Phylogeny
The
aligned amino acid dataset
(PCGs_faa)
included 3,467 sites. The aligned nucleotide sequence datasets included
6,934 sites
(PCG12_fna)
and 9,218 sites (PCG12_fna+2rRNAs), respectively. A total of five
phylogenetic trees were generated, including one Bayesian inference tree
(maxdiff value = 0.11, minimum effective sizes>58) (Figure
S1) and four maximum likelihood trees (Figure S2–S5). As expected, all
phylogenetic analyses robustly supported the monophyly of Typhlocybinae
with high nodal support values
(PP=1,
SH‒aLRT=100,
UFB=100) (Figure 2). Unlike the ML trees (Figure 2, c, e), which support
the subfamilies Cicadellinae and Typhlocybinae as sister group, the
other three phylogenetic trees consistently support that the latter as
sister to Mileewinae (Mileewini) (Figure 2, a, b, d). However, these
alternative phylogenetic arrangements received low to moderate nodal
support. In tribal relationships of Typhlocybinae, the sister-group
pairs Dikraneurini+Erythroneurini
(Figure
3, b) and Alebrini+Empoascini (Figure 3, e) were recovered with moderate
to strong nodal support. However, the results differed in the placement
of Typhlocybini, with some analyses placing this tribe sister to
Alebrini+Empoascini
(Figure
2, a, b) and others placing Typhlocybini sister to
Dikraneurini+Erythroneurini (Figure 2, c, d, e). The included
representatives of Zyginellini (Figure 3, in bold) were never recovered
as a monophyletic group but were derived from within Typhlocybini,
consistent with the five tribe classification adopted by some previous
authors (Ahmed, 1983; Dietrich, 2013a; Zhou et al., 2020).Zyginella , the type genus of Zyginellini, and Limassollawere consistently placed as sister to the rest of Typhlocybini
with
strong nodal support, but other included genera of Zyginellini
(Yangisunda and Paraahimia ) occupied more derived
positions within Typhlocybini with low to moderate nodal support (Figure
3). In addition, the relationships of several deep internal nodes within
Typhlocybini were unstable (Figure S1–S5).
Statistical tests of alternative tree topologies rejected hypotheses a
(sister group relationship of Cicadellinae and Typhlocybinae) and b
(sister group relationship of Typhlocybini and Zyginellini) but failed
to reject either alternative hypothesis of the relationship of
Typhlocybini to the other tribes (Table 2). Thus, the placement of
Typhlocybini must be considered equivocal according to our results. The
recent analysis of Lu et al. (2021) based on sequence data from 5 genes
recovered Typhlocybini as sister to Dikraneurini+Erythroneurini but with
only low to moderate nodal support. All analyses consistently supported
the monophyly of the four tribes for which more than one exemplar was
included: Dikraneurini, Empoascini, Erythroneurini, Typhlocybini (sensu
lato, including Zyginellini). Zyginellini was consistently polyphyletic
with the four included genera each representing an independent branch
(Figure 3). Many branches pertaining to relationships within tribes were
consistently well supported in all analyses but some were unresolved,
particularly within Typhlocybini; for example, relationships among four
major lineages of Typhlocybini were resolved inconsistently across
analyses and form a polytomy in the Bayesian consensus tree (Figure 3).
The phylogenetic results, combined with evidence from morphological
characters (see below) and genetic distances (Table S3) support the
establishment of a new genus, Subtilissimia Yan & Yanggen. nov. , including two new species: Subtilissimia
fulva Yan & Yang sp. nov. and Subtilissimia pelliculaYan & Yang sp. nov. , but suggest that the speciesFarynalasinistraYan & Yang, 2017 and Farynala dextra Yan & Yang, 2017, which
have the male aedeagus identical but with processes curved in opposite
directions (Yan & Yang, 2017, Figs 13–32), should be treated as
synonyms (p-distance < 0.7%, except COX3 gene),Farynala sinistra Yan & Yang, 2017 syn. nov. The
previously sequenced species identified as “Typhlocyba sp.”
(GenBank, KY039138) belongs to the genus Aguriahana Distant, 1918
(COX1 p-distance=1.2%; PP=1, SH‒aLRT=100, UFB=100). Based on previous
study (Qin et al., 2015; Yu et al., 2015) and analyses of phylogeny and
genetic distance, we also show that the species previously identified as
“Empoasca
vitis (GenBank,
NC_024838)”
probably equals Matsumurasca onukii (COX1 p-distance = 0.3%).
Other recent studies have shown that the latter species, a major pest of
tea, has been widely misidentified in China (Qin et al., 2015).
Unfortunately, we were unable to check voucher specimens from previous
studies in order to confirm the identifications suggested by our
results.
Ancestral
charac ter
state reconstruction
Eight typhlocybine morphological characters were traced over the BI
consensus tree and ML tree (PCG12_fna). Overall, however, these
reconstructions indicate that some characters used previously to
diagnose tribes of Typhlocybinae exhibit considerable homoplasy (Figure
4; Figure S6). Ocelli were retained by Alebrini and Empoascini and lost
in the common ancestor of Dikraneurini, Erythroneurini and
Typhylocybini. However, a reversal to the ancestral state occurred in
Typhylocybini corresponding to the genus Hiratettix (Figure 4,
a). The forewing appendix is present in most non-typhlocybine
leafhoppers but absent in all Typhlocybinae except Alebrini; thus
Alebrini has traditionally been viewed as the most “primitive” group
of Typhlocybinae (Figure 4, b). However, the recovered sister-group
relationship between Alebrini and Empoascini suggests that the appendix
may either have been lost independently in Empoascini and the remaining
tribes or lost in the common ancestor of Typhlocybinae and regained in
Alebrini. The bifurcation of hind wing vein CuA vein, shared with the
outgroup, was lost independently in Empoascini and Typhlocybini (Figure
4, f). The distal part of the hind wing submarginal vein (Figure 4, e)
was lost independently in Erythroneurini and Typhlocybini and hind wing
veins RP and MA (Figure 4, g) became confluent and separated multiple
times independently within Typhlocybini. The unique condition of the
submarginal vein connecting to CuA directly traditionally used to
diagnose Zyginellini as a distinct tribe was acquired independently in
three different lineages of Typhlocybini (sensu lato) (Figure 4, h).
Except for the single derivation of the latter “Zyginellini” character
state in the analysis of Lu et al. (2021) our results are consistent
with this prior study.