecology. Ambio, Figure S8. 37, 114–118. Bressan, A., Clair, D., Sem´ etey,´ O. & Boudon-Padieu, E. (2006) Insect Audio S7. Male–female duet of A. diminuta as shown in injection and artificial feeding bioassays to test the vector specificity Figure S8. of Flavescence Doree´ phytoplasma. Phytopathology, 96, 790–796. Audio S8. Male–female duet of A. makarovi as shown in Broughton, W.B. (1963) Method in bioacoustic terminology. Acoustic Behaviour of Animals (ed. by R.G. Busnel), pp. 3–24. 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