Parahiraciini (Hemiptera, Fulgoromorpha, Issidae): one new genus, two new species and three new subtribes

Abstract A new genus Pusulissusgen. nov. with two new species P. phiaoacensissp. nov. and P. coronomensissp. nov. are described respectively from Vietnam and China in the tribe Parahiraciini of the family Issidae. A molecular phylogeny using combined genes (18S, 28S, COX1 and Cytb) confirms its placement in the tribe Parahiraciini. The tribe is divided into three subtribes: Scantiniinasubtribe nov. with the type genus Scantinius Stål, 1866, and Vindilisinasubtribe nov. with type genus Vindilis Stål, 1870 plus Nisoprincessa Gnezdilov, 2017, and the nominal subtribe Parahiraciina Cheng & Yang, 1991 subtribe nov. The characteristics of these subtribes are provided, with a key to identification. Genus Folifemurum Che, Zhang & Wang, 2013 is transferred to HemisphaeriiniMongolianina, and genus Gelastyrella Yang, 1994 is maintained as a valid name.

In fact, in some 15 years since Parahiraciini was recognized again by Gnezdilov (2003), the tribe shows already a rather complex history with up to 26 genera with 82 species (Bourgoin 2020). In this study we retain 26 genera including the new one described. Figure 1 summarizes this already rich and complex chronological account of Parahiraciini generic taxa.
In addition to this synthetic taxonomic review, we also describe a new genus (already mentioned in Wang et al. (2016)'s molecular phylogeny under the name 'Gen. nov. apud Tetricodes') with two new species from Vietnam and China. A molecular phylogeny, using the combined genes (18S, 28S, COX1 and Cytb) of the already sequenced taxa, allows us to place this new genus into Parahiraciini and to briefly discuss this placement. Moreover, the large diversity of the taxa now included in the tribe also allows us to better clarify its morphological characteristics. Accordingly, the tribe is divided into three subtribes: Scantiniinia subtribe nov., Vindilisina subtribe nov. and the nominal subtribe Parahiraciina Cheng & Yang, 1991 subtribe nov., for which morphological characteristics are compared.

Materials and methods
Morphological interpretations and subsequent terminologies follow Bourgoin (1987) and Bourgoin (1993) respectively for male and female genitalia and Bourgoin et al. (2015) for wing venation. The metatibiotarsal formula provides the number of spines on the side of the metatibia -apex of metatibia/apex of first metatarsomere/apex of second metatarsomere. Issidae classification follows Wang et al. (2016)'s phylogeny, adapted in Bourgoin et al. (2020).
All the type specimens of the species described below are deposited in the Museum national d'Histoire naturelle (MNHN), Paris, France or in the China West Normal University (CWNU), Nanchong, Sichuan Province, China.
For genitalia study, the abdomen was separated from the specimen using microscissors, and then boiled in a 10% NaOH solution for several minutes until the muscles were completely dissolved leaving only tegumentary structures. Final dissection and observation of the abdomen was done in glycerin after rinsing in distilled water  Cheng & Yang, 1991. Box colors follows FLOW (Bourgoin 2020) standard colors with pale blue: taxon protonym and green: accepted taxon. Left arrows indicate when a genus was included in the tribe and right ones when excluded. Red and blue arrows refer respectively to taxa excluded or included in Parahiraciini. several times. Genitalia were finally stored in genitalia vials under the corresponding specimen. Photographs of external morphology and genitalia characters were taken using a Leica DFC camera attached to a Leica M205FA stereomicroscope and further refined with the software LAS X.
Total genomic DNA was extracted from the fore or middle leg from a paratype of Pusulissus phiaoacensis sp. nov. and the holotype of Pusulissus coronomensis sp. nov. using a Sangon Ezup column animal genomic DNA purification kit. The DNA of the genes (18S rRNA, 28S rRNA, COXI, Cytb) was amplified using the same primers and amplification procedures as in Wang et al. (2016). DNA sequencing was conducted by the Sangon Company (Shanghai, China). Contigs assembly was made using the software Seqman from package DNAstar v5.01 (www.dnastar.com). All sequences obtained in this study were registered in GenBank with accession numbers mentioned below. MEGA v7.0 (Kumar et al. 2016) was used for performing alignments for a subset of Parahiraciini taxa already analysed in Wang et al. (2016) plus the specimen of Rhombissus sp. analysed in Zhao et al. (2019). Four species (two species in Sarimini and two species in Hemisphaeriini) were chosen as outgroups for the analysis. The related taxon name, collecting location, and GenBank accession numbers are in Table 1. Phylogenetic analysis was performed using the software MrBayes v. 3.2.4 (Ronquist et al. 2012) using the same method as Wang et al. (2016)  Diagnosis. This genus is similar to Tetricodes Fennah, 1956, from which it differs as follows: 1) The absence of the frontal black median tubercle on the disc of frons (Figs 5,28); 2) Median carina of frons well distinct, extending from the dorsal margin almost to the frontoclypeal suture (Figs 5,28), while it is only present in the dorsal part of the frons or invisible in Tetricodes (Zhang and Chen 2009, fig. 21); 3) The forewing distinctly broadest in the basal 1/3 (Figs 6,29), while regularly convex in Tetricodes (Zhang and Chen 2009, fig. 4). This genus is also very similar to Thabena Stål, 1866, but differs by 1) Its vertex with anterior margin in dorsal view very slightly angularly convex (Fig. 4) or straight (Fig. 27), while roundly convex in Thabena (Chen et al. 2014, figs 2-74C); 2) Frons much longer, more than 1.2 times longer in midline than widest part (Figs 5,28), but wider than long in Thabena, less than 0.9 times longer in midline than widest part (Chen et al. 2014, fig. 2-74E).
Etymology. The name is an arbitrary association from the Latin word "pusulosus" which means 'pustulous', referring to the tubercles or pustules present on the frons and "issus" referring to the family. The name is treated as masculine. Coloration. For the dry specimens, general coloration tawny (Fig. 2). Vertex tawny, with two brown circular markings near base of disc (Fig. 4). Compound eyes black, supported by tawny callus (Figs 4,5). Frons brown, dorsal part black (Fig. 5); dorsal and lateral margins carinated and brown, median carina brown (Fig. 5). Frons with two tawny round markings in middle area, dorsal and lateral areas distributed with around ten tawny tubercles on each side (Fig. 5). Antennae brown (Fig. 5). Postclypeus brown (Fig. 5). Genae tawny (Fig. 3). Pronotum tawny, median area with two small brownish impressions on disc (Fig. 4), paranotal lobes tawny (Fig. 5). Mesonotum tawny, with two brown longitudinal bands on disc (Fig. 4). Forewings tawny with veins tawny, basal part of costal area with some irregular black markings, area from basal 1/3 of costal margin extending to middle of forewing surface also with some irregular black markings, apical margin with black linear marking (Fig. 6); some specimens with these black markings almost invisible, but with one extremely large whitish round marking at basal 1/3 of forewing on each side (Fig. 2). Hindwings brown, veins clearer (Fig. 7). Legs tawny (Figs 3,5). For alive and fresh specimens, vertex, pronotum, mesonotum and forewings interspersed with some reddish markings or coloration as mentioned above  Head and thorax. Vertex 2.1 times wider than long in midline, anterior margin very slightly angularly convex (Fig. 4). Frons 1.3 times longer in midline than widest part, 1.3 times wider at widest part than dorsal margin (Fig. 5). Pronotum 1.4 times longer in midline than vertex in midline (Fig. 4). Mesonotum 2.3 times wider along anterior margin than long in midline (Fig. 4). Forewings 2.1 times longer at longest part than widest part (Fig. 6). Hindwing with apical margin of CuP-Pcu-A1 lobe sinuate (Fig. 7). Metatibiotarsal formula: 2-8/5/2.  Male genitalia. Anal tube in lateral view with lateral lobes well visible (Fig. 8); in dorsal view mushroom shaped, widest at middle, as long in midline as widest part, apical margin angularly rounded, lateral margins strongly angularly rounded (Fig. 9); anal opening located near middle of anal tube (Fig. 9). Pygofer in lateral view 2.5 times higher than wide, dorsal margin obviously sloping to the posterior, dorso-lateral angle rounded, posterior margin strongly roundly convex (Fig. 8). Gonostylus in lateral view with dorsal margin slightly sinuate (Fig. 10). Capitulum of gonostylus derived from middle of gonostylus, broad, sharp triangular, directed to anterior; meniscate processes with one side not reaching to anterior margin of capitulum and another side beyond posterior margin of capitulum (Fig. 10). Periandrium in lateral view with dorso-lateral lobe rounded apically (Figs 11, 13). In posterior view, ventral lobe of periandrium with apical margin mostly straight but spinous protruded at middle (Fig. 14). Lateral processes of aedeagus long and slender, hooked, derived from the middle, directed anteriorly to basal 1/4, then curved upward, surpassing the dorsal margin of periandrium (Figs 11,13); in ventral view this pair of processes curved outwards (Fig. 12).

Pusulissus phiaoacensis
Female genitalia. Anal tube in dorsal view broadly ovate, 1.1 times longer in midline than widest part, widest at middle, apical margin nearly straight, lateral margins rounded, anal opening situated slightly below middle (Fig. 15). Anterior connective lamina of gonapophysis VIII with two obscure large teeth in the apex and four small keeled teeth in outer-lateral group (Fig. 20). Gonocoxa VIII subquadrangular (Fig. 20). Hind margin of sternite VII roundly concave, with the median part nearly straight (Fig. 19). Etymology. The name refers to the locality where the new species was found. Habitat. The species was swept from pteridophytes at the margin of the mountainous rainforest (around 1050 m altitude) beside a road (Fig. 24).
Coloration. General appearance brown (Figs 25, 26). Vertex tawny, with two brown circular markings near the base, midline brown, margins black (Fig. 27). Compound eyes brownish dark, supported by tawny callus (Fig. 27). Frons brown, dorsal part black (Fig. 28); dorsal and lateral margins carinated by black, median carina brown (Fig. 28). Frons with two yellowish round markings in middle area, apical and lateral areas distributed with around twelve yellowish tubercles on each side, basal part yellow (Fig. 28). Antennae brown (Fig. 28). Postclypeus brown mixed with some tawny (Fig.  28). Genae tawny (Fig. 26). Pronotum tawny, median area with two brownish small impressions (Fig. 27), paranotal lobes brown mix with some yellow (Fig. 28). Mesonotum tawny (Fig. 27). Forewings tawny with veins tawny, the basal part of costal area with some irregular black markings, the middle area from basal third of costal margin extending to the middle of forewing surface also have some irregular black markings, the apical margin with a black linear marking (Fig. 29); some specimens with the black markings on forewing almost invisible, but with an obscure large paler round marking at basal third on each side of forewing (Fig. 25). Hindwings brown (Fig. 30). Legs tawny (Fig. 26).
Male genitalia. Anal tube in dorsal view mushroom shaped, widest beyond middle, as long in midline as widest part, apical margin rounded (Fig. 32), lateral margins roundly convex in dorsal view (Fig. 32) and visible in lateral view (Fig. 31); anal opening located near the middle of anal tube (Fig. 32). Pygofer in lateral view subrectangular, dorsal margin slightly sloping to the posterior, dorso-lateral angle rounded, posterior margin almost parallel with the anterior margin (Fig. 31). Gonostylus in lateral view with dorsal margin elevated at the basal third (Fig. 33). Capitulum of gonostylus derived after the middle of gonostylus, broad, sharp triangular, directed to anterior; the meniscate processes with one side beyond the base of anterior margin of capitulum and another side beyond the posterior margin of capitulum (Fig. 33). In lateral view, periandrium dorso-lateral lobe and ventral lobe rounded apically (Figs 34, 36). In posterior view, ventral lobe of periandrium spinous protruded at middle in apical margin (Fig.  37). The lateral processes of aedeagus relatively short, hook-shaped, derived from the middle, directed anteriorly to basal fourth, reaching the ventral margin of periandrium (Figs 34,36); in ventral view this pair of processes directed downwards (Fig. 35).
Female genitalia. Anal tube in dorsal view ovate, 1.1 times longer in midline than widest part, widest before mid length, apical margin slightly concave at middle, lateral margins rounded, anal opening situated slightly below middle (Fig. 38). Anterior connective lamina of gonapophysis VIII with two obscure large teeth in the apex but the keeled teeth in outer-lateral margin invisible (Fig. 44). Gonocoxa VIII long, quadrangular (Fig. 44). Hind margin of sternite VII roundly concave, the median part with a small convex protuberance (Fig. 43).
Etymology. Arbitrary euphonic name referring to the crown (latin 'corona') of yellow pustules on the frons.

Pusulissus gen. nov. distribution
With three different species, the new genus Pusulissus appears distributed around the South China in the Guangxi Province (Hezhou, Qichong Natural Reserve) and in North Vietnam (Cao Bang Province: Phia Oac and Vinh Phuc Province: Tam Dao) Figure 45. Distribution map of Pusulissus. The red triangular, blue five-pointed star and purple circular respectively indicate the distribution of species P. phiaoacensis sp. nov., P. coronomensis sp. nov. and Pusulissus sp. (Fig. 45). The Vietnamese specimens were collected in mountainous biotopes at relatively high altitudes (between 750 and 1050 m) while at lower altitude (180 m) in China.

Pusulissus gen. nov. phylogeny
The new genus Pusulissus refers to the taxon "Gen. nov. apud Tetricodes" in Wang et al. (2016)'s molecular phylogenetic analyses, and three different species are observed in this study (Fig. 46). However, only two species are formally described here as the third taxon, Pusulissus sp., corresponding to the one already sequenced as "Gen. nov. apud Tetricodes" in Wang et al. (2016), is represented by only one incomplete female specimen from Vietnam also: Tam Dao, Vinh Phuc Province.

Regard to the tribe Parahiraciini
The Parahiraciini lineage was erected within the Issidae by Cheng and Yang (1991a) for the single genus Parahiracia Ôuchi, 1940 with a subfamily rank. The subfamily recognition was based on the elongate ovate body, the absence of wax plates of abdominal segment VII-VIII (present in other Issidae sec. Fennah 1954) and the presence of respectively 10 and 8 median sensory pits on each side of the meso-and metanotum in the 5 th instar larva, versus 2-5 and 0-2 sensory pits in other issids (Cheng and Yang 1991a, b). This lineage was confirmed as a monophyletic group by several successive molecular analyses (Wang et al. 2016;Bourgoin et al. 2018, unpublished data) while it was not recovered by Gnezdilov et al. (2020), probably due to methodological biases which is out of the scope of this paper.
From a morphological perspective and for the adults, Gnezdilov and Wilson (2007) characterized the tribe by 1) beetle-like conformation, convex, elongate, and apically narrowed forewings not exceeding the length of the abdomen with a net of apical transverse veins and 2) long fore and middle legs. Gnezdilov and Wilson (2007) added a "welldeveloped three-or two lobed (anal lobe more or less reduced) hindwings with a deep notch between remigium and vannus and a net of transversal veins in the distal part", but regarded this last character as plesiomorphic. In their review and identification key to genera, Chen (2012, 2013) used characters of the head capsule (presence or absence of swollen frons, vertex conformation), prothoracic femora and tibiae (flattened or not), forewing conformation and claval suture (present or not) and some genitalia characters. However, none of these characters appear as specific of the tribe. More recently, Gnezdilov (2017) added the "cuspidal apex of clavus" (Gnezdilov 2015) in the diagnosis of the tribe and also retained (Gnezdilov 2018) the narrow anal lobe, the deep cubital cleft, and the cuspidal apex of the forewing clavus as apomorphies of the tribe.
In 2016, Wang et al. (2016) proposed a new classification of the family, confirming Cheng and Yang (1991a)'s separation of Parahiraciini with the recognition of several other new tribes. They put in light the importance of the hindwing conformation in adults that appeared being well characteristic for each lineage. Since, our further studies of many Oriental issid genera have confirmed this view and a clearer figure for Parahiraciini taxa (including several yet non-described new genera) has now emerged. We can now provide a non-ambiguous morphological definition of the tribe Parahiraciini allowing us to review its composition and to divide the tribe into three subtribes: Scantiniina subtribe nov., Vindilisina subtribe nov., and the nominal one Parahiraciina Cheng & Yang, 1991 subtribe nov., although we don't exclude that the first two might represent in the future distinct lineages of higher range value. The following subtribal diagnoses are therefore proposed, mainly based on the hindwing conformation , with a key to Parahiraciini subtribes.
3. A2 lobe with anal area posterior to A1 strongly reduced, much shorter and much thinner than the anterior lobes.
4. Sc-R-MP-CuA and CuP-Pcu-A1 lobes covered with a set of numerous transverse veins.
5. CuA and CuP not merging before the anterior notch. 6. Pcu and A1 1 not merging in basal half of forewing. 7. A2 present, not branched or absent. In some species a transverse a2-a1 connecting A2 with A1 at the level of its basal branching (Tetricodes tamdaoensis Vanslembrouck & Constant, 2018).
Note. Based on this diagnosis, Parahiraciina constitutes a well-defined group supported by several apomorphic characters (reduced anal lobe, numerous transverse veins) and the molecular analysis results. Accordingly, genera Scantinius, Vindilis and Nisoprincessa are moved to separate new subtribes. They probably all belong to the same higher lineage Parahiraciini based on the apomorphic strong cubital notch of the hindwing.
While Folifemurum, was already excluded from Parahiraciini by Wang et al. (2016), this view was not followed by Gnezdilov (2017). Based on its rounded hemisphaeriini general shape, its apomorphic one lobed hind wing, the medio-carinated frons, and particularly according to the molecular analysis result, it is here transferred to Hemisphaeriini Mongolianina. Gelastyrella is here maintained as a valid genus following Chen et al. (2014) versus Gnezdilov (2009)'s synonymy with Thabena, according to the number of small spines on the first metatarsi (more than 35 in Gelastyrella while less than 21 in Thabena), the large corpus connective of the phallic complex bearing a large and obvious ventrad expansion (corpus connective reduced and phallic complex without ventrad expansion in Thabena), and posterior margin of female sternite VII medially quadrate-shaped (triangular in Thabena) (Chen et al. 2014 Diagnosis. The subtribe is identified and separated according to the following list of characters: 1. Hindwing trilobate, strongly notched at CuA2 with CuP-Pcu-A1 lobe slightly longer, as wide as Sc-R-MP-CuA lobe.
Note. This subtribe is currently monogeneric and distributed in Indonesia (Sumatra), in Peninsular Malaysia and Sarawak (Gnezdilov and Wilson 2007). Based on the strong CuA2-CuP notch and the reticulated Sc-R-MP-CuA and CuP-Pcu-A1 lobes, it is tentatively considered as a sister lineage to Parahiraciina, but without molecular data, its place in the phylogeny of the Hemisphaeriinae sec. Wang et al. (2016)  Diagnosis. The subtribe is identified and separated according to the following list of characters: Head capsule with vertex and frons in a slightly convex margin in lateral view. Compound eyes elongated, almost twice as long as wide in lateral view.
Pcu and A1 1 not merging in basal half of forewing. Sc-R-MP-CuA and CuP-Pcu-A1 lobes covered with a set of numerous transverse veins (Figs 51, 52)

Conclusions
Parahiraciini constitutes a well-defined lineage in Issidae Hemisphaeriinae, easily recognized by the apomorphic cubital strong notch of the hindwing. Its monophyly is also fully supported by the molecular analyses at least for the nine genera, including Pusulissus gen. nov., described in this paper (Wang et al. 2016, Bourgoin et al. 2018. Paraphyly reported by Gnezdilov et al. (2020) in the maximum likelihood tree ( fig. 2 pink box) is probably biased, as much as its Bayesian analysis also recovered a 100% supported node for the tribe ( fig. 1). The three new subtribes proposed here are easily separated on morphological characters based on the hindwing conformation but their exact placement in the classification remains to be confirmed by molecular analysis. Within Parahiraciini, the placement of Pusulissus gen. nov. in the phylogeny in respective to the other Parahiraciina genera, remains of course provisional until a wider sampling would be available, but probably is close to the genus Tetricodes.