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\begin{document}

\title{Comparative mitogenome research revealed the phylogenetics and evolution
of the superfamily Tenebrionoidea (Coleoptera: Polyphage)}



\author[1]{Yun-Jian Hu}%
\author[1]{Li Hu              }%
\author[1]{Chuan Wu}%
\author[1]{Tian Tian}%
\author[1]{Ting-Jing Li}%
\author[1]{Bin Chen}%
\affil[1]{Chongqing Normal University}%


\vspace{-1em}



  
  \date{May 27, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Despite the worldwide distribution and rich diversity of the superfamily
Tenebrionoidea, the knowledge of the mitochondrial genomes (mtgenome)
characteristics of the superfamily is still very limited and its
phylogenetics and evolution remains unresolved. In present study,
nineteen species of mtgenomes in Tenebrionoidea are newly sequenced and
annotated, and a total of 90 mtgenomes are analyzed. There exist 37
genes for all 82 species of complete mtgenomes of 16 families
investigated, and their characteristics are identical as reported
mtgenomes of other Tenebrionoids. The Ka/Ks analysis suggests that all
13 PCGs have undergone a strong purifying selection. The phylogenetic
analysis suggests the monophyly of Mordellidae, Ripiphoridae, Meloidae,
Anthicidae, Oedemeridae, Pyrochroidae, Salpingidae, Scraptiidae,
Lagriidae and Tenebrionidae, and the Mordellidae is sister to the
Ripiphoridae. The ``Tenebrionidae clade'' and ``Meloidae clade'' are
monophyletic, and both of them are sister groups. In the ``Meloidae
clade'', Anthicidae is sister to Meloidae. In the ``Tenebrionidae
clade'', the family Lagriidae and Tenebrionidae are sister groups. The
divergence time analysis suggests that Tenebrionoidea originated in
early Jurassic, Mordellidae, Meloidae and Oedemeridae in Cretaceous,
Anthicidae, Lagriidae and Tenebrionidae in the early Cretaceous. The
work lays a base for the study of mtgenome, phylogenetics and evolution
of the superfamily Tenebrionoidea.%
\end{abstract}%



\sloppy


\textbf{Comparative mitogenome research revealed the phylogenetics and
evolution of the superfamily Tenebrionoidea (Coleoptera: Polyphage)}

Yun-Jian Hu, Li Hu, Chuan Wu, Tian Tian, Ting-Jing Li and Bin Chen*

Running title: mtgenome and phylogeny of Tenebrionoidea

Chongqing Key Laboratory of Vector Insects; Institute of Entomology and
Molecular Biology, College of Life Sciences, Chongqing Normal
University, Chongqing 401331, China

*Correspondence: \emph{bin.chen@cqnu.edu.cn} (BC)

\textbf{Abstract} . Despite the worldwide distribution and rich
diversity of the superfamily Tenebrionoidea, the knowledge of the
mitochondrial genomes (mtgenome) characteristics of the superfamily is
still very limited and its phylogenetics and evolution remains
unresolved. In present study, nineteen species of mtgenomes in
Tenebrionoidea are newly sequenced and annotated, and a total of 90
mtgenomes are analyzed. There exist 37 genes for all 82 species of
complete mtgenomes of 16 families investigated, and their
characteristics are identical as reported mtgenomes of other
Tenebrionoids. The Ka/Ks analysis suggests that all 13 PCGs have
undergone a strong purifying selection. The phylogenetic analysis
suggests the monophyly of Mordellidae, Ripiphoridae, Meloidae,
Anthicidae, Oedemeridae, Pyrochroidae, Salpingidae, Scraptiidae,
Lagriidae and Tenebrionidae, and the Mordellidae is sister to the
Ripiphoridae. The ``Tenebrionidae clade'' and ``Meloidae clade'' are
monophyletic, and both of them are sister groups. In the ``Meloidae
clade'', Anthicidae is sister to Meloidae. In the ``Tenebrionidae
clade'', the family Lagriidae and Tenebrionidae are sister groups. The
divergence time analysis suggests that Tenebrionoidea originated in
early Jurassic, Mordellidae, Meloidae and Oedemeridae in Cretaceous,
Anthicidae, Lagriidae and Tenebrionidae in the early Cretaceous. The
work lays a base for the study of mtgenome, phylogenetics and evolution
of the superfamily Tenebrionoidea.

\textbf{Keywords:} Coleoptera, Tenebrionoidea, mtgenome, phylogenetics,
evolution

\textbf{Introduction}

Tenebrionoidea is a large superfamily in Coleoptera, with over 34,000
known species and 28 families (Lawrence, 1995; \selectlanguage{polish}Ś\selectlanguage{english}lipi\selectlanguage{polish}ń\selectlanguage{english}ski \emph{et al} .,
2011). The species in the superfamily have the 5-5-4 tarsal formula in
both sexes, with occasional 4-4-4, 3-3-3 or 3-4-4 in males. Members of
the superfamily demonstrate various types of feeding strategies, the
majority of which are fungivorous, xylophagous, and saprophagous. Some
Tenebrionoidea species are major agricultural and forest pests, which
attack commercial crops or stored products (Song \emph{et al} ., 2018).
They are widely spread throughout all terrestrial habitats from the sea
shore up to dry desert and steppe habitats in all altitudinal belts, and
in all types of forests, and species in arid environments are
conspicuously diverse. Given these diverse feeding strategies and
habitats, the morphology of the superfamily is complicated
(Bouchard\emph{et al.} , 2009). Taxonomic and phylogenetic studies are
essential for understanding their biodiversity and biological
characteristics, and for further protecting, utilizing and controlling
them.

The framework of the classification system for Coleoptera was firstly
established in 1955 and then revised in 1982, in which the
Tenebrionoidea is divided into 5 lineages: 1) Tetratomidae,
Melandryidae, Mordellidae, Ripiphoridae; 2) Synchroidae, Zopheridae,
Prostomidae, Perimylopidae, Chalcodryidae, Tenebrionidae; 3)
Oedemeridae, Stenotrachelidae, Meloidae; 4) Pythidae, Pyrochroidae,
Boridae, Mycteridae, Salpingidae; and 5) Anthicidae, Aderidae,
Scraptiidae (Crowson, 1955; Lawrence \& Newton, 1982). In past years, a
number of studies have been reported to establish the taxonomy system of
Tenebrionoidea using comparative morphology data (Beutel \& Friedrich,
2005; Lawrence \emph{et al} ., 2011). Nowadays, 28 families
(Mordellidae, Ripiphoridae, Anthicidae, Meloidae, Pyrochroidae,
Oedemeridae, Salpingidae, Scraptiidae, Lagriidae, Tenebrionidae and so
on) are widely recognized for Tenebrionoidea (Bouchard \emph{et al} .,
2011). Despite of economic importance, the monophyletic status of many
families and phylogenetic relationships in Tenebrionoidea need to be
elucidated. A comprehensive phylogeny study of beetles showed the three
families Mordellidae, Anthicidae and Meloidae are monophyletic, the
Scraptiidae is paraphyletic based on \emph{18S}rRNA, \emph{16S} rRNA and
\emph{COX1} gene sequences (Hunt \emph{et al} ., 2007). The phylogenetic
analysis of Coleoptera showed that the families Tenebrionidae,
Oedemeridae, Salpingidae, Mordellidae, Meloidae and Scraptiidae are
monophyletic; Pyrochroidae is polyphyletic based on four gene
(\emph{16S} rRNA\emph{, COX1, 28S and 18S} ) sequences (Bocak \emph{et
al} ., 2014). The higher-level phylogenetic analysis of beetles
indicated that the Tenebrionidae, Oedemeridae, Salpingidae,
Pyrochroidae, Meloidae and Scraptiidae are monophyletic, whereas
Anthicidae is paraphyletic based on 95 nuclear protein-coding genes from
373 beetle species (Zhang\emph{et al.} , 2018). Almost all molecular
phylogenetic analyses support the monophyly of Mordellidae and Meloidae,
which is inconsistent with the phylogenetic analyses based on
morphological data (Lawrence\emph{et al} ., 2011). Due to incongruent
phylogenetic relationships from different analysis, the phylogenetic
relationships and monophyly among families are still not well settled,
and the phylogenetic positions among some families are contradictory in
different molecular studies. All of these phylogenetic issues urgently
need to be elucidated.

With the characteristics of conservative gene content, simple genome
organization, maternal inheritance, small genome size and higher
evolution rate, complete mitochondrial genome (mtgenome) has been widely
used in molecular phylogenetics, evolution, and population genetics
study of insects (Cameron \emph{et al} ., 2014). Up to date (October
2021), there have been 71 species of Tenebrionoidea complete mtgenomes
to be reported in GenBank, covering 15 families in Tenebrionoidea.
However, there is no complete mtgenome sequence to be reported from the
families Archeocrypticidae, Stenotrachelidae and Melandryidae, and the
characteristics of the mtgenomes is still little understood in the
superfamily. Up to now, there have been only a few mtgenome-based
phylogenetic studies at high taxa in Tenebrionoidea. A mtgenome-based
phylogenetic inference of beetles indicated that the families
Tenebrionidae, Oedemeridae, Anthicidae, Mordellidae and Meloidae were
monophyletic, whereas Scarptiidae was polyphyletic based on 245
mitochondrial sequences using Bayesian method (Timmermans \emph{et al.}
, 2015). The phylogenetic analysis of 37 beetle mitochondrial genes
supported the Mordellidae and Meloidae to be monophyletic groups, and
the Scraptiidae and Melandryidae to be paraphyletic groups using
Bayesian inference (Song \emph{et al.} , 2018). The mtgenome-based
phylogeny of 51 beetles found that the monophyletic of Meloidae,
Tenebrionidae, and the sister relationship of Meloidae and Tenebrionidae
using Bayesian inference (Tang \emph{et al.} , 2020). As mentioned
above, previous mtgenome-based phylogenetic studies have not well
resolved the phylogeny of Tenebrionoidea, and phylogenetic analysis of
the superfamily need to be further evaluated.

In this study, we newly sequenced and annotated 19 species of complete
mtgenomes, and comparatively analyzed the mtgenome characteristics of a
total of 90 species in Tenebrionoidea. More importantly, we constructed
and discussed the phylogenetic relationships at high taxa level based on
these mtgenome sequences, and inferred the divergence time at main nodes
in the phylogenetics in inference of fossil records. This study lays a
foundation for further understanding of mtgenome characteristics,
phylogenetics and evolution of the Tenebrionoidea.

\textbf{Material and methods}

Sample collection, sequencing and mtgenome assembly

All samples were collected in China (Table S1), and then stored in 95\%
alcohol at -20 \selectlanguage{ngerman}°C until DNA extraction. These samples were identified in
morphology, 19 species were selected for sequencing, and their
identification were confirmed by subsequent \emph{COX1}comparison with
BOLD (http://boldsystems.org/index.php) (Hebert \emph{et al} ., 2003).
The total genomic DNA was extracted from the thorax and leg muscle
tissue by DNeasy Blood and Tissue kit (Qiagen, Duesseldorf, Germany)
according to the instructions of manufacturer. Concentration of
extracted genomic DNA was determined by Qubit 2.0 (Invitrogen, Shanghai,
China). The 350 bp small fragment libraries were constructed, and then
sequenced using the Illumina Hiseq 2500 (San Diego, CA) with 150 bp
paired-end reads in Shenzhen Huitong Biotechnology Co. Ltd (Shenzhen,
China). After removing the adapters, and unpaired, short and low
qualitied reads, clean reads from mtgenomes were extracted using a BLAST
(Altschul \emph{et al.} , 1990) search against known Tenebrionoidea
mtgenome sequences, and then used for \emph{de novo}mtgenome assembly
with SPAdes v. 3.9.0 (Bankevich \emph{et al.} , 2012). The contigs of
mtgenome were extracted and assembled into mtgenomes through searching
against the reference sequences using PRICE (paired-read iterative
contig extension) by NOVOPlasty version 2.6.2 (Dierckxsens \emph{et al.}
, 2016).

Mtgenome annotation and characteristics analysis

The rough annotation of protein-coding genes (PCGs), transfer RNA genes
(tRNAs), ribosomal RNA genes (rRNAs), and CR was initially identified
using MITOS (http://mitos.bioinf.uni-leipzig.de/index.py) (Bernt\emph{et
al.} , 2013), and then determined in comparison of published homologous
mtgenome sequences in phylogeny-close species using MEGAX (Kumar
\emph{et al.} , 2018). The tRNAs secondary structures were predicted
using tRNAscan-SE Search Server v. 1.21
(http://lowelab.ucsc.edu/tRNAscan-SE/) (Lowe \& Eddy, 1997). The
annotation of the mtgenomes was corrected manually using the Geneious v.
4.8.5 (Kearse \emph{et al.} , 2012), and final mtgenomes were submitted
to the GenBank database. The secondary structures of the tRNAs were
visualized and manually edited using VARNA (http://varna.lri.fr)
(Darty\emph{et al.} , 2009). The mtgenomes were visualized using the
Chloroplot online server with default parameters (Zheng \emph{et al.} ,
2020). Base composition and relative synonymous codon usage (RSCU) of 90
species of mtgenome were computed with PhyloSuite desktop platform
(Zhang \emph{et al.} , 2020). AT-skew {[}(A - T) / (A + T){]} and
GC-skew {[}(G - C) / (G + C){]} were estimated to investigate nucleotide
composition bias (Perna \& Kocher, 1995), and three-dimensional
scatterplots of AT-Skew, GC-Skew and AT\% were drawn using Origin Pro v.
9.0 (Mikrajuddin and Khairurrijal, 2009). Selection pressure of the 13
PCGs was analyzed by calculating Ka (non-synonymous mutation rates) and
Ks (synonymous mutation rates) values with DnaSP v. 5.0 (Librado \&
Rozas, 2009), and visualized using RStudio. Sequences saturation was
assessed in DAMBE v. 5.0 (Xia, 2013).

Phylogenetic analysis of Tenebrionoidea

Phylogenetic relationships of 90 species of mtgenomes (including 19
sequenced in this study) in Tenebrionoidea were deduced using three
datasets and two inference methods with \emph{Aiolocaria hexaspilota}and
\emph{Henosepilachna vigintioctopunctata} (Coleoptera: Coccinelloidea)
as outgroups. Taxonomic information for each species investigated and
mtgenome accession numbers are listed in Table 1. Three datasets were
concatenated using PhyloSuite platform: 1) amino acid sequence of 13
PCGs (AA); 2) nucleotide sequence at 1st and 2nd codon position of 13
PCGs (PCG12); 3) PCG12 + 2 rRNAs, respectively with excluding start
codon, stop codon. Nucleotide sequences of 13 PCGs were aligned by
codon-based multiple alignments using the L-INS-i algorithm and the
rRNAs were aligned using the Q-INS-i strategy in MAFFT v. 7.0 (Katoh \&
Standley, 2013), and ambiguously aligned positions were excluded using
Gblocks (Talavera \& Castresana, 2007). The concatenation of aligned
sequences was performed using SequenceMatrix (Vaidya \emph{et al.} ,
2011). The selection of best-fit partitioning schemes and substitution
models for each dataset were calculated using PartitionFinder v. 2.0
(Lanfear \emph{et al.} , 2016) with the settings: branch lengths as
linked, model election as AICc with the greedy algorithm. Partitioning
schemes and models are listed in Table S2. Two methods, maximum
likelihood (ML) and Bayesian inference (BI) were employed for the
deduce. ML-based phylogenetic analyses were conducted using IQ-TREE v.
1.6.8 in PhyloSuite v. 1.2.2 (Zhang \emph{et al.} , 2020). Nodal support
values were inferred with 1 000 bootstrapped replicates (BPs) (Minh
\emph{et al.} , 2013). BI analysis was conducted using MrBayes v. 3.2.6
(Ronquist \emph{et al.} , 2012). A total of 2 000 000 generations with
four chains were sampled every 1 000 generations. Posterior
probabilities (PPs) were computed after discarding the first 25\% of
trees as the burn-in phase. The estimated sample size (ESS)
\textgreater{} 200 and the average deviation of the split frequency of
less than 0.01 indicates that the runs had converged. The phylogenetic
tree was visualized using FigTree v. 1.4.4 and iTOL online tool (Letunic
and Bork, 2016).

Divergence time estimation

Divergence time was estimated using the uncorrelated relaxed clock model
as implemented in BEAST v. 1.6.1 (Drummond and Rambaut, 2007). In order
to limit the numbers of parameter for the estimation, Bayesian tree was
used as a guide tree. The tree prior generated using a Yule speciation
model, and all node calibrations were enforced using Normal
distributions. Constraints on clade ages were enforced using three
fossil calibrations. Mordellidae (\emph{Protoripidius burmiticus} )
diverged from the other lineages in the Tenebrionoidea approximately in
166.6 Mya (Cai \emph{et al} ., 2018), Anthicidae-Meloidae
(\emph{Camelomorpha longicervix} ) split at 145 Mya (Kirejtshuk \emph{et
al} ., 2008), and Tenebrionidae-Lagriidae (\emph{Alphitopsis initialis}
) split at 143.6 Mya (Kirejtshuk \emph{et al} ., 2012). The posterior
time estimation was conducted using a MCMC algorithm, and the MCMC run
was sampled every 1 000 iterations until it achieved 10 000 samples,
after the first 100 000 iterations were discarded as burn-in. The
effective sample sizes of every node age were confirmed using Tracer v.
1.5 until every parameter being \textgreater{}200. The maximum clade
credibility tree was calculated using TreeAnnotator v. 1.6.1, with the
node times scaled to match the mean posterior estimates.

\textbf{Results}

Mtgenome organization

A total of 19 species of mtgenomes in Tenebrionoidea are completely
sequenced in the present study (accession numbers in Table 1), and 11
species of them are reported for the first time with all in Lagriidae.
All 82 species of complete mtgenomes investigated contain the typical 37
genes (including 13 PCGs, 2 rRNAs and 22 tRNAs) and one control region
(CR). There are 22 genes (nine PCGs and 13 tRNAs) located on the
majority coding strand (J-strand), while the other 15 genes (four PCGs,
nine tRNAs and two rRNAs) are on the minority strand (N-strand; Fig. 1).
These mtgenome sequences range in length from 14 777 bp (\emph{Cerogria
kikuchii} ) to 16 861 bp (\emph{Heterotarsus carinula} ) with an average
of 15 763 bp, and the length variation mainly results from the control
region, intergenic overlap and spacers. They all display obvious AT bias
with A+T content ranging from 62.7\% (\emph{Casnonidea terminata} ) to
81.6\% (\emph{Pyrochroidae} sp.) and an average being 73.6\%. AT-skew
values range from -0.141 (\emph{Paramarygmus} sp.) to 0.219
(\emph{Strongylium pinfaense} ), and GC-skew from -0.375 (\emph{S.
pinfaense} ) to 0.366 (\emph{Paramarygmus} sp.) (Fig. 2).

The tRNAs sizes range from 57 bp to 82 bp, and all of the tRNAs can be
folded into a typical clover-leaf structure except for\emph{tRNA}
-\emph{Ser} (AGN), in which the dihydrouridine (DHU) arm is absent and a
UCU anticodon is present (Fig. S1). The most frequently occurred base
mismatches are U-G, U--U and A-G, and the mismatch A-G is only occurred
in \emph{tRNA-Trp} . For rRNAs, \emph{rrnL} is located between the
\emph{trnL1} and \emph{trnV} , ranging from 750 bp (\emph{Anthicidae}
sp.) to 1 323 bp (\emph{Alcidodes juglans} ). The length of the
\emph{rrnS} ranges from 740 bp (\emph{Cerogira popularis} ) to 1 269 bp
(\emph{Uloma} sp.), which is located between the \emph{trnV}and CR
region. The percentage of AT content in rRNAs is 68.3-84.3\%. The CRs
are located between the \emph{rrnS} and\emph{trnI} , and the percentage
of AT content in this region is 72.2-96.1\%.

Rearrangement events

By comparing the composition and structure of the mtgenomes of these 90
Tenebrionoidea species, a total of seven Tenebrionoidea species were
found to have gene rearrangement events. These rearrangement events
occurr in the three different families Lagriidae, Mordellidae and
Pyrochroidae (Fig. 3). In others families, the gene order of the
mtgenome is exactly the same as that of \emph{drosophilid} . The tRNA
genes have the highest frequency of rearrangement
(\emph{trnW-trnC-trnY}and \emph{trnA-trnR-trnN-trnS-trnE-trnF} gene
cluster), followed by protein coding genes. The first one is found in
the\emph{trnW-trnC-trnY} gene cluster, and shuffling of \emph{trnW} gene
and \emph{trnC} gene occurs in three species of \emph{Schizotus
pectinicornis} (Pyrochroidae), \emph{Pyrochroidae} sp. (Pyrochroidae)
and \emph{Anisostira rugipennis} (Lagriidae). The second one in
the\emph{A. rugipennis} of Lagriidae, \emph{trnD} and
\emph{ATP8}transposition to the upstream of \emph{NAD2} , which is the
first rearrangement event of protein coding genes found in the family
Lagriidae. The last one is found in
the\emph{trnA-trnR-trnN-trnS-trnE-trnF} gene cluster, and shuffling
of\emph{trnR} gene and \emph{trnN} gene occurs in four species of the
family Mordellidae: \emph{Mordellidae} sp., \emph{Mordellochroa milleri}
, \emph{Mordella atrata} and \emph{Tomoxia bucephala} .

Codon usage of PCGs and gene selection pressure

Total PCGs nucleotide length ranges from 10 848 bp to 11 142 bp, and the
AT contents ranges from 60.7\% to 81.0\%. Most of the PCGs initiate with
the typical start codon ATN and TTG, whereas the special start codons
AAC, AAT, AAA and TCA are found for \emph{COX1} ; AAA for \emph{COX2} ;
GTG for \emph{ND1} and \emph{ND4L} ; AGG and AAA for \emph{ND2} and GTG
for \emph{ND4} . The most frequently used stop codons are TAA and TAG,
followed by the incomplete stop codons T and TA. The most frequently
used codons are UUA (Leu2), UCU and UCA (Ser2), CGA (Arg), whereas AGC
(Ser1), ACG (Thr), GCG (Ala) and CUG (Leu1) are the least used (Fig. 4).
For each PCG, the Ka/Ks ratio is less than one, and the \emph{ATP8} has
the highest Ka/Ks ratio (0.33-0.67), followed by seven genes (\emph{ND6,
ND5, ND4, ND2, ND4L, ND1, ND3} ) with Ka/Ks ratios of 0.17-0.42. Complex
IV (\emph{COX1, COX2} and \emph{COX3} ), Complex III (\emph{CYTB} )
and\emph{ATP6} have low Ka/Ks ratios with range from 0.01 to 0.17 (Fig.
5). These results imply all of these 13 PCGs experienced purifying
selection, especially Complex IV and Complex III.

Phylogenetic relationships

Substitution saturation tests show no saturation for three datasets AA,
PCG12 and PCG12 + rRNAs (Iss \textless{} Iss.cSym or Iss.cAsym,
\emph{p}\textless{} 0.05) (Table 2), which proposes that these three
datasets be appropriate for phylogenetic construction based on ML and
BI. Six trees generated using these three datasets and both ML and BI
are slightly different in topology (Figs. 6-7; Figs. S3-S6). The Ciidae
is located at base of all phylogenetic trees, followed by families
Mordellidae + Ripiphoridae. The Mordellidae + Ripiphoridae, Mordellidae
and Ripiphoridae all looks monophyletic (PP = 1; BP = 100) and the later
two appear sister groups each other. All remaining families also appear
monophyletic, and the family Aderidae seems sister with ``Meloidae
clade'' + ``Tenebrionidae clade''. Both ``Tenebrionidae clade'' and
``Meloidae clade'' looks monophyletic with both of them being sister
groups (Fig. 6). In the ``Meloidae clade'', the families Meloidae +
Anthicidae look a monophyletic (PP = 1; BP = 100), and it appears sister
to the ``Oedemeridae clade'', and the Meloidae and Anthicidae look
monophyletic (PP = 1; BP = 100), and a sister each other. In the
``Oedemeridae clade'', the Oedemeridae, Pyrochroidae, Salpingidae and
Scraptiidae seem monophyletic (PP = 1; BP = 100), and Salpingidae looks
a sister with Scraptiidae. In the ``Tenebrionidae clade'', the family
Lagriidae and Tenebrionidae are monophyletic (PP = 1; BP = 90-100), and
both of them are sister groups each other. In Lagriidae, the subfamily
Adeliinae is based at the subfamilies Lagriinae and Statininae, both of
which look monophyletic (PP = 1; BP = 100) and are sister groups each
other. In Tenebrionidae, the subfamily Pimeliinae appears monophyletic
group (PP = 1; BP = 100) and is located at the base of Tenebrionidae.
The Alleculinae and Stenochiinae look monophyletic (PP = 1; BP = 100),
and the subfamilies Tenebrioninae and Diaperinae appears polyphyletic
groups.

Six trees generated using AA, PCG12 and PCG12 + 2 rRNAs datasets and
both ML and BI are slightly different in topology. For AA dataset, the
topologies using ML and BI are different in the positions of
Prostomidae. Prostomidae and Tetratomidae are clustered as one clade in
ML tree, but not in BI tree. For PCG12 dataset, two same topologies of
trees from BI and ML differ from two topologies of AA dataset in
phylogenetic relationship of the ``Oedemeridae clade''. For PCG12 + 2
rRNAs dataset, the positions of major families are the same as the four
topologies of AA and PCG12 datasets, with only a few differences in the
``Oedemeridae clade''.

Divergence time

The AA dataset was used to estimate divergence time because AA had
higher node support values than others in the initial phylogenetic
assessment using Bayesian appraoch. Based on three fossil calibrations
points of \emph{Protoripidius burmiticus} (166.6 Mya),\emph{Camelomorpha
longicervix} (145 Mya) and \emph{Alphitopsis initialis} (143.6 Mya)
(filled red cycles in Fig. 8), the superfamily Tenebrionoidea was
inferred to originate in the early Jurassic (192.6 Mya, 95\% confidence
interval (CI): 179.3-208.7 Mya), with most families subsequently
diverging in the Jurassic and early Cretaceous (Fig. 8). The family
Mordellidae and Ripiphoridae is among the earliest diverged families in
the superfamily, and is estimated to originate at 115.7 and 126.6 Mya in
the Cretaceous, respectively. In the ``Meloidae clade'', the family
Meloidae is estimated to be derived at 105 Mya in the Cretaceous, the
Anthicidae at 123.8 Mya in the early Cretaceous, and the Oedemeridae at
100.9 Mya in the middle Cretaceous. In the ``Tenebrionidae clade'', the
Lagriidae is estimated to originate at 134.3 Mya in the early
Cretaceous, and the Tenebrionidae at 128.9 Mya in the early Cretaceous.
In the family Lagriidae, the subfamily Statiriinae diverged 97.6 Mya in
the late Cretaceous, and the Lagriinae diverged 78.6 Mya in the late
Cretaceous. In the family Tenebrionidae, the subfamilies Pimeliinae,
Alleculinae and Stenochiinae originated at 71.1, 53.6 and 53.3 Mya in
the Paleogene, respectively. All of these families/subfamilies are
proposed to be monophyletic and confirmed in relationships in the
phylogenetic analyses using different data or inferring methods, and
others are not determined for their monophyly or relationships or have a
few species included in the phylogenetic analyses, and therefore they
are not given an inferring of divergence time.

\textbf{Discussion}

Characteristics of Tenebrionoidea mtgenomes

These 90 mtgenomes investigated in the present study in the superfamily
Tenebrionoidea have a length variation from 14 777 bp to 16 861 bp, and
the length variation mainly stems from CR, intergenic overlap and
spacers, which is consistent with earlier reports in Tenebrionoids
(Burger \emph{et al.} , 2003). The nucleotide composition for all
species exhibits obvious AT bias with high A+T content, similar as
earlier reports in Tenebrionoids (Jie \emph{et al.} , 2016). All tRNA
genes can form a complete clover secondary structure, except
for\emph{tRNA} -\emph{Ser} (AGN) that lacks the DHU arm, which seem to
be a common feature of Tenebrionoidea (Zhang \emph{et al.} , 2016;
Song\emph{et al.} , 2018). There are some rearrangement events in some
species of the family Mordellidae, Lagriidae and Pyrochroidae. The
shuffling of the \emph{trnR} and \emph{trnN} genes
(\emph{trnA-trnR-trnN-trnS-trnE-trnF} gene cluster) is found in all
specials in the family Mordellidae; the translocation of the\emph{trnD}
and \emph{ATP8} genes is found in the \emph{A. rugipennis}of Lagriidae
for the first time; the shuffling of the \emph{trnC} and\emph{trnW}
genes (\emph{trnW-trnC-trnY} gene cluster) is found in Pyrochroidae and
Lagriidae. These gene rearrangement may be produced by abnormal priming
of mitochondrial replication by a tRNA molecule or tandem duplications,
which can provide an important reference for Tenebrionoidea phylogeny
inference (Boore \& Brown, 1998; Boore \emph{et al.} , 1998; Timmermans
\& Vogler, 2012; Cameron, 2014). The ATN and TTG are mainly used as the
start codon, and TAA and TAG as the stop codon for the 13 PCGs, which is
similar as other mtgenome sequences in Tenebrionoidea (Du \emph{et al.}
, 2017). The Ka/Ks ratio is lower than one for all PCGs, which is
consistent with earlier studies in Tenebrionoidea. The \emph{COX1} gene
has experienced strong evolutionary pressure in order to maintain its
own functional requirements, whereas \emph{ATP8} has experienced weak
evolutionary pressures with allowing more mutations to accumulate in the
mtgenome (Ou \emph{et al.} , 2016; Bai \emph{et al.} , 2018).

Overview of phylogenetic relationships

A total of 16 families are included in the phylogenetics and evolution
analysis in the Tenebrionoidea, in which there are 10 families with at
least two representative species included. The family Ciidae seems to be
earliest derived in these families, followed Mordellidae + Ripiphoridae,
and Aderidae + ``Meloidae clade'' + ``Tenebrionidae clade''. Ciidae was
historily placed in the Cucujoidea (Crowson, 1955) and then to the
superfamily Tenebrionoidea mainly based on characteristics of the
aedeagus and the larval abdomen (Crowson, 1960). It was proposed to be a
monophyly based on 18S and \emph{COX1} genes using ML and BI methods,
and demonstrated to be a either sister to Nitidulidae based on the
reduced sample or at the base of the cucujoid-tenebrionoid assemblage
based on the entire sample (Buder \emph{et al.} , 2008). It was
considered basal tenebrionoids based on 516 adult and larval
morphological characteristics from 359 beetle taxa (Lawrence \emph{et
al} ., 2011). The present study also suggests the family to be the basal
tenebrionoids, but further investigation is necessary to elucidate its
place with the inclusion of more species.

Mordellidae + Ripiphoridae, Mordellidae and Ripiphoridae are all
proposed to be monophyletic, and the two families demonstrate to be
sister groups each other in the present study. The Mordellidae +
Ripiphoridae was also proposed monophyletic in earlier molecular
phylogeny inference based on five nuclear and mitochondrial genes with
300 genera in Tenebrionoidea using ML (Gunter \emph{et al.} , 2014). The
Mordellidae was also proposed to be monophyletic in the study based on
four molecular genes (\emph{18S}rRNA, \emph{28S} rRNA, \emph{rrnL} and
\emph{COX1} ) with 128 species in Tenebrionoidea using ML (Batelka
\emph{et al.} , 2016). The Ripiphoridae was proposed to be monophyletic
from a molecular phylogenetic analysis based on eight nuclear genes with
367 species in Tenebrionoidea using Bayesian method (Mckenna \emph{et
al.} , 2015). However, it was proposed to be paraphyletic in the
molecular phylogenetic study based on four mitochondrial and four
nuclear gene fragments across 404 taxa (including 250 tenebrionid
species) using ML (Kergoat \emph{et al.} , 2014a), which suggests that
the monophyletic status of the Ripiphoridae remains uncertain. The two
families were not proposed to be sister group in the earlier molecular
phylogenetic study (Gunter \emph{et al.} , 2014; Kergoat \emph{et al.} ,
2014a), which due to the monophyletic status of Ripiphoridae remains
uncertain.

Aderidae was proposed to be a monophyletic lineage in the earlier
molecular phylogenetic study (Gunter \emph{et al.} , 2014). There is
only species in Aderidae to be included in the present study, which be
formed a monophyly with ``Meloidae clade'' + ``Tenebrionidae clade'',
and its position and monophyletic status are yet to be resolved with
more species to involved. The ``Meloidae clade'' + ``Tenebrionidae
clade'', ``Meloidae clade'' and ``Tenebrionidae clade'' are all proposed
to be monophyletic, which are consistent with earlier studies based on
five nuclear and mitochondrial genes with 300 genera in Tenebrionoidea
using ML (Gunter \emph{et al.} , 2014) and eight mitochondrial and
nuclear gene with 404 taxa in Coleoptera using ML (Kergoat \emph{et al.}
, 2014a).

Phylogenetic relationships of ``Meloidae clade''

The Meloidae + Anthicidae, Meloidae and Anthicidae are all proposed to
be monophyletic, and the two families demonstrate to be sister groups
each other in the present study. These results are consistent with
earlier studies. The monophyly of Meloidae + Anthicidae was proposed
based on 245 mitochondrial sequences in Coleoptera, including 159 newly
sequenced full or partial mtgenomes using PhyloBayes (Timmermans\emph{et
al.} , 2015). The monophyly of Meloidae was proposed based on 4 818
nuclear genes in 146 species in beetles using ML (Mckenna \emph{et al.}
, 2019). The monophyly of Anthicidae was proposed based on\emph{18S}
rRNA, \emph{16S} rRNA and \emph{COX1} gene sequences from 340-taxa using
BI (Hunt \emph{et al} ., 2007), and also based on other molecular
phylogenetic studies (Kergoat \emph{et al.} , 2014a; Timmermans\emph{et
al.} , 2015; Mckenna \emph{et al.} , 2019). The sister relationship of
Anthicidae and Meloidae was proposed based on the morphology
characteristics of mesothoracic glands (Hemp and Dettner, 1997), and
also based on mitochondrial and nuclear genes (Timmermans\emph{et al.} ,
2015; Mckenna \emph{et al.} , 2019).

In the ``Oedemeridae clade'', the family Prostomidae seems to be located
at the base of ``Oedemeridae clade'', followed Oedemeridae and
Trictenotomidae + Tetratomidae. Prostomidae was proposed a sister to the
``pythid-pyrochroid-lineage'' (including Trictenomatidae, Pyrochroidae,
Salpingidae and so on) based on morphology characteristics of the
maxillary articulatory area, the abdominal tergite IX extending to the
ventral side of the segment, and the strongly pronounced prognathous
condition (Schunger \emph{et al.} , 2003). However, Prostomidae and
Tetratomidae are clustered as one clade in ML tree, and the phylogenetic
position of Prostomidae remained unresolved in the present analyses.

The family Oedemeridae is proposed to be monophyletic, which is
consistent with the earlier phylogenetic study based on four gene
(\emph{16S} rRNA\emph{, COX1, 28S} and \emph{18S} rRNA) sequences from
8441 taxa of Coleoptera, which removed misplaced single specimens and
minor clades (Bocak \emph{et al.} , 2014). However, it was proposed to
be paraphyletic in the phylogenetic study based on mitochondrial and
nuclear genes (Gunter \emph{et al.} , 2014; Zhang \emph{et al.} , 2018),
which suggests that the monophyletic status of the Oedemeridae need be
further determined with more species included. Trictenotomidae and
Tetratomidae are clustered as one clade using BI in this study, whereas
Trictenotomidae was a sister to Boridae in earlier phylogenetic studies
based on nuclear genes (Mckenna\emph{et al.} , 2019). There is only
species in Trictenotomidae and Tetratomidae to be included in the
present study, which suggests that the monophyletic status of the
Trictenotomidae and Tetratomidae remains uncertain, and its position and
monophyletic status are yet to be resolved with more species to
involved.

Zopheridae and Pyrochroidae are clustered as one clade, and Pyrochroidae
seems monophyletic in the present study. The monophyly of Pyrochroidae
was also proposed in the earlier phylogenetic study based on 95 nuclear
protein-coding genes in 373 beetle species using ML and BI (Zhang
\emph{et al.} , 2018), and in other phylogenetic studies based on
mitochondrial and nuclear genes (Gunter \emph{et al.} , 2014; Kergoat
\emph{et al.} , 2014a; Mckenna\emph{et al.} , 2019). Zopheridae was a
sister to Tetratomidae in earlier phylogenetic studies based on
mitochondrial and nuclear genes (Kergoat\emph{et al.} , 2014a), which
suggests that the position and monophyletic status of Zopheridae and
Pyrochroidae need be further determined. The families Salpingidae and
Scraptiidae seem monophyletic, and are sister groups each other in the
present study. The monophyly of Salpingidae and Scraptiidae was also
proposed in the earlier phylogenetic studies based on mitochondrial and
nuclear genes (Bocak \emph{et al.} , 2014; Zhang\emph{et al.} , 2018;
Mckenna \emph{et al.} , 2019), whereas Scraptiidae was proposed a
paraphyletic group in other molecular phylogenetic studies (Hunt
\emph{et al} ., 2007; Kergoat \emph{et al.} , 2014a; Mckenna\emph{et
al.} , 2015). The sister relationship of Salpingidae and Scraptiidae
remains unclear due to the limited inclusion of only two or three
species. Therefore, the position and monophyletic status of Salpingidae
and Scraptiidae need be further determined with more species included.

Phylogenetic relationships of ``Tenebrionidae clade''

In the ``Tenebrionidae clade'', the family Lagriidae and Tenebrionidae
are monophyletic, and both of them are sister groups each other in the
present study. The classification of Lagriidae has been debated over the
years, and some scholars argue that Lagriidae should be classified as a
subfamily within Tenebrionidae. However, the beetles of Lagriidae are
leafivorous like beetles of Chrysomelidae, and morphological
characteristics of Lagriidae adapt much more for free-moving and
leaf-feeding than the beetles from other subfamilies in Tenebrionidae.
The monophyly of Lagriidae was proposed in the earlier morphology study
based on the characteristics of abdominal defense glands, female
reproductive tract, mouthparts morphology and structure of the wings in
Lagriid and Tenebrionoid (Doyen \& Tschinkel, 1982). The monophyly was
also proposed in earlier phylogenetic inference based on mtgenomes and
nuclear genes (Gunter \emph{et al.} , 2014; Kergoat \emph{et al.} ,
2014a). The mtgenome-based phylogeny of 36 species in Tenebrionidae
suggested the monophyly of Lagriidae and Tenebrionidae, and their sister
relationships based on PCG123 datasets using BI and ML (Wu \emph{et al.}
, 2021), which is consistent with the present study. The monophyly of
Tenebrionidae was also proposed in some earlier phylogenetic studies
based on mitochondrial and nuclear genes (Hunt \emph{et al} ., 2007;
Timmermans \emph{et al.} , 2015; Mckenna\emph{et al.} , 2019).

The present study suggests the phylogenetic relationships of Adeliinae +
(Lagriinae + Statininae) in the family Lagriidae, and the monophyly of
Lagriinae and Statininae. The monophyly of Lagriinae was proposed in
earlier phylogenetic studies based on the morphology characteristics
(Doyen, 1989), and also based on mitochondrial and nuclear genes
(Gunter\emph{et al} ., 2014; Wu \emph{et al} ., 2021). The present study
supports the monophyly of the subfamily Statiriinae for the first time.
The Adeliinae is at the base of the family Lagriidae in this study,
which is consistent with the earlier mtgenome-based study (Wu \emph{et
al} ., 2021). The monophyly of Adeliinae in Lagriidae is not yet
determined due to only one species to be included.

In the family Tenebrionidae, the present study supports the monophyly of
the subfamily Pimeliinae, Stenochiinae and Alleculinae, whereas
Tenebrioninae and Diaperinae are recognized as a polyphyly. The
subfamily Pimeliinae was also proposed to be monophyletic in the earlier
studies based on nuclear genes and mitochondrial genes (Gunter \emph{et
al.} , 2014; Wu \emph{et al} ., 2021). The present study supports the
monophyly of the subfamilies Stenochiinae and Alleculinae in
Tenebrionidae, which was also proposed in earlier phylogenetic studies
based on mitochondrial and nuclear genes (Kergoat \emph{et al.} , 2014a;
Wu \emph{et al} ., 2021). The subfamily Tenebrioninae and Diaperinae in
these molecular-based studies are found to be polyphyletic, which is
need to be elucidated with more species involved.

Evolution of Tenebrionoidea

The Tenebrionoidea is inferred to origin in the early Jurassic
(179.3-208.7 Mya) based on the mtgenomes and fossil calibrations points
in the present study, which is consistent with earlier evolution studies
in Coleoptera based on mitochondrial and nuclear genes (Mckenna \emph{et
al.} , 2015; Zhang \emph{et al.} , 2016; Cai \emph{et al.} , 2022). The
present results suggest that most families subsequently diverged in the
Cretaceous. Angiosperms replaced the previously dominant gymnosperms
during the Cretaceous, and the warm and humid environment had been
produced in Cretaceous, which provided food and habitat for the families
in Tenebrionoidea. The family Ciidae seems to be earliest derived in
these families in the present study, which is inconsistent with earlier
evolution studies (Kergoat \emph{et al.} , 2014b). The divergence time
of Ciidae is not yet determined due to only one species to be included.
The family Mordellidae and Ripiphoridae is among the earliest diverged
families in the superfamily, Mordellidae and Ripiphoridae are estimated
to originate at 115.7 and 126.6 Mya in the Cretaceous, which is
consistent with the previous evolution study result based on 95 nuclear
protein-coding genes in 373 beetle species using ML and BI (Zhang
\emph{et al.} , 2018). In the ``Meloidae clade'', the families Meloidae
(105 Mya), Anthicidae (123.8 Mya) and Oedemeridae (100.9 Mya) originated
in the Cretaceous, which is consistent with earlier evolution studies
based on mitochondrial and nuclear genes (Misof \emph{et al.} , 2014;
Kergoat\emph{et al.} , 2014b). In the ``Tenebrionidae clade'', the
family Lagriidae (134.3 Mya) is proposed to be derived in the early
Cretaceous, which is similar with the evolution study based on
mitochondrial and nuclear genes in 404 beetle species (Kergoat \emph{et
al.} , 2014b). The family Tenebrionidae (128.9 Mya) is suggested to be
derived in the early Cretaceous, which is consistent with the results of
the previous evolution study based on 4 818 nuclear genes (Mckenna
\emph{et al.} , 2019).

In the family Lagriidae, the subfamilies Lagriinae (78.6 Mya) and
Statiriinae (97.6 Mya) are proposed to be derived in the late Cretaceous
for the first time. In the family Tenebrionidae, the present study
suggests the origin of the subfamilies Alleculinae (53.6 Mya) and
Stenochiinae (53.3 Mya) in the Paleogene, but is inconsistent with the
results of the earlier evolution study (Kergoat \emph{et al.} , 2014b),
which be due to differences in the taxa included, fossils constraints
and analysis methods applied. In further research, more accurate
estimates of divergence times are necessary with more precise fossil
records for calibration and more complete sampling.

\textbf{Conclusion}

This is the first comprehensive study on the mtgenomes characteristics
and mtgenome-based phylogenetics in Tenebrionoidea. A total of 19
species of mtgenomes in Tenebrionoidea are newly sequenced and
annotated. The comprehensive analysis of 90 mtgenome sequences in
Tenebrionoidea suggests that the AT-skew, length variation, and codon
usage are consistent with other reported mtgenomes in Tenebrionoidea.
The families Mordellidae, Meloidae, Anthicidae, Oedemeridae,
Pyrochroidae, Salpingidae, Scraptiidae, Lagriidae and Tenebrionidae are
suggested to be monophyletic. Ciidae is at the base of the superfamily
of Tenebrionoidea, and the Mordellidae is sister to the Ripiphoridae.
The ``Tenebrionidae clade'' and ``Meloidae clade'' are monophyletic, and
both of them are sister groups. In the ``Meloidae clade'', Anthicidae is
sister to Meloidae. In the ``Tenebrionidae clade'', the family Lagriidae
and Tenebrionidae are sister groups. In Lagriidae, the subfamily
Adeliinae is based at the subfamilies Lagriinae + Statininae. In
Tenebrionidae, the subfamily Pimeliinae, Alleculinae and Stenochiinae
look monophyletic, Tenebrioninae and Diaperinae are polyphyletic. The
divergence time analysis suggests that Tenebrionoidea originated in
early Jurassic, Mordellidae, Meloidae and Oedemeridae in Cretaceous,
Anthicidae, Lagriidae and Tenebrionidae in the early Cretaceous.

\textbf{Availability of data and materials}

All data are available as tables and figures in the main paper and its
supplementary files. The GenBank accession numbers for the 19 mtgenomes
generated in the present study with detailed information in Table S1.

\textbf{Competing interests}

The authors declare that they have no competing
interests.\textbf{Abbreviations}

Mtgenome: mitochondrial genome; PCGs: protein-coding genes; rRNAs:
ribosomal RNA genes; tRNAs: transfer RNA genes; CR: control region;
RSCU: relative synonymous codon usage; BI: Bayesian inference; ML:
Maximum likelihood.

\textbf{Ethics approval and consent to participate}

Not applicable.

\textbf{Funding}

This research was supported by the following, The National Natural
Science Foundation of China (31872262, 31672363), National Key Program
of Science and Technology Foundation Work of China (2015FY210300).

\textbf{Author Contributions}

Conceived and designed the research: BC, YJH, LH, TJL. Performed the
specimens collecting and experiments: BC, CW, TT. Analyzed the data and
wrote the paper: YJH, LH, BC, TJL.

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