Gyponana (Gyponana) mali DeLong, 1942

Trivellone, Valeria, Forte, Vally, Filippin, Luisa & Dietrich, Christopher H., 2021, First records of the North American leafhopper Gyponana mali (Hemiptera: Cicadellidae) invading urban gardens and agroecosystems in Europe, Acta Entomologica Musei Nationalis Pragae (Acta. Ent. Mus. Natl. Pragae) 61 (1), pp. 213-219 : 215-219

Gyponana (Gyponana) mali DeLong, 1942

( Figs 2A–G , 3A–G , 4A–B )

Gyponana mali DeLong, 1942: 44 .

Gyponana (Gyponana) mali : Oආൺඇ (1949): 46; Mൾඍർൺඅൿ (1962): 92; DൾLඈඇǤ & F*©ൾඒඍൺǤ (1964): 96; Hൺආංඅඍඈඇ (1982): 549, 555.

Material examined. SWITZERLAND: T ංർංඇඈ: Agno, 45°59′45.1″N 8°54′09.0″E, 277 m a.s.l., 4.vi.2015, on Nerium oleander (ornamental plant in urban green), 1 ♀. ITALY: Lඈආൻൺඋൽඒ: Varese province: Gaggiolo, 45°49′44.6″N 8°53′22.7″E, 408 m a.s.l., 7.viii.2017, 1 ♀ 1 J (I01/17) on the border of woody area intermixed with annual crop; Milano province: Nosate, 45°32′35.6″N 8°43′19.7″E, 149 m a.s.l., 12.vii.2015, on woody plants along the Ticino riverbank, 5 ♀♀ 1J; same data but 45°32′38.3″N 8°43′22.0″E, 7.vii.2019, 10 ♀♀ (I03/19), 5 JJ (I02/19). V ൾඇൾඍඈ: Treviso province: Gorgo al Monticano, 45°47′58.0″N 12°33′08.5″E, 7 m a.s.l., 3.viii.2016, 1 ♀; 14.ix.2016, on Vitis vinifera ,

1 ♀; 22.vii.2016, on hedgerows surrounding vineyards, 2 JJ, 1 nymph; same data but 45°48′02.5″N 12°33′08.0″E, 6.vii.2017, on Cornus sanguinea in a windbreak row surrounding vineyards, 2 ♀♀ 3 JJ; Vittorio Veneto, 45°58′35.0″N 12°19′11.7″E, 113 m a.s.l., 29.vi.–26.vii.2018, on V. vinifera , 1 ♀ 2 JJ; Carbonera, 45°41′30″N 12°16′57″E, 18 m a.s.l., 29.vi.–26.vii.2018, on V. vinifera , 1 J.

Diagnosis. Species belonging to the subgenus Gyponana s. str. vary from 7 to 13 mm in total length including forewings at rest, with external habitus depressed and pale green in overall coloration (fading to yellow in preserved specimens). Head with anterior margin declivous and subfoliaceous, narrower than pronotum. Pronotum transversely striate and widest posteriorly. Forewings opaque, venation moderately to strongly reticulate ( Figs 2A – B , 3A – B ).

Male ( Fig. 2A , voucher I02/19). Body length 10.0 mm.

Pygofer 1.5 times as long as its maximum height in lateral view, with anteroventral margin rounded and apex abruptly pointed; macrosetae dispersed on the posteroventral quadrant and fine setae present near ventral margin ( Figs 2C – D ). Aedeagus simple, without basal processes, constricted toward the middle, apex enlarged, produced and rounded; a pair of preapical lateral processes curved basally ( Figs 2E, G ). Style elongated, with the tip narrowed beyond

a broad pointed tooth on the ventral margin; connective Y-shaped in dorsal view ( Fig. 2F ).

Female. Body length of specimens examined from Italy ( Fig. 3 , voucher I03/19) and Switzerland (voucher CH 04/15) 11 and 13 mm, respectively. Sternite 7 slightly longer laterally than at midlength, with posterior margin concavely emarginate ( Fig. 3C ). Ovipositor surpassing pygofer apex. Valvula I ( Figs 3D – E ), in lateral view, about 4 times longer than high and slightly convex; apex pointed with fine denticles; dorsal sculptured area strigate and distributed submarginally. Valvula II ( Figs 3F – G ) abruptly broadened medially, with teeth small and rounded, present on distal half.

Comparative notes. Species of Gyponana bear no close resemblance to any other leafhopper taxa recorded in the western Palearctic region until now. In overall size and shape, Gyponana somewhat resembles the widespread Old-World grass-feeding genus Glossocratus Fieber, 1866 ( Cicadellidae : Deltocephalinae ) but is easily distinguishable by the green coloration and placement of the ocelli on the crown distant from the anterior margin and eyes.

Identification of species of Gyponana using morphology can be challenging. In an earlier attempt to review the related genera and subgenera of Gyponini using the genital apparatus, Bൺඅඅ & Rൾൾඏൾඌ (1927) concluded that the aedeagal variation was not suitable to separate the species. Many species originally described based on slight differences in the structure of the male style and aedeagus were synonymized by Hൺආංඅඍඈඇ (1982) who reviewed the genus and provided a pictorial key to species. According to the morphological criteria of Hൺආංඅඍඈඇ (1982) the aedeagal shaft in posterior view is tapered distally in G. mali ( Fig. 4A ), but broadened preapically in G. extenda DeLong, 1942 ( Fig. 4C ). Moreover, the position of the gonopore is preapical on the ventral surface in G. mali , but situated at the apex of the distal extension in G. extenda . However, the male specimens examined from Italy appear to be morphologically intermediate between these two named North American species in the structure of the aedeagus ( Fig. 2G ). For example, in the voucher specimen I02/19, although the aedeagus is expanded well beyond the distal processes ( Fig. 2G ), as in G. extenda (sensu Hൺආංඅඍඈඇ 1982: Fig. 4C and sensu DൾLඈඇǤ & F*©ൾඒඍൺǤ 1964: Fig. 4D ), the gonopore is distinctly preapical, as in G. mali (sensu Hൺආංඅඍඈඇ 1982: Fig. 4A and sensu DൾLඈඇǤ & F*©ൾඒඍൺǤ 1964: Fig. 4B ).

Phylogenetic analysis. The barcoding sequence of mtCOI from the European specimen subjected to BLAST analysis (www.ncbi.nlm.nih.gov/BLAST) revealed 99.54% identity with a sequence of G. mali (accession number KF919958.1) collected from Canada (Ontario). The sequence collected during the present study was aligned with 28 mtCOI sequences downloaded from GenBank belonging to six species of Gyponana ( G. extenda ; G. mali ; G. salsa DeLong, 1942 ; G. parallela DeLong 1942 ; G. cacumina DeLong, 1942 ; and G. striata (Burmeister, 1839)) (Supplementary material: Table S1) showing a percentage of identity higher than 98%. Alignments consisted of 692 nucleotide positions. Relationships among the six species of Gyponana recovered by the mtCOI ML tree ( Fig. 5 ) were not fully congruent with morphological identifications. Our European sample groups with three out of five specimens of G. mali from Canada ( Fig. 5 , Gr. 4), but with low support. No species were recovered as monophyletic, and all grouped with low support ( Fig. 5 , Gr. 1–3), except four specimens of G. extenda from Canada ( Fig. 5 , Gr. 5) and two specimens of G. cacumina ( Fig. 5 , Gr. 6) with support of 87% and 93%, respectively. The comparisons with other sequences showed that the sequence obtained from Italy is very close to the other mtCOI sequences of G. mali (p-distances 0.00), G. extenda (0.01) and G. striata (0.00–0.01), all collected in Ontario ( Canada), with the exception of G. mali (KF919842.1) which showed a higher p-distance (0.02) compared to the other G. mali specimens in Gr. 4. The divergence with specimens of the other three species was also very low, ranging from 0.01 to 0.02 (Supplementary Table S1). The mtCOI ML tree built in RAxML under the GTR + Gamma recovered the same topology and similar branch support (data not shown).

Discussion

Based on our morphological and molecular studies, all of our specimens from Europe can be assigned to the species G. mali . The European specimens studied here are morphologically similar to specimens (including paratypes) identified as this species from its native range in North America and also appear to be genetically similar based on the barcode region of the mitochondrial COI gene. Nevertheless, our analysis of the COI barcode data suggests that this molecular marker may not reliably distinguish some previously named species of Gyponana . This may indicate either that this gene region is not sufficiently variable in the genus, or that some species previously recognized based on slight differences in the male genitalia are not distinct and should be treated as synonyms. Further studies of the genus are needed to determine which molecular or morphological characters are the most useful for delimiting and diagnosing species of Gyponana .

This is the first record of the tribe Gyponini for the Palearctic Region. This group is widely distributed in North and South America, most probably with a Neotropical origin. Other records of Gyponini outside of the western hemisphere are very rare. Kං*©κൺඅൽඒ (1905) described the species Gypona kangrensis Kirkaldy, 1905 based on one specimen from northern India but the identity of this species and its placement in Gyponini have not been confirmed. An unidentified species of the genus Curtara DeLong & Freytag, 1972 was reported from the west coast of Africa, probably representing an anthropogenic introduction (Nංൾඅඌඈඇ & KඇංǤΗඍ 2000), but no further reports of this species have appeared.

In Europe, the first specimen was incidentally collected in Switzerland in 2015 by the first author: it was a female collected using a sweep net on an ornamental plant in a regularly managed urban green area ( Fig. 6A ). During an independent survey, more specimens were collected by an Italian amateur entomologist (Dr. Danilo Mario Piccolino) in 2015 and then regularly until 2019. All the specimens were collected from a riparian habitat located in the Natural Preserve “Parco del Ticino ” which is located about 55 kilometers far away from Agno ( Switzerland) and separated by the Lombardian Prealps (1097–1959 m a.s.l.) and the Ceresio Lake. The co-authors from CREA–VE (Conegliano, Italy) captured more specimens on yellow sticky traps placed in vineyards, routinely investigated to verify the occurrence of leafhopper vectors of quarantine phytoplasmas associated with grapevine yellows diseases (Flavescence dorée). This material remained unidentified until the present study due to the difficulty of species identification in this genus using only morphology.

Following these incidental collections, we planned to extend the survey to verify the host plants in the habitats of occurrences. Despite further surveys in 2017 and 2018, no additional specimens were collected from the site of the first record in Switzerland. However, in Italy, after the first record in 2015, we collected more specimens on grapevine ( Vitis vinifera ) from three additional locations in North Eastern Italy (Gorgo al Monticano, Vittorio Veneto and Carbonera). Using a sweep net, we also inspected every single plant of the windbreak surrounding the vineyard in Gorgo al Monticano ( Acer campestre , Alnus glutinosa , Betula sp. , Cornus sanguinea, Corylus avellana, Crataegus monogyna, Carpinus sp., Platanus sp., Quercus sp. , and Salix sp. ). We collected specimens of G. mali only from Cornus sanguinea ( Fig. 6B ). Further sampling in Gaggiolo, a site located between Agno ( Switzerland) and Nosate ( Italy), yielded additional specimens of the exotic species in the border of a woody patch where C. sanguinea also occurred.

In the native range of North America, G. mali seems to have a narrower distribution than the other closely related species, being recorded in Georgia, Illinois and Ohio ( USA) (Mൾඍർൺඅൿ 1962). The species was commonly recorded from apple ( Malus spp. ) by DൾLඈඇǤ (1942) but is also likely associated with other trees or shrubs. Little information about host plants of most Gyponana (s. str.) species is available in their native area of distribution. Various species have been recorded from woody host plants, including Alnus , Carya , Malus , Pinus , Quercus , Salix , Tsuga , and various herbaceous dicots (Hൺආංඅඍඈඇ 1982).

Altogether, our observations provide evidence for a recent introduction and spreading of this species in southern Switzerland and central northern Italy. We suggest that, after a possible initial introduction with ornamental plants, the species spread across different agroecosystems with a preference for semi-natural, less disturbed habitats and wild plants (e.g., C. sanguinea in the present study). Lately,

several records of exotic leafhoppers introduced from the DMITRIEV D.2003 onward: 3I World Auchenorrhyncha database. Avai- Nearctic or Asian Regions were reported in the literature lable from:http://dmitriev.speciesfile.org (accessed 23 March 2021).

D’URSO V., SÁNCHEZ I. & BELLA S. 2019: Chloropelix canariensis and all of them were presumably linked to the trade of

Lindberg and Balclutha brevis Lindberg (Auchenorrhyncha, Cicaornamental plants (e.g., Sൾඅ*°ൺκ 2013; T*©ංඏൾඅඅඈඇൾ et al. dellidae) two new leafhoppers to continental Europe, with a review 2015, 2017; D’U*©ඌඈ et al. 2019). of the alien Auchenorrhyncha species established in the continent.

Phytoparasitica 47: 163–178.

Acknowledgements FOLMER O., BLACK M., HOEH W., LUTZ R. & VRIJENHOEK R.

1994: DNA primers for amplification of mitochondrial cytochrome We thank Dr. Danilo Mario Piccolino who collected c oxidase subunit I from diverse metazoanin vertebrates. Molecular the material from Nosate Lombardy ( Italy) and kindly Marine Biology and Biotechnology 3: 294–299.

GATINEAU F., LARRUE J., CLAIR D., LORTON F., RICHARD-MOprovided us more specimens to examine and compare with

LARD M.& BOUDON-PADIEU E. 2001:A new natural planthopper our collections. This study was partially supported by the vector of stolbur phytoplasma in the genus Pentastiridius ( Hemiptera : Swiss National Science Foundation (P2NEP3_168526) Cixiidae ). European Journal of Plant Pathology 107: 263–271.

and US NSF grant DEB-1639601. HAMILTON A. 1982: Review of the Nearctic species of the nominate

subgenus of Gyponana Ball (Rhynchota: Homoptera: Cicadellidae ).

Journal of the Kansas Entomological Society 55: 547–562.

Digitally archived data

KIRKALDY G. W. 1905: Neue und wenig bekannte Hemiptera . Wiener Supplementary Table 1. Estimates of evolutionary divergence (unco- Entomologische Zeitung 24: 266–268.

rrected p-distance) of mtCOI sequences of six species of Gyponana . KRISHNANKUTTY S. M., DIETRICH C. H., DAI W. & SIDDAPPAJI Gr. – group; Acc. Number – GenBank accession number. The sequence M. H. 2016: Phylogeny and historical biogeography of leafhopper from the present study was collected in Italy (Acc.Number MH394187.1), subfamily Iassinae ( Hemiptera : Cicadellidae ) with a revised tribal other specimens of G. mali , G. extenda and G. striata were collected in classification based on morphological and molecular data. Systematic Canada (Ontario), G. salsa in Canada (Acc.Number starting for KR) and Entomology 41: 580–595.

USA, Minnesota (KF), G. parallela in USA (Virginia), G. cacumina in KUMAR S., STECHER G. & TAMURA K. 2016: MEGA7: Molecular Canada (KR) and USA, Pennsylvania (KF). Evolutionary Genetics Analysis Version 7.0 for bigger datasets. The.xls file is deposited on the web page of the journal (www.aemnp. Molecular Biology and Evolution 33: 1870–1874.

eu) as well as in the Zenodo scientific archive (https://zenodo.org/) at METCALF Z. P. 1962: General catalogue of the Homoptera. Fascicle https://doi.org/10.5281/zenodo.4733948. VI. Cicadelloidea. Part 3. Gyponidae. United States Department of

Agriculture, Washington, D. C., 229 pp.

References NIELSON M. W. & KNIGHT W. J. 2000: Distributional patterns and

possible origin of leafhoppers (Homoptera, Cicadellidae ). Revista BALL E. D. & REEVES J. A. 1927:Further studies on the genus Gypona Brasileira de Zoologia 17: 81–156.

and its allies (Rhynchota, Homoptera). Annals of the Entomological OMAN P. W. 1949:The Nearctic leafhoppers (Homoptera: Cicadellidae ). Society of America 20: 488–500. A generic classification and check list. Memoirs of the Entomological DE LONG D. M. 1942: A monographic study of the North American spe- Society of Washington 3: 1–253.

cies of the subfamily Gyponinae (Homoptera-Cicadellidae) exclusive OMAN P.W., KNIGHT W.J. & NIELSON M.W.1990: Leafhoppers (Ciof Xerophloea . Graduate Studies, Biological Series, Contributions in cadellidae): A Bibliography, Generic Checklist and Index to the World Zoology and Entomology. Vol. 5. Ohio State University, Columbus, Literature, 1956–1985. CAB International, Wallingford, 368 pp.

xiv + 187 pp., pls. I–XXXV. SELJAK G. 2013:Hishimonus hamatus Kuoh ( Hemiptera : Cicadellidae ): DE LONG D. M. & FREYTAG P. H. 1964: Four genera of the world a new alien leafhopper in Europe. Acta Entomologica Slovenica 21: Gyponinae : a synopsis of the genera Gypona , Gyponana, Rugosana, 123–130.

and Reticana. Bulletin of the Ohio Biological Survey 2 (3): 1–227. STAMATAKIS A. 2014: RAxML version 8: a tool for phylogenetic DIETRICH C. H. 1999: The role of grasslands in the diversification of analysis and post-analysis of large phylogenies. Bioinformatics 30: leafhoppers (Homoptera: Cicadellidae ): a phylogenetic perspective. 1312–1313.

Pp. 44–48. In: WARWICK C. (ed.): Proceedings of the Fifteenth TRIVELLONE V., KNOP E., TURRINI T., ANDREY A., HUMBERT North American Prairie Conference. Natural Areas Association, J. Y. & KUNZ G. 2015: New and remarkable leafhoppers and Bend, Oregon, 255 pp. planthoppers ( Hemiptera : Auchenorrhyncha) from Switzerland. DIETRICH C. H., DMITRIEV D. A., RAKITOV R. A., TAKIYA D. M. Mitteilungen der Schweizerischen Entomologischen Gesellschaft & ZAHNISER J. N.2005: Phylogeny of Cicadellidae (Cicadomorpha: 88: 273–284.

Membracoidea) based on combined morphological and 28S rDNA TRIVELLONE V., MITROVIĆ M., DIETRICH C. H. & TOŠEVSKI I. sequence data. In: PURCELL A. (ed.): Abstracts of Talks and Po- 2017:Osbornellus auronitens ( Hemiptera : Cicadellidae : Deltocephasters: 12th International Auchenorrhyncha Congress. 7–12 August, linae), an introduced species new for the Palaearctic region. Canadian Berkeley, California. University of California, Berkeley, 117 pp. Entomologist 149: 551–559.

Figures

Fig. 2. Gyponana mali DeLong, 1942, male (voucher I02/19). A – dorsal view; B – lateral view; C – pygofer, valve, subgenital plate, and aedeagus, lateral view; D – pygofer, valve, subgenital plate, and aedeagus, dorsal view; E – styles, connective, and aedeagus, lateral view; F – styles, connective, and aedeagus, dorsal view; G – aedeagus, ventral view. Scale bar 1.0 mm.

Fig. 3. Gyponana mali DeLong, 1942, female (voucher I03/19).A – dorsal view; B – lateral view; C – sternite VII, ventral view; D – first valvula, lateral view; E – first valvula, apical portion; F – second valvula, lateral view; G – second valvula, apical portion. Scale bar 1.0 mm.

Fig.4. Gyponana mali DeLong, 1942:A – aedeagus, caudal view (reprinted from Hൺආංඅඍඈඇ 1982); B – aedeagus, ventral view (left) and style, lateral view (right; reprinted from DൾLඈඇǤ & F*©ൾඒඍൺǤ 1964).Gyponana extenda DeLong, 1942: C – aedeagus, caudal view and style, lateral view, with structural variation from “s” to “v” form (reprinted from Hൺආංඅඍඈඇ 1982); D – aedeagus, ventral view (left) and style, lateral view (right; reprinted from DൾLඈඇǤ & F*©ൾඒඍൺǤ 1964).

Fig. 5. Maximum likelihood tree of Gyponana spp. based on mtCOI gene. Bootstrap values are shown if>50. A total of 692 positions were included in the final dataset. The branch marked with “//” was shortened, with the real length of 0.016.Gr.– group. The sequence from the present study (GenBank accession number MH394187.1) is in Gr. 4.

Fig. 6. Two collection sites of Gyponana mali DeLong, 1942 in Europe. A – ornamental plants in privately managed urban green in Switzerland (Agno, Ticino) where G. mali was accidentally collected in 2015; B – plants of Cornus sanguinea in a windbreak row (on the right) surrounding a vineyard plot (on the left) in Italy (Gorgo al Monticano, Treviso) where larvae and adults of G. mali were sampled in 2017.