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Phylogenomics and the evolution of hemipteroid insects

November 2018
Proceedings of the National Academy of Sciences

Authors:

Frank Friedrich

Hamburg University

Rolf Beutel

Friedrich Schiller University Jena

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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.

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Phylogenomics and the evolution of

hemipteroid insects

Kevin P. Johnson

a,1

, Christopher H. Dietrich

, Frank Friedrich

, Rolf G. Beutel

, Benjamin Wipfler

c,d

, Ralph S. Peters

Julie M. Allen

a,e

, Malte Petersen

, Alexander Donath

, Kimberly K. O. Walden

, Alexey M. Kozlov

, Lars Podsiadlowski

f,i

Christoph Mayer

, Karen Meusemann

f,j,k

, Alexandros Vasilikopoulos

, Robert M. Waterhouse

, Stephen L. Cameron

Christiane Weirauch

, Daniel R. Swanson

, Diana M. Percy

o,p

, 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 Urbana–Champaign, 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 Urbana–Champaign, 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,

Germany;

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, People’s Republic of China;

Department of Entomology, China Agricultural University,

100193 Beijing, People’s 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.

phylogeny

systematics

transcriptomes

Hemiptera

Psocodea

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 “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 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 SRR1821891–SRR1821980,SRR2051465–SRR2051515,andSRR921611–SRR921660). 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: kpjohnso@illinois.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.

1073/pnas.1815820115/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1815820115 PNAS Latest Articles

1of6

EVOLUTION

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 (6–9), 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.

Results

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,TablesS1–S4) 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).

2of6

www.pnas.org/cgi/doi/10.1073/pnas.1815820115 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. 22–24)

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)

aretporeteH

-o

ehcu

Aahcnyhrr

Coccoidea (9)

Aphidoidea (6)

Psylloidea (9)

Aleyrodoidea (1)

-on

hcnyhr

aretpimeH

Aeolothripidae (4)

Thripidae (3)

Phlaeothripidae (1)

-nasy

hT ar

Homilopsocidea (7)

Caeciliusetae (4)

Psocetae (4)

Epipsocetae (1)

Philotarsetae (2)

Phthiraptera (8)

Liposcelididae (2)

Sphaeropsocidae (1)

Amphientometae (1)

aedocosP

Trogiomorpha (2)

Holometabola (11)

Polyneoptera (17)

Palaeoptera (5)

Carboniferous Permian Jurassic CretaceousTriassic Paleog. Neo. Q.

100

150200250300 0mya350400

Devonian

ous

mia

iassic

assic

eta

eous

Bootstrap

support

100%

91-99%

76-90%

51-75%

true bugs

leafhoppers/treehoppers

spittlebugs

cicadas

planthoppers

moss bugs

scale insects

aphids

psyllids

thrips

bark lice and

parasitic lice

book lice

bark lice

Gerromorpha

Nepomorpha

Leptopodomorpha

Pentatomomorpha

Cimicomorpha

Cicadomorpha

semi-aquatic bugs

Terebrantia

Tubulifera

Psocomorpha

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

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

3of6

EVOLUTION

rather than addressing relationships among major lineages

(superfamilies).

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, 27–29), 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 401–486) 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 373–451). 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 292–376), with divergence of this

lineage from other insects as early as 404 mya (95% CI 367–451).

Discussion

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 (31–33). 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.

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www.pnas.org/cgi/doi/10.1073/pnas.1815820115 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,

A.Melber,B.Morris,D.Papura,H.Pohl,R.Rakitov,C.Ray,S.Schneider,

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

5of6

EVOLUTION

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

H.M.R.).

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www.pnas.org/cgi/doi/10.1073/pnas.1815820115 Johnson et al.

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Jun 2024

Maria Carolina Viana

Understanding the population dynamics of vectors is crucial for the effective control of diseases transmitted by them. In the semi-arid northeastern region of Brazil, the triatomine insect Triatoma brasiliensis Neiva, 1911 persists as the most significant vector of Chagas disease, often presenting with persistent household infestation. Recent analyses of microsatellites and the mitochondrial gene Cytochrome b (MT-CYB) detected gene flow between T. brasiliensis populations from sylvatic and domiciliary environments in the state of Rio Grande do Norte (RN), raising relevant public health concerns. In the municipality of Currais Novos (RN), a high prevalence of peridomiciliary reinfestations by T. brasiliensis together with high rates of infection by Trypanosoma cruzi were observed, making this a worrying scenario as sylvatic populations represent a focus of reinfestation difficult to access. Therefore, we assessed the distribution of genetic variation via Sanger sequencing of the MT-CYB gene (n = 109) and high-throughput sequencing of single-nucleotide polymorphisms (SNPs, n = 86) to assess gene flow between distinct populations distributed in geographic areas and varied environments, mainly sylvatic and peridomiciliary. The samples available for analysis were previously collected from the rural communities of Currais Novos, within a radius of 16 km, and included 14 sampling points: two domiciliary, eight peridomiciliary, and four sylvatic. Furthermore, we included an external population located 220 km from the study area. Through AMOVA analysis of MT-CYB gene variation, we identified four distinct population groups with statistical significance (FCT= 0.42; p<0.05). Using the method employed to obtain SNP information based on ddRAD-seq genotyping-by-sequencing (GBS), which allows genome-wide analysis to infer genetic variation, we identified a total of 3,013 SNPs, with 11 loci putatively under selection. The variation based on 3,002 neutral loci showed a lack of genetic structure based on low FST values (p>0.05), indicating local panmixis. However, three samples from the external population were assigned to a cluster contrasting with those supposedly under local panmixia (>98%), validating the genomic marker recently applied for studies on population genetics at more precise levels of resolution for T. brasiliensis. The presence of population structure in some of the sampled points, as suggested by the mitochondrial marker, leads us to assume that the infestations were probably initiated by small populations of females, whose demographic event represents a risk of rapid reinfestations. The local panmictic pattern revealed by the GBS marker represents a challenge for vector control measures because reinfestation foci can be distributed over a wide geographic and ecological area. In our dataset, the results demonstrated that the genetic signals of both markers were complementary. Therefore, it is essential to consider the nature and pattern of inheritance of each marker when inferring the pattern of reinfestations.

Diversidad genética de los chinches de agua (Heteroptera: Gerromorpha, Nepomorpha) presentes en el plano inundable del Magdalena Medio, Colombia

Article

Full-text available

Oct 2024

La diversidad genética es una condición básica que determina cómo las especies mantienen sus funciones metabólicas en diversos ecosistemas. Los ecosistemas acuáticos, se enfrentan a diferentes problemáticas, que ponen en riesgo la diversidad genética de varios grupos taxonómicos como los insectos acuáticos, que desempeñan un importante papel ecológico en estos ambientes. El objetivo de esta investigación fue analizar la diversidad genética de chinches acuáticos y semiacuáticos en un sistema cenagoso de la cuenca media del río Magdalena. Se realizó trabajo de campo y se recolectaron los ejemplares mediante barridos con una red entomológica acuática tipo D, en el laboratorio se realizó un análisis de la subunidad 1 del gen Citocromo Oxidasa (COI). Se identificaron 29 ejemplares con la información disponible en el Barcode of Life Data System (BOLD) y GenBank, y se estimaron las distancias y las relaciones genéticas entre Gerromorpha y Nepomorpha. Para el infraorden Gerromorpha se identificaron tres especies, una especie no descrita de Metrobates sp. y once morfotipos agrupados en nueve géneros de cuatro familias y para Nepomorpha se identificó una especie no descrita y tres morfotipos agrupados en tres géneros de la familia Notonectidae. Se obtuvo un nuevo registro del COI para Ovatametra obesa en Colombia. Las distancias genéticas variaron desde 0,05 hasta 0,29 para Gerromorpha y para Nepomorpha los valores fueron menores a 0,24. La alta variación identificada para Gerromorpha y Nepomorpha resalta la diversidad de esta entomofauna presente en la ciénaga, y demuestra la resiliencia ecológica de estos grupos frente a la intervención antrópica.

The expansion and loss of specific olfactory genes in relatives of parasitic lice, the stored-product psocids (Psocodea: Liposcelididae)

Article

Full-text available

Jan 2025
BMC GENOMICS

Background Booklice, belonging to the genus Liposcelis (Psocodea: Liposcelididae), commonly known as psocids, infest a wide range of stored products and are implicated in the transmission of harmful microorganisms such as fungi and bacteria. The olfactory system is critical for insect feeding and reproduction. Elucidating the molecular mechanisms of the olfactory system in booklice is crucial for developing effective control strategies. In this study, we aim to bridge this knowledge gap by leveraging the transcriptome and genome data from five Liposcelis species. Result Using HMMER method and manual annotation, we have identified common gene families associated with olfactory processes, including odorant binding proteins (OBPs), chemosensory proteins (CSPs), odorant receptors (ORs), ionotropic receptors (IRs), and sensory neuron membrane proteins (SNMPs). Specifically, we identified 94, 118, 26, 47, and 34 olfactory-related genes in L. bostrychophila, L. tricolor, L. entomophila, L. decolor, and L. yangi, respectively. Comparison of quantities revealed that the number of ORs and IRs in the genome is significantly higher than those identified in the transcriptome. This discrepancy may be due to the specific expression of these genes in certain tissues or their lack of expression during the experimental stage. Simultaneously, analysis of gene expression profiles across different developmental stages revealed varying periods of peak expression for olfactory-related genes. These results suggest that the identification of olfactory-related genes in booklice on a genome-wide scale is more feasible and reliable than using a transcriptome-based approach. Additionally, compared to parasitic lice, booklice possess significantly more olfactory-related genes. This increase may be due to the inability of parasitic lice to survive without a host, whereas booklice have a wide range of feeding habits and live in complex and variable environments. Furthermore, we observed that the IR gene family in L. tricolor has undergone a certain degree of amplification, which may facilitate its adaptation to diverse environmental conditions. Conclusions We identified olfactory-related genes of five Liposcelis species for the first time, providing valuable insights for future functional investigations into olfactory genes associated with pheromone and odorant recognition. These discoveries present promising targets for effectively managing psocid pests.

Global Invasion History and Genomic Signatures of Adaptation of the Highly Invasive Sycamore Lace Bug

Article

Full-text available

Oct 2024
Dev Reprod Biol

Invasive species cause massive economic and ecological damage. Climate change has resulted in an unprecedented increase in the number and impact of invasive species; however, the mechanisms underlying these invasions are unclear. The sycamore lace bug, Corythucha ciliata, is a highly invasive species originating from North America and has expanded across the Northern Hemisphere since the 1960s. In this study, we assembled the C. ciliata genome using high-coverage Pacific Biosciences (PacBio), Illumina, and high-throughput chromosome conformation capture (Hi-C) sequencing. A total of 15,278 protein-coding genes were identified, and expansions of gene families with oxidoreductase and metabolic activities were observed. In-depth resequencing of 402 samples from native and nine invaded countries across three continents revealed 2.74 million single nucleotide polymorphisms. Two major invasion routes of C. ciliata were identified from North America to Europe and Japan, with a contact zone forming in East Asia. Genomic signatures of selection associated with invasion and long-term balancing selection in native ranges were identified. These genomic signatures overlapped with expanded genes, suggesting improvements in the oxidative stress and thermal tolerance of C. ciliata. These findings offer valuable insights into the genomic architecture and adaptive evolution underlying the invasive capabilities of species during rapid environmental changes.

A transcriptional control model for doublesex-dependent sex differentiation in Nasonia wasps

Preprint

Full-text available

Feb 2025

The transcription factor Doublesex (DSX) orchestrates insect sex differentiation. DSX affects Drosophila melanogaster male and female transcriptome, yet how DSX regulates gene expression in other species is poorly understood. We investigated sex-biased gene expression during the development of the parasitoid wasp Nasonia vitripennis, finding that more than three-quarters of its genes are sex-biased in at least one developmental point. Next, we transiently knocked down dsx expression to infer its role in sex-specific transcriptome regulation, revealing thousands of affected genes in males and a more subtle effect in females. Finally, we performed an in vitro DNA-protein interaction assay to identify DSX binding sites on the genome and primary DSX target genes. By integrating these three datasets, we defined DSX's regulatory function for all genes in N. vitripennis, revealing that DSX acts mainly in males as both an activator and a repressor. This male-centric model for DSX-mediated regulation is likely to apply to many other insect species.

The Genomics Revolution Drives a New Era in Entomology

Article

Full-text available

Jan 2025
ANNU REV ENTOMOL

Thanks to the fast development of sequencing techniques and bioinformatics tools, sequencing the genome of an insect species for specific research purposes has become an increasingly popular practice. Insect genomes not only provide sets of gene sequences but also represent a change in focus from reductionism to systemic biology in the field of entomology. Using insect genomes, researchers are able to identify and study the functions of all members of a gene family, pathway, or gene network associated with a trait of interest. Comparative genomics studies provide new insights into insect evolution, addressing long-lasting controversies in taxonomy. It is also now feasible to uncover the genetic basis of important traits by identifying variants using genome resequencing data of individual insects, followed by genome-wide association analysis. Here, we review the current progress in insect genome sequencing projects and the application of insect genomes in uncovering the phylogenetic relationships between insects and unraveling the mechanisms of important life-history traits. We also summarize the challenges in genome data sharing and possible solutions. Finally, we provide guidance for fully and deeply mining insect genome data.

Phylogenomics and biogeography of the feather lice (Phthiraptera: Ischnocera) of parrots

Article

Mar 2024

Avian feather lice (Phthiraptera: Ischnocera) have undergone morphological diversification into ecomorphs based on how they escape host preening defences. Parrot lice are one prominent example of this phenomenon, with wing, body, or head louse ecomorphs occurring on various groups of parrots. Currently defined genera of parrot lice typically correspond to this ecomorphological variation. Here we explore the phylogenetic relationships among parrot feather lice by sequencing whole genomes and assembling a target set of 2395 nuclear protein coding genes. Phylogenetic trees based on concatenated and coalescent analyses of these data reveal highly supported trees with strong agreement between methods of analysis. These trees reveal that parrot feather lice fall into two separate clades that form a grade with respect to the Brueelia-complex. All parrot louse genera sampled by more than one species were recovered as monophyletic. The evolutionary relationships among these lice showed evidence of strong biogeographic signal, which may also be related to the relationships among their hosts.

Revisiting habitat and lifestyle transitions in Heteroptera (Insecta: Hemiptera): Insights from a combined morphological and molecular phylogeny

Article

Full-text available

Jan 2018

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.

When did the ancestor of true bugs become stinky? Disentangling the phylogenomics of Hemiptera-Heteroptera

Article

Full-text available

Dec 2017

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.

Anchored Hybrid Enrichment-Based Phylogenomics of Leafhoppers and Treehoppers (Hemiptera: Cicadomorpha: Membracoidea)

Article

Full-text available

Oct 2017

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.

Mitochondrial phylogenomics of Hemiptera reveals adaptive innovations driving the diversification of true bugs

Article

Full-text available

Sep 2017
Proc Biol Sci

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.

Contentious relationships in phylogenomic studies can be driven by a handful of genes

Article

Full-text available

Apr 2017
Nat. Ecol. Evol.

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.

Orthograph: A versatile tool for mapping coding nucleotide sequences to clusters of orthologous genes

Article

Full-text available

Feb 2017
BMC BIOINFORMATICS

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 https://mptrsen.github.io/Orthograph. Additionally, we release 24 de novo-sequenced and assembled transcript libraries of apoid wasp species.

Resolving the psyllid tree of life: phylogenomic analyses of the superfamily Psylloidea (Hemiptera): Resolving the psyllid tree of life

Article

Apr 2018

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).

Mitochondrial phylogenomics and genome rearrangements in the barklice (Insecta: Psocodea)

Article

Oct 2017

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.

Wing base structure supports Coleorrhyncha + Auchenorrhyncha (Insecta: Hemiptera)

Article

Jun 2017

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.

Evolutionary History of the Hymenoptera

Article

Apr 2017

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.

Phylogenomics and the evolution of hemipteroid insects

Abstract

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