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Phylogenomics and the evolution of hemipteroid insects



Hemipteroid insects (Paraneoptera), with over 10% of all known insect diversity, are a major component of terrestrial and aquatic ecosystems. Previous phylogenetic analyses have not consistently resolved the relationships among major hemipteroid lineages. We provide maximum likelihood-based phylogenomic analyses of a taxonomically comprehensive dataset comprising sequences of 2,395 single-copy, protein-coding genes for 193 samples of hemi-pteroid insects and outgroups. These analyses yield a well-supported phylogeny for hemipteroid insects. Monophyly of each of the three hemipteroid orders (Psocodea, Thysanoptera, and Hemiptera) is strongly supported, as are most relationships among suborders and families. Thysanoptera (thrips) is strongly supported as sister to Hemiptera. However, as in a recent large-scale analysis sampling all insect orders, trees from our data matrices support Psocodea (bark lice and parasitic lice) as the sister group to the holometabolous insects (those with complete metamorphosis). In contrast, four-cluster likelihood mapping of these data does not support this result. A molecular dating analysis using 23 fossil calibration points suggests hemipteroid insects began diversifying before the Carboniferous, over 365 million years ago. We also explore implications for understanding the timing of diversification , the evolution of morphological traits, and the evolution of mitochondrial genome organization. These results provide a phy-logenetic framework for future studies of the group. phylogeny | systematics | transcriptomes | Hemiptera | Psocodea T he hemipteroid insect orders, Psocodea (bark lice and parasitic lice), Thysanoptera (thrips), and Hemiptera (true bugs and allies; i.e., hemipterans), with over 120,000 described species, comprise well over 10% of known insect diversity. However, the evolutionary relationships among the major lineages of these insects are not yet resolved. Recent phylogenomic analyses questioned the monophyly of this group (1) demanding a reconsideration of the evolution of hemipteroid and holometabolous insects. We assess these prior results, which placed Psocodea as the sister taxon to Holometabola (insects with complete metamorphosis; e.g., wasps, flies, beetles, butterflies), and uncover relationships within and among hemipteroid insect orders by analyzing a large phylogenomic dataset covering all major lineages of hemipteroid insects. Knowledge of the phylogeny of these insects is important for several reasons. First, major transitions between the mandibulate (chewing) mouthpart insect groundplan and "piercing-sucking" mouthparts occurred in this group. In particular, thrips and hemipterans, and some ectoparasite lice in Psocodea, have highly modified mouthparts adapted for feeding on fluids and, hence, differ markedly from their mandibulate ancestors. Through a series of remarkable modifications, hemipteroids acquired a piercing-sucking mode of feeding in both immature and adult stages that enabled them to feed not only on plant vascular fluids, but also on blood and other liquid diets. Resolution of the evolutionary tree of hemipteroid insects is needed to provide a framework for Significance Hemipteroid insects constitute a major fraction of insect diversity, comprising three orders and over 120,000 described species. We used a comprehensive sample of the diversity of this group involving 193 genome-scale datasets and sequences from 2,395 genes to uncover the evolutionary tree for these insects and provide a timescale for their diversification. Our results indicated that thrips (Thysanoptera) are the closest living relatives of true bugs and allies (Hemiptera) and that these insects started diversifying before the Carboniferous period, over 365 million years ago. The evolutionary tree from this research provides a backbone framework for future studies of this important group of insects.
Phylogenomics and the evolution of
hemipteroid insects
Kevin P. Johnson
, Christopher H. Dietrich
, Frank Friedrich
, Rolf G. Beutel
, Benjamin Wipfler
, Ralph S. Peters
Julie M. Allen
, Malte Petersen
, Alexander Donath
, Kimberly K. O. Walden
, Alexey M. Kozlov
, Lars Podsiadlowski
Christoph Mayer
, Karen Meusemann
, Alexandros Vasilikopoulos
, Robert M. Waterhouse
, Stephen L. Cameron
Christiane Weirauch
, Daniel R. Swanson
, Diana M. Percy
, Nate B. Hardy
, Irene Terry
, Shanlin Liu
, Xin Zhou
Bernhard Misof
, Hugh M. Robertson
, and Kazunori Yoshizawa
Illinois Natural History Survey, Prairie Research Institute, University of Illinois at UrbanaChampaign, Champaign, IL 61820;
Institut für Zoologie,
Universität Hamburg, 20146 Hamburg, Germany;
Institut für Zoologie und Evolutionsforschung, Friedrich-Schiller-Universität Jena, 07743 Jena, Germany;
Center of Taxonomy and Evolutionary Research, Arthropoda Department, Zoological Research Museum Alexander Koenig, 53113 Bonn, Germany;
Department of Biology, University of Nevada, Reno, NV 89557;
Center for Molecular Biodiversity Research, Zoological Research Museum Alexander
Koenig, 53113 Bonn, Germany;
Department of Entomology, University of Illinois at UrbanaChampaign, Urbana, IL 61801;
Scientific Computing Group,
Heidelberg Institute for Theoretical Studies, 69118 Heidelberg, Germany;
Institute of Evolutionary Biology and Ecology, University of Bonn, 53121 Bonn,
Evolutionary Biology and Ecology, Institute for Biology I (Zoology), University of Freiburg, 79104 Freiburg, Germany;
Australian National Insect
Collection, Commonwealth Scientific and Industrial Research Organisation National Research Collections Australia, Acton, ACT 2601 Canberra, Australia;
Department of Ecology and Evolution, University of Lausanne and Swiss Institute of Bioinformatics, 1015 Lausanne, Switzerland;
Department of
Entomology, Purdue University, West Lafayette, IN 47907;
Department of Entomology, University of California, Riverside, CA 92521;
Department of Life
Sciences, Natural History Museum, London, SW7 5BD United Kingdom;
Department of Botany, University of British Columbia, Vancouver V6T 1Z4, Canada;
Department of Entomology and Plant Pathology, Auburn University, Auburn, AL 36849;
School of Biological Sciences, University of Utah, Salt Lake City,
UT 84112;
BGI-Shenzhen, Shenzhen, 518083 Guangdong Province, Peoples Republic of China;
Department of Entomology, China Agricultural University,
100193 Beijing, Peoples Republic of China; and
Systematic Entomology, Hokkaido University, Sapporo, 060-8589 Japan
Edited by David M. Hillis, The University of Texas at Austin, Austin, TX, and approved October 25, 2018 (received for review September 13, 2018)
Hemipteroid insects (Paraneoptera), with over 10% of all known
insect diversity, are a major component of terrestrial and aquatic
ecosystems. Previous phylogenetic analyses have not consistently
resolved the relationships among major hemipteroid lineages. We
provide maximum likelihood-based phylogenomic analyses of a
taxonomically comprehensive dataset comprising sequences of
2,395 single-copy, protein-coding genes for 193 samples of hemi-
pteroid insects and outgroups. These analyses yield a well-supported
phylogeny for hemipteroid insects. Monophyly of each of the three
hemipteroid orders (Psocodea, Thysanoptera, and Hemiptera) is
strongly supported, as are most relationships among suborders
and families. Thysanoptera (thrips) is strongly supported as sister
to Hemiptera. However, as in a recent large-scale analysis sam-
pling all insect orders, trees from our data matrices support
Psocodea (bark lice and parasitic lice) as the sister group to the
holometabolous insects (those with complete metamorphosis). In
contrast, four-cluster likelihood mapping of these data does not
support this result. A molecular dating analysis using 23 fossil
calibration points suggests hemipteroid insects began diversify-
ing before the Carboniferous, over 365 million years ago. We also
explore implications for understanding the timing of diversifica-
tion, the evolution of morphological traits, and the evolution of
mitochondrial genome organization. These results provide a phy-
logenetic framework for future studies of the group.
The hemipteroid insect orders, Psocodea (bark lice and para-
sitic lice), Thysanoptera (thrips), and Hemiptera (true bugs and
allies; i.e., hemipterans), with over 120,000 described species,
comprise well over 10% of known insect diversity. However, the
evolutionary relationships among the major lineages of these insects
are not yet resolved. Recent phylogenomic analyses questioned the
monophyly of this group (1) demanding a reconsideration of the
evolution of hemipteroid and holometabolous insects. We assess
these prior results, which placed Psocodea as the sister taxon to
Holometabola (insects with complete metamorphosis; e.g., wasps,
flies, beetles, butterflies), and uncover relationships within and
among hemipteroid insect orders by analyzing a large phylogenomic
dataset covering all major lineages of hemipteroid insects.
Knowledge of the phylogeny of these insects is important for
several reasons. First, major transitions between the mandibulate
(chewing) mouthpart insect groundplan and piercingsucking
mouthparts occurred in this group. In particular, thrips and
hemipterans, and some ectoparasite lice in Psocodea, have highly
modified mouthparts adapted for feeding on fluids and, hence,
differ markedly from their mandibulate ancestors. Through a series
of remarkable modifications, hemipteroids acquired a piercing
sucking mode of feeding in both immature and adult stages that
enabled them to feed not only on plant vascular fluids, but also
on blood and other liquid diets. Resolution of the evolutionary
tree of hemipteroid insects is needed to provide a framework for
Hemipteroid insects constitute a major fraction of insect diversity,
comprising three orders and over 120,000 described species. We
used a comprehensive sample of the diversity of this group
involving 193 genome-scale datasets and sequences from 2,395
genes to uncover the evolutionary tree for these insects and pro-
vide a timescale for their diversification. Our results indicated that
thrips (Thysanoptera) are the closest living relatives of true bugs
and allies (Hemiptera) and that these insects started diversifying
before the Carboniferous period, over 365 million years ago. The
evolutionary tree from this research provides a backbone frame-
work for future studies of this important group of insects.
Author contributions: K.P.J., C.H.D., F.F., R.G.B., B.W., R.S.P., K.M., X.Z., B.M., H.M.R., and
K.Y. designed research; K.P.J., C.H.D., R.G.B., B.W., R.S.P., J.M.A., M.P., A.D., K.K.O.W.,
A.M.K., L.P., C.M., K. M., A.V., R.M.W., S.L. , X.Z., and K.Y. performed re search; K.P.J.,
C.H.D., F.F., B.W., R.S.P., K.M., C.W., D.R.S., D.M.P., N.B.H., I.T., and K.Y. contributed new
reagents/analytic tools; K.P.J., C.H.D., R.G.B., B.W., R.S.P., J.M.A., M.P., A.D., K.K.O.W.,
A.M.K., L.P., C.M., K.M., A.V., R.M.W., and K.Y. analyzed data; and K.P.J., C.H.D., F.F.,
R.G.B., B.W., R.S.P., J.M.A., M.P., A.D., K.K.O.W., A.M.K., L.P., C.M., K.M., A.V., R.M.W.,
S.L.C., C.W., D.R.S., D.M.P., N.B.H., I.T., S.L., X.Z., B.M., H.M.R., and K.Y. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Published under the PNAS license.
Data deposition: Thedata reported inthis paper have beendeposited in NCBI(accession nos.
SRA SRR1821891SRR1821980,SRR2051465SRR2051515,andSRR921611SRR921660). Gene
sets, alignments,trees, quartet likelihood mapping results, morphologicaldata matrices, and
dating analyses results were deposited in Dryad repository, 10.5061/dryad.t4f4g85.
To whom correspondence should be addressed. Email:
This article contains supporting information online at
1073/pnas.1815820115/-/DCSupplemental. PNAS Latest Articles
understanding morphological transitions that occurred in this group, as
well as to provide a timeframe over which these changes occurred.
In addition, several lineages of hemipteroid insects (particularly
thrips and Psocodea) underwent major reorganizations of their
mitochondrial genomes, including the emergence of minicircles
(2). Understanding how these changes in mitochondrial genome
organization occurred requires knowledge of evolutionary rela-
tionships to document in which lineages these changes first arose.
Finally, hemipteroids are among the most abundant insects (3) and
are therefore key components of terrestrial and aquatic food webs
(4). Thus, a robust backbone phylogenetic framework is needed to
place ecological studies in their evolutionary context and for use in
comparative genomic and macroevolutionary analyses.
Despite their importance, relatively few studies have addressed
the relationships among the major groups of hemipteroid insects
[Paraneoptera, sensu stricto (excluding Zoraptera), also termed
Acercaria]. While a recent large transcriptome-based phyloge-
nomic analysis of insects (1) provided a well-resolved and strongly
supported phylogenetic framework for the insect orders in gen-
eral, it did not sample intensively within individual orders and
recovered some unexpected relationships. Among the most puz-
zling was the nonmonophyly of the hemipteroid insects, with
Psocodea as the sister taxon of holometabolous insects rather than
as sister to thrips plus hemipterans (Condylognatha). Although
this result was congruent with one earlier analysis based on three
nuclear protein-coding genes (5), it had not been proposed in
other molecular phylogenetic or morphological studies. Previous
morphological studies indicated monophyly of hemipteroid insects
with Psocodea sister to thrips plus hemipterans (69), or some-
times a group comprising thrips plus Psocodea (10, 11).
Another unexpected relationship recovered by Misof et al. (1)
was the placement of moss bugs (Coleorrhyncha) as sister to a
group comprising leafhoppers, cicadas, and relatives (Auchenor-
rhyncha) instead of sister to true bugs (Heteroptera). A recent
morphological study also found some support for moss bugs sister
to Auchenorrhyncha (12). In contrast, prior analyses based on
morphology (e.g., ref. 9) and DNA sequence data (e.g., ref. 13)
consistently placed moss bugs as sister to true bugs. An analysis of
a reduced gene set from transcriptome data (14) also recovered
moss bugs as sister to true bugs, while the full gene set placed moss
bugs as sister to Auchenorrhyncha. Analysis of mitochondrial
genomes (15) produced an even more unconventional result, with
moss bugs placed as the sister taxon of planthoppers (Fulgor-
oidea), making Auchenorrhyncha paraphyletic. Thus, it is impor-
tant to investigate the placement of moss bugs in more detail with
both expanded taxon and gene sampling.
We evaluated these possible conflicts among analyses by an-
alyzing a more comprehensive dataset comprising an increased
number of clusters of orthologous sequence groups (2,395
protein-coding, single-copy genes) as well as an increased taxon
sample within hemipteroid insects: 160 samples vs. 22 sampled
by Misof et al. (1). We included representatives of all major
hemipteroid lineages (sub- and infraorders). Outgroups com-
prised 33 species of holometabolous and nonholometabolous
insect orders. This dataset enabled us to test the hypothesis of
nonmonophyly of hemipteroid insects and also provides a more
detailed backbone framework for the hemipteroid phylogeny.
We evaluate the implications of this phylogeny for understanding
the evolution of feeding strategy, morphology, and mitochon-
drial genome organization of this major group of insects.
Phylogeny of Hemipteroid Insect Orders. Separate amino acid se-
quence alignments of the 2,395 single-copy genes across 193 ter-
minal taxa (SI Appendix,TablesS1S4) yielded a concatenated
supermatrix of 859,518 aligned amino acid positions, which was
used in subsequent phylogenetic analyses. A concatenated nucleo-
tide sequence supermatrix of only first and second codon positions
resulted in 1.72 million aligned nucleotide sequence sites. Tree
reconstructions based on the nucleotide sequence data supported a
phylogenetic tree (Fig. 1 and SI Appendix,Figs.S1andS2)with172/
190 (90%) of all nodes supported in 100% of bootstrap replicates.
The tree based on amino acid sequence data (SI Appendix,Fig.S3)
was highly concordant with that based on nucleotide data. Analysis
of an optimized amino acid dataset (SI Appendix,Supplemental
Materials and Methods)producedatree(SI Appendix,Fig.S4)that
was identical to that based on all amino acids with respect to re-
lationships among orders, suborders, infraorders, and superfamilies,
but had some minor rearrangements within these groups.
Considering relationships within and among orders in more de-
tail, the thrips (Thysanoptera) were recovered with 100% bootstrap
support as the sister taxon of Hemiptera (i.e., monophyletic Con-
dylognatha), although only 68% of quartets supported this result in
four-cluster likelihood mapping (FcLM) (SI Appendix,TablesS5
and S6). As in the study of Misof et al. (1), Psocodea was placed as
the sister taxon of Holometabola in 100% of bootstrap replicates,
rendering hemipteroid insects paraphyletic. However, only 25% of
quartets supported Psocodea as sister to Holometabola, compared
with 67% of the quartets supporting hemipteroid insect mono-
phyly. Results from the FcLM imply that the placement of
Psocodea as sister to Holometabola is unstable and may be due
to confounding phylogenetic signal (e.g., from heterogeneous
composition of amino acid sequences, nonstationarity of sub-
stitution processes, or nonrandom distribution of missing data)
and is also dependent on the taxon sample. However, permutation
tests of these results suggested the impact of these potential
confounding signals on the topology was minor (SI Appendix,
Table S6). To evaluate whether the parasitic lice in particular
(Phthiraptera), which have elevated substitution rates compared
with other hemipteroids (16), were a possible source of conflicting
signal, we compared quartets with and without these ectoparasitic
insects as the representative of Psocodea. However, the support
from FcLM for monophyly of hemipteroid insects was highly
similar whether parasitic lice were included (66%) or not (67%).
Morphological character mapping over three possible alterna-
tive topologies (SI Appendix,Fig.S5) revealed no apomorphies
supporting Psocodea +Holometabola. In contrast, there are 14
potential apomorphies for the monophyly of Paraneoptera. These
results indicate that there is more agreement between morphology
and the FcLM results, compared with the supermatrix analyses
with all taxa. For Coleorrhyncha (moss bugs), three characters are
apomorphies for a sister relationship to Auchenorrhyncha (leaf-
hoppers and relatives) but two other characters appear to support
a sister relationship to Heteroptera (true bugs).
In general, the phylogenetic results from transcriptomes are
congruent with the generally accepted classification schemes
within these insect orders. Bark lice and parasitic lice (Psocodea)
together are monophyletic. As has been suggested based on both
morphological (17) and molecular (16, 18) analyses, the parasitic
lice are embedded within free-living bark lice, being the sister
taxon of book lice (Liposcelididae), which makes the bark lice
(Psocoptera) paraphyletic. In contrast to results based on 18S
rDNA sequences (18), parasitic lice (Phthiraptera) were sup-
ported as a monophyletic group in our analyses, which included
representatives of all four suborders of parasitic lice.
The thrips (Thysanoptera) were found to be monophyletic. The
thrips family Phlaeothripidae was recovered as the sister taxon to
the remaining thrips (Aeolothripidae +Thripidae), congruent
with previous molecular analyses and the current classification of
Thysanoptera into the suborders Tubulifera (i.e., Phlaeothripidae)
and Terebrantia (all other thrips) (19).
The order Hemiptera was also monophyletic. Within Hemi-
ptera, Sternorrhyncha (whiteflies, psyllids, scales, and aphids) was
recovered as the sister taxon of the remaining hemipterans. Re-
cent classification schemes (20) and prior molecular studies (13,
21) have placed the enigmatic moss bugs as the sister taxon of true
bugs. However, our results recovered moss bugs as the sister
taxon of Auchenorrhyncha (leafhoppers, planthoppers, and rel-
atives), which was also found by Misof et al. (1). In FcLM
analyses, 96% of quartets placed moss bugs with Auchenor-
rhyncha, suggesting little underlying conflict in the data for this
result (SI Appendix, Table S6).
| Johnson et al.
Within Sternorrhyncha, whiteflies (Aleyrodoidea) were sister
to the remainder of the suborder, and psyllids (Psylloidea) were
sister to a clade composed of aphids (Aphidoidea) +scale insects
(Coccoidea), also supported by 91% of quartets in FcLM analyses.
Previous phylogenetic analyses of Sternorrhyncha have tended to
focus within particular superfamilies or families (e.g., refs. 2224)
Lygaeoidea (9)
Coreoidea (3)
Pyrrhocoroidea (3)
Pentatomoidea (9)
Aradoidea (2)
Miroidea (5)
Cimicoidea (2)
Naboidea (2)
Reduvioidea (4)
Saldoidea (1)
Notonectoidea (3)
Naucoroidea (2)
Ochteroidea (1)
Nepoidea (3)
Corixoidea (2)
Gerroidea (4)
Hydrometroidea (1)
Mesoveloidea (1)
Enicocephalomorpha (1)
Dipsocoromorpha (1)
Membracoidea (15)
Cercopoidea (4)
Cicadoidea (2)
Fulgoroidea (13)
Coleorrhyncha (3)
Coccoidea (9)
Aphidoidea (6)
Psylloidea (9)
Aleyrodoidea (1)
Aeolothripidae (4)
Thripidae (3)
Phlaeothripidae (1)
hT ar
Homilopsocidea (7)
Caeciliusetae (4)
Psocetae (4)
Epipsocetae (1)
Philotarsetae (2)
Phthiraptera (8)
Liposcelididae (2)
Sphaeropsocidae (1)
Amphientometae (1)
Trogiomorpha (2)
Holometabola (11)
Polyneoptera (17)
Palaeoptera (5)
Carboniferous Permian Jurassic CretaceousTriassic Paleog. Neo. Q.
150200250300 0mya350400
true bugs
moss bugs
scale insects
bark lice and
parasitic lice
parasitic lice
book lice
bark lice
semi-aquatic bugs
aquatic bugs
shore bugs
litter bugs
unique-headed bugs
assassin bugs
damsel bugs
bed bugs
plant bugs
stink bugs
red bugs
seed bugs
leaf-footed bugs
bark lice
bark lice
bark lice
Fig. 1. Dated phylogeny of hemipteroid insects (Hemiptera, Thysanoptera, and Psocodea) based on maximum likelihood analysis of a supermatrix of first and
second codon position nucleotides corresponding to 859,518 aligned amino acid positions from transcriptome or genome sequences of 193 samples. Colored
circles indicate bootstrap support. Timescale in millions of years (Bottom) estimated from MCMCTree Bayesian divergence time analyses using 23 fossil
calibration points and a reduced dataset. Number of species sampled from each group indicated in parentheses. Higher taxa are indicated as taxon labels and
below branches; most convenient generalized common names are above branches. Images represent five major groups: Heteroptera, Auchenorrhyncha,
Sternorrhyncha, Thysanoptera, and Psocodea.
Johnson et al. PNAS Latest Articles
rather than addressing relationships among major lineages
The earliest molecular phylogenetic analyses of Hemiptera (e.g.,
refs. 25 and 26) failed to recover Auchenorrhyncha as a mono-
phyletic group, as has a more recent analysis of mitochondrial
genomes (15). However, our analyses provided strong support for
monophyly of this group, corroborating results of other studies
based on multiple loci (13, 14). Within Auchenorrhyncha, our re-
sults strongly support the taxonomic status of the two recognized
infraorders Fulgoromorpha (i.e., Fulgoroidea, planthoppers) and
Cicadomorpha (leafhoppers/treehoppers, spittlebugs, and cicadas)
as monophyletic, as found previously (13). However, relationships
among the three superfamilies of Cicadomorpha were inconsis-
tently resolved. Cicadas (Cicadoidea) plus spittlebugs (Cercopoi-
dea) were sister to leafhoppers/treehoppers (Membracoidea) in the
analysis of nucleotide sequences (Fig. 1, FcLM 52% of quartets),
but cicadas were sister to spittlebugs plus leafhoppers/treehoppers
in the analysis of amino acid sequence data (SI Appendix,Fig.S1),
which was also found in 48% of quartets of nucleotide data in
FcLM analyses.
Relationships among the earlier diverging lineages of true bugs
(Heteroptera) have not been resolved consistently across previous
analyses (14, 2729), in which the deepest divergences received low
statistical branch support and recovered different relationships
among infraorders. In our analysis, which included representatives
of all seven currently recognized infraorders, the four infraorders
for which more than one species was included were found to be
monophyletic. Like two recent studies based on combined molec-
ular and morphological data (29) and transcriptome data (14),
we found 100% bootstrap support for (i) a clade comprising litter
bugs (Dipsocoromorpha), unique-headed bugs (Enicocephalo-
morpha), and semiaquatic bugs (Gerromorpha) (also found in
100% of quartets in FcLM analyses) and (ii) shore bugs (Lep-
topodomorpha) as the sister to Cimicomorpha +Pentatomo-
morpha (also found in 100% of quartets in FcLM analyses).
Divergence Time Analysis. The estimate of the root age for our
tree, the split between Paleoptera (dragonflies, damselflies, and
mayflies) and Neoptera (all other insects) at 437 million years
ago (mya) (95% CI 401486) was only slightly older than that
estimated for this node by Misof et al. (1), at 406 mya. Di-
vergence dates for more interior nodes tended to be older than
those estimated by Misof et al. (1) and more similar to those of
Tong et al. (30), possibly due either to much denser sampling of
minimum age fossil calibration points throughout this part of the
insect tree or to different methodology (e.g., MCMCtree versus
BEAST or different prior distributions of expected ages for
Bayesian analyses). Analyses of divergence times postulated a
common ancestor of thrips and hemipterans as early as the Devo-
nian (407 mya, 95% CI 373451). Radiation within Hemiptera is
also inferred to have begun in this period (386 mya, 95% CI 354
427), with radiations within Sternorrhyncha, Auchenorrhyncha, and
Heteroptera having commenced by the late Carboniferous (all be-
fore 300 mya). Radiation within modern Psocodea dates to the
Carboniferous (328 mya, 95% CI 292376), with divergence of this
lineage from other insects as early as 404 mya (95% CI 367451).
Analysis of 2,395 protein-coding, single-copy genes derived from
transcriptomes of hemipteroid insects and outgroups provided
strong support for a backbone tree of hemipteroid insects largely
congruent with previous analyses and classification schemes. In
particular, we recovered with strong support monophyly of the
three orders of hemipteroid insects: Psocodea, Thysanoptera,
and Hemiptera. We also recovered monophyly of most currently
recognized suborders, infraorders, and superfamilies within these
groups as well as resolving relationships among these major
groups. Although the unconventional result of a sister relation-
ship between Psocodea and Holometabola of Misof et al. (1)
appeared to be robust to our substantially increased taxon
sampling based on maximum likelihood bootstrapping, it was not
supported by four-cluster likelihood mapping analyses. FcLM,
which can detect potentially confounding signal, suggests ex-
tensive underlying conflict for this result, with the majority of
quartets placing Psocodea with thrips and hemipterans, which
would imply monophyly of Paraneoptera in rooted trees. How-
ever, permutations appear to rule out several possible types of
confounding signal (e.g., among-lineage heterogeneity or non-
random distribution of missing data) in our dataset. Recent work
has suggested that bootstrap support from very large datasets
may provide an overestimate of confidence for phylogenetic re-
sults (3133). Thus, the position of Psocodea in the insect tree is
still an open question. Monophyly of hemipteroid insects is
supported by several morphological autapomorphies (34); there-
fore, nonmonophyly of the group would imply homoplasy in these
traits. In addition, there is no known morphological apomorphy
supporting Psocodea +Holometabola (SI Appendix,Fig.S5). In
contrast, the other less conventional relationship, a clade com-
prising Coleorrhyncha and Auchenorrhyncha uncovered by Misof
et al. (1), was recovered by our trees with increased taxon sam-
pling and is supported by 96% of quartets in the FcLM analyses
and three morphological apomorphies, suggesting that this result
is robust.
Divergence time estimates using a dense sampling of 23 fossil
calibration points suggest that the radiation of the hemipteroid
insect orders is relatively ancient, beginning before the early
Carboniferous, considerably older than initial expectations based
on available fossils. However, the insect fossil record of this
period is extremely fragmentary, and relatively old fossils of
modern lineages that are used as calibration points imply that
branches uniting these lineages must be older still, given that
fossil ages represent minimum ages.
Implications for Evolution of Feeding Strategy. Our phylogenetic
results generally agree with evidence from the fossil record that the
earliest hemipteroids fed on detritus, pollen, fungi, or spores (as in
most modern bark lice and thrips). Plant-fluid feeding probably
coincided with the origin of Hemiptera and was independently
derived in thrips. Today, Hemiptera is the fifth largest insect order,
surpassed only by the four major holometabolous orders (Hyme-
noptera, Coleoptera, Lepidoptera, and Diptera). It remains one of
the most abundant and diverse groups of plant-feeding insects.
Within Hemiptera, the origin of true bugs apparently coincided
with a shift from herbivory to predation, with subsequent shifts
back to herbivory (29, 35) in the more derived lineages (Pentato-
momorpha and Cimicomorpha). The two other large suborders of
Hemiptera (Auchenorrhyncha and Sternorrhyncha) feed almost
exclusively on vascular plant fluids.
Our results also suggest that the earliest hemipterans fed
preferentially on phloem. Phloem feeding remains predominant in
extant plant-feeding hemipterans, including nearly all Sternor-
rhyncha and most Auchenorrhyncha (36), while modern moss
bugs feed on phloem-like tissues in mosses (37). A shift to xylem
feeding appears to have coincided with the origin of Cicadomor-
pha (at least the crown group of this lineage), in which all cicadas
and spittlebugs retain this preference. This is also supported by the
fossil record in which the earliest leafhoppers had inflated faces
(38), indicating a preference for xylem feeding, despite the pre-
dominance of phloem feeding among modern leafhoppers and
treehoppers (Membracoidea). A shift to phloem feeding appar-
ently occurred early in the evolution of Membracoidea but at least
one reversal to xylem feeding [in Cicadellinae (sharpshooters)] has
been inferred previously (39), consistent with our results.
Implications for Morphological Evolution. Based on the conflicting
statistical support between the supermatrix analysis and four-
cluster likelihood mapping, the position of lice (Psocodea) ap-
pears to be unstable. Morphological evidence, in contrast, supports
the monophyly of hemipteroid insects (Paraneoptera). Our parsi-
mony mapping of 142 morphological characters (SI Appendix,Fig.
S5) found no apomorphies supporting Psocodea +Holometabola
but 14 apomorphies supporting hemipteroid insect monophyly.
| Johnson et al.
Some of these are reductions or losses, including the reduced
number of tarsomeres (three in modern hemipteroids), reduced
number of Malpighian tubules (four), and presence of only one
abdominal ganglionic complex. Nevertheless, these characters, to-
gether with characters of the forewing base, still appear to support
the sister group relationship between Psocodea and thrips plus
hemipterans (11, 34, 40). Thus, the phylogenetic position of Pso-
codea requires further study of morphological and molecular data.
In contrast to the equivocal support for Paraneoptera, Con-
dylognatha is strongly supported not only in the phylogenomic
analyses, but also with six morphological apomorphies. The or-
igin of this group apparently coincided with a distinct shift in
mouthpart morphology and feeding habits toward piercing and
sucking. These changes include anterior shifting of tentorial pits,
elongated and slender mandibles, stylet-like laciniae, and a
narrowed labium (SI Appendix, Fig. S5). Subsequent evolution-
ary transformations led to the very distinct and unique piercing
sucking mouthparts of hemipterans that facilitate ingestion of
liquid from plant or animal tissues.
The sister-group relationship that we found between moss bugs
(Coleorrhyncha) and Auchenorrhyncha has not, to our knowledge,
been proposed previously in any explicit phylogenetic analysis other
than in recent phylogenomic analyses of transcriptomes (1, 14).
Traditionally, moss bugs were treated as one of three suborders of
Homoptera(along with Sternorrhyncha and Auchenorrhyncha),
largely based on the structure of the head. The mouthparts of moss
bugs arise posteroventrally (41), as in leafhoppers and relatives,
rather than anteriorly as in true bugs (42). Nevertheless, morpho-
logical evidence from fossil and living moss bugs, primarily from
wing structure and musculature, suggested a closer relationship to
true bugs (9, 41, 43). However, a recent comparative morphological
study (12) revealed that moss bugs share a unique derived feature of
the wing base with Auchenorrhyncha; a membranous proximal
median plate. The same study also showed that some previously
suggested morphological synapomorphies of moss bugs and true
bugs (SI Appendix,Fig.S5C) are either ambiguous or have been
misinterpreted (12). Prior molecular evidence supporting moss bugs
plus true bugs was also somewhat equivocal [ref. 13: maximum
likelihood (ML) bootstrap 83% and maximum parsimony (MP)
bootstrap 63%]. Our results support those of other transcriptome
studies (1, 14) in placing Coleorrhyncha sister to Auchenorrhyncha.
Implications for Evolution of Mitochondrial Genome Organization.
Several groups of hemipteroid insects have been shown to have
highly rearranged mitochondrial genomes (2). The sister re-
lationship between thrips and hemipterans indicates that the
heightened rates of mitochondrial (mt) genome rearrangements
observed in the lice (44) and thrips (45) are the result of con-
vergence between these two clades. Even if Psocodea is sister to
thrips plus hemipterans, and not to holometabolous insects, re-
cent analyses indicating that the ancestor of all Psocodea had a
generally standard insect mitochondrial gene order still result in
an interpretation involving convergence (46). This phylogenetic
evidence is also consistent with the absence of any shared, de-
rived gene arrangements between Psocodea and thrips, as both
have independently diverged from the inferred ancestral insect
mt genome arrangement (2, 45).
An interpretation involving convergence is also consistent with
the varying degrees of rearrangement observed within each order.
Within Psocodea, mt genomes vary wildly across different taxo-
nomic scales, from a single derived arrangement found in all
Psocomorpha (46), to wide variation within a single genus (Lip-
oscelis, ref. 47), and between closely related species of parasitic
lice. In contrast, for the thrips, mitochondrial genome arrange-
ments are relatively consistent at the family level (with only tRNA
rearrangements observed), albeit still highly rearranged relative to
the ancestral insect mt genome (48). Very few rearrangements of
any type are observed in the Hemiptera, with the vast majority of
families possessing the inferred ancestral arrangement (2).
In summary, although the exact phylogenetic position of
Psocodea remains to be resolved convincingly, our results based
on transcriptomes for hemipteroid insects provide a strong phyloge-
netic framework for future studies of genomic, morphological, eco-
logical, and behavioral characteristics of this important group of insects.
Materials and Methods
Our general approach closely followed methods described previously by
Misof et al. (1) and Peters et al. (49) for phylogenomic analyses of insect
transcriptomes (SI Appendix, Dryad repository, 10.5061/dryad.t4f4g85). Tran-
scriptomes of 140 samples of Paraneoptera were newly sequenced with
100 bp paired-end reads for this study using Illumina HiSeq2000 or HiSeq2500
machines to achieve at least 2.5 Gbp per taxon. The final taxon sample of
193 includes representatives of 97 hemipteroid families with several larger
families represented by multiple subfamilies.
All paired-end reads were assembled with SOAPdenovo-Trans (version
1.01; ref. 50) and the assembled transcripts were filtered for possible con-
taminants (SI Appendix, Table S2) as described in Peters et al. (49). The raw
reads and filtered assemblies were submitted to the NCBI SRA and TSA ar-
chives (SI Appendix, Table S1). We searched the assemblies for transcripts of
2,395 protein-coding genes that the OrthoDB v7 database (51) suggested to
be single copy across the genomes of six species (SI Appendix, Table S3) using
the software Orthograph (version beta4, ref. 52; for results of the orthology
search see SI Appendix, Table S4). Orthologous transcripts were aligned with
MAFFT (version 7.123; ref. 53) at the translational (amino acid) level. Cor-
responding nucleotide multiple sequence alignments were generated with a
modified version of the software Pal2Nal (54) (version 14).
Alignment sections that could not be discriminated from randomly aligned
regions at the amino acid level of each gene were identified with Aliscore version
1.2 (55, 56). To maximize the fit of our substitution models, we identified for
each gene the protein domains (clans, families) and unannotated regions using
the Pfam database (refs. 1 and 57 and SI Appendix,Supplemental Materials and
Methods). The phylogenetic information content of each data block was assessed
with MARE (version 0.1.2-rc) (58), and all uninformative data blocks (IC =0) were
removed. We subsequently used PartitionFinder (developer version 2.0.0-pre14,
ref. 59) to simultaneously infer the best partitioning scheme and amino acid or
nucleotide (removing third positions because of heterogeneity, SI Appendix,Fig.
S6) substitution models, using the rclusterf algorithm.
Phylogenetic treeswere inferred using a maximum likelihood approach with
ExaML version 3.0.17 (60)for both the nucleotide and amino acid datasets. We
performed 50 nonparametric bootstrap replicates mapping the support on the
best ML tree after checking for bootstrap convergence with the default
bootstopping criteria (61). An optimized dataset, which requires the presence
of at least one species from a given taxonomic group (SI Appendix,TableS5)in
each data block of the supermatrix (62), was used for testing the possible
impact of missing data at the partition level. Four-cluster likelihood mapping
(63) was used for assessing the phylogenetic signal for alternative phyloge-
netic relationships (SI Appendix,TablesS5andS6). Permutation tests in these
analyses assessed the impact of heterogeneous amino acid sequence compo-
sition among lineages, nonstationarity of substitution processes, and non-
random distribution of missing data on the inferred phylogenetic tree (1).
To understand the morphological transformations underlying the evolution
of the hemipteroid groups and to identify potential shared derived characters
(synapomorphies),we used the morphological data matrix of Friedemann et al.
(9) with 118 charactersof the entire body (with modifications from ref.12) and
additionally 25 characters associated with the wing base (8). By tracing char-
acters over the tree using maximum parsimony using Winclada (64), we
evaluated three possible phylogenetic alternatives: (i) paraphyletic Para-
neoptera and Coleorrhyncha sister to Auchenorrhyncha (result from ML
analysis of transcriptomes); (ii) monophyletic Paraneo ptera (as sugg ested by
FcLM analyses); and (iii) paraphyletic Paraneoptera, but with Coleorrhyncha
sister to Heteroptera (as suggested in previous literature).
To estimate divergence dates, we used the topology resulting from ML
analysis of first and second position nucleotides as the input tree and assigned
23 ingroup fossil calibration points (65) throughout the tree (SI Appendix,
Table S7). These calibrations were used as minimum ages in soft bound
uniform priors with a root age of 406 mya (1) as a soft bound maximum.
These priors were used in a Bayesian MCMCTree (66) molecular dating
analysis of a first and second position nucleotide dataset for which sites were
present in at least 95% of taxa.
ACKNOWLEDGMENTS. We thank E. Anton, M. Bowser, C. Bramer, T. Catanach,
D. H. Clayton, J. R. Cooley, G. Gibbs, A. Hansen, E. Hdez, A. Katz, K. Kjer, J. Light,
K. Schütte, W. Smith, K.-Q. Song, T. Sota, N. Szucsich, G. Taylor, S. Taylor,
S. Tiwari, and X. Tong for assistance with obtaining specimens; G. Meng and
Beijing Genomics Institute staff for their efforts in data curation; and O. Niehuis
Johnson et al. PNAS Latest Articles
for assistance preparing the ortholog gene set. R.M.W. was supported by Swiss
National Science Foundat ion Grant PP00P3_1706642. K.M. was supported by
David Yeates, the Schlinger Endowment, CSIRO National Research Council
Australia, J. Korb, and the University of Freiburg. This work was also sup -
ported by National Science Foundation DEB-1239788 (to K.P.J., C.H.D., and
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| Johnson et al.
... Booklice are the phylogenetic sister group to parasitic lice and they have been considered a key taxon in determining the origins and evolution of parasitic lice [24][25][26]. The habits of Liposcelis species are similar to those of parasitic lice; for example, they are found in the nests of birds and mammals, indicating a close relationship with their potential host [27][28][29]. ...
... Our results thus indicated a closer relationship of the Paraneoptera clades. The MCMCTREE result indicated a divergence time between booklice and parasitic lice at ∼231 mya, which is similar to the results of several previous studies [25]. ...
Full-text available
Background Booklice (psocids) in the genus Liposcelis (Psocoptera: Liposcelididae) are a group of important storage pests, found in libraries, grain storages, and food-processing facilities. Booklice are able to survive under heat treatment and typically possess high resistance to common fumigant insecticides, hence posing a threat to storage security worldwide. Results We assembled the genome of the booklouse, L. brunnea, the first genome reported in Psocoptera, using PacBio long-read sequencing, Illumina sequencing, and chromatin conformation capture (Hi-C) methods. After assembly, polishing, haplotype purging, and Hi-C scaffolding, we obtained 9 linkage groups (174.1 Mb in total) ranging from 12.1 Mb to 27.6 Mb (N50: 19.7 Mb), with the BUSCO completeness at 98.9%. In total, 15,543 genes were predicted by the Maker pipeline. Gene family analyses indicated the sensing-related gene families (OBP and OR) and the resistance-related gene families (ABC, EST, GST, UGT, and P450) expanded significantly in L. brunnea compared with those of their closest relatives (2 parasitic lice). Based on transcriptomic analysis, we found that the CYP4 subfamily from the P450 gene family functioned during phosphine fumigation; HSP genes, particularly those from the HSP70 subfamily, were upregulated significantly under high temperatures. Conclusions We present a chromosome-level genome assembly of L. brunnea, the first genome reported for the order Psocoptera. Our analyses provide new insights into the gene family evolution of the louse clade and the transcriptomic responses of booklice to environmental stresses.
... were newly sequenced in this study, with the remaining 42 transcriptomes obtained from Misof et al. (2014), Johnson et al. (2018) and Skinner et al. (2019). For newly sequenced individuals, fresh specimens collected in the field were photographed, the abdominal genital segment was removed, and the rest of the insect body was immediately placed into a plastic tube containing RNAlater, with 1 specimen in each tube. ...
... MAFFT 7.471, HMMer 3.3, NCBI BLAST+ 2.2.28, and Exonerate 2.2.0. The reference set of orthologues in this study included 2395 genes previously identified in Paraneoptera by Johnson et al. (2018). ...
The suborder Auchenorrhyncha (“true hoppers”) comprises nearly half of known Hemiptera, with >43,000 known species of sap‐sucking herbivores distributed worldwide, including many important agricultural pests and vectors of plant disease. More than half of the known Auchenorrhyncha belong to superfamily Membracoidea (leaf‐ and treehoppers), which has been a source of phylogenetic contention for many years. To construct an improved backbone phylogeny of this superfamily, we obtained transcriptome data for multiple representatives of all 5 previously established extant families and nearly all subfamilies to test their monophyly and relationships. 138 taxa (132 Membracoidea and 6 outgroups) were sampled with an emphasis on families Cicadellidae and Membracidae, which were paraphyletic as previously defined by most authors, several problematic subfamilies (Aphrodinae, Eurymelinae, Ledrinae, Nicomiinae, Stegaspidinae and Tartessinae). We analysed different combinations of data sets (amino acid, complete nucleotide and degeneracy‐coded nucleotide) using different modelling schemes. The resultant trees based on different analyses are congruent in most nodes. Discordant nodes mainly pertain to relationships among cicadellid subfamilies and tribal relationships within Aphrodinae and Eurymelinae. Analyses of gene‐ and site concordance factors and quartet scores indicate that this instability is largely attributable to an overall lack of informative characters across genes and sites rather than strongly supported conflict among genes. According to the congruent nodes, we make the following revisions: combine Stegaspidinae and Centrotinae into a single subfamily, Centrotinae sensu lato; restore Stenocotini from Tartessinae to its original position in the Ledrinae; and transform Holdgatiella Evans from Nicomiinae to Melizoderinae. In addition, to solve the paraphyly of both Cicadellidae and Membracidae, a preferred option would be to combine all five previously recognized families into a single family, Membracidae sensu lato; the other option could be to render Cicadellidae monophyletic by excluding Megophthalminae and Ulopinae from Cicadellidae and elevating them to status as separate families. The phylogeny of Membracoidea was reconstructed based on a greatly expanded taxon sample of 139 transcriptomes covered all 5 families, 40 subfamilies and 90 tribes of Membracoidea. Most of the Membracoidea relationship was resolved with phylogenomics even though the relationships remain unresolved among a few subfamilies of Cicadellidae and tribes within Aphrodinae and Eurymelinae. The following revisions were made according to well‐resolved and highly supported clades: combined Stegaspidinae and Centrotinae into a single subfamily, Centrotinae sensu lato; restored Stenocotini from Tartessinae to its original position in the Ledrinae; transformed Holdgatiella from Nicomiinae to Melizoderinae.
... Females treated with dsE93 (n=36) also formed an ootheca on AdD8, and 50% of them gave first instar nymphs 18 days later (between 35 and 40 emerged nymphs per ootheca). However, 27.8% dropped the ootheca between ED2 and ED3 (12-17% embryogenesis), whereas 22.2% transported the ootheca beyond day 18, dropping it between ED19 and ED21. No nymphs emerged from these 18 oothecae. ...
The early embryo of the cockroach Blattella germanica exhibits high E93 expression. In general, E93 triggers adult morphogenesis during postembryonic development, but in the cockroach E93 is also crucial in early embryogenesis. Moreover, the embryonic levels of E93 expression are high in hemimetabolan insects, while in holometabolans they are very low. They are also low in Thysanoptera and in Hemiptera Sternorrhyncha with postembryonic quiescent stages, as well as in Odonata, the nymph of which is very different from the adult. In ametabolans, such as the Zygentoma Thermobia domestica, E93 expression levels are very high in the early embryo, whereas during postembryonic development they are medium and relatively constant. Given that embryogenesis of hemimetabolans yields an adultiform nymph, we speculate that E93 plays some sort of adult triggering role in the embryo of these species. We conjecture that the reduction of E93 transcript levels in the embryo has been instrumental in the evolution of insect metamorphosis. The suppression of E93 expression during the nymphal period, and its concentration in the preadult stage, is consubstantial with the emergence of hemimetaboly. As such, attenuation of E93 expression in the embryo could have resulted in a larval genetic program and the emergence of holometaboly. Independent decreases of E93 expression in the embryo of Odonata, Thysanoptera, and different groups of Hemiptera Sternorrhyncha would have allowed the development of modified juvenile stages adapted to specific ecophysiological conditions.
... In this study, we 50 tackle this topic by reconstructing the evolution of male genital size across the order Hemiptera, spanning over 350 m.y. 51 of evolution (Johnson et al. 2018). Following, we link the phenotype with the genotype by estimating the association 52 between genital size and the rates of sequence evolution of several developmental genes. ...
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Our understanding of the genetic architecture of phenotypic traits has experienced drastic growth over the last years. Nevertheless, the majority of studies associating genotypes and phenotypes have been conducted at the ontogenetic level. Thus, we still have an elusive knowledge of how these genetic-developmental architectures evolve themselves and how their evolution is mirrored in the phenotypic change across evolutionary time. We tackle this gap by reconstructing the evolution of male genital size, one of the most complex traits in insects, together with its underlying genetic architecture. Using the order Hemiptera as a model, spanning over 350 million years of evolution, we estimate the correlation between genitalia and three features: development rate, body size, and rates of DNA substitution in 68 genes associated with genital development. We demonstrate that genital size macro-evolution has been largely dependent on body size and weakly influenced by development rate and phylogenetic history. We further revealed significant correlations between mutation rates and genital size for 19 genes. Interestingly, these genes have diverse functions and participate in distinct signaling pathways, suggesting that genital size is a complex trait whose fast evolution has been enabled by molecular changes associated with diverse morphogenetic processes. Our data further demonstrate that the majority of DNA evolution correlated with the genitalia has been shaped by negative selection or neutral evolution. Thus, in terms of sequence evolution, changes in genital size are predominantly facilitated by relaxation of constraints rather than positive selection, possibly due to the high pleiotropic nature of the morphogenetic genes.
... The phylogenetic relationships among species at higher than species level were taken from existing phylogenies that were published most recently. Specifically, we used Misof et al. (2014) for the phylogeny of insect orders, McKenna et al. (2019) for the phylogeny of beetles, Johnson et al. (2018) and Jung and Lee (2012) for the phylogeny of Hemiptera, and Wheeler et al. (2017) for the phylogeny of spiders. The relationships at lower than genus level were reconstructed using new molecular data: For 36 species, sequences of the COI molecular marker were available from GenBank, and 34 species were bar-coded de novo. ...
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Myrmecomorphy is the most frequent type of Batesian mimicry. Myrmecomorphic species differ in the accuracy with which they resemble ants, however, the hypothesis of the co-evolution of mimetic traits has been rarely tested. Here, we measured dozens of traits of colour, shape, size, and behaviour, and quantified objectively the resemblance between dozens of arthropod mimics and ants. In all traits, the mimics were more similar to ants than to closely related non-myrmecomorphic species. We found that mimics resemble ants mainly in colour and behaviour, and less in size and body shape. We found that the mimetic accuracy in four trait categories show divergent co-evolutionary patterns. Mimetic accuracy in colour was positively correlated to shape and size in insects but negatively in spiders, presumably reflecting developmental constraints. Accuracy in shape was negatively related to movement in both insects and spiders supporting the motion-limited discrimination hypothesis.
... Enicocephalomorpha, commonly called as unique-headed bugs, is a poorly known group of true bugs. It has long been recognized as one of the basal infraorders within Heteroptera on the basis of morphological and molecular evidence (Schuh 1979;Schuh and Slater 1995;Li et al., 2017;Johnson et al., 2018;Wang et al., 2019). This infraorder comprises two families, Enicocephalidae and Aenictopecheidae. ...
A new genus and species of unique-headed bug, Cretocephalus stysi gen. et sp. nov., is described based on a well-preserved specimen from mid-Cretaceous amber from northern Myanmar. Habitus photographs of the male holotype, photographs and drawings of detailed characters of head, legs, thorax, and photographs under epifluorescence of abdomen and terminalia are provided. The new genus exhibits remarkable differences in the diagnostic characters of all four known aenictopecheid subfamilies. However, it also shares several important characters with Aenictopecheinae, Maoristolinae, and Nymphocorinae. The morphological characters of Cretocephalus in pronotum, leg armature, and forewing may provide new insights on the morphological diversity and the higher classification system of this family. Additionally, the fossil genera Paenicotechys (Aenictopecheidae) and Enicocephalinus (Enicocephalidae) are briefly reviewed and the latter is transferred to Aenictopecheidae.
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Capturing phylogenetic signal from a massive radiation can be daunting. The superfamily Chalcidoidea is an excellent example of a hyperdiverse group that has remained recalcitrant to phylogenetic resolution. Chalcidoidea are mostly parasitoid wasps that until now included 27 families, 87 subfamilies and as many as 500,000 estimated species. We combined 1007 exons obtained with Anchored Hybrid Enrichment with 1048 Ultra-Conserved Elements (UCEs) for 433 taxa including all extant families, over 95% of all subfamilies and 356 genera chosen to represent the vast diversity of the superfamily. Going back and forth between molecular results and our collective morphological and biological knowledge, we detected insidious bias driven by the saturation of nucleotide data and highlighted morphological convergences. Our final results are based on a concatenated analysis of the least saturated exons and UCE data sets (2054 loci, 284,106 sites). Our analyses support a sister relationship with Mymarommatoidea. Seven of the previously recognized families were not monophyletic, so foundations for a new classification are discussed. Biology appears potentially more informative than morphology, as illustrated by the elucidation of a clade of plant gall associates and a clade of taxa with planidial first-instar larvae. The phylogeny suggests a shift from smaller soft-bodied wasps to larger and more heavily sclerotized wasps. Deep divergences in Chalcidoidea coincide with an increase in insect families in the fossil record, and an early shift to phytophagy corresponds with the beginning of the “Angiosperm Terrestrial Revolution”. Our dating analyses suggest a Middle Jurassic origin of 174 Ma (167.3-180.5 Ma) and a crown age of 162.2 Ma (153.9–169.8 Ma) for Chalcidoidea. During the Cretaceous, Chalcidoidea underwent a rapid radiation in southern Gondwana with subsequent dispersals to the Northern Hemisphere. This scenario is discussed with regard to knowledge about host taxa of chalcid wasps, their fossil record, and Earth’s paleogeographic history.
Mycorrhizal plants mediate interactions between their root‐associated fungal symbionts and insect herbivores. Arbuscular mycorrhizal (AM) associations tend to boost phloem sap sucking insects and host specialist chewing insects but depress host generalist chewing insects. In turn, insect herbivores have only small negative effects on the abundance of AM fungi. Ectomycorrhizal (EM) associations, less studied, have no consistent effect on phloem sap‐sucking or chewing insects, but their insect herbivores have strong negative effects on EM fungal abundance. Spittlebugs (Hemiptera: Cercopoidea) feed on xylem sap and are disproportionately associated with EM plants, probably because many EM fungi are especially well equipped to access nitrogen (N) from complex soil organic matter. Assimilated N is transported in host plant xylem sap as amino acids, the primary food source for xylem‐feeding insects. Although EM plants account for only 2% of all vascular plants, about 19% of spittlebugs have EM primary hosts. This compares to 9% of hosts that are rhizobial or actinorhizal N‐fixing plants, alternative modes of root‐associated N acquisition that also attract spittlebugs. Notable spittlebug EM host plants include Pinus and Abies (Pinaceae), Quercus, Alnus and Betula (Fagales), Salix (Salicaceae), Eucalyptus (Myrtaceae) and the gymnosperm Gnetum (Gnetales). Some EM plants, including several Pinus species, host spittlebug pests and some play an important role in the ecology of spittlebug vectors of the bacterial plant pathogen Xylella fastidiosa. The fossil record and dated molecular phylogenies suggest that spittlebugs initially evolved in parallel with EM Pinaceae.
The thoracic musculature of the insect order Psocodea has been examined in only a few species of a single suborder to date. In the present study, we examined the thoracic musculature of species selected from all three suborders of Psocodea to elucidate the ground plan of the order and to examine the phylogenetic utility of the character system. The sister‐group relationship between the suborders Troctomorpha and Psocomorpha received support from two novel nonhomoplasious synapomorphies, although the support from other morphological characters for this relationship is ambiguous. The sister‐group relationship between the infraorders Epipsocetae and Psocetae also received support from one nonhomoplasious synapomorphy, although no other morphological characters supporting this relationship have been identified to date. The present examination revealed the potential of thoracic muscle characters for estimating deep phylogeny, possibly including interordinal relationships. We examined the thoracic musculature of all three suborders of Psocodea to elucidate the ground plan of the order and to examine the phylogenetic utility of this character system. The sister‐group relationship between the suborders Troctomorpha and Psocomorpha received support from two novel nonhomoplasious synapomorphies. The present examination revealed the high potential of thoracic muscle characters for estimating deep phylogeny, possibly including interordinal relationships.
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Mammals host a wide diversity of parasites. Lice, comprising more than 5,000 species, are one group of ectoparasites whose major lineages have a somewhat patchwork distribution across the major groups of mammals. Here we explored patterns in the diversification of mammalian lice by reconstructing a higher-level phylogeny of these lice, leveraging whole genome sequence reads to assemble single-copy orthologue genes across the genome. The evolutionary tree of lice indicated that three of the major lineages of placental mammal lice had a single common ancestor. Comparisons of this parasite phylogeny with that for their mammalian hosts indicated that the common ancestor of elephants, elephant shrews and hyraxes (that is, Afrotheria) was the ancestral host of this group of lice. Other groups of placental mammals obtained their lice via host-switching out of these Afrotherian ancestors. In addition, reconstructions of the ancestral host group (bird versus mammal) for all parasitic lice supported an avian ancestral host, indicating that the ancestor of Afrotheria acquired these parasites via host-switching from an ancient avian host. These results shed new light on the long-standing question of why the major groups of parasitic lice are not uniformly distributed across mammals and reveal the origins of mammalian lice. Mammals host a diversity of parasites including lice. Using cophylogenetics and phylogenetic comparative methods, the authors show that the main lineages of placental mammal lice had a single common ancestor and find that all parasitic lice had an avian ancestral host.
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Heteroptera, the true bugs, are part of the largest clade of non-holometabolous insects, the Hemiptera, and include > 42 000 described species in about 90 families. Despite progress in resolving phylogenetic relationships between and within infraorders since the first combined morphological and molecular analysis published in 1993 (29 taxa, 669 bp, 31 morphological characters), recent hypotheses have relied entirely on molecular data. Weakly supported nodes along the backbone of Heteroptera made these published phylogenies unsuitable for investigations into the evolution of habitats and lifestyles across true bugs. Here we present the first combined morphological and molecular analyses of Heteroptera since 1993, using 135 taxa in 60 families, 4018 aligned bp of ribosomal DNA and 81 morphological characters, and various analytical approaches. The sister-group relationship of the predominantly aquatic Nepomorpha with all remaining Heteroptera is supported in all analyses, and a clade formed by Enicocephalomorpha, Dipsocoromorpha and Gerromorpha in some. All analyses recover Leptopodomorpha + (Cimicomorpha + Pentatomomorpha), mostly with high support. Parsimony- and likelihood-based ancestral state reconstructions of habitats and lifestyles on the combined likelihood phylogeny provide new insights into the evolution of true bugs. The results indicate that aquatic and semi-aquatic true bugs invaded these habitats three times independently from terrestrial habitats in contrast to a recent hypothesis. They further suggest that the most recent common ancestor of Heteroptera was predacious, and that the two large predominantly phytophagous clades (Trichophora and Miroidea) are likely to have derived independently from predatory ancestors. We conclude that by combining morphological and molecular data and employing various analytical methods our analyses have converged on a relatively well-supported hypothesis of heteropteran infraordinal relationships that now requires further testing using phylogenomic and more extensive morphological datasets.
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The phylogeny of true bugs (Hemiptera: Heteroptera), one of the most diverse insect groups in terms of morphology and ecology, has been the focus of attention for decades with respect to several deep nodes between the suborders of Hemiptera and the infraorders of Heteroptera. Here, we assembled a phylogenomic data set of 53 taxa and 3102 orthologous genes to investigate the phylogeny of Hemiptera–Heteroptera, and both concatenation and coalescent methods were used. A binode-control approach for data filtering was introduced to reduce the incongruence between different genes, which can improve the performance of phylogenetic reconstruction. Both hypotheses (Coleorrhyncha + Heteroptera) and (Coleorrhyncha + Auchenorrhyncha) received support from various analyses, in which the former is more consistent with the morphological evidence. Based on a divergence time estimation performed on genes with a strong phylogenetic signal, the origin of true bugs was dated to 290–268 Ma in the Permian, the time in Earth's history with the highest concentration of atmospheric oxygen. During this time interval, at least 1007 apomorphic amino acids were retained in the common ancestor of the extant true bugs. These molecular apomorphies are located in 553 orthologous genes, which suggests the common ancestor of the extant true bugs may have experienced large-scale evolution at the genome level.
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A data set comprising DNA sequences from 388 loci and >99,000 aligned nucleotide positions, generated using anchored hybrid enrichment, was used to estimate relationships among 138 leafhoppers and treehoppers representative of all major lineages of Membracoidea, the most diverse superfamily of hemipteran insects. Phylogenetic analysis of the concatenated nucleotide sequence data set using maximum likelihood produced a tree with most branches receiving high support. A separate coalescent gene tree analysis of the same data generally recovered the same strongly supported clades but was less well resolved overall. Several nodes pertaining to relationships among leafhopper subfamilies currently recognized based on morphological criteria were separated by short internodes and received low support. Although various higher taxa were corroborated with improved branch support, relationships among some major lineages of Membracoidea are only somewhat more resolved than previously published phylogenies based on single gene regions or morphology. In agreement with previous studies, the present results indicate that leafhoppers (Cicadellidae) are paraphyletic with respect to the three recognized families of treehoppers (Aetalionidae, Melizoderidae, and Membracidae). Divergence time estimates indicate that most of the poorly resolved divergence events among major leafhopper lineages occurred during the lower to middle Cretaceous and that most modern leafhopper subfamilies, as well as the lineage comprising the three recognized families of treehoppers, also arose during the Cretaceous.
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Hemiptera, the largest non-holometabolous order of insects, represents approximately 7% of metazoan diversity. With extraordinary life histories and highly specialized morphological adaptations, hemipterans have exploited diverse habitats and food sources through approximately 300 Myr of evolution. To elucidate the phylogeny and evolutionary history of Hemiptera, we carried out the most comprehensive mitogenomics analysis on the richest taxon sampling to date covering all the suborders and infra-orders, including 34 newly sequenced and 94 published mitogenomes. With optimized branch length and sequence heterogeneity, Bayesian analyses using a site-heterogeneous mixture model resolved the higher-level hemi-pteran phylogeny as (Sternorrhyncha, (Auchenorrhyncha, (Coleorrhyncha, Heteroptera))). Ancestral character state reconstruction and divergence time estimation suggest that the success of true bugs (Heteroptera) is probably due to angiosperm coevolution, but key adaptive innovations (e.g. prog-nathous mouthpart, predatory behaviour, and haemelytron) facilitated multiple independent shifts among diverse feeding habits and multiple independent colonizations of aquatic habitats.
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Phylogenomic studies have resolved countless branches of the tree of life, but remain strongly contradictory on certain, contentious relationships. Here, we use a maximum likelihood framework to quantify the distribution of phylogenetic signal among genes and sites for 17 contentious branches and 6 well-established control branches in plant, animal and fungal phylogenomic data matrices. We find that resolution in some of these 17 branches rests on a single gene or a few sites, and that removal of a single gene in concatenation analyses or a single site from every gene in coalescence-based analyses diminishes support and can alter the inferred topology. These results suggest that tiny subsets of very large data matrices drive the resolution of specific internodes, providing a dissection of the distribution of support and observed incongruence in phylogenomic analyses. We submit that quantifying the distribution of phylogenetic signal in phylogenomic data is essential for evaluating whether branches, especially contentious ones, are truly resolved. Finally, we offer one detailed example of such an evaluation for the controversy regarding the earliest-branching metazoan phylum, for which examination of the distributions of gene-wise and site-wise phylogenetic signal across eight data matrices consistently supports ctenophores as the sister group to all other metazoans.
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Background: Orthology characterizes genes of different organisms that arose from a single ancestral gene via speciation, in contrast to paralogy, which is assigned to genes that arose via gene duplication. An accurate orthology assignment is a crucial step for comparative genomic studies. Orthologous genes in two organisms can be identified by applying a so-called reciprocal search strategy, given that complete information of the organisms' gene repertoire is available. In many investigations, however, only a fraction of the gene content of the organisms under study is examined (e.g., RNA sequencing). Here, identification of orthologous nucleotide or amino acid sequences can be achieved using a graph-based approach that maps nucleotide sequences to genes of known orthology. Existing implementations of this approach, however, suffer from algorithmic issues that may cause problems in downstream analyses. Results: We present a new software pipeline, Orthograph, that addresses and solves the above problems and implements useful features for a wide range of comparative genomic and transcriptomic analyses. Orthograph applies a best reciprocal hit search strategy using profile hidden Markov models and maps nucleotide sequences to the globally best matching cluster of orthologous genes, thus enabling researchers to conveniently and reliably delineate orthologs and paralogs from transcriptomic and genomic sequence data. We demonstrate the performance of our approach on de novo-sequenced and assembled transcript libraries of 24 species of apoid wasps (Hymenoptera: Aculeata) as well as on published genomic datasets. Conclusion: With Orthograph, we implemented a best reciprocal hit approach to reference-based orthology prediction for coding nucleotide sequences such as RNAseq data. Orthograph is flexible, easy to use, open source and freely available at Additionally, we release 24 de novo-sequenced and assembled transcript libraries of apoid wasp species.
Understanding evolutionary relationships in the superfamily Psylloidea is challenging due to the lack of clear morphological synapomorphies for many groups. Some families and many of the genera, including the two largest, Cacopsylla Ossiannilsson and Trioza Foerster, have long been acknowledged as nonmonophyletic and the circumscription of natural groups has remained fluid. We present the best phylogenetic hypothesis to date for Psylloidea and provide a working systematic framework to better reflect evolutionary relationships. A shotgun sequencing approach using mixed pool DNAs for more than 400 species resulted in recovery from de novo assemblies of near‐complete mitogenomes (≥10 kb) for 359 species, and partial genomes (5–10 kb) for an additional 40 species. The resulting phylogeny improves and clarifies the family classification and resolves some of the longstanding uncertainties in relationships within and between genera. A whole‐nuclear‐genome scan approach (yielding data from an estimated 373 nuclear genes) using the anchored hybrid enrichment method for a representative subset of taxa confirms the placement of major groupings and overall tree topology recovered with the mitochondrial data. The data generated represent a major increase in molecular resources for this superfamily. In addition, we highlight areas of remaining uncertainty that require further sampling and/or additional sources of data. The phylogeny provides new insights for both evolutionary and applied research, and a backbone constraint tree allows the placement of taxa of particular interest or concern (e.g. pest taxa) with only small fragments of sequence available (e.g. DNA barcodes).
The mitochondrial genome arrangement in the insect order Psocodea (booklice, barklice, and parasitic lice) is extremely variable. Genome organization ranges from the rearrangement of a few tRNAs and protein coding genes, through extensive tRNA and protein coding gene rearrangements, to subdivision into multiple mini-chromosomes. Evolution of the extremely modified mitochondrial genome in parasitic lice (Phthiraptera) has been the subject of several studies, but limited information is available regarding the mitochondrial genome organization of the more plesiomorphic, free-living Psocodea (formerly known as the "Psocoptera"). In particular, the ancestral state of the psocodean mitochondrial genome arrangement and the evolutionary pathway to the rearranged conditions are still unknown. In this study, we addressed mitochondrial evolutionary questions within the Psocodea by using mitochondrial genome sequences obtained from a wide range of Psocoptera, covering all three suborders. We identified seven types of mitochondrial genome arrangements in Psocoptera, including the first example in Psocodea of retention of the ancestral pancrustacean condition in Prionoglaris (Prionoglarididae). Two methods (condition-based parsimony reconstruction and common-interval genome distances) were applied to estimate the ancestral mitochondrial arrangement in Psocodea, and both provided concordant results. Specifically, the common ancestor of Psocodea retained the ancestral pancrustacean condition, and most of the gene arrangement types have originated independently from this ancestral condition. We also utilized the genomic data for phylogenetic estimation. The tree estimated from the mitochondrial genomic data was well resolved, strongly supported, and in agreement with previously estimated phylogenies. It also provided the first robust support for the family Prionoglarididae, as its monophyly was uncertain in previous morphological and molecular studies.
The phylogenetic placement of the moss bugs (Insecta: Hemiptera: Coleorrhyncha) has been highly controversial. Many apparent morphological apomorphies support the close relationship between Coleorrhyncha and Heteroptera (=true bugs). However, a recent phylogenomic study strongly supported a sister group relationship between Coleorrhyncha and Auchenorrhyncha (planthoppers, leafhoppers, treehoppers, spittlebugs, and cicadas). To test these two alternative hypotheses, we examined the fore- and hindwing base structure of the only known extant macropterous species of Coleorrhyncha using binocular and confocal laser scanning microscopes and analyzed the data selected from the wing base phylogenetically. When full morphological data including the wing base characters were analyzed, the sister group relationship between Coleorrhyncha + Heteroptera was supported, agreeing with previous consensus based on morphology. In contrast, when only wing base characters were analyzed separately, the clade Coleorrhyncha + Auchenorrhyncha was recovered, in agreement with the result from the phylogenomic study. The membranous condition of the proximal median plate in the forewing was identified as a potential synapomorphy of the latter grouping, and the absence of the tegula was excluded as a potential synapomorphy of Coleorrhyncha and Heteroptera.
Hymenoptera (sawflies, wasps, ants, and bees) are one of four mega-diverse insect orders, comprising more than 153,000 described and possibly up to one million undescribed extant species. As parasitoids, predators, and pollinators, Hymenoptera play a fundamental role in virtually all terrestrial ecosystems and are of substantial economic importance. To understand the diversification and key evolutionary transitions of Hymenoptera, most notably from phytophagy to parasitoidism and predation (and vice versa) and from solitary to eusocial life, we inferred the phylogeny and divergence times of all major lineages of Hymenoptera by analyzing 3,256 protein-coding genes in 173 insect species. Our analyses suggest that extant Hymenoptera started to diversify around 281 million years ago (mya). The primarily ectophytophagous sawflies are found to be monophyletic. The species-rich lineages of parasitoid wasps constitute a monophyletic group as well. The little-known, species-poor Trigonaloidea are identified as the sister group of the stinging wasps (Aculeata). Finally, we located the evolutionary root of bees within the apoid wasp family "Crabronidae." Our results reveal that the extant sawfly diversity is largely the result of a previously unrecognized major radiation of phytophagous Hymenoptera that did not lead to wood-dwelling and parasitoidism. They also confirm that all primarily parasitoid wasps are descendants of a single endophytic parasitoid ancestor that lived around 247 mya. Our findings provide the basis for a natural classification of Hymenoptera and allow for future comparative analyses of Hymenoptera, including their genomes, morphology, venoms, and parasitoid and eusocial life styles.