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Anchored hybrid enrichment provides new insights into the phylogeny and evolution of longhorned beetles (Cerambycidae)

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Cerambycidae is a species-rich family of mostly wood-feeding (xylophagous) beetles containing nearly 35 000 known species. The higher-level phylogeny of Cerambycidae has never been robustly reconstructed using molecular phylogenetic data or a comprehensive sample of higher taxa, and its internal relationships and evolutionary history remain the subjects of ongoing debate. We reconstructed the higher-level phylogeny of Cerambycidae using phylogenomic data from 522 single copy nuclear genes, generated via anchored hybrid enrichment. Our taxon sample (31 Chrysomeloidea, four outgroup taxa: two Curculionoidea and two Cucujoidea) included exemplars of all families and 23 of 30 subfamilies of Chrysomeloidea (18 of 19 non-chrysomelid Chrysomeloidea), with a focus on the large family Cerambycidae. Our results reveal a monophyletic Cerambycidae s.s. in all but one analysis, and a polyphyletic Cerambycidae s.l. When monophyletic, Cerambycidae s.s. was sister to the family Disteniidae. Relationships among the subfamilies of Cerambycidae s.s. were also recovered with strong statistical support except for Cerambycinae being made paraphyletic by Dorcasomus Audinet-Serville (Dorcasominae) in the nucleotide (but not amino acid) trees. Most other chrysomeloid families represented by more than one terminal taxon – Chrysomelidae, Disteniidae, Vesperidae and Orsodacnidae – were monophyletic, but Megalopodidae was rendered paraphyletic by Cheloderus Gray (Oxypeltidae). Our study corroborates some relationships within Chrysomeloidea that were previously inferred from morphological data, while also reporting several novel relationships. The present work thus provides a robust framework for future, more deeply taxon-sampled, phylogenetic and evolutionary studies of the families and subfamilies of Cerambycidae s.l. and other Chrysomeloidea.
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Systematic Entomology (2018), 43, 68– 89 DOI: 10.1111/syen.12257
Anchored hybrid enrichment provides new insights into
the phylogeny and evolution of longhorned beetles
(Cerambycidae)
STEPHANIE HADDAD1,*, SEUNGGWAN SHIN1,ALANR.
LEMMON2, EMILY MORIARTY LEMMON3, PETR SVACHA4,
BRIAN FARRELL5,ADAM ´
SLIPI ´
NSKI6, DONALD WINDSOR7
and DUANE D. MCKENNA1
1Department of Biological Sciences, University of Memphis, Memphis, TN, U.S.A., 2Department of Scientic Computing, Florida
State University, Dirac Science Library, Tallahassee, FL, U.S.A., 3Department of Biological Science, Florida State University,
Tallahassee, FL, U.S.A., 4Institute of Entomology, Biology Centre, Czech Academy of Sciences, Ceske Budejovice, Czech Republic,
5Museum of Comparative Zoology, Harvard University, Cambridge, MA, U.S.A., 6CSIRO, Australian National Insect Collection,
Canberra, Australia and 7Smithsonian Tropical Research Institute, Ancon, Republic of Panama
Abstract. Cerambycidae is a species-rich family of mostly wood-feeding
(xylophagous) beetles containing nearly 35 000 known species. The higher-level
phylogeny of Cerambycidae has never been robustly reconstructed using molecular
phylogenetic data or a comprehensive sample of higher taxa, and its internal relation-
ships and evolutionary history remain the subjects of ongoing debate. We reconstructed
the higher-level phylogeny of Cerambycidae using phylogenomic data from 522 single
copy nuclear genes, generated via anchored hybrid enrichment. Our taxon sample
(31 Chrysomeloidea, four outgroup taxa: two Curculionoidea and two Cucujoidea)
included exemplars of all families and 23 of 30 subfamilies of Chrysomeloidea (18 of
19 non-chrysomelid Chrysomeloidea), with a focus on the large family Cerambycidae.
Our results reveal a monophyletic Cerambycidae s.s. in all but one analysis, and a
polyphyletic Cerambycidae s.l. When monophyletic, Cerambycidae s.s. was sister to
the family Disteniidae. Relationships among the subfamilies of Cerambycidae s.s. were
also recovered with strong statistical support except for Cerambycinae being made
paraphyletic by Dorcasomus Audinet-Serville (Dorcasominae) in the nucleotide (but
not amino acid) trees. Most other chrysomeloid families represented by more than one
terminal taxon – Chrysomelidae, Disteniidae, Vesperidae and Orsodacnidae – were
monophyletic, but Megalopodidae was rendered paraphyletic by Cheloderus Gray
(Oxypeltidae). Our study corroborates some relationships within Chrysomeloidea
that were previously inferred from morphological data, while also reporting several
novel relationships. The present work thus provides a robust framework for future,
more deeply taxon-sampled, phylogenetic and evolutionary studies of the families and
subfamilies of Cerambycidae s.l. and other Chrysomeloidea.
Correspondence: Stephanie Haddad, Department of Biology, Rhodes
College, 2000 North Parkway, Memphis, TN, 38112-1690, U.S.A.
E-mail: stephanyhaddad@gmail.com and Seunggwan Shin, Department
of Biological Sciences, University of Memphis, 3700 Walker Avenue,
Memphis, TN, 38152, U.S.A. E-mail: sciaridae1@gmail.com
Present address: Department of Biology, Rhodes College, Memphis,
TN, U.S.A.
Introduction
Longhorned beetles (family Cerambycidae Latreille) comprise
one of the most species-rich families of animals, with an
estimated 4000 genera and 35 000 described extant species
(Monné et al., 2009; Svacha & Lawrence, 2014). Cerambyci-
dae s.s. (see Fig. 1 for representatives) is usually divided into
eight subfamilies: Lamiinae (>20 000 species), Cerambycinae
68 © 2017 The Royal Entomological Society
Cerambycidae phylogeny 69
(11 000 species), Lepturinae (1500 species), Prioninae
(>1000 species), Dorcasominae (>300 species), Parandrinae
(119 species), Spondylidinae (100 species) and Necydalinae
(70 species) (Svacha & Lawrence, 2014). Cerambycidae s.s.
plus the families Disteniidae (>300 species), Oxypeltidae (three
species) and Vesperidae (80 species) comprise the informal
grouping Cerambycidae s.l., equivalent to the Cerambycidae or
Longicornia of most earlier authors, the Cerambycoidea of, for
example, Böving & Craighead (1931) or Svacha & Danilevsky
(1987), the cerambycid lineage/Cerambycidae s.l. of Reid
(1995) or the cerambyciform assemblage of Svacha et al. (1997).
Cerambycidae s.l. occur worldwide but attain maximum
species richness in the tropics, where Lamiinae, Cerambycinae,
and Prioninae typically dominate, although Dorcasominae is the
second largest subfamily in Madagascar (Berkov & Tavakilian,
1999; Svacha & Lawrence, 2014). Cerambycidae s.l. belongs
to the informal grouping Phytophaga, a clade of mostly phy-
tophagous beetles consisting of the sister superfamilies Cur-
culionoidea Latreille (weevils s.l. including bark beetles) and
Chrysomeloidea Latreille (leaf beetles, longhorned beetles and
relatives). According to ´
Slipi´
nski et al. (2011), the clade Phy-
tophaga contains 125 237 described species (61 854 described
species in Curculionoidea and 63 383 described species in
Chrysomeloidea).
The earliest fossil Cerambycidae s.l. (Wang et al., 2013; pos-
sibly Yu et al., 2015) are from the Early Cretaceous. The earliest
known unambiguous fossil angiosperms (e.g. Friis et al., 2006)
are also from that epoch. The diversity of Chrysomeloidea and
other phytophagous insects has been attributed by some to their
co-diversication with angiosperms (e.g. Farrell, 1998; Mitter &
Farrell, 1991; McKenna & Farrell, 2006; McKenna et al., 2009;
McKenna, 2011). This seems only partially true for ceramby-
cids. Although they quite possibly pre-date angiosperms, whose
increasing diversity later contributed to cerambycid diversity,
cerambycids (even at low taxonomic levels) are often highly
polyphagous in the larval stage and some species may even feed
on both gymnospermous and angiospermous plants. There were
undoubtedly multiple host switches between gymnosperms and
angiosperms in cerambycid evolution.
Larvae of Cerambycidae s.l. are mostly internal borers in
woody plants (xylophagous in the broadest sense) and feed on
living or dead (including rotten and fungus-infested) plant tis-
sue, although larvae of some Cerambycidae s.s. feed in herbs,
and those of some Lamiinae, Prioninae, Lepturinae and all Ves-
peridae are terricolous and feed externally on plant roots. Unlike
in Chrysomelidae (leaf beetles), no free-living cerambycid lar-
vae (such as defoliators or external bark grazers) are known.
Most wood-boring species feed on nutrient-rich subcortical tis-
sues (inner bark, cambium and immature xylem), with some
species feeding on nutrient-poor outer bark, sapwood, heart-
wood and pith (Linsley, 1959; Hanks, 1999). Adult feeding
(Butovitsch, 1939; Linsley, 1959) is often poorly known; some
Cerambycidae s.s. and possibly all Vesperidae apparently do
not feed, whereas adults of the subfamily Lamiinae undergo
obligate maturation feeding, which is thought to be an apo-
morphy for the subfamily (Svacha & Lawrence, 2014). Adult
food includes various plant parts (leaves, conifer needles and
cones, bark, stems of herbs or owers in Lamiinae), pollen
and nectar (some Lepturinae, Necydalinae, Dorcasominae and
Cerambycinae), sap (some Prioninae, Cerambycinae and Lep-
turinae), fruit (e.g. various Cerambycinae and Lamiinae), and
fungal spores or fruiting bodies (known in some Lepturinae and
Lamiinae).
Adult feeding usually has little impact on the environment
by itself, but some Lamiinae may transfer pathogens or para-
sites such as the nematode Bursaphelenchus xylophilus (Steiner
and Buhrer) Nickle causing Pine Wilt Disease (e.g. Togashi
& Shigesada, 2006). The extensive larval feeding makes cer-
ambycids an important component of both natural and man-
aged forest ecosystems. They are major recyclers of dead wood,
and through their feeding activities they create access routes
into wood for other decomposing agents, such as fungi, and
other invertebrates (´
Slipi´
nski & Escalona, 2013). However, lar-
val feeding can also seriously damage or even kill the host plant,
either directly, or when the larval tunnels and adult emergence
holes provide access points for pathogenic fungi (e.g. Schowal-
ter, 2009). For instance, some Cerambycinae and Lamiinae are
especially notorious pests of trees in urban, suburban and forest
ecosystems (Linsley, 1959). Some Cerambycinae are capable of
infesting dry seasoned wood and may seriously damage struc-
tural timber. Compared to the subfamilies Lepturinae, Prioni-
nae and Parandrinae, whose larvae mostly develop in decaying
wood, species of Cerambycinae and Lamiinae also have higher
capacities for introduction in living or freshly dead plants or con-
struction wood and wood products (Cocquempot & Lindelöw,
2010; Raje et al., 2016). For instance, the Asian longhorned bee-
tle, Anoplophora glabripennis Motschulsky, has recently shown
the potential to spread after introduction and cause signicant
ecological and economic damage (Nowak et al., 2001; Meng
et al., 2015; McKenna et al., 2016). Also, some terricolous root
feeders (such as Philus Saunders or Migdolus Westwood of Ves-
peridae) may cause considerable damage to forest or agricultural
plantations (Svacha et al., 1997; Machado & Habib, 2006).
As in other similarly extensive and diverse groups, it is often
extremely difcult to even distinguish cerambycid higher taxa
on a worldwide scale based on classical adult characters. Lar-
val morphology appears more promising in this group (perhaps
because of the universally concealed larval habits resulting in
lower morphological diversity), but particularly in some exotic
regions, the larvae are known only for a fraction of described
species. Under such conditions, it is problematic to use the
‘exemplar’ approach in morphological phylogenetic studies (as
done by Napp, 1994, who sampled 12 of the current 14 subfami-
lies; Vesperinae and Dorcasominae were not included), particu-
larly if the exemplars are few. As a result of the obvious multiple
parallelisms, Napp was forced to somewhat arbitrarily polarize
characters by rooting with a ‘hypothetical ancestor’, and she also
rather subjectively coded some characters. Despite this, Napp
(1994) remains the only modern phylogenetic study specically
focused on resolving higher-level relationships within Ceram-
bycidae s.l. Subsequent phylogenetic studies (whether based on
morphological data, molecular data or employing a combined
approach) did not specically address the relationships among
the families and subfamilies of Cerambycidae s.l. They all lack
© 2017 The Royal Entomological Society, Systematic Entomology,43, 68–89
70 S. Haddad et al.
Fig. 1. Representatives of the subfamilies of Cerambycidae s.s. (a) Cerambyx cerdo Linnaeus (Cerambycinae; image courtesy of Stefano Trucco), (b)
Lamia textor Linnaeus (Lamiinae; image courtesy of Serhey Ruban), (c) Dorcasomus sp. (Dorcasominae; image courtesy of Petr Malec), (d) Rutpela
maculata Poda (Lepturinae; image courtesy of Paul Mitchy), (e) Necydalis mellita Say (Necydalinae; image courtesy of Jeff Gruber), (f) Parandra
polita Say (Parandrinae; image courtesy of John and Kendra Abbott/Abbott Nature Photography), (g) Prionus coriarius Linnaeus (Prioninae; image
courtesy of Nikola Rahmé), (h) Spondylis buprestoides Linnaeus (Spondylidinae; image courtesy of Serhey Ruban). [Colour gure can be viewed at
wileyonlinelibrary.com].
some important higher taxa and/or the relationships within Cer-
ambycidae s.l. lack consistent resolution and strong statistical
support (see Haddad & McKenna, 2016 for review) whether
the studies focused on Phytophaga (Farrell, 1998; Marvaldi
et al., 2009), Chrysomeloidea (Reid, 1995; Farrell, 1998; Far-
rell & Sequeira, 2004; Gómez-Zurita et al., 2007, 2008; Wang
et al., 2013), Coleoptera (Hunt et al., 2007; Lawrence et al.,
2011; Bocak et al., 2014; McKenna et al., 2015) or even Cer-
ambycidae s.s. (Raje et al., 2016). Consequently, many aspects
of chrysomeloid classication and evolution, particularly those
concerning Cerambycidae s.l., remain the subject of consider-
able debate: the monophyly of Cerambycidae s.s. and s.l.;the
phylogenetic positions of Megalopodidae and Orsodacnidae rel-
ative to Cerambycidae s.l.; possible Southern (?Gondwanan)
versus Northern (?Laurasian) origins of the subfamilies of Cer-
ambycidae s.s. (see Svacha & Lawrence, 2014); early host plant
associations (gymnosperms versus angiosperms); etc. Without
a reliable phylogeny, morphological ground plans of individual
subtaxa cannot be reconstructed. This also makes it difcult to
interpret fossils that are not clearly related to extant taxa, on
account of the limited set of characters available for fossils.
Due to recent methodological advances (both analytical and
wet-lab), a decrease in the cost of DNA sequencing, and a
concomitant increase in available genomic resources, it is now
possible to efciently obtain and analyse DNA sequence data
from hundreds of genes/loci from all kinds of organisms (e.g.
Niehuis et al., 2012; Lemmon & Lemmon, 2013; Hulcr et al.,
2014; Misof et al., 2014; Young et al., 2016). Recent studies
have effectively employed whole genome sequencing (WGS;
e.g. Niehuis et al., 2012; Prum et al., 2015), transcriptome
sequencing (e.g. Bi et al., 2012; Kawahara & Breinholt, 2014;
Misof et al., 2014; Kjer et al., 2015; Lei & Dong, 2016) or target
enrichment (e.g. Faircloth et al., 2012; Lemmon et al., 2012;
Smith et al., 2014; Brandley et al., 2015; Eytan et al., 2015;
Prum et al., 2015; Hamilton et al., 2016; Young et al., 2016;
Breinholt et al., 2017), to generate large-scale phylogenomic
datasets for use in phylogeny reconstruction, particularly in
groups for which relationships have been difcult to resolve
using smaller samples of molecular data (see Lemmon &
Lemmon, 2013 for a review of alternative approaches). WGS
produces the most data, but is the most expensive of these
approaches and also carries a high bioinformatic and compu-
tational burden. Transcriptome sequencing requires tissues that
are preserved for RNA and such tissues are often not available
for all taxa of interest. Therefore, in cases where high-quality
RNA is unobtainable, hybrid enrichment is typically the
© 2017 The Royal Entomological Society, Systematic Entomology,43, 68–89
Cerambycidae phylogeny 71
preferred approach (Lemmon & Lemmon, 2013). Hybrid
enrichment has also demonstrated utility across a broad spec-
trum of taxonomic scales, and is an extremely cost-effective
and efcient strategy (e.g. Faircloth et al., 2012; Lemmon et al.,
2012; Lemmon & Lemmon, 2013). However, this approach
requires that at least some ‘model’ DNA sequences are avail-
able for designing probes to target the loci of interest. Hybrid
enrichment is a comparatively versatile approach, and has been
successfully used with museum specimens (Bi et al., 2013) and
ancient DNA (Burbano et al., 2010; Carpenter et al., 2013).
Anchored Hybrid Enrichment (AHE) (Lemmon et al., 2012)
is a relatively recent approach for hybrid enrichment that is
now being used rather widely in phylogenomic studies of
vertebrates (e.g. Brandley et al., 2015; Eytan et al., 2015; Prum
et al., 2015) and most recently invertebrates (Diptera: Young
et al., 2016; Arachnida: Hamilton et al., 2016; Lepidoptera:
Breinholt et al., 2017). It is a fast and cost-efcient method for
enriching hundreds of aprioritargeted loci for use in phylogeny
reconstruction. Ideally, organisms with sequenced genomes or
other genomic resources are used to design enrichment probes
that capture sequence data from organisms lacking genomic
resources. These probes are designed based on select conserved
‘anchor regions’ in the model organisms’ genomes that are
adjacent to less conserved regions. As such, these probes can
be used to obtain conserved anchor regions in addition to the
more rapidly evolving anking regions. The resulting captured
fragments are then sequenced using high-throughput Next Gen-
eration Sequencing (NGS) methods, assembled into contigs,
evaluated for orthology and used in phylogenetic analyses
(Lemmon et al., 2012).
This is the rst study to use hybrid enrichment on nuclear
protein coding genes in beetles, the rst molecular phyloge-
netic study focused on resolving relationships among the fam-
ilies and subfamilies of Cerambycidae s.l. and s.s., and the
rst study to determine the phylogenetic placement of Mega-
lopodidae and Orsodacnidae in the context of a higher-level
phylogeny of Chrysomeloidea. Although the sampling within
the subfamilies had to be limited because of the demanding
methodology, we believe that the present work will serve as a
robust framework for future, more deeply taxon-sampled phy-
logenetic studies of the families and subfamilies of the super-
family Chrysomeloidea. It will also facilitate future evolutionary
studies of Cerambycidae (e.g. pertaining to morphology, host
associations, biogeographic origins, pheromones, mimicry), and
promote studies on the biological control, monitoring and con-
servation of this ecologically and economically signicant group
of beetles.
Materials and methods
Taxon sampling
Exemplars were selected based on the availability of speci-
mens suitable for DNA, and type genera and species of families
and subfamilies were sampled when possible (see Table 1; refer
to Table S1 for the number of recovered loci for each taxon used
in this study). We included 14 taxa representing all eight subfam-
ilies of Cerambycidae s.s., seven exemplars from other families
of Cerambycidae s.l. (two Disteniidae, one Oxypeltidae, four
Vesperidae), four exemplars of presumed close relatives in the
families Megalopodidae and Orsodacnidae, and six exemplars
representing six subfamilies of Chrysomelidae. Four outgroups
were sampled, including two Curculionoidea (sister group to
Chrysomeloidea; McKenna, 2014) and two Cucujoidea s.s. (sis-
ter group to Phytophaga; Robertson et al., 2015). All trees were
rooted with the Cucujoidea [Aethina Erichson (Nitidulidae) and
Cucujus Fabricius (Cucujidae)] based on recent studies (e.g.
McKenna et al., 2015).
DNA extraction, library preparation and sequencing
DNA extraction, library preparation, enrichment and sequenc-
ing were performed for all but two taxa in Table 1 (the sequenced
genome of Anoplophora glabripennis and the transcriptome
shotgun assembly from NCBI of Aethina tumida Murray were
used in our phylogenetic analyses). Depending on the size of
the specimen, genomic DNA was extracted from one to six legs,
thoracic muscle, a piece of larval tissue or the whole body of
the specimen. Specimens were live-frozen, alcohol-preserved
(80–100% ethanol), or pinned/dry. Total genomic DNA was
extracted from air-dried specimens using the Omniprep™ kit
(G-biosciences, St. Louis, MO, U.S.A.) and treated with RNase
A. The recommended minimum amount of DNA required for
library prep is 200 ng, which can be readily obtained from cer-
ambycids of any size. Genomic DNA QC statistics were gener-
ated for each extracted specimen/sample using a Qubit uorom-
eter, and DNA quality (fragmentation/degradation and/or con-
tamination with RNA) was further assessed via gel electrophore-
sis. Remaining specimen parts (intact and/or ground pieces)
are preserved in 99% ethanol and retained in the McKenna
Lab (University of Memphis) as vouchers. Extracted DNA was
sent to the Center for Anchored Phylogenomics at Florida State
University, Talahassee, FL (www.anchoredphylogeny.com) for
library preparation, hybrid enrichment and DNA sequencing.
Protocols for library preparation, enrichment, sequencing and
probe design followed Lemmon et al. (2012). Genomic DNA
samples were sonicated to a fragment size of 150 –350 bp
using a Covaris E220 Focused-ultrasonicator with Covaris
microTUBES. Library preparation and indexing were per-
formed on a Beckman-Coulter Biomek FXp liquid-handling
robot following a protocol modied from Meyer & Kircher
(2010), that included size-selection after blunt-end repair using
SPRIselect beads (Beckman-Coulter Inc.; 0.9×ratio of bead to
sample volume). Indexed samples were then pooled at equal
quantities (12–16 samples/pool), and enrichments were per-
formed on each multi-sample pool using an Agilent Custom
SureSelect kit (Agilent Technologies), which contained probes
designed for anchored loci from the selected model beetle
genomes/transcriptomes. After enrichment, the three enrich-
ment pools were pooled in equal quantities for sequencing in
three PE150 Illumina HiSeq 2000 lanes, shared with samples
from other projects (a total of 27 714 941 100 bp were collected
© 2017 The Royal Entomological Society, Systematic Entomology,43, 68–89
72 S. Haddad et al.
Tabl e 1. Nomenclature and terminal taxa used in this study. Note that Cerambycidae s.l. were polyphyletic and Megalopodidae paraphyletic in all
analyses because the genus Cheloderus (Oxypeltidae) was invariably recovered as sister to Palophagoides (Megalopodidae: Palophaginae).
SuperfamilyFamilySubfamily (Type genus)Genus Species
“Phytophaga”
Chrysomeloidea
Cerambycidae s.l.
Cerambycidae
s.s.
Cerambycinae (Cerambyx)Callisphyris Newman macropus Newman
Megacyllene Casey robiniae (Forster)
Dorcasominae (Dorcasomus)Dorcasomus Audinet-Serville mirabilis Quentin & Villiers
Prioninae (Prionus)Prionus Geoffroy coriarius (Linnaeus)
Tragosoma Audinet-Serville depsarium (Linnaeus)
Parandrinae (Parandra)Acutandra Santos-Silva araucana (Bosq)
Lepturinae (Leptura)Desmocerus Dejean palliatus (Forster)
Rutpela Nakane & Ohbayashi maculata (Poda)
Necydalinae (Necydalis)Necydalis Linnaeus formosana Kano
Lamiinae (Lamia)
Lamia Fabricius textor (Linnaeus)
Anoplophora Hope glabripennis (Motschulsky)
Tetraopes Dalman tetrophthalmus (Forster)
Spondylidinae (Spondylis)Asemum Eschscholtz striatum (Linnaeus)
Spondylis Fabricius buprestoides(Linnaeus)
Disteniidae Disteniinae (Distenia)
Cyrtonops White punctipennisWhite
Distenia Le Peletier &
Audinet-Serville japonica Bates
Vesperidae
Vesperinae (Vesperus)Vesperus Dejean sanzi Reitter
Philinae (Philus)Philus Saunders pallescens Bates
Anoplodermatinae (Anoploderma)
MigdolusWestwood fryanus Westwood
Mysteria Thomson darwini (Lameere)
Oxypeltidae Oxypeltinae (Oxypeltus)Cheloderus Gray childreniGray
Orsodacnidae
Orsodacninae (Orsodacne)Orsodacne Latreille cerasi (Linnaeus)
Aulacoscelidinae (Aulacoscelis)Aulacoscelis Duponchel &
Chevrolat costaricensis Bechyne
Megalopodidae Zeugophorinae (Zeugophora)Zeugophora Kunze varians Crotch
Palophaginae (Palophagus)PalophagoidesKuschel vargasorum Kuschel
Chrysomelidae
Bruchinae (Bruchus)Caryobruchus Bridwell gleditsiae (Linnaeus)
Sagrinae (Sagra)Sagra Fabricius femorata (Drury)
Criocerinae (Crioceris)Lilioceris Reitter lilii (Scopoli)
Galerucinae (Galeruca)Diabrotica Chevrolat undecimpunctata
Mannerheim
Cryptocephalinae
(Cryptocephalus)Neochlamisus Karren bebbianae(Brown)
Cassidinae (Cassida)Chelobasis Gray perplexa (Baly)
Curculionoidea Nemonychidae Rhinorhynchinae (Rhinorhynchus)Rhynchitomacerinus Kuschel kuscheli (Voss)
Attelabidae Rhynchitinae (Rhynchites)Merhynchites Sharp bicolor (Fabricius)
Cucujoidea Nitidulidae Nitidulinae (Nitidula)Aethina Erichson tumida Murray
Cucujidae Cucujinae (Cucujus)Cucujus O. F. Müller clavipes Fabricius
© 2017 The Royal Entomological Society, Systematic Entomology,43, 68–89
Cerambycidae phylogeny 73
for the samples used in this study). Sequencing was performed
by the Translational Science Laboratory in the College of
Medicine at Florida State University.
Probe design and identication of conserved orthologous loci
Probes for Coleoptera were designed using methods sim-
ilar to those used for a recently published probe set for
Diptera (Young et al., 2016) and Lepidoptera (Breinholt et al.,
2017). Specically, we obtained nucleotide alignments of 4485
protein-coding genes for 13 insect species from Niehuis et al.
(2012). These alignments included 11 species of Holometabola
from ve orders (Diptera, Hymenoptera, Lepidoptera, Strep-
siptera and Coleoptera) and two nonholometabolous outgroups
from the insect orders Anoplura and Hemiptera (See Young
et al., 2016, Table 1 for details). A preliminary set of loci
was then selected containing greater than or equal to six taxa,
and at least one consecutive 120bp region with >50% pair-
wise sequence identity. Exon boundaries were then identied
using custom scripts that identied matches between the beetle
model genomes/transcriptomes and the genomes obtained from
Niehuis et al. (2012) using 40-mers (see Young et al., 2016 for
details and scripts).
In order to develop the beetle probe kit, 26 taxa (see Table 2)
with sequenced genomes and/or transcriptomes representing the
major lineages of interest and near outgroups in Cucujiformia
(McKenna et al., 2015) were chosen for consideration: four
cerambycids, six chrysomelids, ve curculionids, one brentid,
one carabid, one coccinellid, one hydroscaphid, one cupedid,
one byturid, one clerid, one cryptophagid, one tenebrionid, one
mengenillid and one corydalid. We refer to these taxa as the
model species.
Enrichment probes were developed targeting 941 orthologous
nuclear loci (average length 440 bp) pre-determined to be useful
for phylogeny reconstruction in beetles based on their location
in conserved anchor regions (anked by less conserved regions)
of the genomes/transcriptomes of the model species, and their
status as 1:1 orthologs in the model species. The 941 target
loci were selected from a pool of 1200 candidate loci that
constituted the intersection of a genome-based dataset (4485
1:1 orthologs from Holometabola; Niehuis et al., 2012) with a
transcriptome-based dataset (1478 1:1 orthologs from 139 rep-
resentative Arthropoda, mostly insects; Misof et al., 2014). They
comprise a core set of 236 loci with utility across Arthropoda,
but primarily focused on Insecta, plus 705 loci selected from a
1:1 ortholog set for Neuropteroidea (Coleoptera +Strepsiptera
and Neuropterida) (McKenna & Farrell, 2010; Beutel &
McKenna, 2016; McKenna, 2016). The 941 target loci were
sought in each of the genomes and transcriptomes of the model
species to conrm their presence and further assess their phy-
logenetic utility (e.g. copy number, length, % identity, GC
content). Alignments for the 941 target loci containing the 26
model species were used to identify enrichment probes. Probes
were tiled approximately every 50bp for each of 26 model
species (2.4×coverage per species), starting at the beginning
of the alignment. Final alignments and probe sequences will be
made available on Dryad (the beetle probes are continuously
being rened; Contact D. D. McKenna for the latest versions).
Read processing, assembly and orthology assessment
Quality-ltered sequencing reads were processed following
the methods described in Lemmon et al. (2012) and Prum
et al. (2015). In short, reads were quality ltered and demulti-
plexed (with no mismatches tolerated), and overlapping reads
were identied and merged following Rokyta et al. (2012).
Reads were then assembled following Prum et al. (2015), except
that the following models were used as references: Calloso-
bruchus maculatus Fabricius (genome), Leptinotarsa decem-
lineata Say (transcriptome), Diabrotica undecimpunctata Lin-
naeus (genome), Rhamnusium bicolor Schrank (transcriptome),
Anoplophora glabripennis (genome) and Tribolium castaneum
Herbst (genome; Richards et al., 2008). T. castaneum covered
100% of the target loci which is why it was selected as the
main reference gene set. The remaining reference taxa covered
approximately 8292% of the target loci, with the lowest being
C. maculatus at 72.3%. After assembly, we checked for possible
cross contamination using an all-vs.-all blast search for each
taxon (Camacho et al., 2009).
Orthologous genes were identied using Orthograph (Petersen
et al., 2017), a protein-based orthology search pipeline. It
removes possible paralogous genes using Hidden Markov
Model (HMM)-based orthology searches of protein-translated
sequences. Three ofcial gene sets (OGS) of three insect taxa
from OrthoDB7 (online database of orthologous protein-coding
genes across major radiations in the tree of life) were used as
references for orthology prediction: Danaus plexippus Lin-
naeus (Lepidoptera: Danaidae) (Zhan et al., 2011), Nasonia
vitripennis Walker (Hymenoptera: Pteromalidae) (Werren et al.,
2010) and T. castaneum (Coleoptera: Tenebrionidae) (Richards
et al., 2008). The OGS for T. castaneum is the only beetle
OGS available in OrthoDB7, and the other two insect taxa
were chosen based on their high-quality genomes and OGSs.
OrthoDB7 (Waterhouse et al., 2013; Kriventseva et al., 2015)
was used to generate a table of clusters of orthologous groups
(COGs) for each of the three chosen OGSs. In particular, the
941 AHE reference locus set for T. castaneum was remapped
by BLASTX (E-value threshold <1e6) against the reference
OGS for T. castaneum (OGS 3.0; Richards et al., 2008). This
recovered 663 genes, each an assembly of sequences from one
or more of the targeted AHE loci. AHE reference assemblies
can generate duplicates of single-copy orthologs if the anking
regions of different targeted loci (different exons from the same
gene) overlap. Based on the  results, these duplicate loci
were removed. Thus, we settled on 522 COGs that matched
with all single-copy COGs of the aforementioned three OGSs
in the Orthograph pipeline (141 of the 663 genes represented
by our 941 target loci were excluded because they had multiple
copies in at least one of the three OGSs).
The resulting COG tables and OGS sequences were loaded
into Orthograph as the reference database for subsequent strict
protein-based orthology searches. First, all DNA sequences
© 2017 The Royal Entomological Society, Systematic Entomology,43, 68–89
74 S. Haddad et al.
Tab l e 2 . Data used to design anchored hybrid enrichment probes for Coleoptera.
Family Subfamily Genus Species Data Type Source
Cerambycidae Cerambycinae Phymatodes amoenus Genome Robert Mitchell (University of WI Oshkosh)
Cerambycidae Cerambycinae Xylotrechus colonus Genome Robert Mitchell (University of WI Oshkosh)
Cerambycidae Lamiinae Anoplophora glabripennis Genome Asian Longhorned Beetle Genome Project
(https://www.hgsc.bcm.edu/arthropods/
asian-long-horned-beetle- genome-
project)
Cerambycidae Lepturinae Rhamnusium bicolor Transcriptome 1000 Insect Transcriptome Evolution project
(http://www.1kite.org/)a
Chrysomelidae Bruchinae Callosobruchus maculatus Genome http://www.beanbeetles.org/genome/
Chrysomelidae Chrysomelinae Chrysomela tremulae Transcriptome Yannick Pauchet (Max Planck Institute of
Chemical Ecology)
Chrysomelidae Chrysomelinae Gastrophysa viridula Transcriptome Yannick Pauchet (Max Planck Institute of
Chemical Ecology)
Chrysomelidae Chrysomelinae Leptinotarsa decemlineata Transcriptome Yannick Pauchet (Max Planck Institute of
Chemical Ecology)
Chrysomelidae Donaciinae Donacia marginata Transcriptome 1000 Insect Transcriptome Evolution project
(http://www.1kite.org/)
Chrysomelidae Galerucinae Diabrotica undecimpunctata Genome Hugh Robertson (University of Illinois at
Urbana-Champaign)
Brentidae Brentinae Arrhenodes sp. Transcriptome 1000 Insect Transcriptome Evolution project
(http://www.1kite.org/)
Curculionidae Dryophthorinae Sitophilus oryzae Transcriptome Yannick Pauchet (Max Planck Institute of
Chemical Ecology)
Curculionidae Dryophthorinae Rhynchophorus ferrugineus Transcriptome GenBank: PRJNA79205
Curculionidae Molytinae Pissodes strobi Transcriptome GenBank: PRJNA186387
Curculionidae Scolytinae Dendroctonus ponderosae Transcriptome GenBank: PRJNA178770
Curculionidae Scolytinae Ips typographus Transcriptome 1000 Insect Transcriptome Evolution project
(http://www.1kite.org/)
Cryptophagidae Atomariinae Atomaria fuscata Transcriptome 1000 Insect Transcriptome Evolution project
(http://www.1kite.org/)
Tenebrionidae Tenebrioninae Tribolium castaneum Genome http://beetlebase.org/ Richards et al. (2008)
Carabidae Carabinae Calosoma scrutator Genome D. D. McKenna (Unpublished data)
Coccinellidae Coccinellinae Harmonia axyridis Genome D. D. McKenna (Unpublished data)
Hydroscaphidae Hydroscaphinae Hydroscapha redfordi Genome D. D. McKenna (Unpublished data)
Cupedidae Cupedinae Priacma serrata Genome D. D. McKenna (Unpublished data)
Byturidae Byturinae Byturus ochraceus Transcriptome 1000 Insect Transcriptome Evolution project
(http://www.1kite.org/)
Cleridae Clerinae Thanasimus formicarius Transcriptome 1000 Insect Transcriptome Evolution project
(http://www.1kite.org/)
Mengenillidae Mengenillinae Mengenilla moldryzki Genome GenBank: PRJNA181027
Corydalidae Chauliodinae Chauliodes pectinicornis Genome D. D. McKenna (Unpublished data)
aAn earlier version of the 1KITE assembly was used (as in Misof et al., 2014) for all 1KITE references. The latest assembly version (E3) is not yet
available. Umbrella Comparative Genomics project https://www.ncbi.nlm.nih.gov/bioproject/1832
were translated in all 6 possible reading frames, then the
resulting library of amino acid (AA) sequences was searched
using prole HMMs (pHMMs) that were trained by the three
OGSs previously selected from OrthoDB7. Each of the 522
genes was then assessed for orthology in Orthograph using a
reciprocal blast search, and this was done for each taxon-based
result from the Orthograph pipeline. Results were stored in
both AA and nucleotide (NT) format for each taxon-based
result (following Petersen et al., 2017). The resulting fasta
formatted NT les for each species were screened for vector
contamination using UniVec (Cochrane & Galperin, 2010).
Finally, an all-vs.-all blast search was used to further assess our
dataset for cross-contamination.
Phylogenomic pipeline
Orthograph generates fasta les that include OrthoDB7 IDs for
every taxon and gene and include descriptive information con-
cerning the results in their headers. They also include three OGS
sequences for each gene. As a result, we employed a custom
bioinformatics pipeline to process these les. First, headers of
all les were modied using orthograph2hamstrad.pl (Perl script
provided from the Orthograph package; Petersen et al., 2017).
Second, reference genes (OGSs) are removed for each le, such
that each of them contained only one target sequence for each
gene and a clear taxon name (or taxon code) that includes an
OrthoDB7 ID. Next, fasta les for each taxon were combined
© 2017 The Royal Entomological Society, Systematic Entomology,43, 68–89
Cerambycidae phylogeny 75
using Perl script summarize_orthograph_results.pl (from Ortho-
graph package; Petersen et al., 2017). Genes were then aligned
in  v7 (Katoh & Standley, 2013) with L-ins-i and default
options. The resulting le names were then changed from the
HaMStRad header to simple taxon names using the custom
script change_taxon_names.sh (requires an input le of custom
taxon names/codes to be created by the user). Finally, aligned
les were concatenated with a modied AMAS.sh (alignment
manipulation and summary statistics) script executed in AMAS
0.97 (Borowiec, 2015). Custom scripts used in this pipeline will
be made available in Shin et al. (submitted).
Phylogenetic analysis
Phylogenetic analyses were conducted using maximum-
likelihood (ML) and Bayesian inference (BI) for both nucleotide
(781 446 bp) and amino acid (257 303 aa) data. ML analy-
ses were performed in x 8.1.5 (Stamatakis, 2014) and
Bayesian analyses were performed in  3.2.5 (Ronquist
et al., 2012). Most analyses were carried out on the HPC (high
performance computing) cluster at the University of Memphis.
We chose to carry out both partitioned (Figs 2, 3) and unparti-
tioned (Figures S1 and S2) ML analyses for both the amino acid
and nucleotide datasets. Trees resulting from the partitioned
ML analyses are our preferred trees. Partitioning optimizes the
selection of appropriate models of molecular evolution and has
been determined to account for among-site heterogeneity in
the rates and patterns of evolution of sequence alignments (e.g.
Lanfear et al., 2012) which has been shown to improve various
aspects of phylogenetic inference (e.g. see Poux et al., 2008;
Rota & Wahlberg, 2012; Leavitt et al., 2013).
Partitioned analyses were carried out using 
2.0.0 (PF; Lanfear et al., 2012) with the x option to deter-
mine optimal partitioning schemes and best-t models of substi-
tution for both AA and NT data. For the AA PF analysis (Fig. 3),
we specied use of the strict hierarchal clustering algorithm
(hcluster) to search for the best partitioning scheme using the
Bayesian Information Criterion (BIC). PF recommended both
LG +G (60 subsets) and WAG+G (nine subsets) models for
the AA data, but only 13% of the data was tted to the WAG +G
model, so we used the LG +G model for the AA ML analysis.
For the NT PF analysis (Fig. 2), we also specied the use
of the h-cluster algorithm to search for the best partitioning
scheme using the BIC criterion. The GTR+I+G model was
recommended by PF for all of the NT data (16 subsets), so we
used that model for the NT ML analysis.
The AA and NT datasets were analysed separately in x
(10 replicate ML searches and 1000 rapid bootstrap replicates).
Results from the bootstrap analyses were mapped onto the
resulting ML trees. A node with a ML bootstrap support
(MLBS) value greater or equal to 95% was considered strongly
supported.
The Bayesian analyses for both unpartitioned AA (Figure
S4) and NT (Figure S3) datasets were also performed on
the HPC cluster of the University of Memphis. Concatenated
datasets were analysed using the GTR +I+G model for the NT
dataset and mixed models for the AA dataset; 24 chains were
executed using the MPI (Message Passing Interface) version of
 for both datasets respectively, starting with a random
tree and running for 1 million MCMC (Markov Chain Monte
Carlo) generations, with trees sampled every 1000 generations.
Burn-in was set at 25% of the sampled number of trees
(250 000 generations). We used  1.6 (Rambaut et al.,
2014) to monitor convergence of the MCMC runs, which was
also conrmed by monitoring the value of split frequencies
between runs (value fell below 0.01). The runs converged at
or before approximately 100 000 generations. A 50% majority
rule consensus tree was constructed from the remaining (post
burn-in) trees to estimate posterior probability (PP) values, with
nodes having PP 0.95 considered strongly supported.
Results
The nomenclature and terminal taxa used in this study are shown
in Table 1. For clarity, in the following text, we usually refer to
taxa by their family-level names. We also do this for the families
or subfamilies represented only by a single terminal taxon (such
as Oxypeltidae by Cheloderus childreni Gray), although the
monophyly of such family-level taxa remains entirely untested.
Relationships recovered from all analyses
Within the Phytophaga, at the superfamily and fam-
ily level, all trees (Figs 2, 3, Figures S1S4) recovered
the following clades (which had maximum support in all
trees, except where noted): (1) Curculionoidea sister to
Chrysomeloidea; (2) Chrysomelidae sister to all remaining
Chrysomeloidea (the latter had 99% MLBS in the unparti-
tioned ML tree of NT data); (3) Orsodacnidae; (4) a clade
containing Oxypeltidae and paraphyletic Megalopodidae [(Zeu-
gophorinae +(Palophaginae +Oxypeltidae))] (99% MLBS
in the unpartitioned ML tree of NT data); Cerambycidae s.l.
were thus polyphyletic in all analyses; (5) Vesperidae [as
((Vesperinae +Philinae) +Anoplodermatinae)] (MLBS for
Vesperidae was 95% and 94% in the partitioned and unpar-
titioned ML trees of AA data, respectively, and the clade
Vesp e ri n ae +Philinae had 99% MLBS in the partitioned ML
tree of AA data); and (6) Disteniidae.
Cerambycidae s.s. were monophyletic (although with low
support in the ML trees of AA data) and sister to Disteniidae
in all trees except for the Bayesian AA tree (Figure S4) where
they were rendered paraphyletic by the disteniids. Whether
monophyletic or paraphyletic, Cerambycidae s.s. consistently
contained the following clades (with maximum support in all
trees, except where noted): (1) Spondylidinae sister to Lami-
inae; (2) Lepturinae sister to Necydalinae; (3) a clade containing
Dorcasominae and a paraphyletic (NT trees) or monophyletic
(AA trees) Cerambycinae; and (4) Prioninae sister to Paran-
drinae. Clade 3 had maximum support in all trees, but the
internal relationship of Callisphyris Newman (whether with
Dorcasomus in the NT trees or with Megacyllene Casey in the
© 2017 The Royal Entomological Society, Systematic Entomology,43, 68–89
76 S. Haddad et al.
s.s.
Fig. 2. Maximum-likelihood phylogeny for nucleotide data partitioned in  v2.0 with the x option, and using the hcluster
algorithm to search for the best partitioning scheme using the BIC criterion. Maximum-likelihood bootstrap support (MLBS) is shown only for nodes
with MLBS <100%. Information regarding the systematics of the sampled exemplars is indicated on the right of the tree. [Colour gure can be viewed
at wileyonlinelibrary.com].
AA trees) did not, except in the Bayesian trees where both alter-
natives had maximum support (Figures S3 and S4). In addition,
the relationship among the clades 24 was always (2 +(3 +4))
with maximum support, and within the monophyletic Ceram-
bycidae s.s., clade 1 (Spondylidinae +Lamiinae) was the most
basal.
Comparing the NT and AA trees
The trees obtained from the three different analyses of
NT data (Fig. 2, Figures S1 and S3) were identical in topo-
logy but varied slightly in nodal support. The tree result-
ing from the Bayesian analysis of NT data (Figure S3) had
maximal support for all nodes, and the greatest variation
in nodal support among the three NT trees was within the
very selectively sampled chrysomelid clade. Notably, all NT
analyses showed maximal support (100% MLBS and PP=1)
for Cerambycidae s.s., Vesperidae and all of its internal clades,
and for the relationships (Orsodacnidae +(Vesperidae +(Dis-
teniidae +Cerambycidae s.s.))). This latter group was sister to
a clade containing Oxypeltidae and a paraphyletic Megalopodi-
dae with high support (MLBS of 99100% and PP =1). Within
the Cerambycidae s.s., Cerambycinae (represented by two very
different genera Callisphyris and Megacyllene) was rendered
paraphyletic by Dorcasomus (Dorcasominae). Relationships
within the Chrysomelidae were recovered as (((Bruchinae +
Sagrinae) +Criocerinae) +((Cryptocephalinae +Cassidinae) +
Galerucinae)), but none of the internal clades except for
Cryptocephalinae +Cassidinae had maximum support in the
ML trees.
The analyses of AA data recovered slightly different results.
The partitioned (Fig. 3) and unpartitioned (Figure S2) ML
© 2017 The Royal Entomological Society, Systematic Entomology,43, 68–89
Cerambycidae phylogeny 77
s.s.
Fig. 3. Maximum-likelihood phylogeny for amino acid data partitioned in  v2.0 with the x option, and using the hcluster
algorithm to search for the best partitioning scheme using the BIC criterion. Maximum-likelihood bootstrap support (MLBS) is shown only for nodes
with MLBS <100%. Information regarding the systematics of the sampled exemplars is indicated on the right of the tree. Branches in red indicate
differences in relationships between this phylogeny and that in Fig. 2. [Colour gure can be viewed at wileyonlinelibrary.com].
analyses resulted in identical tree topologies with a mono-
phyletic but poorly supported Cerambycidae s.s. (63% and
50% MLBS for the partitioned and unpartitioned analyses,
respectively). The sister-group relationship between Ceramby-
cidae s.s. and Disteniidae was maximally supported in both.
The Cerambycidae s.s. +Disteniidae clade was sister (with
maximal nodal support) to a likewise maximally supported
clade comprised of the Orsodacnidae (sister to the following
two), Vesperidae, and the clade containing Oxypeltidae and
a paraphyletic Megalopodidae. Within Cerambycidae s.s.,
the two Cerambycinae (Callisphyris +Megacyllene) grouped
together but with low support (MLBS of 65% and 61% in the
partitioned and unpartitioned trees, respectively). Relationships
within the Chrysomelidae were recovered as (((Bruchinae +
(Sagrinae +Criocerinae)) +(Cryptocephalinae +Cassidinae))
+Galerucinae); the Sagrinae +Criocerinae and Cryptocephali-
nae +Cassidinae clades had maximum support in the ML trees.
The Bayesian AA tree (Figure S4) had an identical topology to
the ML trees of AA data, except for recovering a paraphyletic
Cerambycidae s.s. due to Disteniidae becoming sister to all
cerambycid subfamilies except Spondylidinae and Lamiinae;
this clade had PP =0.9807 (the only node with support lower
than 1 in both Bayesian trees). All remaining nodes in the
Bayesian AA tree had maximum support.
Discussion
This study was not aimed at elucidating relations between the
Curculionoidea and Chrysomeloidea or within the Chrysomel-
idae s.s. Both the Curculionoidea and Chrysomelidae were
© 2017 The Royal Entomological Society, Systematic Entomology,43, 68–89
78 S. Haddad et al.
sampled very selectively. Yet all the trees (rooted by two cucu-
joids) reliably recovered Curculionoidea as a sister group to
Chrysomeloidea, and Chrysomelidae s.s. as a monophyletic
group (even if the internal relationships were variable) sister to
all remaining Chrysomeloidea. The following discussion will
be mostly restricted to the non-chrysomelid Chrysomeloidea,
particularly to the Cerambycidae s.l.
Brief summary of chrysomeloid classication
It is beyond the scope of this paper to review the rich tax-
onomic history of the Chrysomeloidea in the present broad
sense and of the Cerambycidae s.l. (Cerambycoidea, Longi-
cornia, etc.) of various authors. For the Chrysomeloidea, see
Crowson (1955), Kuschel & May (1990) and Reid (1995, 2000,
2014), unlike Crowson, the latter authors focused mainly on the
chrysomelid cluster in the traditional sense including the for-
mer Bruchidae). For the Cerambycidae s.l., see Crowson (1955),
Linsley (1961), Napp (1994) or Svacha & Lawrence (2014).
Briey, within the chrysomelid cluster, the long-reigning sys-
tem of the families Chrysomelidae (including the present Orso-
dacnidae and Megalopodidae, e.g. Seeno & Wilcox, 1982) and
Bruchidae has been, in recent literature, almost universally
replaced by another (supported also by our results), placing
Bruchinae as a chrysomelid subfamily (exceptions are uncom-
mon, e.g. Kingsolver, 2002, accepted Bruchidae as a family),
whereas Megalopodidae (including Palophaginae as a subfam-
ily following Kuschel & May, 1990) and Orsodacnidae (includ-
ing Aulacoscelidinae as a subfamily following Kuschel & May,
1990 and Reid, 1995) are separate families (e.g. Clark &
Riley, 2002; Riley et al., 2003; Clark et al., 2004; Lawrence
&´
Slipi´
nski, 2013, 2014a,b; Löbl & Smetana, 2010; Bouchard
et al., 2011).
The situation is much more unsettled within Cerambycidae
s.l. We had to disregard the enigmatic Mexican Vesperoctenus
ohri Bates in our study due to the lack of necessary data
(larvae unknown, no material available for molecular study).
Earlier authors had placed it in Lepturinae, and it was later
preliminarily classied as a taxon (genus or monogeneric
tribe Vesperoctenini Vives) incertae sedis close to Ves p e ru s
Dejean or within the present Vesperidae (Svacha et al., 1997;
Vives, 2001, 2005; Svacha & Lawrence, 2014). Additionally, it
was moved for unexplained reasons to Prioninae by Bousquet
et al. (2009) and Bouchard et al. (2011). All remaining current
families and subfamilies of the Cerambycidae s.l. (Table 1),
except for Dorcasominae, had already been mentioned either
as cerambycid subfamilies or at least as taxa of problematic
taxonomic status (Oxypeltini, Ves p e r u s ) by Crowson (1955).
Subsequent authors raised the subfamilies Oxypeltinae (Duffy,
1960), Dorcasominae (Danilevsky, 1979b, as Apatophysinae,
misspelling of Apatophyseinae, and containing the single genus
Apatophysis Chevrolat) and Vesperinae (Crowson, 1981, within
his broad Disteniidae and including Oxypeltinae and Philinae,
but excluding Anoplodermatinae; the family should have been
named Vesperidae for priority reasons). Svacha & Danilevsky
(1987, pp. 14, 66) added Dorcasomus [whose larva, unlike
the pupa, was considered ‘unquestionably lepturine’ by Duffy
(1957), although it lacks several typical lepturine characters]
to Apatophyseinae but failed to appropriately rename the sub-
family to Dorcasominae (this was formally done by Özdikmen,
2008). Svacha & Danilevsky (1987) also suggested that nearly
all Afrotropical and Madagascan taxa that were then classied
in Lepturinae might belong to Apatophyseinae. This was later
conrmed by larval morphology (Svacha et al., 1997, p. 364).
Following those changes, the number and extent of subfam-
ilies recognized within the Cerambycidae s.l. were relatively
stable in major cerambycid publications. The exceptions were
the gradual but now prevailing acceptance of the subfamily
Dorcasominae (the bulk of which were previously classied
in Lepturinae), and the occasional recognition of separate
subfamilies Spondylidinae and Aseminae (e.g. Napp, 1994);
such division is neither supported by larval morphology (Duffy,
1953; Svacha & Danilevsky, 1987) nor molecular studies where
any genera of Spondylidini (Spondylis Fabricius, Neospondylis
Sama or Scaphinus LeConte) were included together with sev-
eral Asemini or even other tribes of Spondylidinae (S´
ykorová,
2008, where Spondylidinae is well sampled; Raje et al., 2016,
where Spondylidinae is monophyletic but both Asemini and
Spondylidini are not). Based on the remark by Danilevsky (in
Löbl & Smetana, 2010, p. 48) who rejected the inclusion of
Apatophysis in Dorcasominae, and retained the name Apato-
physeinae, Bouchard et al. (2011) accepted separate subfamilies
Apatophyseinae and Dorcasominae. This was considered unsup-
ported by Svacha & Lawrence (2014, p. 143) even with the lack
of a phylogeny and reliable synapomorphies for Dorcasominae.
Svacha & Lawrence (2014) also summarized some conrmed
or suspected misclassications at the subfamily level.
However, even if the number and denition of subfamilies
within the Cerambycidae s.l. gradually stabilize, their relation-
ships and rank are unclear or not agreed upon and various
alternatives can be found in recent literature. The system of
families and subfamilies as proposed predominantly on lar-
val characters by Svacha et al. (1997) and used in Bouchard
et al. (2011) and Svacha & Lawrence (2014) was used in the
present study. Various intermediate solutions exist and partic-
ularly some primarily nomenclatural or cataloguing publica-
tions (such as Bousquet et al., 2009, or Löbl & Smetana, 2010)
place all subfamilies within a single broad family Cerambyci-
dae. In addition, relationships of Cerambycidae with Orsodac-
nidae and Megalopodidae, even if occasionally indicated (e.g.
Crowson, 1955, p. 150, 1960; Schmitt, 1994a; Svacha et al.,
1997, p. 360; Svacha & Lawrence, 2014, p. 59), have not been
studied using a sufciently representative taxon sample and
the monophyly of Cerambycidae s.l. has not been convincingly
demonstrated.
Discrepancies among our trees
Although our analyses mostly recover the same monophyletic
families and subfamilies (the only exceptions being the para-
phyletic Cerambycidae s.s. in the Bayesian AA tree and the
paraphyletic vs monophyletic Cerambycinae in the NT and AA
© 2017 The Royal Entomological Society, Systematic Entomology,43, 68–89
Cerambycidae phylogeny 79
trees, respectively), there is clearly some incongruity in support
and topology between the AA and NT analyses, which is not
uncommon (e.g. Regier et al., 2010). On the one hand, generally,
in protein-coding sequences, NT data are more informative than
AA data for phylogenetic reconstruction of recent divergences
due to the increased likelihood of substitutions at synonymous
sites (Rota-Stabelli et al., 2013). On the other, in phylogenetic
reconstructions of deep-level relationships, the NT data may
suffer from saturation and convergence affecting those variable
synonymous sites, which may result in poorly supported or false
phylogenies (Rota-Stabelli et al., 2013). In such cases, either
AA data are preferentially used, or the NT data are modied
to alleviate those issues (e.g. by removing third codon posi-
tions or via R-Y recoding). Nevertheless, studies continue to
disagree over which data type should be prioritized for phy-
logenetic reconstruction, with analyses of AA data oftentimes
preferred for reconstructing phylogenies of deeper-level rela-
tionships, despite some studies arguing for the preference of NT
data in all cases for phylogenetic analysis (e.g. Townsend et al.,
2008, for vertebrates, but see discussion therein; Holder et al.,
2008). Some recent studies argue that incongruence between AA
and NT analyses is the result of poor modelling methods for AA
analyses, and that NT data can still be utilized but require better
methods of nucleotide degeneracy coding (Zwick et al., 2012),
whereas others suggest that given the current state of things, AA
data should be preferred (Rota-Stabelli et al., 2013). Taking all
this into consideration, we included separate analyses for both
AA and NT data with the intention of comparing the resulting
phylogenetic hypotheses. We consider cases of congruence in
topology across the different analyses to reect the robustness
of those recovered relationships, whereas cases of incongruence
require further investigation.
Families of Chrysomeloidea and their relationships
Chrysomeloid families listed in Table 1 were represented by
more than one terminal taxon except for Oxypeltidae, but both
known oxypeltid genera (Cheloderus and Oxypeltus Blanchard)
have similar adult and larval morphology with some unique
synapomorphies (Svacha & Lawrence, 2014). As such, there
is little doubt of the monophyly of Oxypeltidae. Oxypeltus and
Cheloderus were recovered as sister taxa in Bocak et al. (2014;
Cheloderus was misplaced and miscoloured in their gure S1 as
a curculionoid).
All of our analyses (Figs 2, 3, Figures S1 S4) divide
Chrysomeloidea into two sister clades, Chrysomelidae and all
remaining Chrysomeloidea. The same two clades were obtained
in McKenna et al. (2015) based on different molecular data,
but not in any previous molecular or morphological analy-
ses. Within the non-chrysomelid Chrysomeloidea, four clades
are universally recovered: (1) paraphyletic Megalopodidae
including Oxypeltidae as sister to Palophaginae, (2) Orsodac-
nidae, (3) Vesperidae, and (4) Disteniidae and Cerambycidae
s.s. as sister groups except for the Bayesian AA tree where
Cerambycidae s.s. is paraphyletic (Figure S4). However, the
relationships of those four clades differ between the NT trees
(1 +(2 +(3 +4))) and AA trees ((2 +(1 +3)) +4). Speci-
cally, each of the four clades has a different sister group in
the NT versus AA trees. Cerambycidae s.l. is not recovered
as monophyletic either in the NT trees (Oxypeltidae and a
clade containing Vesperidae, Disteniidae and Cerambyci-
dae s.s.) or in the AA trees (Oxypeltidae, Vesperidae, and a
clade containing Disteniidae and Cerambycidae s.s.). Thus,
the higher-level relationships within the non-chrysomelid
group particularly require a more focused and better
sampled analysis.
As mentioned in the above section on chrysomeloid clas-
sication, Megalopodidae and Orsodacnidae were historically
treated within Chrysomelidae, but are currently widely accepted
as separate families and thought to occupy transitional (but
uncertain) positions between Chrysomelidae and Cerambyci-
dae or even being closer to the latter (e.g. Crowson, 1955,
p. 150; Schmitt, 1994a; Reid, 1995, 2000). Orsodacnidae and
Megalopodidae occur in variable positions in recent molecu-
lar phylogenetic studies, either recovered closer in afnity to
Chrysomelidae or to Cerambycidae (e.g. Farrell, 1998; Far-
rell & Sequeira, 2004; Gómez-Zurita et al., 2007, 2008; Hunt
et al., 2007; Marvaldi et al., 2009; Bocak et al., 2014; McKenna
et al., 2015). Our analyses place both Orsodacnidae and (para-
phyletic) Megalopodidae reliably in the non-chrysomelid clade
of Chrysomeloidea together with Cerambycidae s.l. All of
our analyses reliably recovered a monophyletic Orsodacnidae
including Aulacoscelidinae as proposed by Kuschel & May
(1990) and Reid (1995). However, Megalopodidae in the clas-
sical sense was equally reliably rendered paraphyletic by
Oxypeltidae (placed as sister to Palophaginae). Both families,
but particularly Megalopodidae (24 genera and an estimated 450
species; Lawrence & ´
Slipi´
nski, 2014a) should be better sampled
in future phylogenetic studies.
The sister-group relationship of Palophaginae and Oxypelti-
dae has been recovered in several molecular studies to date:
Prado et al. (2012), McKenna et al. (2015), and in part
in Bocak et al. (2014), who recovered a clade ((Oxypel-
tus +Cheloderus)+Palophagus Kuschel) with Palophagoides
Kuschel recovered outside it, but within the same small clade
of nine taxa. However, the results of these studies are difcult
to interpret either due to the poor sampling of Cerambycidae
s.l., or (in Bocak et al., 2014 where Cerambycidae s.l. is well
sampled) because the two oxypeltids and two palophagines
were recovered in an improbable clade sister to all other
Chrysomeloidea and containing also three species of Zeu-
gophora Kunze (other species were elsewhere in the tree), a
species of Aulacoscelis Duponchel & Chevrolat (again not
the only one in the tree) and an obviously misidentied taxon
labelled as the genus Collops Erichson of Melyridae. However,
there is not a single molecular study containing both oxypeltids
and palophagines that has not recovered them as sister taxa,
or at least in very close phylogenetic positions (Palophagoides
in Bocak et al., 2014). To verify whether purely mitochondrial
DNA provides the same signal, we performed a ML analysis of
COI sequence data obtained from GenBank (not shown here)
in which we included an exemplar for every chrysomeloid
subfamily for which COI sequence data were available, and we
© 2017 The Royal Entomological Society, Systematic Entomology,43, 68–89
80 S. Haddad et al.
also recovered Oxypeltidae and Palophaginae as sister groups.
The Oxypeltidae, although placed as a possible sister group
to Vesperidae on very doubtful characters, were considered
‘perhaps the greatest jeopardy for the monophyly of the cer-
ambyciform lineage’ (=Cerambycidae s.l.) by Svacha et al.
(1997). Oxypeltidae and Palophaginae share a fused immov-
able larval clypeolabrum (possible synapomorphy; Svacha &
Lawrence, 2014), an apomorphy absent in all other Phytophaga
except the cryptocephaline cluster (‘Camptosomata’) within
Chrysomelidae where the larval head morphology is very
different and the character undoubtedly arose because of con-
vergent evolution. Kuschel & May (1990, p. 702) mentioned the
shared clypeolabral fusion in Palophaginae and Oxypeltidae,
but considered it a parallelism. They were apparently not aware
that their key to families of Chrysomeloidea based on male and
female genitalia did not t the peculiar genitalia of Oxypeltidae
(see Fragoso, 1985; Svacha & Lawrence, 2014). Oxypeltidae
and Palophaginae share a ‘Gondwanan’ distribution (southern
South America, Palophaginae also occur in Australia) and their
known hosts – Araucaria and probably Agathis (Araucari-
aceae) for Palophaginae (Lawrence & ´
Slipi´
nski, 2014a) and
Nothofagus (Nothofagaceae) for Oxypeltidae – are currently
also restricted to the Southern Hemisphere. Thus, despite the
dissimilar adults and biology (tree boring vs pollen feeding
larvae), the relationship of Oxypeltidae and Palophaginae is
supported, or at least not contradicted, by a variety of data in
addition to the consistent support from molecular data.
The family Vesperidae in the present sense (Table 1; see
taxonomic overview above for comments on the Mexican
Vesperoctenus Bates not sampled here) was rst proposed by
Svacha et al. (1997) on larval characters and biology. The family
continues to be almost impossible to dene based on adult char-
acters when it includes the Neotropical Anoplodermatinae (the
extraordinary genus Hypocephalus Desmarest makes almost
any group it would be included in difcult to dene), although
a close relationship between Ve s p e r u s (Mediterranean) and
Philinae (basically Oriental with one species in tropical Africa)
has been proposed previously (e.g. Gahan, 1906; Saito, 1990,
on female genitalia). Anoplodermatinae have previously been
associated with the Prioninae and Parandrinae based on adult
characters (e.g. Napp, 1994), but many of those characters are
inconsistent (Svacha & Lawrence, 2014, p. 133) and anoploder-
matines differ from all Prioninae and Parandrinae, for example
by the internally and externally closed procoxal cavities (the
only known prionine with internally closed procoxal cavities,
the genus Anoeme Gahan, has them broadly open externally).
Ves p e r u s (and Vesperoctenus) were most often placed with
the cerambycid subfamily Lepturinae because of the posteri-
orly constricted head, and the position of Philinae was very
unstable in different systems. The monophyly of Vesperidae
sensu Svacha et al. (1997) has been repeatedly questioned,
particularly with respect to adult characters (e.g. Svacha &
Lawrence, 2014; Vives, 2001). Vesperidae were not recovered
monophyletic based on morphological characters by Napp
(1994; Philus and Anoploderma Guérin-Méneville sampled),
Reid (1995) or Lawrence et al. (2011); Ve s p e r u s and Sypilus
Guérin-Méneville sampled), and were not monophyletic in the
only two molecular phylogenies which included Anoploder-
matinae and at least one other vesperid subfamily (Bocak et al.,
2014; McKenna et al., 2015; see Table 1 in Haddad & McKenna,
2016). Our data recovered a monophyletic Vesperidae with high
to maximum support in all analyses. Svacha et al. (1997)
proposed a (Vesperinae +(Philinae +Anoplodermatinae))
relationship of vesperid subfamilies, whereas our results
clearly place Philinae and Vesperinae as sister groups which
agrees with the morphology of female genitalia (unlike the
females of Vesperinae and Philinae studied by Saito, 1990, the
anoplodermatine females dissected by Svacha & Lawrence,
2014 had distinct sclerotized presumably plesiomorphic sper-
matheca). This suggests that either the larva of Ves p e r u s is
strongly derived and the proposal of Svacha et al. (1997) was
based on larval plesiomorphies, or that the larval characters
joining Philinae and Anoplodermatinae were parallelisms
(Svacha & Lawrence, 2014, p. 35).
There were attempts in earlier literature to divide the broad
Cerambycidae into more than one family (usually either Prion-
idae or Lamiidae were raised to family level; see Crowson, 1955)
and some authors effectively treated cerambycids as a superfam-
ily rather than a family. Disteniidae was the rst group in the
modern cerambycid taxonomic history to be given the family
rank (Linsley, 1961, 1962), in part on larval characters (mainly
the absence of the ventral external sclerotized cranial closure, the
gula and more retracted ventral mouthparts; rst described by
Craighead, 1923, and Böving & Craighead, 1931). The absence
of a larval gula is very probably plesiomorphic as it is shared
with virtually all other Phytophaga, except Cerambycidae s.s.
(where the gula is invariably present), and thus cannot be used
to establish disteniid relationships. Disteniid larvae are unfortu-
nately known only in a few genera of the nominotypical tribe
Disteniini, but all known larvae are very similar and unlike any
other Chrysomeloidea. None of the adult characters listed by
Linsley (1962, p. 1) can dene disteniids on a worldwide basis
because they occur in some Cerambycidae s.s. (clypeus oblique
to frons, ‘non-hylecoetoid’ metendosternite, wing crossvein r4
without spur, truncate ‘scalpriform’ mandibular apex; the lat-
ter character is also not universally present within Disteniidae).
Gahan (1906), who dened the group (as a cerambycid sub-
family) in the present extent, listed some additional characters
but none are universal and exclusive. A possible morpholog-
ical character exclusive to Disteniidae (and undoubtedly apo-
morphic because it is unknown in any other chrysomeloids) is
the presence of a row of very long setae (lying in a groove at
repose) along at least some antennal agellomeres (mentioned
by Gahan but not by Linsley). However, those setae are absent
in Cyrtonops White and reduced in Dynamostes Pascoe (Gahan,
1906; Svacha & Lawrence, 2014).
All of our analyses recovered Disteniidae (Distenia Le Peletier
& Audinet-Serville +Cyrtonops) in a monophyletic clade with
Cerambycidae s.s. The latter family was paraphyletic in the
Bayesian AA tree (Figure S4; note that the clade containing
disteniids and cerambycids other than Spondylidinae and Lami-
inae was the only one in both Bayesian trees which did not
have maximum support). A sister-group relationship between
Disteniidae and Cerambycidae s.s. (recovered in all remaining
© 2017 The Royal Entomological Society, Systematic Entomology,43, 68–89
Cerambycidae phylogeny 81
trees) was proposed by Svacha et al. (1997) based on larval
characters, notably the identical and apomorphic fusion of the
larval postclypeus with frons (forming the so-called epistomal
margin bearing epistomal – originally postclypeal setae) and
possibly the annular-multiforous spiracles in later-instar lar-
vae (annular in Vesperidae and apparently in the only known
later-instar orsodacnid larva described by Prado et al., 2012;
annular-biforous in Megalopodidae and Oxypeltidae). The rst
character is difcult to evaluate in some other chrysomeloid
larvae (e.g. in the Palophaginae and Oxypeltidae with fused
clypeolabrum), and although annular-multiforous spiracles may
be a groundplan character in Cerambycidae s.s., its spira-
cle morphology is very variable (annular, annular-biforous,
annular-multiforous). Svacha et al. (1997) also remarked that
Disteniidae was the only chrysomeloid group sharing the dead
wood feeding habit widespread (and possibly plesiomorphic) in
Cerambycidae s.s. There are no known adult synapomorphies
indicating a relationship between Disteniidae and any other
group within Chrysomeloidea (Svacha & Lawrence, 2014);
Napp (1994) retained Disteniidae as a separate family requir-
ing further study, and those subsequent phylogenetic studies that
included disteniids (see Haddad & McKenna, 2016) recovered
the group in rather variable positions.
Monophyly of Cerambycidae s.s. and relationships within
the family
For a summary of cerambycid classication, adult and lar-
val morphology, and distribution and biology see Svacha &
Lawrence (2014) and references therein. Our results do not all
rmly support the monophyly of Cerambycidae s.s. with respect
to Disteniidae, and the maximal versus poor nodal support (or
even paraphyly in the Bayesian AA tree) is a major difference
between the NT versus AA trees. Mann & Crowson (1981);
key to chrysomeloid higher taxa), Svacha & Danilevsky (1987),
and Svacha et al. (1997) proposed the larval gula as a possible
synapomorphy for Cerambycidae s.s. As a summary of classical
and (partly unpublished) molecular data, Svacha & Lawrence
(2014) proposed a preliminary phylogeny of Cerambycidae s.s.
as an unresolved basal trichotomy with three branches: Prion-
inae and Parandrinae (with no synapomorphies available for
the former which could thus be paraphyletic), Dorcasominae
and Cerambycinae (no clear synapomorphies known for the for-
mer), and a third branch (which they and others suspect may not
be monophyletic) including the remaining four subfamilies as
(Spondylidinae +(Lepturinae +Necydalinae) +Lamiinae).
In our present analyses, the eight subfamilies of Ceram-
bycidae s.s. (Table 1, Figs 2, 3, Figures S1 S4) form four
maximally supported clades composed of two subfamilies
each (see Results): (1) Spondylidinae +Lamiinae, (2) Lepturi-
nae +Necydalinae, (3) Dorcasominae and Cerambycinae, the
latter paraphyletic in NT trees, and (4) Prioninae +Parandrinae.
The relationship of those four clades is (1 +(2 +(3 +4))) in all
trees where Cerambycidae s.s. is monophyletic, and would be
the same also in the Bayesian AA tree (where Cerambycidae
s.s. is rendered paraphyletic by Disteniidae; Figure S4) if the
disteniids were disregarded.
Clade 4. The relationship of Parandrinae to Prioninae is cur-
rently widely accepted. The two subfamilies have previously
been recovered as sister groups in some trees resulting from
molecular or combined phylogenetic analyses with limited sam-
pling (Farrell, 1998; Farrell & Sequeira, 2004; Gómez-Zurita
et al., 2007, 2008; Marvaldi et al., 2009; Wang et al., 2013;
McKenna et al., 2015). In fact, it is possible that the rst may
be an ingroup of the second (Nearns, 2013). A close rela-
tionship between the two subfamilies has been consistently
suggested based on larval morphology; either the two groups
formed a single subfamily (Craighead, 1915, 1923) or, at the
very least, were considered closely related. The previous pro-
posal of some authors of Parandrinae being the most basal group
of Cerambycidae s.l., or even related to other Cucujiformia has
been discussed and criticised (e.g. by Crowson, 1955) and is
today virtually abandoned. Prioninae and Parandrinae have often
been associated with Anoplodermatinae based on adult mor-
phology (Crowson, 1955; Napp, 1994), but such an associa-
tion greatly contradicts larval characters (Svacha & Danilevsky,
1987; Svacha et al., 1997) and our results support the larval con-
clusions in placing Anoplodermatinae within Vesperidae and
not near Cerambycidae s.s. Adults of Parandrinae and Prioninae
share the internally open procoxal cavities (a character virtually
unique in Chrysomeloidea; the prionine genus Anoeme Gahan is
the single known exception with the procoxal cavities internally
closed), the absence of a mesonotal stridulatory plate (present in
at least some taxa of all other subfamilies of Cerambycidae s.s.
and in Disteniidae), and the presence of lateral pronotal carina
in most species (also present in some other Chrysomeloidea but
very seldom in other Cerambycidae s.s. and never in Disteni-
idae). Larvae possess the combination of a short gula and arela-
tively broad rm tentorial bridge (unique in Chrysomeloidea),
and in later instars, the at (not conical) antennal sensorium
which is nearly unknown in remaining Cerambycidae s.s. and
in Disteniidae.
Clade 3. Most Dorcasominae were previously classied
within or near Lepturinae (or its equivalent) mainly based on
the similarity in the supercial adult morphology of some ori-
colous species. Danilevsky (1979b) proposed a close relation-
ship with Cerambycinae based on larval characters when he
raised the subfamily Dorcasominae (as Apatophysinae) based
on the genus Apatophysis, and this was conrmed for all dorca-
somine larvae discovered since then (Svacha et al., 1997; Svacha
& Lawrence, 2014; P. Svacha, unpublished). Our analyses con-
rm a close relationship to Cerambycinae for Dorcasomus;we
did not sample any exemplars for the largest dorcasomine tribe
Apatophyseini (we attempted the Madagascan genus Logisti-
cus Waterhouse but DNA obtained from the specimen was not
suitable) and we thus cannot evaluate the monophyly of Dorca-
sominae. S´
ykorová (2008) also recovered dorcasomine genera
belonging to Apatophyseini (Apatophysis and Tsivoka Villiers)
in a cluster containing Cerambycinae, Prioninae and Parandri-
nae (and away from Lepturinae). Additional sampling of cer-
ambycine species is needed to clarify the contradiction between
the subfamily’s paraphyly and monophyly in the NT versus AA
trees. Nodal support for Callisphyris +Dorcasomus in the NT
© 2017 The Royal Entomological Society, Systematic Entomology,43, 68–89
82 S. Haddad et al.
trees was reasonably high (91% and 96% for partitioned and
unpartitioned ML analyses, respectively) whereas support for
a monophyletic Cerambycinae (Callisphyris +Megacyllene)in
the AA trees was low (65% and 61% for partitioned and unparti-
tioned ML analyses, respectively). A nonmonophyletic Ceram-
bycinae would be unexpected because the subfamily appears to
have distinct larval apomorphies (such as a constricted clypeus
or round ‘gouge-like’ mandibles; see Svacha & Lawrence,
2014). Dorcasomines were mostly absent from the taxon sam-
ple of recent phylogenetic studies (see Table 1 in Haddad &
McKenna, 2016); they have no obvious larval or adult apomor-
phies supporting their monophyly, and there are no reliable adult
characters distinguishing them from cerambycines (Svacha &
Lawrence, 2014). Clade 3 (Dorcasominae and Cerambycinae) is
difcult to dene by apomorphies. Both subfamilies invariably
lack the wedge cell in the wing (it may be present in all three
remaining clades, namely in almost all Prioninae, some Lep-
turinae and some Spondylidinae), but it was lost several times
in parallel in cerambycids. Both subfamilies lack the characters
listed for Clade 4 above, and although adult dorcasomines are
frequently habitually similar to lepturines, Clade 3 universally
lacks the mandibular molar sclerite present in most species of
Clade 2 (Lepturinae and Necydalinae) (see Svacha & Lawrence,
2014, gs 2.4.12K, L). Larvae almost universally differ from
Clades 2 and 1 (Lamiinae and Spondylidinae) by the presence
of a postnotal fold (if it is absent in a few Cerambycinae, the
cerambycine larval apomorphies mentioned above are present).
Lamiinae has a number of unique larval and adult apomorphies,
but it is difcult to distinguish some cerambycine adults with
rich wing venation from those spondylidine taxa lacking the
wing wedge cell, and misclassications between Cerambycinae
and Spondylidinae on external adult morphology were common
(such confusion is impossible in the case of larval morphology).
Clade 2. The close relationship between Necydalinae and
Lepturinae is also widely accepted. It is possible that necy-
dalines may be a lepturine ingroup and some authors placed
Necydalini as a tribe of Lepturinae (e.g. Linsley, 1940; Lins-
ley & Chemsak, 1972; Monné, 2006). Napp (1994) consis-
tently recovered Necydalis Linnaeus within Lepturinae, but on
debatable characters and with poor sampling. A separate sub-
family Necydalinae prevails in recent literature. Necydalinae is
a morphologically distinct subfamily whose adults have very
short elytra and exposed hindwings and both morphologically
and behaviorally mimic certain Hymenoptera. Similar mimics
occur in some other subfamilies (particularly in Cerambycinae)
and were occasionally incorrectly associated with necydalines.
Most of those misclassications have already been corrected,
but some persist. Svacha & Lawrence (2014, p. 152) suggested
that the subfamily Necydalinae includes only two genera (Necy-
dalis and Ulochaetes LeConte), which are nearly completely
restricted to the Northern Hemisphere. They also noted that
the ten additional American (predominantly Neotropical) gen-
era placed in Necydalinae (most recently in Bezark, 2016) are
Cerambycinae that are misclassied based on adult parallelisms
(larvae are known for two of them, Callisphyris and presumed
Hephaestion Newman, and bear the characteristic cerambycine
apomorphies; Duffy, 1960, Appendix; Svacha & Lawrence,
2014 and g.2.4.24D therein). Callisphyris, the most ‘Necy-
dalis-like’ of those Neotropical genera, was included in our
study and was reliably recovered within the clade also con-
taining Dorcasomus (Dorcasominae) and Megacyllene (Ceram-
bycinae), thus conrming that the placement of Callisphyris
in Necydalinae is a misclassication. We sampled only one
species of Necydalis, but the only molecular study that contained
both Necydalis (three species) and Ulochaetes (S´
ykorová, 2008)
recovered a monophyletic Necydalinae, even if often with poor
support. In Lepturinae, we sampled a typical member of the
nominotypical tribe Lepturini (Rutpela Nakane & Ohbayashi)
and the Nearctic genus Desmocerus Dejean which has usually
been classied by American authors in a separate tribe Desmo-
cerini (e.g. Linsley & Chemsak, 1972; Bezark, 2016). How-
ever, Desmocerus also clusters in Lepturini s.s. in S´
ykorová
(2008). Thus, additional sampling of taxa is needed to inves-
tigate the monophyly of the Lepturinae. Svacha & Lawrence
(2014) suggested a possible larval apomorphy for each of Lep-
turinae (reduction or absence of larval pronotal lateral furrows)
and Necydalinae (duplicate lateral impressions of the ventral
larval ambulatory ampullae) that would distinguish their other-
wise relatively similar larvae. Adults of Necydalinae are highly
apomorphic (see above), but it would be difcult to name any
apomorphies for the relatively large Lepturinae with an esti-
mated 200 described extant genera (Svacha & Lawrence, 2014).
Both subfamilies share some characters that could be consid-
ered plesiomorphic compared with all other Cerambycidae s.s.:
in adults, the mandibular molar sclerite (very seldom rudimen-
tary); in larvae, the relatively long legs bearing (with the excep-
tion of the Oriental Pyrocalymma Thomson) a distinct mesal
pretarsal seta or the almost always broadly separate dorsal epi-
cranial halves. However, as none of these characters occur in
the basalmost Spondylidinae +Lamiinae clade and the long legs
with pretarsal seta also do not occur in Disteniidae (plus the
mandibular mola in adult disteniids, when present, is constructed
somewhat differently), at least some of those characters might
be in fact synapomorphies of the present clade. In addition, this
clade lacks all characters listed for Clade 4, the larval postnotal
fold almost universally present in Clade 3, several characteristic
larval and adult apomorphies of the Lamiinae (see below), and
the long distinct larval lateral pronotal furrows of the Spondylid-
inae (although they are somewhat intermediate in necydalines).
Clade 1. The only pair of subfamilies which is currently not
commonly accepted is Spondylidinae +Lamiinae. This relation-
ship has previously been proposed (e.g. by Crowson, 1955,
based on some adult characters, even if those mentioned by
him also occur in some other cerambycid subfamilies or may
be plesiomorphic) and larval characters do not contradict such
a relationship (Danilevsky, 1979a; Svacha & Danilevsky, 1987,
p. 15). One larval synapomorphy might be the split retractors
of dorsal ambulatory ampullae in the spondylidine and lami-
ine groundplan causing two pairs of lateral impressions (Svacha
& Danilevsky, 1987), although lamiines show a vast array of
various modications and there is only one pair of impressions
in the spondylidine genus Pectoctenus Fairmaire. Two pairs of
© 2017 The Royal Entomological Society, Systematic Entomology,43, 68–89
Cerambycidae phylogeny 83
impressions also occur in Necydalinae, but on both dorsal and
ventral ampullae and the muscle split may have occurred inde-
pendently. Although the spondylidine larval and adult ground-
plan is one of a relatively generalized cerambycid, the huge
subfamily Lamiinae is highly derived in both adult and larval
characters: in larvae, the (at most) rudimentary legs, elongate
cranium jointly rounded posteriorly and bearing a deep dorso-
median intracranial crest, and the reduced immovable maxil-
lary cardo; in the adult groundplan, the perpendicular or reced-
ing frons (more or less prognathous forms appear to be sec-
ondary reversals), the asymmetrical mesonotal carina, a peculiar
bilobed or bidentate prominent sclerite at base of tibial exor
apodeme, and a few others. This makes Lamiinae easy to dene,
but at the same time, difcult to associate with its closest rela-
tives based on morphological characters. It would be difcult
to list any adult or larval synapomorphies for Spondylidinae,
and although spondylidine larvae can be easily distinguished
from all other subfamilies, we lack reliable adult distinguishing
characters. Some Spondylidinae were frequently misclassied
in Cerambycinae, and vice versa, and even the generally richer
spondylidine wing venation has not been entirely reliable in
dening the group (see Clade 3 and Svacha & Lawrence, 2014).
Both larval morphology and molecular data make it impossi-
ble to accept separate subfamilies Spondylidinae and Aseminae
(see section on classication). S´
ykorová (2008) recovered the
subfamily (whose relationships to other subfamilies were unfor-
tunately inconclusive) as composed of two distinct branches,
one comprising Spondylis (tribe Spondylidini) and the usually
paraphyletic Asemini, and the other including the Anisarthrini,
Saphanini and Atimiini. However, the subfamily gradually came
together only after including a sufcient sample of taxa (see her
g. 8a and b with 11 and 4 spondylidine species, respectively).
Our study includes only two genera of the Spondylidini-Asemini
branch (type genera of both tribes) and inclusion of taxa from
the other branch would be desirable. However, Spondylidinae
was also recovered sister to Lamiinae in at least some trees by
Gómez-Zurita et al. (2007, 2008), Marvaldi et al. (2009) and
Wan g et al. (2013), and in the rst and last of these, spondy-
lidines were represented by Drymochares Mulsant (a genus of
Saphanini). Lamiinae was often associated with Cerambycinae
(gs 2.4.19AD in Svacha & Lawrence, 2014), but generally
based on problematic and nonexclusive or misinterpreted adult
characters. In addition to the characters of Napp (1994) critically
discussed in Svacha & Lawrence (l. c., p. 133), the ‘undivided’
mesonotal stridulatory plate was occasionally listed as a shared
character (e.g. Linsley, 1961), but whereas the typical ‘undi-
vided’ plate in Cerambycinae resulted from disappearance of the
median mesonotal endocarina, in most Lamiinae that endoca-
rina is present but shifted sidewise and asymmetrical (Crowson,
1955; g. 2.4.14G in Svacha & Lawrence, 2014) and the endo-
carina is present in some Cerambycinae (Svacha & Lawrence,
2014; some photographs in ´
Slipi´
nski & Escalona, 2016).
Although none of the latter four recovered clades are very
surprising, their relationships as recovered in this study (disre-
garding for the moment the invasion of disteniids in the Bayesian
AA tree, Figure S4, which requires further investigation) are
far from commonly accepted. We cannot comprehensively
discuss even the more recent classication systems as they are
too many (for an incomplete sample, see gs 2.4.19A– F in
Svacha & Lawrence, 2014, and references therein). Briey,
Parandrinae and Prioninae were traditionally considered basal
(and occasionally associated with Anoplodermatinae based on
adult characters) in the Cerambycidae s.l.,oratleastbasalin
Cerambycidae s.s. after removal of some taxa as separate fami-
lies (e.g. Crowson, 1955; Linsley, 1961; Svacha & Danilevsky,
1987; Napp, 1994; Svacha et al., 1997) based on the following:
the laterally margined pronotum, broad fore coxae or absence of
mesonotal stridulatory plate in adults if considered plesiomor-
phic, and the occipital foramen apparently divided in two parts
by a broad and ventrally visible tentorial bridge in larvae. Both
subfamilies also held basal (although in details different) posi-
tions in some molecular or combined phylogenies (e.g. Farrell,
1998; Hunt et al., 2007). Conversely, our results place Prioninae
and Parandrinae in a more derived phylogenetic position within
Cerambycidae s.s. (and far from Anoplodermatinae). This is
similar to the phylogenetic placement of Prioninae as recov-
ered in other recent molecular phylogenetic studies (some clado-
grams in Gillett, 2006; Raje, 2012; Nearns, 2013; Raje et al.,
2016). This would necessitate re-evaluation of some prionine
and parandrine adult characters which have been so far treated as
plesiomorphic or of uncertain polarity. The questionable homol-
ogy of lateral pronotal margins in various chrysomeloid taxa
was pointed out by Reid (1995), for example. The absence of
a mesonotal stridulatory le should be apomorphic as the le
occurs (even if not universally) in all other cerambycid subfam-
ilies, in Disteniidae, some Vesperidae (always absent in Anoplo-
dermatinae), in Megalopodidae, perhaps in some Chrysomelidae
(Schmitt, 1994b), and in some Nemonychidae of Curculionoidea
(Kuschel & May, 1990). Broad largely exposed fore coxae also
occur in some other subfamilies and their almost universal pres-
ence in Prioninae and Parandrinae may be associated with the
internally open procoxal cavities which is an apparently unique
character within the entire Chrysomeloidea (even if the closing
bridge is very narrow in some other groups) and thus also proba-
bly an apomorphy. The larval tentorial bridge is discussed in the
following paragraph.
The relationship between the prionine-parandrine clade and
the dorcasomine-cerambycine clade is also a nontraditional fea-
ture. In addition to our results, some recent molecular studies
also indicate a similar relationship (although most of them did
not sample dorcasomines): some cladograms in Gillett (2006),
Nearns (2013), and Raje et al. (2016). Almost no such earlier
opinions exist except for Danilevsky (1979a; see g. 24.19F in
Svacha & Lawrence, 2014) who proposed a close relationship
between Cerambycinae and the Prioninae-Parandrinae clade
based on the ‘divided’ larval occipital foramen (later found also
in many Dorcasominae): the tentorial bridge lies on the ven-
tral cranial plane (i.e. is not sunken into the cranial cavity) well
behind the gula and thus divides the occipital foramen into a
smaller anterior part and a larger posterior part. However, as
long as the broad larval tentorial bridge in all known Disteniidae,
Vesperidae, Oxypeltidae, and Prioninae and Parandrinae (of
Cerambycidae s.s.) was regarded as a possible symplesiomor-
phy lost in the six remaining subfamilies of the Cerambycidae
© 2017 The Royal Entomological Society, Systematic Entomology,43, 68–89
84 S. Haddad et al.
s.s. (most recently Svacha & Lawrence, 2014; see the gures of
larval heads therein), it was impossible to accept the proposition
of Danilevsky because the bridge in Cerambycinae (and Dor-
casominae) is narrow and thus a potential synapomorphy with
the subfamilies Spondylidinae, Lamiinae, Lepturinae and Necy-
dalinae.However, the conformation of our present trees suggests
that the groundplan of Cerambycidae s.s. might include a nar-
row and possibly partially internalized tentorial bridge with dis-
tinct metatentorial pits (a narrow bridge occurs in Clades 1– 3,
distinct metatentorial pits in clades 1 and 2 and in some Dor-
casominae). Thus, the broad bridge in Prioninae and Parandri-
nae may be an independent apomorphic parallelism, and con-
sequently the proposal by Danilevsky (1979a) may be correct.
If the broad larval tentorial bridge of the prionine-parandrine
clade is independent and apomorphic, then so is the very
short larval gula (shortest of all cerambycids) in those two
subfamilies.
The ‘second most basal’ Clade 2 (Lepturinae +Necydalinae)
is difcult to comment on with respect to classical characters.
Its position is neither expected nor surprising, but it would
be difcult to propose any synapomorphies with Clades 3+4.
Given the unorthodox relationships recovered in this study, the
morphology of Cerambycidae s.s. will have to be carefully
revisited.
The basalmost Clade 1 (Spondylidinae +Lamiinae) comprises
two morphologically and biologically different subfamilies.
Spondylidinae (some taxa are pronouncedly relict) is the second
smallest subfamily. Its adults are typically somber-coloured and
nonfeeding, and usually crepuscular or nocturnal. Its larvae
are uniformly wood-boring or subcortical. Taking our results
into consideration, the subfamily has no obvious larval or
adult apomorphies. Lamiinae (containing more than half of
described cerambycid species) has diversied larvae and adults,
obligate adult maturation feeding on plant tissues or fungi, many
different types of larval feeding (including terricolous taxa),
and numerous adult and larval apomorphies (see above). As
such, the biology and morphology of Lamiinae would not aid
in reconstructing the cerambycid groundplan. Thus, considering
the present results and the difculty of dening Spondylidinae
based on adult characters, the morphology of the surviving
spondylidine taxa needs to be carefully revised.
After taking into consideration the distribution, preliminary
molecular phylogenetic results, the geographical distribution of
plesiomorphic characters (e.g. in wing venation) in the world-
wide subfamilies, and the presence of related low-rank taxa on
different continents, Svacha & Lawrence (2014, p. 135) pro-
posed that the subfamilies Prioninae, Parandrinae, Dorcasom-
inae and Cerambycinae might be of ‘southern’ (Gondwanan)
origin (Dorcasominae has since been found in Australia, unpub-
lished record), whereas the remaining four subfamilies (Spondy-
lidinae, Lamiinae, Lepturinae and Necydalinae; placed as a
questionable monophylum in the preliminary phylogenetic tree
of Svacha and Lawrence) could be of ‘northern’ (Laurasian)
provenience. Interestingly, the proposed ‘southern’ subfamilies
form a monophyletic and phylogenetically advanced group in
our cladograms whereas the presumably ‘northern’ subfamilies
remain as a paraphyletic base.
Relationships within Chrysomelidae
Resolving relationships within the Chrysomelidae was not
within the scope of this study. Nonetheless, our analyses corrob-
orate some previous ndings on the phylogenies of Chrysomel-
idae and Chrysomeloidea. Chrysomelidae was recovered mono-
phyletic with maximum nodal support in all our analyses (Figs 2,
3, Figures S1S4) and including Bruchinae, which is consis-
tent with many previous phylogenetic studies (e.g. Reid, 1995,
2000; Farrell, 1998; Duckett et al., 2004; Farrell & Sequeira,
2004; Gómez-Zurita et al., 2007, 2008; Hunt et al., 2007; Mar-
valdi et al., 2009; Lawrence et al., 2011; Bocak et al., 2014;
McKenna et al., 2015). We sampled only six of the approxi-
mately 12 currently recognized chrysomelid subfamilies (classi-
cations differ), each represented by a single species (Table 1).
Their interrelationships differed between NT and AA trees (with
some clades not maximally supported) with the following points
in common: Criocerinae, Sagrinae and Bruchinae formed one
clade in both NT and AA trees, although Sagrinae was sis-
ter to Bruchinae in the former but to Criocerinae in the latter.
Cassidinae was sister to Cryptocephalinae and thus the subfam-
ilies Cassidinae, Criocerinae, Sagrinae and Bruchinae whose
members share the bid tarsal setae (see Stork, 1980; Mann
& Crowson, 1981) did not form a monophyletic group, sim-
ilar to Gómez-Zurita et al. (2008) and Marvaldi et al. (2009).
The position of Galerucinae differed between NT trees (sister to
Cassidinae +Cryptocephalinae) and AA trees (sister to all other
Chrysomelidae).
Practical classication
The families and subfamilies of Cerambycidae s.l. are rela-
tively clear-cut based on larval morphology (what is known so
far), but often difcult or impossible to dene based on adult
morphology. For details of the characters mentioned in this
section see Svacha & Lawrence (2014).
The practical classication system is subjective, particularly
concerning the extent and rank of higher taxa. However, if the
relationships reliably recovered in all our analyses are sound,
the following should be accepted: (1) Cerambycidae s.l. in the
present extent (Table 1) is polyphyletic and should be aban-
doned. The non-chrysomelid Chrysomeloidea (Cerambycidae
s.l., Orsodacnidae and Megalopodidae) form a monophyletic
group in all our analyses and could be classied as a single
broad family Cerambycidae sensu latissimo (as was in fact
suggested by Schmitt, 1994a on some morphological charac-
ters, but his proposal was not accepted by subsequent authors).
Such a nomenclatural change would represent a major depar-
ture from all systems currently in use and would profoundly
inuence cataloguing and retrieval of information among other
things. The group would also be more difcult to characterize
based on morphological characters as many of its members bear
some plesiomorphies compared with most or all Chrysomel-
idae. Therefore, we suspect that such a proposal would not
be favourably accepted by chrysomeloid workers. Addition-
ally, Cerambycidae and Megalopodidae originate from the same
© 2017 The Royal Entomological Society, Systematic Entomology,43, 68–89
Cerambycidae phylogeny 85
paper (Latreille, 1802) and we would have to establish prior-
ity. (2) Oxypeltidae cannot be accepted as a family so long as
Palophaginae is included in Megalopodidae; either Oxypelti-
nae should be included as a subfamily of Megalopodidae, or
Palophaginae should be placed as a subfamily of Oxypeltidae.
Our study does not include any member of the nominotypical
subfamily Megalopodinae, but both molecular and morpholog-
ical analyses indicate that it is closer to Zeugophorinae than to
Palophaginae (e.g. Reid, 1995, 2000; Duckett et al., 2004; Far-
rell & Sequeira, 2004; Marvaldi et al., 2009) and some include
Zeugophorinae in Megalopodinae (Kuschel & May, 1990) or at
least suspect that Megalopodinae without Zeugophorinae may
be paraphyletic (Reid, 1995, p. 605). Thus, both the above alter-
natives would be phylogenetically acceptable. Considering the
generally accepted placement of Palophaginae in Megalopodi-
dae, we would prefer the former solution (Oxypeltinae as a
subfamily of Megalopodidae sister to Palophaginae). (3) Ves-
peridae (and any of its subgroups sampled here) should not be
placed in Cerambycidae s.s. when Disteniidae is accepted as
a separate family. (4) Because the results recovered from the
AA analyses would make inclusion of Vesperidae in Cerambyci-
dae possible only if Cerambycidae included all non-chrysomelid
Chrysomeloidea (a solution questioned above), we recommend
retaining Vesperidae as a separate family (containing the sub-
families Vesperinae, Philinae and Anoplodermatinae) until its
relationships are claried by additional analyses. (5) Disteniidae
(we sampled two of its four tribes, Disteniini and Cyrtonopini)
could be included in the Cerambycidae as a subfamily because
our cladograms place Disteniidae as sister group or even ingroup
(Bayesian analysis of AA data) of Cerambycidae s.s. How-
ever, it may be acceptable to classify Disteniidae and Ceram-
bycidae as separate families because Disteniidae as a separate
family has already become relatively widespread and the apo-
morphic presence of the larval gula conveniently (and without
any known exceptions) denes the Cerambycidae s.s. (whereas
disteniid larvae, like those of all other Phytophaga, lack
the gula).
Our results basically support the current division of Ceram-
bycidae s.s. into eight subfamilies (except for the paraphyletic
Cerambycinae in the NT trees), although their relationships are
in part unexpected and enforce revision of some widespread
assumptions (see above). Further research may reveal Paran-
drinae and Necydalinae as ingroups of Prioninae and Lepturi-
nae respectively (our sampling is insufcient to test that), but
presently, we recommend accepting the former two as sepa-
rate subfamilies to emphasize their uncertain status. Currently,
there are no clear synapomorphies available for the subfam-
ily Dorcasominae, only larval synapomorphies for the sub-
family Cerambycinae, and no reliable distinguishing characters
between adults of the two subfamilies (Svacha & Lawrence,
2014). We recommend retaining both Dorcasomini and Apato-
physeini (the latter not sampled here), plus some related tribes
of uncertain status (Protaxini if not considered synonymous
with Apatophyseini; Trigonarthrini; see Svacha & Lawrence,
2014), in one broad subfamily Dorcasominae, at least until its
limits and relationships to the Cerambycinae can be further
investigated.
Supporting Information
Additional Supporting Information may be found in the online
version of this article under the DOI reference:
10.1111/syen.12257
Figure S1. Unpartitioned maximum-likelihood phyloge-
netic tree for nucleotide data inferred in x 8.1.5.
Maximum-likelihood bootstrap support (MLBS) is shown
only for nodes with MLBS <100%. Information regarding
the systematics of the sampled exemplars is indicated on the
right of the tree.
Figure S2. Unpartitioned maximum-likelihood phylogenetic
tree for amino acid data inferred in x 8.1.5. Maxi-
mum likelihood bootstrap support (MLBS) is shown only for
nodes with MLBS <100%. Information regarding the sys-
tematics of the sampled exemplars is indicated on the right of
the tree. Branches in red indicate differences in relationships
between this phylogeny and that in Figure S1.
Figure S3. Phylogeny based on Bayesian inference for
nucleotide data inferred in  3.2.5. All nodes had a
Bayesian posterior probability (PP) value of 1. Information
regarding the systematics of the sampled exemplars is indi-
cated on the right of the tree.
Figure S4. Phylogeny based on Bayesian inference for
amino acid data inferred in  3.2.5. Bayesian pos-
terior probability (PP) values are shown only for nodes with
PP <1. Information regarding the systematics of the sampled
exemplars is indicated on the right of the tree. Branches in red
indicate differences in relationships between this phylogeny
and that in Figure S3.
Tabl e S 1 . Number of loci analysed for each taxon used in
this study.
Supplementary material. AHE probes and nal
sequence alignments will be uploaded to Dryad, doi:
10.5061/dryad.v0b7v.
Acknowledgements
The authors would like to thank M. Krull (University of Mem-
phis) H. Ralicki (Florida State University) and M. Kortyna
(Florida State University) for their assistance with lab work, and
R. Mitchell (University of Wisconsin, Oshkosh), H. Robertson
(University of Illinois at Urbana-Champaign) and Y. Pauchet
(Max Planck Institute for Chemical Ecology) for providing
unpublished genomes and/or transcriptomes to assist with probe
design. We thank the following colleagues for helpful discus-
sions and/or for specimens used in this study: G. Monteith, A.
Marvaldi, A. Ray, D. Funk and M. Monné. We thank A. Donath,
X. Zhou, K. Kjer, J. Kjer, B. Misof, R.S. Peters, J. Beller, M.
Kubiak, E. Anton, K. Meusemann and the 1KITE Coleoptera
subproject for allowing access to unpublished transcriptome
assemblies. The authors would also like to thank D. Clarke
© 2017 The Royal Entomological Society, Systematic Entomology,43, 68–89
86 S. Haddad et al.
(University of Memphis) for his valuable comments on earlier
versions of this manuscript, and S. Lingafelter for helpful dis-
cussions and contribution of specimens. P. Svacha acknowledges
support from the Institute of Entomology (RVO:60077344).
This work was supported by the University of Memphis FedEx
Institute (to D.D.M.); the United States National Science Foun-
dation (grant number DEB1355169 to D.D.M.; IIP-1313554
to A.R.L. and E.M.L.) and the United States Department of
Agriculture-Animal and Plant Health Inspection Service coop-
erative agreement (grant number 15-8130-0547-CA to D.D.M.).
The authors declare no conict of interest.
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Accepted 21 May 2017
First published online 18 August 2017
© 2017 The Royal Entomological Society, Systematic Entomology,43, 68–89
... The family Cerambycidae (Coleoptera), also known as longhorned beetles, is significant due to its economic importance, complex behaviours, ecological interactions (e.g. suitable indicators of saproxylic communities), and evolutionary history (Švácha and Lawrence, 2014;Haddad et al., 2018;Karpiński et al., 2021). Among its subfamilies, Cerambycinae is the second most diverse one following Lamiinae (Lee and Lee, 2020;Jin et al., 2022), including more than 12,500 known extant species classified under 119 tribes (Tavakilian and Chevillotte, 2023). ...
... Cerambycinae can generally be identified by the absence of a distinct pronotal margin that distinguishes it from Parandrinae and most Prioninae and the absence of a wedge cell that distinguishes it from Prioninae, some Lepturinae and Spondylidinae (Švácha and Lawrence, 2014).Š vácha and Lawrence (2014) paved a path by proposing the Cerambycidae phylogeny as a trichotomy, in which the first branch covers Prioninae + Parandrinae, the second covers Dorcasominae + Cerambycinae, and the third covers Spondylidinae + Lepturinae (including Necydalinae) + Lamiinae. Haddad et al. (2018) recovered the Dorcasominae + Cerambycinae clade sister to the Prioninae + Parandrinae, while Nie et al. (2021) recovered a 'cerambycine' clade [Cerambycinae + Prioninae s. l. (including Parandrinae) + Dorcasominae]. Lee and Lee (2020) and Sutherland et al. (2021) retrieved monophyletic Cerambycinae, as did Jin et al. (2022) who downgraded Dorcasominae to the tribe Dorcasomini of Cerambycinae. ...
... Reconstructing a more precise phylogenetic tree depends on obtaining adequate taxon sampling and molecular data; however, it can be challenging, especially when the taxa studied are as diverse as beetles. It has mostly been achieved at higher taxonomic levels using 95 nuclear protein-coding genes by Zhang et al. (2018), 522 single-copy nuclear genes by Haddad et al. (2018), 4818 nuclear genes by McKenna et al. (2019), and 13 mitochondrial protein-coding genes by Nie et al. (2021). Deeper-level relationships are still being widely studied with marker sequences for longhorned beetles (Lee and Lee, 2020;Karpiński et al., 2021;Zamoroka, 2021;Soydabaş-Ayoub et al., 2023). ...
... Chrysomeloidea are one of the seven Coleoptera superfamilies of the Series Cucujiformia and are considered a sister-group of Curculionoidea; the two superfamilies con-stitute a clade informally known as Phytophaga, with more than 125,000 species (Haddad and McKenna 2016). With approximately 63,000 described extant species (Ślipiński et al. 2011), Chrysomeloidea include Cerambycidae, Disteniidae, Vesperidae, Orsodacnidae, Megalopodidae and Chrysomelidae (Bouchard et al. 2011, Reid 2014a, Haddad et al. 2018. All Chrysomeloidea families occur in Brazil (Monné 2012) and constitutes the most species rich superfamily representing 30% of the Brazilian Coleoptera fauna (Caron et al. 2024) and 8.5% of the Brazilian animal fauna (Boeger et al. 2024). ...
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The leaf beetles (Chrysomelidae) are one of the most species-rich family of herbivorous beetles with about 45,000 species worldwide. Based on the contributions of chrysomelidologists to the Taxonomic Catalog of the Brazilian Fauna - CTFB, the family comprises 6,079 species in 562 genera of which 951 species are endemic to Brazil, standing out as the most diverse, representing 4.8% of the Brazilian fauna and 17.1% of the beetle species. Chrysomelidae has twelve subfamilies with nine reported to Brazil: Galerucinae, the richest with 1,916 species in 202 genera, followed by Cassidinae, Eumolpinae, Cryptocephalinae, Chrysomelinae, Bruchinae, Criocerinae, Lamprosomatinae and Sagrinae - this with only one species. Most of these subfamilies need urgent revision, since many species are poorly characterized, and polymorphism is frequent in some groups. The Czech couple Jan and Bohumila Bechyně were the researchers who described most species from Brazil. Furthermore, despite the increase of research on biology, natural history, host plants, genetics, ecology from 1980’s much still need to be investigated to better known the Brazilian Chrysomelidae and probably many new species are yet to be discovered. KEY WORDS: Brazilian fauna; CTFB; biodiversity; leaf beetles; phytophagous
... Phylogenetic analyses based on DNA sequences rely on the assumption that closely related species share a more recent common ancestor than distantly related ones. By comparing DNA sequences, researchers can construct phylogenetic trees that depict the branching patterns of species, reflecting their evolutionary relationships [27] . This tree not only elucidates the evolutionary history of the Hesperophanini species but also provides a framework for understanding their taxonomic classifications, ecological interactions, and evolutionary processes. ...
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The fig stem borer Trichoferus fissitarsis Sama, Fallahzadeh, and Rapuzzi, 2005 (Coleoptera: Cerambycidae) considered the widely distributed and harmful fig insect in the Kurdistan Region-Iraq. Larvae are dangerous, and create large-diameter exit holes and tunnels that can pointedly reduce the tree's vitality. Incontrast to the adult beetle, there is little evidence of larval identification. During the survey, 15 larval specimens were collected. mtDNA sequence of the COI specimens was obtained and analyzed for the species reported in NCBI Gen Bank from Kurdistan Region-Iraq. Besides, local distribution, taxonomy, host plants, and economic impact of the species were demonstrated. Moreover, the last larval instar and pupa morphologically had been described. Trunk significance and dried wood as a possible pathway for damaging wood borer species were highlighted. https://creativecommons.org/licenses/by-nc/4.0/
... exons) and more variable, generally, non-coding flanking regions (e.g. introns or intergenic regions) located on flanks of the probe region (Lemmon et al., 2012;Haddad et al., 2018;Shin et al., 2018). Following the pipeline, we trimmed flanking regions with 1.5 entropy and 50% density cutoffs at each site in the nucleotide sequence alignments . ...
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... exons) and more variable, generally, non-coding flanking regions (e.g. introns or intergenic regions) located on flanks of the probe region (Lemmon et al., 2012;Haddad et al., 2018;Shin et al., 2018). Following the pipeline, we trimmed flanking regions with 1.5 entropy and 50% density cutoffs at each site in the nucleotide sequence alignments . ...
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eLife assessment Through anchored phylogenomic analyses, this important study offers fresh insights into the evolutionary history of the plant diet and geographic distribution of Belidae weevil beetles. Employing robust methodological approaches, the authors propose that certain belid lineages have maintained a continuous association with Araucaria hosts since the Mesozoic era. Although the biogeograph-ical analysis is somewhat limited by uncertainties in vicariance explanations, this convincing study enhances our understanding of Belidae's evolutionary dynamics and provides new perspectives on ancient community ecology. Abstract The rise of angiosperms to ecological dominance and the breakup of Gondwana during the Mesozoic marked major transitions in the evolutionary history of insect-plant interactions. To elucidate how contemporary trophic interactions were influenced by host plant shifts and palaeogeographical events, we integrated molecular data with information from the fossil record to construct a time tree for ancient phytophagous weevils of the beetle family Belidae. Our analyses indicate that crown-group Belidae originated approximately 138 Ma ago in Gondwana, associated with Pinopsida (conifer) host plants, with larvae likely developing in dead/decaying branches. Belids tracked their host plants as major plate movements occurred during Gondwana's breakup, surviving on distant, disjunct landmasses. Some belids shifted to Angiospermae and Cycadopsida when and where conifers declined, evolving new trophic interactions, including brood-pollination mutualisms with cycads and associations with achlorophyllous parasitic angiosperms. Extant radiations of belids in the genera Rhinotia (Australian region) and Proterhinus (Hawaiian Islands) have relatively recent origins.
... Phylogenetic analyses based on DNA sequences rely on the assumption that closely related species share a more recent common ancestor than distantly related ones. By comparing DNA sequences, researchers can construct phylogenetic trees that depict the branching patterns of species, reflecting their evolutionary relationships [27] . This tree not only elucidates the evolutionary history of the Hesperophanini species but also provides a framework for understanding their taxonomic classifications, ecological interactions, and evolutionary processes. ...
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The fig stem borer Trichoferus fissitarsis Sama, Fallahzadeh, and Rapuzzi, 2005 (Coleoptera: Cerambycidae) considered the widely distributed and harmful fig insect in the Kurdistan Region-Iraq. Larvae are dangerous, and create large-diameter exit holes and tunnels that can pointedly reduce the tree's vitality. Incontrast to the adult beetle, there is little evidence of larval identification. During the survey, 15 larval specimens were collected. mtDNA sequence of the COI specimens was obtained and analyzed for the species reported in NCBI Gen Bank from Kurdistan Region- Iraq. Besides, local distribution, taxonomy, host plants, and economic impact of the species were demonstrated. Moreover, the last larval instar and pupa morphologically had been described. Trunk significance and dried wood as a possible pathway for damaging wood borer species were highlighted.
... exons) and more variable, generally, non-coding flanking regions (e.g. introns or intergenic regions) located on flanks of the probe region (Lemmon et al., 2012;Haddad et al., 2018;Shin et al., 2018). Following the pipeline, we trimmed flanking regions with 1.5 entropy and 50% density cutoffs at each site in the nucleotide sequence alignments . ...
Preprint
The rise of angiosperms to ecological dominance and the breakup of Gondwana during the Mesozoic marked major transitions in the evolutionary history of insect-plant interactions. To elucidate how contemporary trophic interactions were influenced by host plant shifts and palaeogeographical events, we integrated molecular data with information from the fossil record to construct a timetree for ancient phytophagous weevils of the beetle family Belidae. Our analyses indicate that crown-group Belidae originated approximately 138 Ma ago in Gondwana, associated with Pinopsida (conifer) host plants, with larvae likely developing in dead/decaying branches. Belids tracked their host plants as major plate movements occurred during Gondwana’s breakup, surviving on distant, disjunct landmasses. Some belids shifted to Angiospermae and Cycadopsida when and where conifers declined, evolving new trophic interactions, including brood-pollination mutualisms with cycads and associations with achlorophyllous parasitic angiosperms. Extant radiations of belids in the genera Rhinotia (Australian region) and Proterhinus (Hawaiian Islands) have relatively recent origins.
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