ABSTRACT
The subfamily Typhlocybinae is a ubiquitous, highly diverse group of mostly tiny, delicate leafhoppers. The tribal classification has long been controversial and phylogenetic methods have only recently begun to test the phylogenetic status and relationships of tribes. To shed light on the evolution of Typhlocybinae, we performed phylogenetic analyses based on 28 newly sequenced and 19 previously sequenced mitochondrial genomes representing all currently recognized tribes. The results support the monophyly of the subfamily and its sister group relationship to Mileewinae. The tribe Zyginellini is polyphyletic with some included genera derived independently within Typhlocybini. Ancestral character state reconstruction suggests that some morphological characters traditionally considered important for diagnosing tribes (presence/absence of ocelli, development of hind wing submarginal vein) are homoplastic. Divergence time estimates indicate that the subfamily arose during the Middle Cretaceous and that the extant tribes arose during the Late Cretaceous. Phylogenetic results support establishment of a new genus, Subtilissimia Yan & Yang gen. nov. , with two new species,SubtilissimiafulvaYan & Yang sp. nov. and Subtilissimia pelliculaYan & Yang sp. nov. ; but indicate that two previously recognized species of Farynaladistinguished only by the direction of curvature of the processes of the aedeagus are synonyms, i.e., Farynala dextra Yan & Yang, 2017 equals Farynala sinistra Yan & Yang, 2017 syn. nov. A key to tribes of Typhlocybinae is provided.
KEYWORDS classification; high-throughput sequencing; Membracoidea; microleafhopper; mitochondrial genes; phylogenetic analysis
INTRODUCTION
The subfamily Typhlocybinae (Hemiptera, Membracoidea, Cicadellidae) is a large group of mostly tiny, delicate leafhoppers that feed primarily on leaf parenchymal cell contents of their host plants, thus differing from the phloem- or xylem-feeding (Cicadellinae) preferences exhibited by most other leafhoppers (Dietrich, 2013a; Bartlett et al., 2018). This group is distributed worldwide and comprises numerous agricultural pests (Nault & Ammar, 1989; Sun, 2004; Wearing et al., 2011; Shi et al., 2018). For example, the nymphs and adults of Empoasca fabaeand Matsumurasca onukii attack crops of potato and tea, respectively (Backus et al., 2005; Chasen et al., 2014). However, the vast majority of species, which feed on a wide variety of plants, appear to be of little or no economic importance. The group remains poorly studied, partly because their delicate nature makes them difficult to preserve for morphological studies. Based on the large number of described species (approximately 5000), Typhlocybinae is the second largest cicadellid subfamily (after Deltocephalinae Bartlett et al., 2018) but the actual diversity of the group is probably much higher (Dietrich, 2013a). Typhlocybinae is readily distinguished from other leafhoppers by the following morphological characters: forewing fully developed without closed anteapical cells; hind tarsomere Ⅰ acuminate, without transverse row of blunt setae (Dietrich, 2005).
Since Kirschbaum (1868) first recognized the subfamily, various authors employed different concepts of Typhlocybinae and its tribes (Table S1), with as few as four and as many as eleven tribes recognized (Metcalf, 1968; Mahmood & Ahmed, 1968; Dworakowska, 1979; Ahmed, 1983; Oman et al., 1990; Hamilton, 1998; Gebicki & Szwedo, 2006; Dietrich, 2013a; Dietrich et al., 2017). Dworakowska’s (1979) six-tribe typhlocybine classification including Alebrini, Empoascini (synonym, Jorumini and Helionini), Erythroneurini (with synonym Bakerini), Dikraneurini (with synonym Forcipatini), Typhlocybini (with synonym Eupterygini) and Zyginellini has been followed by most recent authors. However, the status of Zyginellini has remained controversial, with recent authors regarding it either as a synonym of Typhlocybini (Ahmed 1984, Balme, 2007; Dietrich, 2013a; Zhou et al., 2020), or as an independent and valid tribe (Zhang, 1990; Song, 2010; Dietrich et al., 2017; Lu et al., 2021).
The current tribal classification of Typhlocybinae is based almost entirely on a few characters of the wing venation. Compared with other cicadellid tribes, typhlocybines have the venation of the fore- and hind wings relatively reduced. Thus, particular patterns of reduction and loss or consolidation of veins have traditionally been used to define the different tribes. Alebrini, the tribe traditionally considered to be the most “primitive”, is the only tribe with an appendix on the forewing, a trait shared with non-typhlocybine leafhoppers. Empoascini lack the section of the hind wing submarginal vein that extends along the costal margin. Erythroneurini and most Typhlocybini have the hind wing submarginal vein completely lacking at the wing apex. Erythroneurini and some Dikraneurini have hind wing vannal vein unbranched but Dikraneurini retain a complete submarginal vein.
Young (1965) transferred Mileewini (including genera Amahuaka andUjna ) into Typhlocybinae based on intuitive morphological criteria but he later suggested that Mileewinae should be treated as a separate subfamily (Young, 1968), a classification that has been followed by most subsequent authors. Phylogenetic analyses of Membracoidea based on morphology and DNA sequence data yielded inconsistent results. The concatenated ML analysis of anchored-hybrid data by Dietrich et al. (2017) placed Eurymelinae (sensu lato) as sister to Typhlocybinae but with only 54% bootstrap support. An earlier morphology-based analysis of Cicadellidae (Dietrich 1999) placed Mileewinae (in part, Mileewini) as sister to Typhlocybinae but the first molecular phylogeny of this family (Dietrich et al., 2001) did not consistently recover Typhlocybinae as monophyletic and its relationships to other subfamilies were also inconsistently resolved. A subsequent combined analysis of morphological and 28S rDNA sequence data supported the monophyly of Typhlocybinae and its sister relationship to Mileewini (Dietrich et al., 2005). This sister-group relationship was also recovered in the recent multi-locus analysis of Lu et al. (2021) with moderate ML bootstrap support.
Most prior research on typhlocybine systematics has focused on species descriptions. New typhlocybine taxa continue to be described at a rapid pace, but the phylogeny of the group remains little explored. Previous analyses have supported the monophyly of Typhlocybinae but its sister-group has remained uncertain. Zhang (1990) proposed an intuitive morphology-based hypothesis of tribal relationships within Typhlocybinae, considering Alebrini to be the earliest diverging lineage based on the retention of an appendix (shared with other leafhoppers) in the forewing, with Dikraneurini sister to Empoascini based on the relatively well-developed submarginal vein (smv) of the hind wing. In an unpublished dissertation, Balme (2007) conducted the first explicit cladistics analysis of the group, using 73 discrete morphological characters and two molecular markers (16S rRNA and Histone 3) and proposed a classification including four tribes, Alebrini + (Typhlocybini + Empoascini) + Dikraneurini, with Erythroneurini treated as a synonym of the latter tribe. The anchored hybrid phylogenomic analysis of Membracoidea based on 388 genetic loci and more than 99,000 aligned nucleotides (Dietrich et al., 2017) included only 1-2 representatives of each tribe but recovered Alebrini as sister to Empoascini and this clade sister to a clade comprising Typhlocybini and Erythroneurini + Dikraneurini with strong support. This dataset did not include a taxon sample large enough to test the monophyly of individual tribes or examine relationships among genera within tribes. Most recently, Lu et al. (2021) analyzed a much larger sample of typhlocybine taxa using data from fragments of three nuclear and two mitochondrial genes, recovering the same tribal relationships found by Dietrich et al. (2017) and also recovering Zyginellini as sister to Typhlocybini but with low support.
Here we use sequence data from complete mitochondrial genomes in an attempt to improve resolution of phylogenetic relationships within Typhlocybinae and examine the evolution and stability of wing characters traditionally used for the classification of typhlocybine tribes. A total of 110 leafhopper mitochondrial genome assemblies have been previously deposited in GenBank. Among them, only 19 typhlocybine species are included representing the tribes Typhlocybini, Empoascini, Erythroneurini and Zyginellini. Data for species of Alebrini and Dikraneurini were not previously available. Therefore, prior to our study, mitogenome data for Typhlocybinae remained insufficient to facilitate a comprehensive phylogenetic analysis of the subfamily.
For this study, we assembled and annotated 28 new typhlocybine mitogenomes using next-generation sequencing (NGS) data, and conducted a comprehensive phylogenetic analysis to examine relationships among major lineages of the subfamily. In addition, we performed ancestral character state reconstruction (ACSR) to examine the evolution of key morphological characters. We also used molecular divergence time methods to estimate the times of origin of various typhlocybine clades.