A new genus of leafhopper subtribe Paraboloponina (Hemiptera: Cicadellidae) with molecular phylogeny of related genera

A new leafhopper genus Chandra and species Chandra dehradunensis gen. nov., sp. nov. are described, illustrated from India and placed in the subtribe Paraboloponina (Cidadellidae: Deltocephalinae: Drabescini). This genus is closely associated with the genus Parabolopona Webb but differs in shape of the head, placement of antennae, male genitalia and molecular analysis using Histone H3 and COI genes confirmed the difference. The taxonomic and phylogenetic position of Chandra is discussed using morphological characters and preliminary molecular evidence of the new genus and related genus Parabolopona.


Introduction
Paraboloponini, primarily distinguished by the striated fore margin of the head and long antennae situated near middle to upper corners of eyes, was subsequently placed as a subtribe Paraboloponina of Drabescini of Deltocephalinae by Dmitriev [1] and Zahniser and Dietrich [2,3]. Zahniser and Dietrich [3] revised the classification of Deltocephalinae based on the molecular and morphological data and provided a revised interpretation of Drabescini with two subtribe Drabescina and Paraboloponina, including 38 genera, out of which 36 genera belong to Paraboloponina with 132 species worldwide. Paraboloponina can be distinguished from Drabescina by the antennal ledges which are weak or absent, antennae usually longer, 1.5x width of head or longer, texture of the frontoclypeus shagreen, protibia rounded dorsally or rarely somewhat flattened, and forewing appendix broad [3].
Work on this group since Zhang and Webb [4], Webb [5], Viraktamath [6] and Meshram, et al. [7] has lead to description of many new taxa. So far, Paraboloponina contains 14 known genera, including 37 species from India. In the present work we describe a new leafhopper genus and species Chandra dehradunensis gen. nov. and sp. nov., and discuss its phylogenetic position within Drabescini, as inferred from available molecular data.

Collection of samples and morphological study
Collections were not done from any national park or other protected area of land or sea, or on any private land, hence no permission was required. No specific permissions were required for any of the collection localities/activities, as the collections were done in and around ICAR research Institutes. The field studies did not involve any endangered or protected species Specimens were collected through Mercury vapour lamp light trap from Dehradun (India: Himachal Pradesh), were processed by series of steps like sorting, cleaning and mounting.
Male genitalia dissection was carried out as described by Oman [8] and Knight [9]. 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. Afterwards, 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 stereozoom automontage microscope.
Molecular phylogenetic analyses. Histone H3 and COI sequences in FASTA format were imported into the sequence alignment application of MEGA 6.05 [13] software package and multiple sequence alignments were performed with the ClustalW [14] algorithm using default parameters. The Basic Local Alignment Search Tool (BLAST) [15] was used to query the National Center for Biotechnology Information (NCBI) non-redundant nucleotide database and protein database with other leafhopper Histone H3 and COI sequence data in blastn and blastx searches, respectively. The sequences were submitted to NCBI for GenBank Accessions (Tables 1 and 2).
Sequence divergences between selected leafhoppers were calculated using the Kimura 2-Parameter distance model [16] and graphically displayed in a neighbor-joining (NJ) tree [17] by the program MEGA 6.06 [13]. Tree robustness was evaluated by bootstrapping (Felsenstein, 1985) with 2,000 replicates with the Xestocephalus desertorum for Histone H3 and Aphrodes diminuta for COI sequence as outliers.

Nomenclatural acts
The electronic edition of this article conforms to the requirements of the amended International Code of Zoological Nomenclature, and hence the new names contained herein are available under that Code from the electronic edition of this article. 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: FAF956DF-D6C5-4153-85D5-8F2ACD572260. The electronic edition of this work was published in a journal with an ISSN, and has been archived and is available from the following digital repositories: PubMed Central, LOCKSS.

Depositories of material
Type material is deposited in the National Pusa Collection, Division of Entomology, Indian Agricultural Research Institute, New Delhi, (NPC) and University of Agricultural Sciences Bangalore (USAB), India. posteroventral margins of pygofer with spine like ventral pygofer process arising somewhat in the middle, below which small digitate lobe arises ( Fig 1G); Subgenital plate tapering from mid-posteriorly to membranous fingerlike, glabrous ( Fig 2F); Aedeagal shaft with pair of apical and middleteeth (Fig 2D-2E).  Diagnosis. External morphology, as in generic description. Male genitalia, as in generic description, pygofer with lobe (Fig 2A) broadly truncate in lateral view. Connective Y-shaped, stem and arms subequal (Fig 2C). Style with beak-like apophysis (Fig 2B). Aedeagal shaft with pair of apical and middle teeth (Fig 2D).
Etymology. The species was named after the place of collection Dehradun in Uttarakhand India.

Taxonomic position
The systematic position of Chandra dehradunensis Meshram gen. nov. and sp. nov. as suggested by morphological evidence due to the presence of a peculiar combination of characters of tribe Drabescini. This genus can be distinguished from its closely associated genus Parabolopona by the following combination of characters: vertex in dorsal view strongly concave ( Fig  1D); prothoracic femur, with AM setae present ( Fig 1E); mesothoracic femur with AD1and PD1 setae; posteroventral margins of pygofer with spine like ventral pygofer process arising somewhat in the middle, below which small digitate lobe present ( Fig 1G); subgenital plate tapering from mid-posteriorly to membranous, fingerlike, glabrous ( Fig 2F); aedeagal shaft with pair of apical and middle (Fig 2D-2E). Most of these characters also separate Chandra gen. nov. from other known deltocephaline groups. Our preliminary molecular analysis using Histone H3 gene and Cytochromne oxidase I (Figs 3 and 4) agrees with the morphological and biogeographic phylogeny of Zahniser and Dietrich (2013) and places them in the subtribe Paraboloponina of tribe Drabescini.
To test the actual phylogenetic position of the new genus in the context of the morphologically related genera of Drabescini, we performed a preliminary molecular analysis using available material of a series of taxa within Drabescini from NCBI GenBank (Table 1). Histone H3 analysis resulted to be the most closely (Parabolopona, Bhatia, Drabescus and Osbonellus) or the most distantly related (Phlogotettix, Scaphoidophyses and Xestocephalus) (S1 Table). The final data matrix of our preliminary phylogenetic analysis (Table 1) included 11 terminals (10 ingroup taxa belonging to 4 genera of Deltocephalinae and 1 outgroup taxon). The availability of mtCOI sequence information is very limited for species of Drabescini. Due to this reason we have selected Chandra gen. nov., Parabolopona, Scaphoideus, Osbornellus, Phlogotettix, Mimotettix and Aphrodes (Table 2) for phylogenetic analysis (S2 Table).
Analysis of Histone H3 revealed that the sequence variation between new genus and Parabolopona 8.80 percent (S1 Table). The other closest genera are Bhatia, Drabescus and Osbonellus, all these four genera will share the same tribe (Drebescini) and two of them Parabolopona, Bhatia sharing same subtribe (Paraboloponina). Therefore, molecular data complement the morphological data of placing new genus into Paraboloponina. Furthermore, analysis of COI data places new genus with Parabolopona zhangi and consistent with Histone H3 analysis.

Conclusions
The combined morphological and molecular analysis clearly indicate and confirm that Chandra dehradunensis Meshram gen. nov., sp. nov., is near the distinct genus Parabolopona. Our molecular analyses also suggest that there is a large variation between new genus and other related genera of tribe Drabescini. Phylogeny estimated from the molecular data for new genus follows a previous phylogeny for subfamily Deltocephalinae from Zahniser and Dietrich (2013) and places Chandra dehradunensis gen. nov., sp. nov. in the subtribe Paraboloponina. There is a need for further detailed study using more molecular data to establish intricacies within the tribe Drabescini.
Supporting information S1