Tribe reassessment of the subhimalayan leafhopper genus Pseudosubhimalus (Homoptera: Cicadellidae) based on molecular phylogeny

The phylogeny of the Pseudosubhimalus were investigated using of two different data sets, including 91 taxa and 3853 aligned nucleotide positions from the histone H3, 28S rDNA (D2 & D9–10 region). The results suggest the placement of genus in the tribe Ciacadulini, as it was clustered with Cicadulini genera. Relationships between genera of the Cicadulini were strongly supported and leads placement to tribe Cicadulini from Athysanini. Along with this, genus Pseudosubhimalus Ghauri is revised, and P. trilobatus sp. nov. (Himachal Pradesh: Katrain) is added, described from Indian subcontinent and deposited to National Pusa Collection, IARI, New Delhi, with repository number RRS1.


INTRODUCTION
Genus Pseudosubhimalus are medium sized, ochraceous to dark brown with black spots on crown, distributed in Himalayan and Subhimalayan region. The species of genus were established by Ghauri (1974), with type species Ophiola bicolor Pruthi (Pruthi, 1936), as a replacement name for Ophiola Edwards. This genus was replaced to present name due to its general coloration which superficially resembles Subhimalus Ghauri (Ghauri, 1971). This genus was earlier placed in the tribe Deltocephalini (Oman, Knight & Nielson, 1990) of Deltocephalinae. Recently, Zahniser & Dietrich (2013), based on the molecular and morphological aspects of Deltocephalinae, placed it in the tribe Athysanini. Pseudosubhimalus is recorded from higher altitudes (8,000-12,000 ft), and mostly feed and breeds on grasses. This genus is distinguished from its related genera by aedeagal shaft with a pair of subapical processes; subgenital plates triangular, apophysis of style slender, tapering distally. Pruthi (1936) described the two species P. bicolor from India and P. yatungensis from Tibet.
In spite of the colour patterns and male genitalia character that characterize Pseudosubhimalus, the composition and placement of the genus have been problematic. Molecular data potentially provide numerous additional characters useful for phylogenetic How to cite this article Niranjana GN, Meshram NM, Shashank PR, Stuti, Hashmi TR. 2019. Tribe reassessment of the subhimalayan leafhopper genus Pseudosubhimalus (Homoptera: Cicadellidae) based on molecular phylogeny. PeerJ 7:e7162 http://doi.org/10.7717/peerj.7162 hypotheses. Zahniser & Dietrich (2013) revised the classification of Deltocephalinae based on the molecular and morphological data and provided a revised interpretation of tribe Athysanini and placed this genus under this tribe. Here, we replaced Pseudosubhimalus to the tribe Cicadulini based on histone H3, 28S rDNA (D2 & D9-10 region). Along with this morphological characterization of Pseudosubhimalus with a new species from India is provided.

Collection of samples & 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 traps from different location in Himachal Pradesh, India were processed by a series of steps such as sorting, cleaning, and mounting. Male genitalia dissections were carried out as given by Oman (1949) and Knight (1965). The abdomen was removed by inserting a sharp pin between the abdomen and thorax and with gentle piercing. The abdomen will be 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.
Type material is deposited in National Pusa Collection, IARI, New Delhi, with repository number RRS1.
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:DB2CE2D9-1238-4199-96A4-A7FDCD98DD8B. 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
Total genomic DNA, from the legs of each specimens, were extracted with the help of the DNA Sure Tissue Mini Kit, following the manufacturer protocol. The extracted DNA was stored at −20 • C for further processing. The amplification of the desired product was done with the help diagnostic PCR reactions, using universal Histone H3 primers (HEX-AF 5 -ATGGCTCGTACCAAGCAGACGGC-3 and HEX-AR 5 -ATATCCTTGGGCATGATGGTGAC-3 ) (Ogden & Whiting, 2003) and 28S rDNA primers (for D2 region 5 -AGTCGKGTTGCTTGAKAGTGCAG-3 & 5 -TTCGGGT CCCAACGTGTACG-3 ) and for D9-D10 region 5 -GTAGCCAAATGCCTCGTCA-3 & 5 -CACAATGATAGGAAGAGCC-3 (Dietrich et al., 2001). The PCR protocol for Histone H3 followed Zahniser & Dietrich (2010) and 28S gene was amplified in 25 µl reactions using DNA polymerase (Fermentas GmBH, St. Leon-Rot, Germany) under the following cycling protocol: 4 min. hot start 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 was carried out at 72 • C for 8 min in a C1000 TM Thermal cycler (Meshram, Shashank & Sinha, 2017). The reactions were combined (as described by KOD FX puregene TM manufacturer protocol) of DNA template 4 µl, 2× PCR buffer 12.5 µl, 2 mM dNTP 10 µl, TAQ (KODFX) enzyme 1 unit, and forward and reverse primers were 0.3 µM each at final concentration. The products were checked on 1% agarose gel and visualized under UV using Alphaview R software version 1.2.0.1. 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 (Table 1).

Pseudosubhimalus Ghauri
Head including eyes 1.09× width of pronotum (Fig. 1A), in dorsal view obliquely rounded in front, crown length 0.35× width across eyes; face length 0.9× width of face. Ocelli near anterior margin of crown and distance between eye and ocellus equal to diameter of ocellus. Pronotum 0.5× as long as width and 0.8× length of scutellum. Genitalia. Male. Pygofer ( Fig. 2A) is longer 1.4× than broad, with long setae on posterior half, dorsal and ventral posterior margin with minute serrations. Subgenital plate (Fig. 2B) triangular with wide base, sharply narrowed posteriorly, with setae 10 along lateral margin. Connective Y-shaped (Fig. 2F) with stem shorter 0.6× than arm. Style (Fig. 2C) broad at base with well-developed preapical lobe, apophysis long, slender, with blunt apex, 0.33 of total style length. Aedeagus in lateral view (Fig. 2E) narrowed basally, pointed apically and moderately broad medially with two small traingluar subapical processes. Gonopore subapical at the base of the processes. Diagnosis. P. trilobatus sp. nov. resembles P. bicolor (Pruthi) in coloration and external morphology but can be distinguished by certain male genitalia characters like pygofer dorsal and ventral posterior margin without serrations. Aedeagal shaft narrowed apically, with trilobed apex in dorsal view. Gonopore subapical placed above base of the processes. Description. Male: Dark brown color, Crown, pronotum and scutellum marked with irregular dark brown spot. Face completely black without any marking. Compound eyes black with reddish tinge and ocelli orange color. Forewings dark brown with hyaline venations with dark brown mottling.
Head including eyes 1.1× width of pronotum (Fig. 1C), in dorsal view obliquely rounded in front, crown length 0.35× width across eyes; face length 0.87× width of face. Ocelli near anterior margin of crown and distance between eye and ocellus equal to diameter of ocellus. Pronotum 0.5× as long as width and 0.8× length of scutellum. Genitalia. Male: Pygofer is longer than broad (Fig. 4A), posterior margin conically rounded, macrosetae confined to posterior half, dorsal and ventral posterior margin without serrations. Valve broadly triangular (Fig. 4B), subgenital plate long (Fig. 4B), triangular uniseriate macrosetae and fringe of very long fine setae along. Connective Y-shaped (Fig. 4F); stem narrowed, 0.6× smaller than arms. Style broad basally (Fig. 4C), apophysis long, slender, with blunt apex, 0.33 of total style length. Aedeagus, in lateral view (Fig. 4D), broadly C-shaped, shaft narrowed apically, with trilobed apex in dorsal view, with two subapical processes, length of process is 0.33 of total length in dorsal view (Fig. 4E). Gonopore subapically placed above base of the processes. Measurement (mm).

Molecular analysis
Maximum Parsimony Bootstrap analysis of the 91 taxa and 3853 aligned nucleotide positions from the histone H3 and 28S rDNA gene (D2 & D9-10 region) by PAUP*4.0b10 was done and yielded strict consensus tree (Fig. 5). The genus Pseudosubhimalus, which was sister to Elymana well-supported clade (>50 MPBS). This suggest the placement of the genus in the tribe Cicadulini. Our combined analysis resulted to be the most closely (Cicadula, Proceps, and Elymana), which resolved the placement of the genus to the tribe Ciacaulini from Atthysanini.
In Pseudosubhimalus anal tube elongate, long, well sclerotized which strongly suggest the placement of this genus to tribe Cicadulini. To confirm actual phylogenetic position of the Pseudosubhimalus in the context of the subfamily Deltocephalinae, we performed a preliminary molecular analysis (Maximum Parsimony Bootstrap analysis) by using available material of a series of taxa belonging to different tribes of Deltocephalinae from NCBI GenBank (Table 1). Our molecular results exclude Pseudosubhimalus from a close relationship among the genera of the previously placed tribe Athysanini (Zahniser &