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Phylogenomic relationships of bioluminescent elateroids define the 'lampyroid' clade with clicking Sinopyrophoridae as its earliest member


Abstract and Figures

Bioluminescence has been hypothesized as aposematic signalling, inter-sexual communication and a predatory strategy, but origins and relationships among bioluminescent beetles have been contentious. We reconstruct the phylogeny of the bioluminescent elateroid beetles (i.e. Elateridae, Lampyridae, Phengodidae and Rhagophthalmidae), analysing genomic data of Sinopyrophorus Bi & Li, and in light of our phylogenetic results, we erect Sinopyrophoridae Bi & Li, stat.n. as a clicking elaterid-like sister group of the soft-bodied bioluminescent elateroid beetles, that is, Lampyridae, Phengodidae and Rhagophthalmidae. We suggest a single origin of bio-luminescence for these four families, designated as the 'lampyroid clade', and examine the origins of bioluminescence in the terminal lineages of click beetles (Elateridae). The soft-bodied bioluminescent lineages originated from the fully sclerotized elateroids as a derived clade with clicking Sinopyrophorus and Elateridae as their serial sister groups. This relationship indicates that the bioluminescent soft-bodied elateroids are modified click beetles. We assume that bioluminescence was not present in the most recent common ancestor of Elateridae and the lampyroid clade and it evolved among this group with some delay, at the latest in the mid-Cretaceous period, presumably in eastern Laurasia. The delimitation and internal structure of the elaterid-lampyroid clade provides a phylogenetic framework for further studies on the genomic variation underlying the evolution of bioluminescence.
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Systematic Entomology (2020), DOI: 10.1111/syen.12451
Phylogenomic relationships of bioluminescent
elateroids define the ‘lampyroid’ clade with clicking
Sinopyrophoridae as its earliest member
1Laboratory of Biodiversity and Molecular Evolution, Palacky University, Olomouc, Czech Republic, 2State Key Laboratory of
Genetic Resources and Evolution, Kunming Institute of Zoology CAS, Kunming, China, 3Department of Biology, Monte L. Bean
Museum, Brigham Young University, Provo, UT, U.S.A. and 4Zoological Research Museum Alexander Koenig, Centre of Molecular
Biodiversity Research, Bonn, Germany
Abstract. Bioluminescence has been hypothesized as aposematic signalling, inter-
sexual communication and a predatory strategy, but origins and relationships among
bioluminescent beetles have been contentious. We reconstruct the phylogeny of the
bioluminescent elateroid beetles (i.e. Elateridae, Lampyridae, Phengodidae and
Rhagophthalmidae), analysing genomic data of Sinopyrophorus Bi & Li, and in light
of our phylogenetic results, we erect Sinopyrophoridae Bi & Li, stat.n. as a clicking
elaterid-like sister group of the soft-bodied bioluminescent elateroid beetles, that is,
Lampyridae, Phengodidae and Rhagophthalmidae. We suggest a single origin of bio-
luminescence for these four families, designated as the ‘lampyroid clade’, and examine
the origins of bioluminescence in the terminal lineages of click beetles (Elateridae).
The soft-bodied bioluminescent lineages originated from the fully sclerotized elateroids
as a derived clade with clicking Sinopyrophorus and Elateridae as their serial sister
groups. This relationship indicates that the bioluminescent soft-bodied elateroids are
modied click beetles. We assume that bioluminescence was not present in the most
recent common ancestor of Elateridae and the lampyroid clade and it evolved among
this group with some delay, at the latest in the mid-Cretaceous period, presumably
in eastern Laurasia. The delimitation and internal structure of the elaterid-lampyroid
clade provides a phylogenetic framework for further studies on the genomic variation
underlying the evolution of bioluminescence.
Bioluminescence has been intensively studied by numerous
researchers (Costa, 1975; Branham & Wenzel, 2003; Nakatsu
et al., 2006; Fallon et al., 2018). The production of light occurs
Correspondence: Ladislav Bocak, Laboratory of Biodiversity and
Molecular Evolution, Palacky University, Olomouc, Czech Republic,
E-mail:; and Xueyan Li, State Key Laboratory
of Genetic Resources and Evolution, Kunming Institute of Zoology,
Chinese Academy of Sciences, Kunming, Yunnan, 650223, China,
These authors contributed equally to the study.
sporadically in insects (Watkins et al., 2018; Martin et al., 2019);
nevertheless, beetle bioluminescence is well known to both
researchers and the general public. Most luminous beetles
belong to Elateroidea and we know of 2000 rey species
(Lampyridae, Fig. 1F– J), 300 glow-worms or railroad-worm
beetles (Phengodidae, Rhagophthalmidae), as well as >100 bio-
luminescent click beetle species (Elateridae; Fig. 1AD), espe-
cially in the Neotropical region (Costa, 1975, 1984). With the
recent discovery of the rst Palearctic bioluminescent click-
ing beetle, the number of bioluminescent beetle lineages has
increased (He et al., 2019; Bi et al., 2019; Fig. 1DE).
The phylogenetics of Elateroidea have been studied with
morphology since the late 1980s, where the bioluminescent
© 2020 The Royal Entomological Society 1
2D. Kusy et al.
Fig 1. Morphological diversity of the elaterid-lampyroid clade. Elateridae: (A) Denticollis sp.; (B) Agrypnus murinus (L.); (C) click beetle larva;
(D) Ampedus sp. The lampyroid clade. Sinopyrophoridae: (D, E) Sinopyrophorus schimmeli Bi & Li; Lampyridae: (F) Asymmetricata circumdata
(Motschulsky); (G) Lampyris noctiluca (L.), larva; (H) ditto, in copula; (I, J) Lamprohiza splendidula (L.), female. Photographs by M. Motyka (B, D,
G– J), L. Bocak (A,C) and Z.-W. Dong (F); (D, E) from Bi et al., 2019. [Colour gure can be viewed at].
soft-bodied ‘cantharoids’ and fully sclerotized click beetles were
merged in a single superfamily, Elateroidea (Lawrence, 1988;
Branham & Wenzel, 2003; Lawrence et al., 2011). Later
studies using DNA data supported showed that the soft-bodied
elateroids were polyphyletic (Bocakova et al., 2007). Some
relationships among these groups remained inconclusive
when topologies were inferred from a few widely used
molecular markers and the controversies persisted even
when protein-coding nuclear fragments were employed
(McKenna et al., 2015), or when taxon sampling was sub-
stantially increased (Kundrata et al., 2014; Bocak et al., 2016;
Linard et al., 2018) (Fig S1AC). A clade of bioluminescent
elateroids, that is, Elateridae, Lampyridae, Phengodidae and
Rhagophthalmidae, was rst identied by the analyses of 13
mitochondrial genes (Timmermans et al., 2010) and conrmed
by further analyses of these mitogenomes but with more exten-
sive taxon sampling (Amaral et al., 2016; Bocak et al., 2016).
Nevertheless, the analyses were relatively limited by the volume
of data and resulted in ambiguous support for critical clades and
contradictory results (Kundrata et al., 2014; Linard et al., 2018).
Recent progress has been made possible by high throughput
sequencing, transcriptomes and whole-genome analyses. Zhang
et al. (2018a) used 99 nuclear markers and Kusy et al. (2018a)
analysed 4000 orthologous genes. Both studies provided
evidence for a clade that included ve elateroid families with
at least some bioluminescent taxa. However, these two studies
either used a low number of orthologs or restricted taxon sam-
pling. A phylogenomic analysis for all of Coleoptera provided
a timeframe for the evolution of the order based on multi-
ple calibration points, conrming the elateroid backbone but
contained only six elateroid terminals (McKenna et al., 2019)
(Fig S1D,E).
Recently, the subfamily Sinopyrophorinae Bi & Li was pro-
posed in Elateridae for the bioluminescent Sinopyrophorus
schimmeli Bi & Li. Bi et al. (2019) included homologous
fragments of S. schimmeli in a phylogenetic reconstruction
from earlier published click beetle rRNA and mtDNA genes
(Bocakova et al., 2007; Timmermans et al., 2010, 2016; Kun-
drata et al., 2014; Amaral et al., 2016) and used glow-worms
as outgroups. They recovered Sinopyrophorus with the elaterid
subfamilies Hemiopinae and Oestodinae, but with limited statis-
tical support for many relationships among elaterid subfamilies.
Herein, several thousand orthologs of S. schimmeli are used
to investigate the evolution of the clade of bioluminescent
© 2020 The Royal Entomological Society, Systematic Entomology, doi: 10.1111/syen.12451
Phylogenomics of luminescent elateroids 3
elateroid families. Large genomic datasets are proving empiri-
cally necessary if the relationships among old lineages can be
recovered with some level of condence (Misof et al., 2014;
Kusy et al., 2018a; McKenna et al., 2019). Based on these
results, we discuss (i) the origins of bioluminescence in
Elateroidea, (ii) loss of the clicking mechanism, and (iii)
loss of a fully sclerotized body in most bioluminescent
elateroids. This phylogeny will enable further evaluation of
the similarity of the luciferase genes and track their evolution
(Fallon et al., 2018).
Genomic data
The genomic DNA of a single male adult of S. schimmeli from
Yunnan Province (Collecting data: Husa village, 1770 m a.s.l.,
Longchuan County, 15– 25 Jun 2017, coll. Wenxuan Bi) was
shotgun-sequenced on the Illumina HiSeq4000 by Novogene
Co., Ltd. (Tianjing, China) for 150 bp paired-end reads and 30
Gbp of total data (Bi et al., 2019). Raw paired-end reads were
ltered using fastp v.0.20.0 (Chen et al., 2018) and low-quality
reads were removed. The draft genome was assembled using
SPAdes v.3.13.1 (Bankevich et al., 2012), with k-mer sizes of 21,
33, 55, 77 and 99. Contigs were used to train ‘Augustus’ (Stanke
& Waack, 2003) for species-specic gene models with BUSCO
v.3 (Waterhouse et al., 2018). Predicted models were used for ab
initio gene predictions and protein-coding sequences were used
for analyses. Basic statistics of genome assembly were evaluated
with QUAST v.5 (Mikheenko et al., 2018). We performed k-mer
counts on the ltered data in Jellysh 2.2.10 using 17 and 21-mer
sizes (Marcais & Kingsford, 2011). Based on the distribution
of k-mer occurrences, we estimated the genome size using
GenomeScope (Vurture et al., 2017).
We compiled the phylogenomic dataset using Sinopyrophorus
and 42 publicly available transcriptomes or genomes (Poelchau
et al., 2014; Sanders & Hall, 2015; Amaral et al., 2017,
2019; Wang et al., 2017; Fallon et al., 2018; Ye et al., 2018;
Kusy et al., 2018b, 2019; McKenna et al., 2019) (Table S1).
The single-copy ortholog set was collated by searching the
OrthoDB v.9.1 database (Zdobnov et al., 2016) (Tables S2,
S3). We carried out Orthograph v.0.6.3 searches (Petersen
et al., 2017) on assembled transcriptomes and protein-coding
gene sets. Terminal stop codons were removed and internal
stop codons at the translational and nucleotide levels were
masked using the Perl script
(Petersen et al., 2017). The amino acid sequences were aligned
using Mafft v.7.407 with the L-INS-i algorithm (Katoh &
Standley, 2013). Resulting alignments from each ortholog
group were checked for the presence of outliers using the
script and earlier reported meth-
ods (Misof et al., 2014; Peters et al., 2017). Outlier sequences
were removed from alignments. Then, the non-elateriform taxa
were removed. The multiple sequence alignments of nucleotides
were generated using Pal2Nal (Suyama et al., 2006) and Alis-
core v.2.2 (Misof & Misof, 2009; Kück et al., 2010) was used
to identify ambiguous and randomly similar aligned sections.
Aliscore was invoked with a custom –r 1027 option, with a
scoring approach for gap-lled amino acid sites (option -e).
After that, we used (Misof et al., 2014) to create a
list of corresponding codons to be removed from nucleotide
alignments. Identied random or ambiguous similarities were
masked using ALICUT v.2.3 (Kück et al., 2010). In each gene
alignment, the short randomly aligned fragments were replaced
with gaps and sequences with 80% missing data were removed
using Python scripts (Zhang et al., 2020). We used MARE
v.0.1.2-rc (Misof et al., 2013) to calculate the information con-
tent of each gene partition. Partitions with zero information con-
tent were removed.
For the remaining 4199 genes, we used AMAS
(Borowiec, 2016) to calculate statistics (alignment length,
GC content, number of missing taxa, number of parsimony
informative sites) and individual gene alignments were retained
for coalescent analyses. The concatenated datasets were gen-
erated using FasConCat-G v.1.4 (Kück & Longo, 2014).
From the 4199 genes, we generated the datasets A-4199-AA
and B-4199-NT (designation: name-number of taxa-amino
acid or nucleotide level, Table S4). To reduce the effect
of saturation (Breinholt & Kawahara, 2013), we created
dataset C-4199-NT12, with third-codon positions excluded. To
increase the data decisiveness, we constructed additional super-
matrices using only partitions with all 43 species present:
datasets D-968-AA and E-968-NT. The degree of missing
data and overall completeness scores across all datasets was
inspected using AliStat v.1.7 (
Compositional heterogeneity tests
To explore the effect of compositional heterogeneity, we
inspected the dataset A-4199-AA with BaCoCa v.1.105 (Kück
& Struck, 2014). We considered compositional heterogeneity
among species in a given partition to be high when the overall
RCFV value was 0.1 (Fernandez et al., 2016; Vasilikopoulos
et al., 2019). Heterogeneous partitions were excluded from
the dataset A-4199-AA to generate dataset F-2195-AA. We
used Maximum Symmetry Test (Naser-Khdour et al., 2019)
to exclude the deviating genes from the dataset B-4199-NT
(P-value cutoff <0.05) and the dataset G-958-NT contains only
partitions that passed the test. The software SymTest v.2.0.49
( was used to calculate the
deviation from stationarity, reversibility and homogeneity
(Jermiin et al., 2008) (SRH). Heatmaps were generated
for all datasets to visualize the pairwise deviations from
SRH conditions. To eliminate synonymous signal (Kawa-
hara et al., 2011), we used Degen v.1.4 (Zwick et al., 2012)
( All
sites with synonymous substitutions were replaced by the
corresponding ambiguity codes. The synonymous signal was
removed from dataset B-4199-NT and H-4199-DEGEN-NT
was generated.
© 2020 The Royal Entomological Society, Systematic Entomology, doi: 10.1111/syen.12451
4D. Kusy et al.
Phylogenetic analyses
Phylogenetic reconstructions were performed using maxi-
mum likelihood (ML) criterion with IQ-TREE v.2.0-rc2 (Minh
et al., 2020). Model selection for each gene was performed
with ModelFinder (Chernomor et al., 2016; Kalyaanamoorthy
et al., 2017) using the -MFP option. For amino acid superma-
trices, the substitution models LG, DCMUT, JTT, JTTDCMUT,
DAYHOFF, WAG, and free rate models LG4X and LG4M were
tested and all combinations of rate heterogeneity among sites
were allowed (options: -mrate E,I,G,I+G,R -gmedian -merit
AICc). We used the edge-linked partitioned model for tree
reconstructions (-spp option) allowing each gene to have its
own rate. The optimized partition schemes and best-tting
model were inferred for the datasets A-4199-AA, B-4199-NT,
D-968-AA, and E-968-NT using -m MFP+MERGE merit
AICc -gmedian options and considering the same substitu-
tion models as above. The fast-relaxed clustering algorithm
was used (Lanfear et al., 2017). The top 10% of partitions
schemes –rclusterf 10 and maximum 10 000 partitions pairs
rcluster-max 10 000 were considered for all datasets except for
D-968-AA and E-968-NT, where –rclusterf 10 rcluster-max
5000 were used. Ultrafast bootstrap (Hoang et al., 2018) and
SH-like approximate likelihood ratio test (SH-aLRT) were cal-
culated using options -bb 3000 and -alrt 10 000.
To account for variation among gene trees owing to incom-
plete lineage sorting and to account for potential gene tree
heterogeneity and discordance (Degnan & Rosenberg, 2006;
Edwards, 2009; Mirarab et al., 2016), the datasets A-4199-AA,
B-4199-NT and C-4199-NT12 were analysed using the
coalescent-based species-tree method. For every single-gene
partition, we calculated an ML gene tree, with 1000 ultra-
fast bootstrap replicates (-bb option) and using the same
substitution models as earlier. For coalescent species tree
estimation, the Accurate Species Tree Algorithm was used
[ASTRAL-III v.5.6.3 (Zhang et al., 2018b)]. ASTRAL accu-
racy is reduced when poorly resolved gene trees are included
(Barrow et al., 2018). Therefore, we calculated average ultrafast
bootstrap and branch length for every gene tree using an R script
and reduced gene trees datasets were created. To account for
very poorly resolved branches on gene trees, branches with
ultra-fast bootstrap 10 were collapsed using Newick utilities
v.1.6 (Junier & Zdobnov, 2010) in every ASTRAL analysis.
Local posterior probabilities (Erfan & Mirarab, 2016) and
quartet frequencies of the internal branches in every species
tree were calculated using the parameter ‘-t =2’. Furthermore,
we used DiscoVista v.1.0 (Erfan et al., 2018) to visualise
gene-tree quartet frequencies of three topologies around
focal internal branches of the inferred ASTRAL nucleotide
species tree in the datasets A-4199-AA, B-4199-NT, and
C-4199-NT12. Here, the following regularly recovered mono-
phyletic groups were considered: nine elateriformian outgroups,
Throscidae, Lycidae, Cantharidae, Elateridae (except Elateri-
nae), Elaterinae, Sinopyrophorus, Lampyridae, Phengodidae,
Analyses of alternative relationships
We used Four-cluster Likelihood mapping (Strimmer &
von Haeseler, 1997; Misof et al., 2013) (FcLM) analysis to
investigate alternative topologies in the datasets A-4199-AA,
B-4199-NT and C-4199-NT12. The analysis determines if
incongruent or confounding signals are present, which may be
obscured in a multispecies phylogenetic tree. The tree-likeness
graph for the three possible quartet topologies shows the support
for each topology. Additionally, we tested positions of Elaterinae
as (i) a sister to clade of Sinopyrophorus, Lampyridae, Phen-
godidae, Rhagophthalmidae (paraphyletic Elateridae) and (ii)
a sister to other Elateridae (monophyletic Elateridae) by eval-
uating which gene partitions of the datasets A-4199-AA and
B-4199-NT favour the alternatives. We calculated log-likelihood
scores and differences of each pL score for each gene partition
using option -wpl in IQ-TREE (Minh et al., 2020).
66-gene dataset
We assembled a dataset from earlier published data (Kusy
et al., 2018a,b, 2019; Zhang et al., 2018a) and newly produced
homologs (Table S5). Sequences of 84 elateroids and outgroups
were used as an input to Orthograph, 66 beetle orthologs were
extracted, and aligned at an amino acid level using mafft v.7.394
with the L-INS-i algorithm (Katoh & Standley, 2013). Multi-
ple sequence alignments of nucleotides were generated using
Pal2Nal (Suyama et al., 2006) to produce datasets J-66-NT and
K-66-AA. We then manually checked all alignments for the
presence of outliers and alignment errors. The matrices for both
amino acid and corresponding nucleotide alignments were gen-
erated using FasConCat-G v.1 (Kück & Longo, 2014). The soft-
ware SymTest v.2.0.49 ( was
used to calculate the overall SRH deviation. The fully parti-
tioned reconstructions were performed using the ML criterion
with IQ-TREE. Full methods and the complete list of analyses
are provided in File S1.
Newly generated shotgun DNA reads of S. schimmeli produced
a genome assembly with 190x read coverage (Fig S34A).
Despite fragmentation, the assembly contained a sufcient
amount of information for ortholog extraction and downstream
phylogenomic analyses (Table S3). The genome completeness
and assembly statistics are summarized in Figs S33A, B, S34A.
The genome size of S. schimmeli was estimated to 171.8 and
190.7 million base pairs (Mbp) (Fig S34B, C) using k-mer sizes
17 and 21, respectively.
Denition and relationships of lineages
Two sets of topologies were produced which differ in the
density of sampling and the number of orthologs. The rst
© 2020 The Royal Entomological Society, Systematic Entomology, doi: 10.1111/syen.12451
Phylogenomics of luminescent elateroids 5
Fig 2. Phylogenetic relationships. The topology recovered by the ASTRAL coalescent phylogenetic method applied to the full set of single-gene trees
inferred at nucleotide level from the dataset B-4199-NT; only the elaterid-lampyroid clade shown, see the full trees in Figs S17, S18. [Colour gure can
be viewed at].
set was based on 4199 orthologs and 43 taxa, nine of them
as outgroups; the topologies were inferred under the ML cri-
terion and the coalescent method by the analyses of amino
acids and nucleotides (Fig. 2). The second set was based on
the 66-orthologs, 83 elateroids and the topologies were inferred
by ML analyses (Figs 3A, B, S2S22). All analyses recovered
a clade containing exclusively the families with at least some
taxa that produce light, that is, Elateridae (2% of taxa biolumi-
nescent, in three subfamilies), Sinopyrophoridae (Sinopyropho-
rus, 1 sp., bioluminescent in an adult stage, larvae unknown),
Phengodidae, Rhagophthalmidae and Lampyridae (all biolumi-
nescent at least in the larval stage, except Phengodidae: Cydis-
tinae for which larvae and females are unknown and males are
non-luminescent). Hereafter, these widely accepted families and
Sinopyrophorus are designated as the ‘elaterid-lampyroid clade’
(Figs 24).
Sinopyrophorus was found outside of Elateridae and as a sister
to Lampyridae, Phengodidae and Rhagophthalmidae. The topol-
ogy was stable regardless of the dataset and inference method
employed (Figs 2– 3, S2 S22). Its position was also supported
by the FcLM analysis (Figs 5A, B, S25, Table S6) and alter-
native topologies were rejected by an approximately unbiased
test (AU test, Table S7). All phylogenomic analyses indicate
that phenotypically elaterid-like Sinopyrophorus (originally Ela-
teridae: Sinopyrophorinae, Fig. 6) and the here redened Ela-
teridae do not share a common exclusive ancestor and form a
serial paraphylum towards the branch of Lampyridae, Rhagoph-
thalmidae and Phengodidae. The clade of Sinopyrophorus and
the soft-bodied bioluminescent families is here designated as the
‘lampyroid clade’.
In contrast to the relatively well-supported lampyroid clade,
we found confounding signal for some relationships among ela-
terid lineages (Figs 2, 3, 5B, D). Most analyses of genomic
datasets suggested the paraphyly of Elateridae with Elaterinae
rooted as a sister to the lampyroid clade and other elaterids
as a sister to both of them (Figs S233). Such a result was
inferred from all ML analyses and the coalescent analysis of the
amino acid data, but not by the coalescent method applied to
the nucleotide dataset (Fig. 2). The distribution of the signal for
alternative topologies prefers the monophyly of Elateridae. The
FcLM analysis of the nucleotide dataset B-4199-NT returned
58.7% for the monophyly of Elateridae versus 39.6% for their
paraphyly (Figs 5B, S25). Similarly, the DiscoVista relative fre-
quency analysis of the B-4199-NT dataset (node 9; Fig. 5D)
preferred the monophyly of Elateridae, but paraphyly is sup-
ported by the datasets A-4199-AA and C-4199-NT12 (Figs S23,
24). The 66-gene amino acid dataset suggests Elateridae without
Sinopyrophorus as a serial paraphylum of three elaterid groups
to the lampyroid clade (Fig. 3A), but the same dataset produced
a monophyletic Elateridae at the nucleotide level (Fig. 3B).
With regard to the positions of the clicking and non-clicking
forms in the elaterid-lampyroid clade, our analyses always
recovered the clicking forms as the earliest splits. Biolu-
minescent taxa are always recovered in a derived position
within the elaterid-lampyroid clade, regardless of monophyly
or paraphyly of click beetles inferred from various analyses
(Figs 23), that is, the most recent common ancestor of the
elaterid-lampyroid clade was clicking and non-bioluminescent.
All topologies and additional information on data are shown
in Figs S2S34.
© 2020 The Royal Entomological Society, Systematic Entomology, doi: 10.1111/syen.12451
6D. Kusy et al.
Fig 3. (A) The topology recovered by the maximum likelihood analysis of the 66-gene dataset J-66-AA at the amino acid level. (B) The topology
recovered by the maximum likelihood analysis of the 66-gene dataset J-66-NT at the nucleotide level. The dark blue branches are signicantly supported
(both SH-aLRT >80% and UFboot >95%). [Colour gure can be viewed at].
Sinopyrophoridae Bi & Li, 2019, new status
Sinopyrophorinae Bi & Li, 2019 in Bi et al., 2019: 83.
Type genus: Sinopyrophorus Bi & Li, 2019 in Bi et al.,
2019: 89.
=Sinopyrophorus He et al., 2019: 565, unavailable name
due to the absence of a description in the study where the
name was proposed (International Committee for Zoological
Nomenclature, 1999).
Based on the recovered relationships, Sinopyrophorus (earlier
Elateridae: Sinopyrophorinae) cannot be a member of Elateri-
dae. Despite its morphological similarity and a shared clicking
mechanism, its phylogenetic position requires it to be classi-
ed as a separate taxon of the same rank as true click beetles,
that is, family. The proposed rank fulls the requirement of the
reciprocal monophyly of all taxa and, simultaneously, keeps tra-
ditionally recognized families valid.
Phylogenomic relationships
The uncertain homology of characters in phenotypically dis-
parate elateroids, ambiguities in length variable alignments and
low information content in the Sanger data have produced con-
tradicting phylogenies (Bocakova et al., 2007; Sagegami-Oba
et al., 2007; Lawrence et al., 2011; Kundrata et al., 2014;
McKenna et al., 2015). Here, we present analyses based on more
than 4000 orthologs that support three principal relationships
that have not been well supported in earlier studies:
1 The monophyly of the elaterid-lampyroid clade (Figs 2 4;
Timmermans et al., 2010; Amaral et al., 2016; Bocak
et al., 2016; Kusy et al., 2018b; McKenna et al., 2019) is
preferred over alternative hypotheses (Bocakova et al., 2007;
Sagegami-Oba et al., 2007; Kundrata et al., 2014; McKenna
et al., 2015; Zhang et al., 2018a).
2 We prefer the monophyly of redened click beetles, that
is, including Elaterinae and Lissominae without Sinopy-
rophorus, as suggested by the B-4199-NT dataset and the
coalescent method, the J-66-NT dataset and ML methods,
and as additionally supported by the FcLM analyses, Disco-
Vista analyses and AU test (Figs 2, 3B, 5B, D, S20, S22B,
S23S26; Table S7) over a paraphyletic Elateridae obtained
from all ML analyses of the genomic datasets, from the
coalescent method analyses of C-4199-NT12/A-4199-AA
datasets, and the ML analysis of the I-66-AA dataset
(Figs 3A, S2S19, S21, S22A). The paraphyletic or poly-
phyletic Elateridae were recovered in earlier estimates where
datasets representing all Coleoptera were analysed (McKenna
© 2020 The Royal Entomological Society, Systematic Entomology, doi: 10.1111/syen.12451
Phylogenomics of luminescent elateroids 7
Fig 4. The summary cladogram of Elateroidea with bars showing the distribution of estimated origin of Elateroidea, the earliest split within the
bioluminescent clade, and the splits within the [Lampyridae (Rhagophthalmidae, Phengodidae)] clade. The origin of Sinopyrophoridae is supposed
after the origin of the bioluminescent clade and before the earliest split between Lampyridae and Rhagophthalmidae +Phengodidae. [Colour gure can
be viewed at].
et al., 2015, 2019; Zhang et al., 2018a). In this case, only
a slight majority of genes recover the monophyly of click
beetles over their paraphyly (Figs 5B, S26). Generally, the
amino acid and nucleotide rst +second codon position
sequences are highly conservative when the set of orthologs
for whole Coleoptera is assembled and possibly, due to
incomplete lineage sorting, the analyses of these datasets
do not support the monophyly of click beetles is analysed
using the maximum likelihood approach (Figs 3A, S2– 16).
The ASTRAL coalescent phylogenetic method applied to the
full set of gene trees inferred the monophyly of Elateridae
from the dataset B-4199-NT at the nucleotide level (Fig. 2).
Additionally, the FcLM and DiscoVista relative frequency
analyses of the dataset B-4199-NT support, albeit weakly,
the monophyly of Elateridae (Figs 5B, D, S24, 25). Similarly,
the 66-gene dataset is based on highly conservative genes and
Elateridae are split into three serial groups of the lampyroid
clade if amino acids are analysed (Figs 3A, S17), but the
family is monophyletic when the tree is inferred with the
maximum likelihood analysis at the nucleotide level (Figs 3B,
S18). The earlier 99-gene (among them the here employed
66 orthologs) analysis of the whole Coleoptera by Zhang
et al. (2018a) suggested the polyphyly of click beetles when
Lissominae was the sister to net-winged beetles and the rest
of Elateridae formed two serial sister groups to the lampyroid
families. We are aware that further analyses of a much larger
dataset will be needed to test the monophyly of Elateridae
but, based on the current results, we prefer to accept the
monophyly of Elateridae. Although the genome-scale data
offer a powerful tool for phylogenetics, the relationships
of elaterid subfamilies remain poorly supported and need
rigorous testing with a dense sampling of taxa and a higher
number of orthologs specically designed for this question.
3 Sinopyrophoridae is decisively placed as a sister to Lampyri-
dae, Phengodidae and Rhagophthalmidae (Figs 2, 3A,
S2S25), or as a sister to Lampyridae (Fig. 3B) rather than
a member of the click beetles (Bi et al., 2019). Therefore,
we propose to accept Sinopyrophorus as a member of
the ‘lampyroid clade’, that is, the ancient lineage closely
related to reies and glow-worms (Figs 2, 3, S2– S26).
The elaterid-like morphology of Sinopyrophorus supports
the idea that the characteristic well-sclerotized body form
and clicking escape mechanism were abandoned multiple
times during the evolution of Elateroidea. Here, a single
shift to incomplete sclerotization in the common ancestor of
soft-bodied reies and glow-worms was recovered by all
analyses (Figs 2, 3). Such a process was earlier inferred for
drilids and omalisids now included in Elateridae (Kundrata
et al., 2014; Kusy et al., 2018a). The clicking mechanism is
unique, relatively complex and we can assume that it evolved
once if the probabilities of the origin and the loss of the
clicking mechanism are substantially different (Trueman
et al., 2004).
Formal classication
The elevation of Sinopyrophoridae as a separate family is our
preferred, but not a single possible solution of the formal clas-
sication. Our decision is conservative in the sense that it keeps
the family rank for all earlier designated reciprocally mono-
phyletic lineages, that is, click beetles, reies, glow-worms and
© 2020 The Royal Entomological Society, Systematic Entomology, doi: 10.1111/syen.12451
8D. Kusy et al.
Fig 5. Four cluster Likelihood Mapping tests of the selected phylogenetic hypothesis applied at the nucleotide level of the dataset B-4199-NT. (A) The
test of the position of Sinopyrophorus. (B) The test of the monophyly of Elateridae. (C) Evaluated topology. (D) DiscoVista relative frequency analyses
of the dataset B-4199-NT. [Colour gure can be viewed at].
railroad-worm beetles. There are several alternative solutions.
We could merge Sinopyrophoridae, Phengodidae, Rhagoph-
thalmidae and Lampyridae under a single family, that is, the here
recovered lampyroid clade will be called Lampyridae Latreille,
and contain ve subfamilies. Lampyridae in a new wide sense
would contain several morphologically distinct lineages with
differing natural history and the whole internal classication
of reies would be down-ranked. The formal requirement for
the monophyly of all named taxa would also be fullled by the
redenition of Elateridae sensu lato that would contain eight
traditional families Elateridae, Drilidae, Omalisidae, Plastoceri-
dae, Lampyridae, Phengodidae, Rhagophthalmidae and Sinopy-
rophorinae. (Kusy et al., 2018a,b; Bi et al., 2019). Considering
also the logical extreme, we could accept whole Elateroidea
as a single family (sans the artemotopodid clade) as recently
mentioned by Muona & Taräväinen (2020). The clade would
be dened by the clicking mechanism that was secondarily lost
in half of its members. Nevertheless, for convenience, we pre-
fer to keep the family status for all traditional, widely accepted
families whenever possible. Click beetles and reies are known
to non-specialists and they are intuitively distinguished also by
the general public, for example, naturalist involved in the Fire-
y Citizen Science Project by the Natural History Museum of
Utah among others. Therefore, the family rank is appropriate for
their separation and the expansion of reies to a heterogeneous
assemblage of biologically and morphologically disparate forms
would not be practical.
Origins of the bioluminescence
Our analyses indicate a unique origin of bioluminescence
in the common ancestor of the clade Sinopyrophoridae,
Rhagophthalmidae, Phengodidae and Lampyridae clade and
further independent origin of bioluminescence within click
beetles as dened here, that is, without Sinopyrophorus.The
relatively distant position of bioluminescent taxa in the formal
classication was conrmed by the earliest molecular phyloge-
nies (Bocakova et al., 2007; Sagegami-Oba et al., 2007) and has
been inferred due to the genetic structure around the luciferase
genes from the genomes of Photinus,Aquatica and Ignelater
(Fallon et al., 2018). The position of photic organs is variable
in adult click beetles: prothoracic spots are reported in Balgus,
Campyloxenus, and Pyrophorini: Nyctophyxina, the prothoracic
and abdominal organs in Pyrophorini: Hapsodrilina and most
Pyrophorina, and only the abdominal photic organs in the
pyrophorine genus Hifo (Costa, 1975, 1984). Unlike these, only
abdominal photic organs are shared by all adult bioluminescent
forms in the lampyroid clade (Branham & Wenzel, 2003; Bi
et al., 2019).
© 2020 The Royal Entomological Society, Systematic Entomology, doi: 10.1111/syen.12451
Phylogenomics of luminescent elateroids 9
Fig 6. The summary of Elateroidea relationships with the distribution of clicking, soft-bodied, and bioluminescent forms. Photographs J. Klvá ˇ
cek and
authors. [Colour gure can be viewed at].
About 2300 species of the lampyroid clade inherited bio-
luminescence from their most recent common ancestor and
we must suppose that if this ancestor was a fully sclerotized
elaterid-like beetle, the origin of bioluminescence preceded
the shift to being soft-bodied (Fig. 6). Among bioluminescent
groups, only lampyrids are species-rich today (2000 spp.)
and the second largest bioluminescent clade are glow-worms
(>200 spp.). Both are common in the Neotropical region along
with bioluminescent elaterids (>100 spp.) (Costa, 1975, 1984).
The effectiveness of bioluminescence as an aposematic signal
depends on the number of taxa and individuals sharing the sig-
nal. As ashing can serve as an isolating mechanism (Branham
& Wenzel, 2003), the intensive interactions among biolumines-
cent elateroids might have played a role in the high diversity of
extant reies, glow-worms and luminous click beetles in the
Neotropical region (Ellis & Oakley, 2016).
Since South America is a major epicentre for bioluminescent
species, the region has been hypothesized as the ancestral region
for the diversication of reies (Amaral et al., 2016). The sister
group position of the elaterid-like East Asian Sinopyrophori-
dae (Figs 2, 3), the predominantly Laurasian distribution of two
of the deepest subfamilies of reies, Ototretinae and Lucioli-
nae (Kundrata et al., 2014; Martin et al., 2017, 2019; Zhang
et al., 2018a) (Fig. 3), Southeast Asian Rhagophthalmidae, and
the presence of Phengodidae in Burmese amber (unpublished
data) suggest as an alternative hypothesis that the early diver-
sication of the bioluminescent lineages took place in eastern
Laurasia and was followed by subsequent intensive radiations
of reies in the Neotropical region.
Bioluminescence is scattered among some terminal lineages
of click beetles, each of them being fully sclerotized and with
a clicking mechanism. Further investigation of the origins of
bioluminescent elaterids will need denser sampling of all biolu-
minescent genera, including as many species as possible. Nev-
ertheless, we support the earlier proposed hypothesis that ela-
terid photic organs evolved several times (Bocakova et al., 2007;
Sagegami-Oba et al., 2007; Fallon et al., 2018). The known
examples of elaterid bioluminescence are placed in three dif-
ferent subfamilies (Costa, 1975, 1984), and a non-luminescent
most recent common ancestor and sister taxa have already
been recovered for Balgus (Thylacosterninae) and Pyrophorini
(Agrypninae) (Bocakova et al., 2007; Kundrata et al., 2014).
How old is elateroid bioluminescence?
We do not attempt a formal dating analysis as our data
partly overlap with the datasets used earlier for the analyses
of all beetles with multiple fossil calibration points (McKenna
et al., 2015, 2019; Bocak et al., 2016; Toussaint et al., 2017;
Kusy et al., 2018a; Zhang et al., 2018a). Most calibration points
would not be available if we attempt an analysis restricted
only to elateroids. Previous studies and the elateroid fossil
records already serve as a guide to estimating of the origins
of both bioluminescence and Sinopyrophoridae. The recov-
ered topologies place Sinopyrophorus between the rst split
of the elaterid-lampyroid clade and the deepest split between
soft-bodied bioluminescent reies, railroad and glow-worms
(Figs 2, 3). Most published analyses dated the earliest split
© 2020 The Royal Entomological Society, Systematic Entomology, doi: 10.1111/syen.12451
10 D. Kusy et al.
within the elaterid-lampyroid clade to the lower Cretaceous peri-
ods, 135 Ma (Toussaint et al., 2017; Zhang et al., 2018a;
McKenna et al., 2019) (Ma; Fig. 4). More shallow estimations
of 115125 Ma (McKenna et al., 2015; Fallon et al., 2018), as
well as a deeper one at 165 Ma (Kusy et al., 2018a,b) are also
hypothesized. The estimations for the earliest split between the
reies and glow-worm clade are similarly inconclusive, at 98
Ma (Zhang et al., 2018a), 122 Ma (Martin et al., 2019), and
140 Ma (Kusy et al., 2018a,b), but the presence of a lucioline
rey in Burman amber supports older dates (Kazantsev, 2015).
If we consider median estimations, the elaterid-lampyroid clade
would have originated in the lower Cretaceous and the subse-
quent split of the Lampyridae, Rhagophthalmidae and Phengo-
didae clade in the mid-Cretaceous. As a result, the ancestor of
Sinopyrophoridae had to have split from their closest relative
120 Ma (Fig. 4). That period must also be considered as a
moment when bioluminescence evolved in Elateroidea for the
rst time.
Dating analyses using different taxon sampling, analysed
markers, applied models and calibrations often come to dif-
ferent age estimates (Fig. 4). Some studies recovered the early
origins of the bioluminescent elateroids (Bocak et al., 2016;
Kusy et al., 2018a) and we suggest these should be inspected as
a possibility. The shallower estimates (McKenna et al., 2015,
2019; Toussaint et al., 2017; Fallon et al., 2018; Zhang et al.,
2018a) set the origin of the elaterid-lampyroid clade of which
click beetles is a principal branch to 115140 Ma in contrast
with the upper Jurassic 152158 Ma old Karatau deposits
contain a rich click beetle fauna (Doludenko et al., 1990).
Additionally, Elaterophanes (Whalley, 1985) was assigned to
Elateridae, and even if it is not a true click beetle but an ances-
tral non-artematopodid elateroid lineage, its age is older than
some estimates of the origin of Elateroidea (Fig. 4). Similarly,
false click beetles, Eucnemidae, were reported from the lower
Jurassic Xiwan deposits (Lin, 1986) and multiple species are
known also from the upper Jurassic– lower Cretaceous period
(Chang et al., 2011). These fossils also support quite an early
origin of the Elateroidea. Consequently, an earlier origin of
bioluminescence is also possible.
The phylogenetic studies dealing with Elateroidea produce
conicting results (Bocakova et al., 2007; Sagegami-Oba
et al., 2007; Lawrence et al., 2011) and similarly, the placement
of Sinopyrophorus as a terminal clade within Elateridae is
ambiguous (Bi et al., 2019; He et al., 2019). Phylogenomic data
presented here provide strong evidence that Sinopyrophoridae,
stat.n., is the sister group of reies, railroad and glow-worm
beetles, despite its morphological similarity to the extant click
beetles (Fig. 6). Our nding suggests that lampyrids and their
closest relatives are in fact modied click beetles. The denition
of the Sinopyrophoridae+Phengodidae+Rhagophthalmidae+
Lampyridae clade supports an early origin of bioluminescence
in their common clicking ancestor, likely 120 Ma, in the lower
Cretaceous, the delayed shift to being soft-bodied and further
radiation and signal diversication in the lineages which already
used some form of luminescence.
Author contributions
DK analysed genomic data, carried out sequence alignments
and phylogenetic analyses, XYL and JWH produced genomic
data, MM and JWH participated in analyses, all co-authors
contributed to the draft of the manuscript; WXB collected the
specimen, DK, LB, LP, and SB conceived and designed the
study, XYL and LB coordinated the study, LB, DK, SB, XYL
and JWH drafted the manuscript. All authors commented the
drafts and gave nal approval for publication.
Supporting Information
Additional supporting information may be found online in
the Supporting Information section at the end of the article.
Figure S1. Classication of Elateroidea: an overview of
hypothesized phylogenies.
Figure S1. An overview of Elateroidea topologies recovered
by earlier studies.
Figure S2– S22. Topologies recovered by individual analy-
Figure S23– S24. DiscoVista relative frequency analyses.
Figure S25. Four cluster likelihood mapping (FcLM) analy-
Figure S26. Calculated log-likelihood difference analyses.
Figure S27– S32. AliStat and SymTest analyses.
Figure S33. BUSCO3 and QUAST assessment for draft
genome assembly.
Figure S34. K-mer coverage depth of assembled Spades
Table S1. The list of taxa, accession numbers.
Table S2. Overview of gene sets used for ortholog assess-
Table S3. Descriptive statistics and results of the orthology
Table S4. Detailed information and statistics of each gener-
ated dataset.
Table S5. The list of taxa included in the 66-gene datasets.
Table S6. Results of four-cluster likelihood mapping and
approximately unbiased test.
Table S7. Result of approximately unbiased (AU) test for the
dataset A-4199-AA.
© 2020 The Royal Entomological Society, Systematic Entomology, doi: 10.1111/syen.12451
Phylogenomics of luminescent elateroids 11
We are obliged for the generous advice and pipeline sharing
to all colleagues from the Museum of A. Koenig in Bonn
and R. Bilkova is cordially acknowledged for her assistance.
J. Klváˇ
cek. and Z.-W. Dong generously provided their pho-
tographs. We specially thank Wen Wang and other members in
his laboratory for their support. We thank to the editor and two
anonymous reviewers for invaluable comments on the earlier
version of the manuscript. The study was funded by the GACR
and IGA projects (18-14942S; LB, DK, and MM; PrF-2020;
DK), the projects of NNSF China (31472035; X-YL) and CAS
(‘Light of West China’; X-YL), the NSF project (DEB-1655981;
SMB). The authors declare no conicts of interest.
Data availability statement
The data that support the ndings of this study are openly
available in GenBank, accession number PRJNA64573 and the
datasets are openly available in the Mendeley Data repository at
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Supplementary resource (1)

... The systematic position of Cantharidae within the superfamily Elateroidea is unsettled. Some studies have placed cantharids amongst other groups with soft-bodied adults, such as Lampyridae, Lycidae, Omethidae and Phengodidae in a group formerly referred to as Cantharoidea, or more closely related to hard-bodied groups of Elateridae (e.g., Crowson 1972;Bocakova et al. 2007;Lawrence et al. 2011;Kundrata et al. 2014;McKenna et al. 2015McKenna et al. , 2019Zhang et al. 2018;Douglas et al. 2021;Kusy et al. 2021;Cai et al. 2022). However, recent molecular studies converge in results showing Cantharidae as sister to Elateridae (sometimes therein included 'lampyroid' lineages) (Zhang et al. 2018;Kusy et al. 2021;Douglas et al. 2021;Cai et al. 2022), with members of either Malthininae or Chauliognathinae as sister to the remaining cantharids. ...
... Some studies have placed cantharids amongst other groups with soft-bodied adults, such as Lampyridae, Lycidae, Omethidae and Phengodidae in a group formerly referred to as Cantharoidea, or more closely related to hard-bodied groups of Elateridae (e.g., Crowson 1972;Bocakova et al. 2007;Lawrence et al. 2011;Kundrata et al. 2014;McKenna et al. 2015McKenna et al. , 2019Zhang et al. 2018;Douglas et al. 2021;Kusy et al. 2021;Cai et al. 2022). However, recent molecular studies converge in results showing Cantharidae as sister to Elateridae (sometimes therein included 'lampyroid' lineages) (Zhang et al. 2018;Kusy et al. 2021;Douglas et al. 2021;Cai et al. 2022), with members of either Malthininae or Chauliognathinae as sister to the remaining cantharids. The uncertainty about the placement of cantharids impacts the interpretations on the evolution of morphological characters. ...
... In some sense, larval characters corroborate the results based on recent molecular phylogenies (e.g., Bocak et al. 2018;Zhang et al. 2018;Kusy et al. 2021;Douglas et al. 2021;Cai et al. 2022) that show Cantharinae and Silinae as sister groups. The similarity of larval features between these subfamilies (Table 3) supports this hypothesis, despite the limited knowledge on Silinae larvae. ...
Dysmorphocerinae is a subfamily of Cantharidae erected for a group of genera with a mainly gondwanan distribution whose adult forms could not be reliably assigned to any other subfamily. The systematic position and monophyly of Dysmorphocerinae remains questionable, as recent molecular and morphological studies have produced conflicting results. Despite the importance of immature morphology for characterising lineages of Cantharidae, so far, the larvae of only two dysmorphocerine species had been briefly described: Neoontelus sp., from New Zealand, and Afronycha picta (Wiedemann), from South Africa. Their morphologies considerably differ from one another, and the larvae cannot be readily attributed to any subfamily, as usually occurs with cantharid larvae. Here, we fully describe for the first time the larvae of Asilis Broun (New Zealand) and Plectonotum laterale Pic (Brazil) and redescribe Neoontelus Wittmer (New Zealand). We also diagnose larvae of Heteromastix Boheman (Australia) and A. picta. Dysmorphocerinae cannot be clearly diagnosed because each genus has a unique combination of features, though Neoontelus is the most divergent. We conclude that the Dysmorphocerinae may not be monophyletic with Plectonotum laterale, Asilis, Neoontelus, Heteromastix showing a closer relationship to Malthininae and Afronycha more aligned with Silinae or Cantharinae. The double gland openings present on the body of Neoontelus reported by Crowson (1972) are reinterpreted as a complex character involving a single posterior pore linked to a gland and an anterior sensillum that may serve as a trigger for the release of defensive chemicals. These are also reported in Asilis and Heteromastix and may be a potential synapomorphy for part of the Dysmorphocerinae. Neoontelus has a series of unique features, including a cotyliform glandular pore on abdominal segment IX.
... Besides research on the diversity, systematics, and morphology of Rhagophthalmidae, many studies in the 21 st century have focused on their bioluminescence Ohba 2004a;Chen et al. 2010;Oba et al. 2011;Oba 2015;Liu et al. 2020) and embryogenesis (Kobayashi et al. 2001(Kobayashi et al. , 2002(Kobayashi et al. , 2003. Additionally, the rapid development of molecular phylogenetic methods in the last decades has enabled scientists to test the phylogenetic placement of Rhagophthalmidae within Elateroidea using one or several markers (e.g., Suzuki 1997;Bocakova et al. 2007; Sagegami- Oba et al. 2007;Stanger-Hall et al. 2007; Kundrata and Bocak 2011b;Kundrata et al. 2014;McKenna et al. 2015), mitogenomes (Li et al. 2007;Amaral et al. 2016;Chen et al. 2019), or a phylogenomic approach (Zhang et al. 2018;Amaral et al. 2019;Douglas et al. 2021;Kusy et al. 2021;Cai et al. 2022). ...
... The number of genera included in Rhagophthalmidae and also their placement within Elateroidea classification vary by source (e.g., McDermott 1966;Crowson 1972;Lawrence and Newton 1995;Kawashima et al. 2010;Kundrata and Bocak 2011a). In the last decade, Elateroidea systematic research has accelerated and the classification of the superfamily has experienced many taxonomic changes (e.g., Kundrata et al. 2014Bocak et al. 2018;Kusy et al. 2018bKusy et al. , 2021, including the discoveries of two new recent families Rosa et al. 2020) and one new extinct family (Li et al. 2021b). However, only six new species of Rhagophthalmidae were described in three taxonomic papers in the same period (Ho et al. 2012;Kazantsev 2012;Yiu 2017). ...
... The phylogenetic placement of Rhagophthalmidae within Elateroidea has been controversial based on morphology only (Crowson 1972;Lawrence 1988;Branham and Wenzel 2001;Lawrence et al. 2011), and Rhagophthalmidae were often placed either in or close to Lampyridae or Phengodidae. Molecular phylogenetic analyses using various datasets and analytical approaches repeatedly confirmed that Rhagophthalmidae are sister to Phengodidae Kundrata and Bocak 2011b;Kundrata et al. 2014;Zhang et al. 2018;Douglas et al. 2021;Kusy et al. 2021;Cai et al. 2022). Both families share soft-bodied males with large eyes, often bipectinate antennae with 12 antennomeres, leathery elytra which are usually shortened and narrowed, larviform females, and larvae that possess bioluminescent organs and feed on millipedes (Kawashima et al. 2010;Zaragoza-Caballero and Hernández 2014;. ...
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Rhagophthalmidae are a small beetle family known from the eastern Palaearctic and Oriental realms. Rhagophthalmidae are closely related to railroad worms (Phengodidae) and fireflies (Lampyridae) with which they share highly modified paedomorphic females and the ability to emit light. Currently, Rhagophthalmidae include 66 species classified in the following 12 genera: Bicladodrilus Pic, 1921 (two spp.), Bicladum Pic, 1921 (two spp.), Dioptoma Pascoe, 1860 (two spp.), Diplocladon Gorham, 1883 (two spp.), Dodecatoma Westwood, 1849 (eight spp.), Falsophrixothrix Pic, 1937 (six spp.), Haplocladon Gorham, 1883 (two spp.), Menghuoius Kawashima, 2000 (three spp.), Mimoochotyra Pic, 1937 (one sp.), Monodrilus Pic, 1921 (two spp. in two subgenera), Pseudothilmanus Pic, 1918 (two spp.), and Rhagophthalmus Motschulsky, 1854 (34 spp.). The replacement name Haplocladon gorhami Kundrata, nom. nov. is proposed for Diplocladon hasseltii Gorham, 1883b (described in subgenus Haplocladon) which is preoccupied by Diplocladon hasseltii Gorham, 1883a. The genus Reductodrilus Pic, 1943 is tentatively placed in Lampyridae: Ototretinae. Lectotypes are designated for Pseudothilmanus alatus Pic, 1918 and P. marginalis Pic, 1918. Interestingly, in the eastern part of their distribution, Rhagophthalmidae have remained within the boundaries of the Sunda Shelf and the Philippines demarcated by the Wallace Line, which separates the Oriental and Australasian realms. This study is intended to be a first step towards a comprehensive revision of the group on both genus and species levels. Additionally, critical problems and prospects for rhagophthalmid research are briefly discussed.
... of Martin et al. [20] and Kusy et al. [21]. One origin of bioluminescence in the common ancestor of fireflies and their relatives (Lampyridae, Rhagophtalmidae, Phengodidae and Sinopyrophoridae) was supported by Kusy et al. [21], and a second independent origin in click beetles (Elateridae). ...
... of Martin et al. [20] and Kusy et al. [21]. One origin of bioluminescence in the common ancestor of fireflies and their relatives (Lampyridae, Rhagophtalmidae, Phengodidae and Sinopyrophoridae) was supported by Kusy et al. [21], and a second independent origin in click beetles (Elateridae). Evidence for these two origins is corroborated by Fallon et al. [22], who provided genomic evidence that strongly supports the independent evolution of elaterid and lampyrid luciferases. ...
... A limited fossil record, the difficulty of placing extinct taxa, and the immense extant diversity for a group like Elateroidea (21 000 described species), have led to continued disagreement for the ages of these groups. In addition, the systematics of the group has a level of uncertainty (see [21,37]). This broader-scale uncertainty, however, Proc. ...
We understand very little about the timing and origins of bioluminescence, particularly as a predator avoidance strategy. Understanding the timing of its origins, however, can help elucidate the evolution of this ecologically important signal. Using fireflies, a prevalent bioluminescent group where bioluminescence primarily functions as aposematic and sexual signals, we explore the origins of this signal in the context of their potential predators. Divergence time estimations were performed using genomic-scale datasets providing a robust estimate for the origin of firefly bioluminescence as both a terrestrial and as an aerial signal. Our results recover the origin of terrestrial beetle bioluminescence at 141.17 (122.63–161.17) Ma and firefly aerial bioluminescence at 133.18 (117.86–152.47) Ma using a large dataset focused on Lampyridae; and terrestrial bioluminescence at 148.03 (130.12–166.80) Ma, with the age of aerial bioluminescence at 104.97 (99.00–120.90) Ma using a complementary Elateroidea dataset. These ages pre-date the origins of all known extant aerial predators (i.e. bats and birds) and support much older terrestrial predators (assassin bugs, frogs, ground beetles, lizards, snakes, hunting spiders and harvestmen) as the drivers of terrestrial bioluminescence in beetles. These ages also support the hypothesis that sexual signalling was probably the original function of this signal in aerial fireflies.
... Our molecular clock estimates corroborate the controversial idea, famously portrayed in numerous palaeontological reconstructions, that coprophagous beetles, namely geotrupids (dung beetles) and scarabaeoids (scarabs), may have been associated with Cretaceous herbivorous dinosaurs [23,116,117]. Our analyses also corroborate a Cretaceous origin of the bioluminescent lampyroid clade [118], temporally overlapping with the diversification of visually hunting predators such as anurans and stem-group birds during the KTR [119]. At the same time, some Mesozoic beetle families have their last appearance in the fossil record during the KTR, highlighting complex dynamics of transitioning from a gymnosperm-to angiosperm-dominated world [120]. ...
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Beetles constitute the most biodiverse animal order with over 380 000 described species and possibly several million more yet unnamed. Recent phylogenomic studies have arrived at considerably incongruent topologies and widely varying estimates of divergence dates for major beetle clades. Here, we use a dataset of 68 single-copy nuclear protein-coding (NPC) genes sampling 129 out of the 193 recognized extant families as well as the first comprehensive set of fully justified fossil calibrations to recover a refined timescale of beetle evolution. Using phylogenetic methods that counter the effects of compositional and rate heterogeneity, we recover a topology congruent with morphological studies, which we use, combined with other recent phylogenomic studies, to propose several formal changes in the classification of Coleoptera: Scirtiformia and Scirtoidea sensu nov., Clambiformia ser. nov. and Clamboidea sensu nov., Rhinorhipiformia ser. nov., Byrrhoidea sensu nov., Dryopoidea stat. res., Nosodendriformia ser. nov. and Staphyliniformia sensu nov., and Erotyloidea stat. nov., Nitiduloidea stat. nov. and Cucujoidea sensu nov., alongside changes below the superfamily level. Our divergence time analyses recovered a late Carboniferous origin of Coleoptera, a late Palaeozoic origin of all modern beetle suborders and a Triassic–Jurassic origin of most extant families, while fundamental divergences within beetle phylogeny did not coincide with the hypothesis of a Cretaceous Terrestrial Revolution.
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The study analyzes the relationships of click beetles (Elateridae) Paulusiella Löbl, 2007, and Analestesa Leach, 1824 (=Cebriognathus Chobaut, 1899), both incapable of jumping, with soft-bodied habitus, and unknown females. Due to divergent morphology, their positions have been an uncertain issue. We use mitochondrial genomes to test their current placement in Cebrionini (=Cebriognathini) and Elaterinae incertae sedis, respectively. We recover Paulusiella as a sister to Hemiops Laporte, 1838 (Hemiopinae) and Analestesa as one of the serially splitting branches in Cardiophorinae, both with robust support. Paulusiellinae subfam. nov. is proposed for Paulusiella. Analestesa is transferred to Cardiophorinae, and Cebriognathini Paulus, 1981, an earlier synonym of Elaterinae: Cebrionini, is a synonym of Cardiophorinae Candèze, 1859. The click beetles affected by ontogenetic modifications converge to similar forms lacking derived states. As a result, their phylogenetic position cannot be reliably inferred by morphological analyses and needs to be validated by molecular data. Paulusiellinae and Analestesa represent two additional cases of the shift to incomplete sclerotization in elaterids raising the total number to six. The present transfers of extant taxa between subfamilies call for a cautious interpretation of morphology in other soft-bodied groups, including the taxa described from amber deposits.
We present a genome assembly from an individual female Podabrus alpinus (soldier beetle; Arthropoda; Insecta; Coleoptera; Cantharidae). The genome sequence is 777 megabases in span. Most of the assembly is scaffolded into seven chromosomal pseudomolecules, including the assembled X sex chromosome. The mitochondrial genome has also been assembled and is 18.8 kilobases in length. Gene annotation of this assembly on Ensembl identified 30,955 protein coding genes.
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Anthropogenic light pollution is a novel environmental disruption that affects the movement, foraging and mating behaviour of nocturnal animals. Most of these effects are sublethal, and their net impact on reproductive fitness and population persistence is often extrapolated from behavioural data. Without dedicated tracking of wild individuals, however, it is impossible to predict whether populations in light-polluted habitats will decline or, instead, move to shaded refuges. To disentangle these conflicting possibilities, we investigated how artificial light affects mating and movement in North American Photinus, a genus of bioluminescent fireflies known to experience courtship failure under artificial light. The degree to which artificial light reduced mate success depended on the intensity of the light treatment, its environmental context, and the temporal niche of the species in question. In the laboratory, direct exposure to artificial light completely prevented mating in semi-nocturnal Photinus obscurellus. In the field, artificial light had little impact on the movement or mate success of local Photinus pyralis and Photinus marginellus but strongly influenced mate location in Photinus greeni; all three species are relatively crepuscular. Our nuanced results suggest greater appreciation of behavioural diversity will help insect conservationists and dark sky advocates better target efforts to protect at-risk species.
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Fireflies (Lampyridae, Coleoptera) are fascinating creatures that emanate beautiful bright light hence called "living light producers". In spite of their tiny body size, they can create flashes of light due to the presence of photogenic organs located in the abdominal segments 6&7 in males, 7 th in females. In this article, a brief idea about the light production mechanism will be given.
Fireflies are one of the best-known bioluminescent organisms, and the reaction mechanism and ecological utility of bioluminescence have been well-studied. Genome assemblies of six species of bioluminescent beetles have recently been published. These studies have focused on the evolution of novelties; luciferase, and the biosynthesis of luciferin and defensive chemicals. For example, clustering of the luciferase gene with acyl-CoA synthetase genes on a chromosome in luminous beetle genomes suggests the involvement of tandem gene duplications and neofunctionalization during the evolution of beetle bioluminescence. Several candidate genes for critical roles in beetle bioluminescence have been identified, but their functional analyses are still ongoing. The establishment of a long-term mass-rearing system and strain will be the key for the post-genome research on bioluminescent beetles. Lastly, the application of contemporary chromosome-scale genome assembly techniques to luminous beetles will help resolve outstanding evolutionary questions, such as how many times bioluminescence evolved in this clade.
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The larval characters of Eucnemidae are re-evaluated. Tribe Schizophilini Muona, 1993 is considered to merit subfamily rank as Schizophilinae Muona new status. Larval characters congruent with previously used adult morphological characters support the earlier published sister-group hypotheses for lignicolous eucnemids with one exception, Pseudomeninae, which is here split resulting in the following evolutionary hypothesis: (Pseudomeninae (Schizophilinae (Paleoxeninae (Melasinae, Eucneminae, Macraulcinae)))). The position of three remaining clades, Anischiinae, Perothopinae and Phyllocerinae remain ambiguous.
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IQ-TREE (, last accessed February 6, 2020) is a user-friendly and widely used software package for phylogenetic inference using maximum likelihood. Since the release of version 1 in 2014, we have continuously expanded IQ-TREE to integrate a plethora of new models of sequence evolution and efficient computational approaches of phylogenetic inference to deal with genomic data. Here, we describe notable features of IQ-TREE version 2 and highlight the key advantages over other software.
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Fireflies (Lampyridae Rafinesque) are a diverse family of beetles which exhibit an array of morphologies including varying antennal and photic organ features. Due in part to their morphological diversity, the classification within the Lampyridae has long been in flux. Here we use an anchored hybrid enrichment approach to reconstruct the most extensive molecular phylogeny of Lampyridae to date (436 loci and 98 taxa) and use this phylogeny to evaluate the higher-level classification of the group. None of the currently recognized subfamilies were recovered as monophyletic with high support. We propose several classification changes supported by both phylogenetic and morphological evidence: 1) Pollaclasis Newman, Vestini McDermott (incl. Vesta Laporte, Dodacles Olivier, Dryptelytra Laporte, and Ledocas Olivier), Photoctus McDermott, and Araucariocladus Silveira & Mermudes are transferred to Lampyridae incertae sedis, 2) Psilocladinae Mcdermott, 1964status novum is reestablished for the genus Psilocladus Blanchard, 3) Lamprohizini Kazantsev, 2010 is elevated to Lamprohizinae Kazantsev, 2010status novum and Phausis LeConte is transferred to Lamprohizinae, 4) Memoan Silveira and Mermudes is transferred to Amydetinae Olivier, and 5) Scissicauda McDermott is transferred to Lampyrinae Rafinesque.
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The order Coleoptera (beetles) is arguably the most speciose group of animals, but the evolutionary history of beetles, including the impacts of plant feeding (herbivory) on beetle diversification, remain poorly understood. We inferred the phylogeny of beetles using 4,818 genes for 146 species, estimated timing and rates of beetle diversification using 89 genes for 521 species representing all major lineages and traced the evolution of beetle genes enabling symbiont-independent digestion of lignocellulose using 154 genomes or transcriptomes. Phylogenomic analyses of these uniquely comprehensive datasets resolved previously controversial beetle relationships, dated the origin of Coleoptera to the Carboniferous, and supported the codiversification of beetles and angiosperms. Moreover, plant cell wall-degrading enzymes (PCWDEs) obtained from bacteria and fungi via horizontal gene transfers may have been key to the Mesozoic diversification of herbivorous beetles—remarkably, both major independent origins of specialized herbivory in beetles coincide with the first appearances of an arsenal of PCWDEs encoded in their genomes. Furthermore, corresponding (Jurassic) diversification rate increases suggest that these novel genes triggered adaptive radiations that resulted in nearly half of all living beetle species. We propose that PCWDEs enabled efficient digestion of plant tissues, including lignocellulose in cell walls, facilitating the evolution of uniquely specialized plant-feeding habits, such as leaf mining and stem and wood boring. Beetle diversity thus appears to have resulted from multiple factors, including low extinction rates over a long evolutionary history, codiversification with angiosperms, and adaptive radiations of specialized herbivorous beetles following convergent horizontal transfers of microbial genes encoding PCWDEs.
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In phylogenetic inference we commonly use models of substitution which assume that sequence evolution is stationary, reversible and homogeneous (SRH). Although the use of such models is often criticized, the extent of SRH violations and their effects on phylogenetic inference of tree topologies and edge lengths are not well understood. Here, we introduce and apply the maximal matched-pairs tests of homogeneity to assess the scale and impact of SRH model violations on 3,572 partitions from 35 published phylogenetic datasets. We show that roughly one quarter of all the partitions we analysed (23.5%) reject the SRH assumptions, and that for 25% of datasets, the topologies of trees inferred from all partitions differ significantly from those inferred using the subset of partitions that do not reject the SRH assumptions. This proportion of significantly different topologies is actually even greater when evaluating trees inferred using the subset of partitions that rejects the SRH assumptions, as compared to trees inferred from all partitions. These results suggest that the extent and effects of model violation in phylogenetics may be substantial. They highlight the importance of testing for model violations and possibly excluding partitions that violate models prior to tree reconstruction. Our results also suggest that further effort in developing models that do not require SRH assumptions could lead to large improvements in the accuracy of phylogenomic inference. The scripts necessary to perform the analysis are available in, and the new tests we describe are available as a new option in IQ-TREE (
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The new subfamily Sinopyrophorinae within Elateridae is proposed to accommodate a bioluminescent species, Sinopyrophorus schimmeli Bi & Li, gen. et sp. nov., recently discovered in Yunnan, China. This lineage is morphologically distinguished from other click-beetle subfamilies by the strongly protruding frontoclypeal region, which is longitudinally carinate medially, the pretarsal claws without basal setae, the hind wing venation with a well-defined wedge cell, the abdomen with seven (male) or six (female) ventrites, the large luminous organ on the abdominal sternite II, and the male genitalia with median lobe much shorter than parameres, and parameres arcuate, with the inner margin near its apical third dentate. Molecular phylogeny based on the combined 14 mitochondrial and two nuclear genes supports the placement of this taxon far from other luminescent click-beetle groups, which provides additional evidence for the multiple origin of bioluminescence in Elateridae. Illustrations of habitus and main diagnostic features of S. schimmeli Bi & Li, gen. et sp. nov. are provided, as well as the brief description of its luminescent behavior.
Chalcidoidea (Hymenoptera) are a megadiverse superfamily of wasps with astounding variation in both morphology and biology. Most species are parasitoids and important natural enemies of insects in terrestrial ecosystems. In this study, we present a transcriptome‐based phylogeny of Chalcidoidea; we found that poorly resolved relationships could only be marginally improved by adding more genes (a total of 5591) and taxa (a total of 65), proof‐checking for errors of homology and contamination, and decreasing missing data. Concatenation analyses consistently place Mymaridae and Trichogrammatidae sister to remaining Chalcidoidea. However, our coalescent analyses provide a different hypothesis with a grouping of (Mymaridae (((Trichogrammatidae, Eulophidae), (Encyrtidae, Aphelinidae)), remaining Chalcidoidea)). This hypothesis complicates our hypothesis of egg parasitism as being the ancestral state in Chalcidoidea. At the deeper nodes, the results uncovered a wide spectrum of gene discordance in the transcriptomic markers and identified a strong signal of functional bias in genes supporting alternative phylogenies. These deeper nodes of the phylogeny are thus strongly influenced by biased support from different functional gene complexes. Shallower nodes showed similar gene discordance, but without strong functional bias. Understanding and identifying mechanisms that result in gene tree discordance may be beneficial and even essential for elucidating deeper relationships, especially for groups that have undergone extremely rapid radiation. Concatenation and coalescent analyses recover competing backbone relationships of Chalcidoidea, which complicates our hypothesis of egg parasitism as being the ancestral state in Chalcidoidea. A wide spectrum of conflicting signal accounts for the poorly supported backbone phylogeny of Chalcidoidea. Functional bias is present in genes supporting alternative relationships at the deeper nodes of the chalcidoid phylogeny.
Net-winged beetles (Coleoptera: Lycidae) are a diverse group of elateroids known for aposematism and neoteny. Phylogenetic analyses of morphological and molecular data have revealed different results with respect to within-group relationships. In this study, we recovered a highly supported phylogenomic phylogeny and identified seven subfamilies: Dexorinae stat.n., Calochrominae stat.n., Erotinae, Ateliinae, Lyci-nae, Lyropaeinae stat.n. and Metriorrhynchinae stat.n. Our results suggest that female neoteny evolved multiple times. Therefore, the development of similar morphological modifications in neotenics may be linked and may have produced characteristics such as body miniaturization, structural simplification, i.e. reduction of mouthparts, fewer antennomeres and palpomeres, uniquely shaped terminal palpomeres, shortened elytra, the loss of coadaptation between the elytra and pronotum, and others. Additional traits evolved in parallel due to similarities in biology, function and sexual selection. These characteristics include mimetic similarities, the presence of the rostrum, pronotal carinae and elytral costae, and the structure of male genitalia. By comparing the phylogenomic topology with the evolution of morphological characters, we were able to identify evolutionary trends in lycids and compare them with analogues for other neotenic elateroids. These traits have not been accepted as homoplasies due to the ambiguous phylogenetic signal from Sanger sequencing markers.
The beetle superfamily Dytiscoidea, placed within the suborder Adephaga, comprises six families. The phylogenetic relationships of these families, whose species are aquatic, remain highly contentious. In particular the monophyly of the geographically disjunct Aspidytidae (China and South Africa) remains unclear. Here we use a phylogenomic approach to demonstrate that Aspidytidae are indeed monophyletic, as we inferred this phylogenetic relationship from analyzing nucleotide sequence data filtered for compositional heterogeneity and from analyzing amino-acid sequence data. Our analyses suggest that Aspidytidae are the sister group of Amphizoidae, although the support for this relationship is not unequivocal. A sister group relationship of Hygrobiidae to a clade comprising Amphizoidae, Aspidytidae, and Dytiscidae is supported by analyses in which model assumptions are violated the least. In general, we find that both concatenation and the applied coalescent method are sensitive to the effect of among-species compositional heterogeneity. Four-cluster likelihood-mapping suggests that despite the substantial size of the dataset and the use of advanced analytical methods, statistical support is weak for the inferred phylogenetic placement of Hygrobiidae. These results indicate that other kinds of data (e.g. genomic meta-characters) are possibly required to resolve the above-specified persisting phylogenetic uncertainties. Our study illustrates various data-driven confounding effects in phylogenetic reconstructions and highlights the need for careful monitoring of model violations prior to phylogenomic analysis.