A new genus Vittaliana belonging to the tribe Opsiini (Hemiptera: Cicadellidae) from India and its molecular phylogeny

The new leafhopper genus Vittalianareticulata gen. nov., sp. nov., is described from India, and placed in the tribe Opsiini based on ocelli close to eyes, without carina on anterior margin of the face and bifurcate aedeagus with two gonopores. Phylogenetic analysis with maximum likelihood (ML) using IQtree v1.4.1 of combined data (Histone H3 and 28S rDNA) reveals that the new genus Vittaliana belongs to a clade consisting of Opsius versicolor (Distant, 1908), Opsiini gen. sp., Libengaia sp., Hishimonus phycitis (Distant, 1908) and Yinfomibus menglaensis Du, Liang & Dai (2019) with good branch support, and that the tribe Opsiini is paraphyletic. This resolves the placement of a new genus in the tribe Opsiini under Deltocephalinae.


INTRODUCTION
The Cicadellide is the largest family of the suborder Auchenorrhyncha, and the Deltocephalinae is the largest and most economically important subfamily of leafhoppers, including at least 6700 described species grouped into 39 tribes (Zahniser & Dietrich, 2013;Dai et al., 2017). The tribe Opsiini is distinguished from other tribes by the face oblique, not strongly depressed, not concave in profile; anterior margin of the head without carinae; antennal bases near middle or posteroventral (lower) corner of eyes; gena not extended onto dorsum behind eyes; the stem of connective longer and bifurcated aedeagus with two gonopores (Emeljanov, 1962;Zahniser & Dietrich, 2013). This tribe is economically important as vectors of viral, bacterial, phytoplasma, and spiroplasma phytopathogens (Nielson, 2002). Zahniser & Dietrich (2013) revised the classification of Deltocephalinae based on molecular and morphological data, and provided a revised interpretation of Opsiini with four subtribes Achaeticina, Circuliferina, Eremophlepsiina, and Opsiina. These subtribes comprises of 40 genera, out of which 29 genera belong to Opsiina with more than 230 species worldwide. The subtribe Opsiina can be differentiated from the others by ovipositor not protruding far beyond pygofer apex and subgenital plates with a lateral row of macrosetae; aedeagal shafts divided near to base (Zahniser & Dietrich, 2013).

For morphological studies
Data was collected as previously described in Meshram, Shashank & Sinha (2017) specifically, in and around ICAR research institutes, Vittal, Kasargod (Kerala: India), with a mercury vapor lamp. Hence, no specific permissions were required for any of the collection localities/activities. Specimens were processed by a series of steps like sorting, cleaning, and mounting. Male genitalia dissections were carried out as described by Oman (1949) and Knight (1965) as follows, the abdomen was removed by inserting a sharp pin between the abdomen and thorax with gentle piercing. The abdomen was treated in 10% KOH for 2∼4 h to remove unsclerotized material by gently prodding the abdomen with the head of a pin. Afterward, the abdomen was rinsed thoroughly in water. The internal structures were then removed by a hooked pin, before being stored in glycerol vials for study.
Photographs were taken with a Leica DFC 425C digital camera on the Leica M205FA stereo zoom automontage microscope.
The electronic version of this article in Portable Document Format (PDF) will represent a published work according to the International Commission on Zoological Nomenclature (ICZN), and hence the new names contained in the electronic version are effectively published under that Code from the electronic edition alone. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix http://zoobank.org/. The LSID for this publication is: urn:lsid:zoobank.org:pub:228D17FC-590C-41C7-9434-60554F753DBA. The online version of this work is archived and available from the following digital repositories: PeerJ, PubMed Central, and CLOCKSS.

DNA extraction and PCR amplification
The DNA was extracted from the head and thorax of specimens according to manufacturer protocols using DNASure R Tissue Mini Kit. The isolated DNA was stored at −20 • C until required. The amplification of the desired product was done with the help diagnostic PCR reactions, using universal histone H3 primers: HEXAF (forward) 5 -ATGGCTCGTACCAAGCAGACGGC-3 and HEX-AR (reverse) 5 -ATATCCTTGGGCATGATGGTGAC-3 (Ogden & Whiting, 2003) and 28S rDNA primers (for D2 region 5 -AGTCGKGTTGCTTGAKAGTGCAG-3 & 5 -TTCGGGTCCCAACGTGTACG-3 ) and for D9-D10 region 5 -GTAGCCAAATGCCTCGTCA-3 &5 -CACAATGATAGGAAGAGCC-3 (Dietrich et al., 2001). The PCR protocol for Histone H3 was followed from Zhaniser & Dietrich (2010) under the following cycling protocol: 4 min at 94 • C, 35 cycles of denaturation for 30 s at 94 • C, annealing for 60 s at 47 • C, elongation for 50 s at 72 • C and a final extension 72 • C for 8 min in a C1000 TM Thermal cycler. The PCR reactions consist of 12.5 µl hot start PCR master mix (Thermo Scientific), 8.5 µl of molecular grade water, 1 µl each forward and reverse primer and 2 µl of genomic DNA (Hashmi et al., 2018). The products were checked on 1% agarose gel and visualized under UV using a gel documentation system (DNr, Bio-Imaging, MiniLumi). The amplified products were sequenced at AgriGenome Pvt. Ltd. (Cochin, India). The quality sequences were assembled with BioEdit version 7.0.0 and deposited in NCBI GenBank.

Alignment and phylogenetic analyses
For phylogenetic analysis, the majority of species sequences were taken from Zahniser & Dietrich (2013) and Zhaniser & Dietrich (2010) and Du, Liang & Dai (2019). A dataset consisting of the newly sequenced taxa and 76 sequences of Deltocephalinae species. The outgroups consist of two species from Aphrodinae and one species from Euacanthellinae ( Table 1).
The histone H3 and 28S rDNA sequences aligned separately with the MUSCLE application in MEGA 6 (Tamura et al., 2013a;Tamura et al., 2013b). The aligned sequences of the two gene regions were concatenated into one dataset using Sequence Matrix 1.7.8 (Vaidya, Lohman & Meier, 2011) and obtained NEXUS data block for combined data set as follows: #NEXUS begin data; dimensions ntax = 80 nchar = 6974; format datatype = dna; gap = −; missing = ?; matrix; end; The NEXUS file used in the phylogenetic analysis deposited in public repository TreeBASE (Study ID S26664; https://treebase.org).

MN816385
(continued on next page)  (Anisimova et al., 2011) were used in the analysis to assess branch support and obtained tree was visualized in FigTree v1.4.2.

Diagnosis
This genus is placed in the subtribe Opsiina of Opsiini based on all these characters macropterous; ovipositor not protruding far beyond pygofer apex and subgenital plates with a lateral row of macrosetae; aedeagal shafts divided near to base. The new genus can be differentiated from all related genera in this subtribe by a combination of the following characters: Body and face whitish with yellow, and brown mottling; anterior margin of crown with five white patches, slightly produced, slightly concave; pronotum with concave posterior margin; aedeagal shafts arising from near to base, aedeagus distinctive outward curved apically, without processes arising from base, with a pair of medial processes arising from mid-length of shafts, apex without processes.

Description
Colour. Body and face whitish with yellow and brown mottling. Crown anterior margin with five white patches (Fig. 1D). Pronotum brown mottled with white patches. Scutellum yellow with an orange basal triangle (Fig. 1D). Eyes red; forewing white speckled with brown patches (Fig. 1B). Body length. Male 3.6 mm long; 1.4 mm wide across eyes. Female 3.8 mm long; 1.46 mm wide across eyes. Head. Anterior margin of head slightly produced, head in dorsal view as wide as pronotum; crown length 2/3rd as long as median length of the pronotum, anterior margin produced with concave posterior margin; face with brown and white irregular mottling; ocelli small, close to eyes on anterior margin of crown; clypellus 2.8x as long as wide; gena obtusely incised laterally (Fig. 1C). Thorax. Pronotum anterior margin convex, 2x as broad as long, hind margin slightly concave; scutellum 1.5x as broad as long, and 0.6x as long as width of pronotum, with distinct scutoscutellar suture (Fig. 1D). Forewing elongate, veins raised, three subapical and four apical cell, claval vein raised with 2 crossveins, appendix extended around the apex (Fig. 1I). Hind wing veination complete, appendix broad (Fig. 1J). Legs. Prothoracic femur with AM1 seta only; intercalary row with one row of more than 5 fine setae; AV row with 2-3 macrosetae, AV1 seta long; AD setae small and sparsely arranged. Prothoracic tibia on dorsal surface rounded, AD row with 4-5 setae long, distributed widely; AV setae moderately dense and long (Fig. 1F). Mesothoracic femur with AD setae small; AV row with basal half short setae and rest with macrosetae; AM seta present; intercalary row with one row of more than 5 fine setae (Fig. 1G). Metathoracic femur with setal formula 2+2+1; lateral surface area broadened distally; metathoracic tibia flattened, tibial row AD setae long and densely arranged, PD with long macrosetae placed equidistantly, AV setae small and densely arranged, PV with macrosetae moderately arranged, Metatarsomere I length equals to tarsomere II and tarsomere III combined (Fig.  1H). Male genitalia. Pygofer longer than wide with macrosetae in midlateral region, with anal tube long, 3/4th membranous from the base ( Fig. 2A). Valve triangular with broad base (Fig. 2B). Subgenital plate triangular, basally broad, posterior half gradually narrowed towards apex, with 7-8 submarginal macrosetae, 7 microsetae medially on distal 2/3rd (Fig. 2F). Style broadly bilobed basally, subapical angle not prominent with few setae (Fig. 2C). Connective Y-shaped, stem 1.3x as long as arms (Fig. 2G). Aedeagus with well-developed dorsal apodeme, with a pair of medial processes arising from mid-length of shafts, gonopores subapical (Fig. 2D). Female genitalia. Female seventh sternite trapezoid in shape, sternite 2x as wide as median length, hind margin with sinuate with shallow notch medially (Fig. 3I); first pair of valvulae wider beyond the base and narrowed at apex, with irregular sculpture on apical 1/2th, dorsal hyaline area restricted to basal half (Fig. 3F); second pair of valvulae, with small teeth and sculpting on apical half (Fig. 3H).

Etymology
The species name, ''reticulata'' is based on the reticulated forewing venation.

DISCUSSION
The most important diagnostic character to the tribe Opsiini is the presence of bifurcate aedeagus with paired shafts and gonopores, although this character is also found in Alocoelidia of the tribe Acostemini (Zahniser & Nielson, 2012) and some genera of the tribe Scaphytopiini and Mukariini (Zahniser & Dietrich, 2013). Vittaliana gen. nov. the best fits into the tribe Opsiini because it lacks the diagnostic morphological characters that define the above tribes, i.e., Mukariini have a depressed body form, with the face nearly the horizontal and the anterior margin usually transversely carinate, and Scaphytopiini has the head strongly produced with gena extended onto dorsum behind eyes (Du, Liang & Dai, 2019). Vittaliana gen. nov. has the face oblique, not strongly depressed in profile, the stem of the connective is long, as in many other Opsiini and its aedeagal shafts arising from near to base, aedeagus distinctive outward curved apically, without processes arising from the base with a pair of medial processes arising from mid-length of shafts, apex without processes, and gonopore open subapically.  (1), this confirms the species of Opsiini resolve as paraphyletic. Our study is not consistent with the previous phylogenetic study of new genus Yinformibus Du, Liang & Dai (2019) based on combined Histone H3 and 28S rDNA resolve tribe Opsiini as monophyletic with moderate bootstrap support (85%). In contrast, our study combined dataset of histone H3 and 28S rDNA resolve the tribe opsiini as paraphyletic with low UFB (>50) and aBayes (1) which may be due to the addition of more members of Opsiini in the phylogenetic analysis may diverge as paraphyletic. However, our study consistent with the previous phylogenetic analysis of Deltocephalinae including combined data from the 28S rDNA, Histone H3 and morphological data (Zahniser & Dietrich, 2013) with approximately similar (<50% ML) bootstrap but the more detailed phylogenetic analysis is needed. Inclusion of more species of Opsiini and more gene addition may resolve the relationship of Opsiini in highly diverse Deltocephalinae.

CONCLUSION
The present study reveals that new genus Vittaliana reticulata gen. nov., sp. nov. belongs in the tribe Opsiini and subtribe Opsiina by morphological characters and molecular phylogenetic analysis. This genus differs from closely related genera by morphological characters and based on available molecular data analysis establish that this genus and species was closely related to the type genus of Opsiini and also indicated that the tribe Opsiini is paraphyletic. However further study is needed by adding more genes to see its evolutionary significance.
Type materials are deposited in National Pusa Collection, IARI, New Delhi, with repository number RRS2, RRS3, RRS4 and RRS5.

Supplemental Information
Supplemental information for this article can be found online at http://dx.doi.org/10.7717/ peerj.9515#supplemental-information.