Morphological and molecular data reveal a new genus of the tribe Issini from Southern China (Hemiptera, Fulgoromorpha, Issidae)

Abstract A new genus Sinonissus gen. n. of the tribe Issini (Issidae, Issinae) with a new species Sinonissus brunetus sp. n. from Chongqing municipality and Sichuan Province, China are described. Barcode of the species is provided. A molecular analysis combined with morphological characters confirms its placement into the Issini. Distribution of this new genus in the Oriental realm is briefly discussed in regard of other Issinae taxa in China.

The lineage was firstly separated as a subtribe Issina Spinola, 1839 by Gnezdilov (2002: 609), later abandoned (Gnezdilov 2016a: 343), but confirmed valid by different molecular analyses (Gnezdilov et al. 2015, Wang et al. 2016) and treated as a separate tribe of Issinae by Wang et al. (2016). In this last treatment, Issini is typically distributed in the Wallace Palaearctic (western area particularly) and Oriental regions, and more precisely into Holt's (2013) Oriental, Sino-Japanese, Palaearctic, and Saharo-Arabian zoogeographic realms (Gnezdilov et al. 2015, Bourgoin 2018. Issus and Latissus genera are natively distributed in the Mediterranean area of the Western Palaearctic region (Gnezdilov et al. 2014). They form the "Issus group" which is thought to have diverged early from the other Western Palaearctic taxa and was one of the first groups to colonise the proto-Mediterranean communities of the ancient Mediterranean in the Eocene (Gnezdilov 2016a(Gnezdilov , 2016b. Issini are characterised by the presence of paired digitate processes on the inner side of the dorsolateral lobes of the periandrium (Gnezdilov 2016a), and not exclusive to the lineage, the veins run in parallel, and according to the schema "R2, M2, CuA2" until the tip of the tegmina (Gnezdilov 2003).
A new genus Sinonissus gen. n. is described from southwest China in the Oriental realm, represented by the new species Sinonissus brunetus sp. n. from Chongqing and Sichuan, for which both morphological data and molecular phylogeny place the taxon into the Issini sec. Wang et al. (2016).

Materials and methods
Type specimens are deposited in College of Life Science, China West Normal University, Nanchong, Sichuan Province, China. The abdomen of specimen was separated from the body, and then boiled in a 10% NaOH solution for 5 minutes until muscles were completely dissolved leaving tegumentary structures. After rinsing in distilled water for several times, the abdomen was subsequently transferred to glycerine for final dissection and observation. Terminalia were conserved under the specimen in genital vials. Photographs for external morphology and terminalia characters were taken using Leica DFC495 camera attached to Leica M205C stereomicroscope and further refined with LAS V3.8 and Helicon Focus v3.10 software. Morphological interpretations and subsequent terminologies for male genitalia follow Bourgoin (1987), for female genitalia Bourgoin (1993) and for wing venation Bourgoin et al. (2015).
The total genomic DNA was extracted from leg of holotype specimen (♂) using the TransGen EasyPure Genomic DNA Kit. COI gene was amplified using the same primers and amplification procedure as Wang et al. (2016). The DNA sequencing was conducted at Sangon Company (Shanghai, China). Software Seqman from package DNAstar v5.01 (www.dnastar.com) was used for checking sequence chromatograms and assembling contigs. Mega v7.0 (Kumar et al. 2016) was used for performing alignments. IQtree v1.4.1 (Nguyen et al. 2015) was used for maximum likelihood phylogenetic analysis using 10000 ultrafast bootstrap (Minh et al. 2013) (Gnezdilov et al. 2011, fig. 4;Gnezdilov et al. 2014, figs 13d-f ) in general appearance, but differs by: 1) vertex without median carina but with carina in Latissus; 2) tubercles on frons very tiny and obscure but large and elevated in Latissus; 3) Pcu and A 1 fused at basal half of clavus but fused at apical 1/3 in Latissus.
Etymology. This name is derived from the Latin prefix word "sino" freely associated with the generic name "Issus", referring to the special distribution of this genus representing the rarity of Issini in China. The gender is masculine.
Remarks. The new genus differs from Issus by the presence of a wide hypocostal plate, also present in Latissus, and from both genera by its rudimentary hindwings. It shows that this last character is not characteristic of the tribe, for which the diagnosis should be modified accordingly. Additionally, the Issina forking schema 'R2, M2, CuA2' indicating the number of main vein terminals in the tegmen appears to be modified in Sinonissus with two to four terminals in CuA: accordingly a 'R2, M2, CuA(2-4)' schema should be retained for Issini sec. Wang et al. (2016)'s diagnosis. In reverse, Sinonissus shares particularly with them the presence of paired digitate processes on the dorsolateral lobes of periandrium, two lateral and 5-9 apical metatibial spines. Molecular phylogeny analysis confirms also the placement of the taxon as sister to the two others, according to the schema (Sinonissus + (Latissus + Issus)) with node value of 90 (Fig. 19). Diagnosis. This new species looks similar to Latissus dilatatus (Fourcroy, 1785), but differs by: frons much longer, 1.2 times longer in midline than broad at widest part (only 0.9 times in L. dilatatus); anal tube of male 1.4 times longer in midline than widest part (2.2 times in L. dilatatus); male genitalia less robust, the digitate processes near apex of periandrium slender and curved (broad and straight in L. dilatatus). Coloration. Vertex brown, margins carinated and dark brown (Fig. 1). Compound eyes dark grey, supported by tawny callus (Fig. 1). Frons brown, apical and lateral margins carinated and dark brown, brown median carina extending from apex near to base, but not reaching the frontoclypeal sulcus (Figs 3, 16); lateral area of frons with some tawny inconspicuous tubercles on each side near the lateral margins (Figs 3, 16). Postclypeus brown, rostrum light brown (Fig. 3). Gena brown (Fig. 2). Antennae dark brown (Fig. 3). Pronotum brown, margins carinated and dark brown, lateral area with three unconspicuous light yellow tubercles on each side (Fig. 1). Mesonotum brown, lateral carinae dark yellow (Fig. 1). Forewings brown, longitudinal veins dark brown and transverse veins grey (Figs 1, 2). Legs brown (Figs 2, 3).
Female terminalia. Anal tube in dorsal view ovate, widest at middle, 1.2 times longer in midline than widest part, apical margin and lateral margins rounded; epiproct long, approximately 1/3 length of anal tube, anal opening situated at basal 1/4 (Fig. 8). Anterior connective lamina of gonapophysis VIII with two or three teeth at apex and three keeled teeth on the outer lateral margin, inner lateral margin without teeth (Fig. 14). Endogonocoxal process developed, slightly sclerotised in basal half and membranous in distal one (Fig. 14), apex of endogonocoxal process with two-digitate processes. Posterior connective lamina of gonapophysis IX in lateral view long and narrow, boat-shaped, tip pointed, dorsal margin roundly convex at base (Fig. 12); in dorsal view basal half broader than apical half, the apical half narrower to apex in outer lateral margins, bifurcate at apical 1/3 in inner part, basal half with outer margins nearly parallel, lateral area sclerotised (Fig. 11). Gonospiculum bridge small and short, in lateral view rectangular with needle-like ventrally (Fig. 12). Gonoplacs fused near base, outer lateral margins roundly convex (Fig. 10), in lateral view rectangular (Fig. 9).
Etymology. The Latin name brunetus, referring to the dark brown colour of the general appearance of this species.

Discussion
The sub-family Issinae currently includes two tribes: Issini and Hysteropterini . They are characteristically distributed in Wallace's Palaearctic region (Bourgoin 2018), with exceptional distributions from Palaearctic Africa (= Saharo-Arabian Holt's 2013 realm) to Afrotropical or Oriental regions (= Sino-Japanese and Oriental Holt's 2013 realm). However, most true Issinae's non-Palaearctic occurrences need confirmation as these records are based on old observations, probably misidentified, or still not formally correctly assigned to the correct tribe (e.g., Eupilis Walker, 1857 in the Hysteropterini was shown to be close to Tempsa in the Sarimini by Gnezdilov 2016c).
In China, Issinae are rare but Hysteropterini were already reported from Xinjiang, Ningxia, Qinghai, Inner Mongolia and Gansu: Celyphoma Emeljanov, 1971 by Meng and Wang (2012), Chen et al. (2014) and Sichuan: Hysteropterum boreale = Potaninum boreale (Melichar, 1902) by Gnezdilov (2017). Issini have also been reported from Hong Kong (Issus quadriguttatus Walker, 1851 = Issus coleoptratus (Fabricius, 1781) by Gnezdilov et al. (2004). Sinonissus is therefore the second genus of the Issini to be reported from China. It appears to be a new genus of this rare group of taxa which has crossed Palaearctic and Sino-Japanese realm frontiers to evolve into the Oriental realm in the southwest areas of Chongqing and Sichuan in China. Gnezdilov (2016a: 333 and fig. 43) supposed that the Issina lineage (sec. Gnezdilov, 2002 = Issini sec. Wang et al. 2016) had diverged early in the tree of the Western Palaearctic taxa as sister taxa to all other western Palaearctic genera, being one of the first groups to colonise the proto-Mediterranean communities of the ancient Mediterranean in the Eocene (Gnezdilov 2016b). The discovery of Sinonissus in the Issini lineage shows that radiation of Issinae in the Palaearctic is probably more complex than expected. Most Issinae genera still need to be molecularly tested to enable their possible placement in the phylogeny and is probably that other genera should join this tribe Issini to fill gaps in this paradoxical geographical distribution.