Phylogeny, character evolution and tribal status
Our study indicates that mitogenome sequences are useful for resolving
phylogenetic relationships of subfamily Typhlocybinae from the species
to the tribal level. Analyses of both amino acid and nucleotide
sequences yielded similar topologies with the main differences among
analyses occurring among a few branches that received low bootstrap
support in one or more analysis. Our results agree with the result of a
recent analysis by Lu et al. (2021) that incorporated partial sequence
data from three nuclear and two mitochondrial genes in recovering
Typhlocybinae as a well-supported monophyletic group sister to
Mileewinae (Mileewini). A comprehensive analysis of Membracoidea based
on anchored hybrid data (Dietrich et al., 2017) failed to recover this
relationship but the relationship of Typhlocybinae to other leafhopper
subfamilies was poorly resolved by that study, so our results require
further confirmation. Within Typhlocybinae, our results consistently
supported the monophyly of the tribes Dikraneurini, Empoascini,
Erythroneurini and Typhlocybini (Alebrini was represented by a single
species in our dataset).
Lu et al. (2021) recovered the three species of Zyginellini included in
their dataset as a monophyletic sister group of Typhlocybini (sensu
stricto) but our larger sample of Zyginellini genera indicates that this
tribe is polyphyletic. This supports the treatment of some recent
authors (e.g., Hamilton, 1983; Dietrich, 2013) of Zyginellini as a
junior synonym of Typhlocybini. Based on our results, the hind wing
character traditionally used to separate Typhlocybini from Zyginellini
(absence of the distal segment of vein CuA; Figure 6, f6) is
homoplasious
and has been derived independently in at least three unrelated lineages.
Another hind wing character (veins RP and MA separate versus confluent;
Figure 4, character g) was previously used to distinguish Eupterygini
from Typhlocybini but our results also indicate considerable homoplasy
for this trait.
Details of relationships within individual tribes are difficult to
compare between our results and those of Lu et al. (2021) given that the
taxon samples of the two studies overlap only partially. Nevertheless,
we note that several branches pertaining to relationships among genera
of Typhlocybini, as well as a few branches within the other tribes, were
extremely short and received low bootstrap support in both studies. This
suggests that neither complete mitogenome data nor data from a few
nuclear and mitochondrial genes will suffice to completely resolve
typhlocybine phylogeny with high confidence, although increased taxon
sampling may also help improve phylogenetic resolution in this group.
Our results are generally consistent with morphology-based
classifications proposed recently. Young (1965) moved Mileewini (as
Mileewanini) to Typhlocybinae but this was rejected by Mahmood (1967).
Young (1968) later suggested treating Mileewinae as a separate
subfamily. Nevertheless, Mileewinae are similar to Typhlocybinae in
having relatively small, slender bodies, reduced forewing venation and
slender hind tarsi (Dietrich, 2011). Previous phylogenetic studies have
not reached a clear consensus on the sister-group relationship of
Typhlocybinae (Balme, 2007;
Dietrich, 2013b; Wang et al., 2017; Dietrich et al., 2017). Our results
are also equivocal in this regard. Two analyses (Figure 2, c, e)
recovered a sister-group relationship between Typhlocybinae and
Cicadellinae (sensu stricto), but all other analyses (Figure 2, a, b, d)
and the tree topology tests (Table 2)
consistently
support Typhlocybinae as sister to Mileewinae. Considering the
morphological similarities shared by Typhlocybinae and Mileewinae, the
latter hypothesis seems to be more plausible.
The tribal relationships in Typhlocybinae have long been controversial
(Wagner, 1951; Mahmood & Ahmed, 1968; Zhang, 1990; Balme, 2007;
Dietrich et al., 2017). Our analyses consistently support a sister-group
relationship between Alebrini and Empoascini, as well as between
Erythroneurini and Dikraneurini, in agreement with other recent analyses
(Dietrich et al., 2017; Lu et al., 2021). The former relationship is
also consistent with Wagner’s intuitive morphology–based hypothesis
(1951), but not with those of Mahmood & Ahmed (1968) and Zhang (1990).
The main area of uncertainty appears to be the relationship of
Typhlocybini to the other tribes and the relationships of deep internal
nodes within Typhlocybini. Different analyses recovered Typhlocybini
either as sister to Alebrini + Empoascini (Figure 2, a, b) or as sister
to Erythroneurini + Dikraneurini (Figure 2, c, d, e), thus this
relationship remains equivocal. Detailed morphology-based cladistics
analyses of Typhlocybinae have not been attempted but there appears to
be some morphological support for the latter hypothesis
(Typhlocybini+(Dikraneurini+Erythroneurini)). For example, all
Erythroneurini and most Typhlocybini (except a few Neotropical taxa)
have the hind wing submarginal vein absent at the wing apex. The male
subgenital plates of Typhlocybini, Dikraneurini and Erythroneurini also
have relatively few macrosetae compared to those of Alebrini and
Empoascini. These traits are potential synapomorphies supporting the
sister relationship of Typhlocybini to Dikraneurini+Erythroneurini
recovered in most of our analysis as well as the analyses of Lu et al.
(2021) and Dietrich et al. (2017).
Ancestral state reconstructions of key morphological characters
previously used to define and diagnose tribes within Typhlocybinae
(Evans, 1963, 1971; Hamilton, 1983; Dworakowska, 1979, 1993; Zhang,
1990) (Table 3) indicate that some wing characters traditionally used to
diagnose tribes are highly stable. Our analyses generally support the
monophyly of tribes Empoascini, Dikraneurini, Erythroneurini and
Typhlocybini as defined morphologically by most authors. Thus, our
analyses suggest that the wing vein characters traditionally used to
define these groups are reliable. Nevertheless, our results also
indicate that some morphological characters have undergone
homoplastic changes during the
evolution of the group. Ocelli may be present or absent within
Empoascini and Typhlocybini. In Empoascini, all genera have ocelli,
except Beamerana and Paulomanus . The latter genera, which
have hind wing venation identical to that of many Empoascini,
nevertheless grouped with Typhlocybini in a recent morphology-based
phylogeny and were tentatively placed in the latter tribe (Xu et al.,
2021). Few genera in Typhlocybini have ocelli, e.g., Hiratettixand Caknesia , but we were able to include only the former in our
analyses and it occupies a relatively derived position within the tribe,
suggesting that ocelli were lost and regained at least once in this
tribe (Figure 4, a). Loss of the hind wing submarginal vein is also
apparently homoplastic with independent losses apparently occurring in
Erythroneurini and Typhlocybini (Figure
4,
e). Hind wing veins RP and MA
have
also apparently become confluent in the common ancestor of
Erythroneurini and Dikraneurini and independently in Empoascini and
Typhlocybini (Table 3). Loss of the branched hind wing anal vein occurs
only in the Dikraneurini + Erythroneurini lineage but further analyses
with a larger sample of taxa will be needed to determine the extent of
homoplasy in this character.
The molecular divergence time estimates indicate that Typhlocybinae and
extant tribes originated during the Middle and Late Cretaceous,
respectively. Thus, diversification of major lineages in this group
seems to have roughly coincided with the
diversification of angiosperms
during the Cretaceous (Hamilton, 1992, 1994; Ledyard, 1974; Foster et
al., 2016). Our divergence time estimates for tribes of Typhlocybinae
are considerably younger than those reported for the same branches by
Dietrich et al. (2017), possibly due to the denser taxon sample of our
study. Thus, in our study, the splits between Erythroneurini and
Dikraneurini, and between Alebrini and Empoascini are both estimated at
76 MYA, compared to the 95 MYA and 112 MYA, respectively, reported by
Dietrich et al. (2017). 95% confidence intervals of both studies are
wide and broadly overlap, indicating that the available methods and data
are only able to provide very approximate estimates of the times of
origin of these major leafhopper lineages. Future analyses incorporating
additional fossil evidence (unavailable at present), diversified clock
models, dating methods, and rate priors may yield improved estimates.