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.