A new species of the genus Euxaldar Fennah, 1978 (Hemiptera, Fulgoromorpha, Issidae) from China and revision on the molecular phylogeny of the family

Abstract A new species Euxaldar daweishanensis Yang, Chang & Chen, sp. nov. is described and illustrated from southwestern China. The female genitalia of the genus Euxaldar is described and presented for the first time. A checklist and key to the known species of the genus are provided. A revised molecular phylogenetic analysis of the family Issidae based on combined partial sequences of 18S, 28S, COI, and Cytb is provided using both Maximum likelihood and Bayesian inference analyses.

Below, we describe and illustrate a new species of Euxaldar from Yunnan Province in China, provide a checklist and key to Euxaldar species, and describe and photograph the female genitalia of the new species. The partial DNA sequences (16S, 28S (d6-d7), COI, Cytb) of the new species are briefly analyzed. A revised molecular phylogeny is analyzed by Bayesian and Maximum likelihood based on seven sequences of four genes (18S, 28S, COI and Cytb), providing molecular evidence of phylogenetic relationships within the Issidae and enabling a revaluation of the current classification of the family Issidae by Wang et al. (2016), Zhao et al. (2019) and Gnezdilov et al. (2020).

Materials and methods
The morphological terminology used for body appearance follows Chan and Yang (1994) and Anufriev and Emeljanov (1988). Forewing venation pattern follows Bourgoin et al. (2015). The terminologies of male and female genitalia follow Bourgoin (1987Bourgoin ( , 1993 and Chang et al. (2015). Body length (included forewings) is given in millimeters (mm).
The genital segments of the specimens were macerated in a boiling solution of 10% NaOH for about 5 minutes, washed in distilled water, then immersed in glycerine for observation, dissection, drawing, and photography. They were stored in a micro vial in glycerol for further examination. A Leica MZ 12.5 stereomicroscope was used for illustrations. A KEYENCE VHX-1000C was used to acquire photographs. All specimens studied are deposited in the Institute of Entomology, Guizhou University, Guiyang, China (GUGC).
The molecular phylogenetic study included 71 species belonging to 48 genera as ingroups from Issidae (Wang et al. 2016;Zhao et al. 2019;Gnezdilov et al. 2020) and five species as outgroups from the families Cixidae, Caliscelidae, Delphacidae, Dictyopharidae, Tropiduchidae. Data for the 71 included species were downloaded from NCBI. Five ingroup species including the new species were newly sequenced, for which total DNA was extracted using the Animal Tissue Genomic DNA Kit (Tiangen Biotech Company, Beijing, China). Primers and PCR procedures are listed in Tables 1, 2 and carried out in 30 μl volume reaction. Accession numbers for species used in the phylogenetic analysis are shown in Table 4.  (Clary and Wolstenholme 1985) 16S-PF GCCTGTTTATCAAAAACAT 16S-PR CCGGTCTGAACTCAGATCA Cytb (Bourgoin et al. 1997) Cytb-PF TATGTACTACCATGAGGACAAATATC Cytb-PR ATCTTAATGCAATAACTCCTCC 28S d6-d7 (Cryan et al. 2000) 28S EE CCGCTAAGGAGTGTGTAA 28S MM GAAGTTAGGGATCTARTTTG 28S d3-d5 (Belshaw and Quicke 2002) 28S Ai GACCCGTCTTGAAACACG 28S D4D5r GTTACACACTCCTTAGCGGA   Coryphe about 3 times as wide as long in the middle. Male anal tube enlarging from base to apical margin and deeply concave at posteromedial part in dorsal view (Gnezdilov et al. 2017a: fig. 6) Diagnosis. This species differs from other Euxaldar species by the following characters: (1) coryphe about 2.3 times wider than long (less, or more than 2.3 times as wide as long in other species of Euxaldar); (2) first metatibiotarsal of hind leg with 8 intermediate spines (other species of Euxaldar with first metatarsomere of hind leg with 6 or 7 intermediate spines); (2) penis with 3 different ribbon-shaped processes at middle (Figs 16, 17, pp, paed), dorsal lobe of periandrium with 2 asymmetrical sword-shaped subapical processes in apical half (Figs 16, 17, sap) (other species without sword-shaped subapical processes in apical half of dorsal lobe of periandrium).
Etymology. This new species is named after the type locality, Mt. Daweishan National Nature Reserve, Yunnan Province, China.
Distribution. China (Yunnan Province) Remark. This new species resembles Euxaldar jehucal but differs from the latter by the following combined features: Anal tube with apical margin convex in the middle, lateral margin with a small triangular process in each side (anal tube wide, apical margin deeply concave medially in E. jehucal); periandrium with two asymmetrical subapical processes sword-shaped in apical half (periandrium with subapical processes not as sword-shaped in E. jehucal); aedeagus with one medial dagger-like process on lateral margins (aedeagus without any processes on lateral margins in E. jehucal).

Discussion
According to our analysis, the tribe Thioniini was recovered as monophyletic, split from the subfamily Issinae sensu Gnezdilov et al. (2020), and placed in the subfamily Figure 28. Bayesian 50% consensus tree based on combined dataset. Nodes of the major clades are numbered and refer to text. Each node is documented with its posterior probability (PP) value.
The monophyletic tribe Hemisphaeriini Melichar, 1906 is confirmed by our data, characterized by hemispherical forewings and single-lobed or rudimentary hind wings (Gnezdilov et al. 2020 Gnezdilov (2017c) and Zhao et al. (2019), but we still consider it incertae sedis until more evidence is presented.
The third lineage of Mongolianina (Zhao et al. 2019) is recovered only in our ML analysis. Euxaldar daweishanensis sp. nov. and E. lenis are grouped into a different cluster in our research: the genera of the cluster ((E. daweishanensis sp. nov. + Retaldar) + (Clypeosmilus + Eusudasina)) share a protruded clypeus, and forewings CuP clear; another cluster (E. lenis + Macrodaruma) recovered by Zhao et al. (2019) share a smooth metope without pustules, and sexual dimorphism. Euxaldar lenis probably belongs to a new genus.
Euxaldar is similar to the genus Paramongoliana Chen, Zhang & Chang, 2014 which is here formally placed in the subtribe Mongolianina according to Wang et al. (2016), but differs by the following characters: metope smooth or with pustules (metope roughly corrugated, without pustules in The genus Euxaldar is also similar to the genus Clypeosmilus (Gnezdilov et al. 2017b) in having forewings with reticulate venation and a distinct claval suture, but can differ from the latter in the following characters: postclypeus with complete median carina and anteclypeus with distinct median carina (Clypeosmilus with postclypeus large, flattened laterally, bearing a thick chisel-like median carina); periandrium asymmetrical (periandrium symmetrical, with pair of long and narrow subapical processes directed apically).
Euxaldar daweishanensis sp. nov., E. jehucal, and E. guangxiensis share several compelling characters: 1) E. daweishanensis sp. nov., E. jehucal, and E. guangxiensis share a metope disc with relatively weak pustules distributed in a row along the lateral margins; and 2) E. daweishanensis sp. nov. and E. guangxiensis have an anal tube with a triangular process on each lateral margin ( Fig. 13; Zhang et al. 2018: figs 12, 13). Other noteworthy characters: 1) E. guangxiensis exhibits a vestigial hind wing; 2) E. lenis has a smooth metope without pustules, and sexual dimorphism. All species of this genus probably belong to different species groups or even different genera. More molecular data and other convincing morphological evidence are expected in the future, enabling further discussion of the taxonomic status of Euxaldar.