G3, 2024, 14(2), jkad266
https://doi.org/10.1093/g3journal/jkad266
Advance Access Publication Date: 13 December 2023

Genome Report

Göran Arnqvist,1,* Ivar Westerberg,2,3 James Galbraith,4,5 Ahmed Sayadi,6 Douglas G. Scofield
Remi-André Olsen,9 Elina Immonen,7 Franziska Bonath,10 Philip Ewels,11 Alexander Suh2

,7,8

1

Animal Ecology, Department of Ecology and Genetics, Uppsala University, Uppsala SE75236, Sweden
Systematic Biology, Department of Organismal Biology, Uppsala University, Uppsala SE75236, Sweden
3
Department of Ecology, Environment and Plant Sciences, Stockholm University, Stockholm SE10691, Sweden
4
School of Biological Sciences, University of Adelaide, Adelaide 5005, Australia
5
Faculty of Environment, Science and Economy, University of Exeter, Cornwall TR10 9FE, UK
6
Rheumatology, Department of Medical Sciences, Uppsala University, Uppsala SE75236, Sweden
7
Evolutionary Biology, Department of Ecology and Genetics, Uppsala University, Uppsala SE75236, Sweden
8
Uppsala Multidisciplinary Center for Advanced Computational Science, Uppsala University, Uppsala SE75236, Sweden
9
Science for Life Laboratory, Department of Biochemistry and Biophysics, Stockholm University, Stockholm SE10691, Sweden
10
Science for Life Laboratory, Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm SE10691, Sweden
11
Seqera Labs, Barcelona 08005, Spain
2*Corresponding author: Department of Ecology and Genetics, Uppsala University, Uppsala SE75236, Sweden. Email: goran.arnqvist@ebc.uu.se

Callosobruchus maculatus is a major agricultural pest of legume crops worldwide and an established model system in ecology and evo­lution. Yet, current molecular biological resources for this species are limited. Here, we employ Hi-C sequencing to generate a greatly
improved genome assembly and we annotate its repetitive elements in a dedicated in-depth effort where we manually curate and classify
the most abundant unclassified repeat subfamilies. We present a scaffolded chromosome-level assembly, which is 1.01 Gb in total
length with 86% being contained within the 9 autosomes and the X chromosome. Repetitive sequences accounted for 70% of the total
assembly. DNA transposons covered 18% of the genome, with the most abundant superfamily being Tc1-Mariner (9.75% of the gen­ome). This new chromosome-level genome assembly of C. maculatus will enable future genetic and evolutionary studies not only ofthis important species but of beetles more generally.
Keywords: Chrysomelidae; chromosome conformation capture; X chromosome assembly; transposable elements; Tc1-Mariner

Introduction
The introduction of long-read sequencing techniques has dramat­
ically improved our ability to generate de novo genome assem­
blies. Yet, for large, repeat-rich, and structurally complex
genomes, the resulting assemblies are still typically very fragmen­ted, which restricts the utility of genome assemblies for certain
types of analyses (Sedlazeck et al. 2018). In systems where linkage
groups cannot easily be identified, chromosome conformation
capture techniques such as Hi-C now offer dramatic improve­
ment of assembly contiguity and scaffold length (Burton et al.
2013; Dudchenko et al. 2017). These techniques retain long-range
genomic information, through crosslinking of chromatin and se­quencing of proximal pairs of sequences, which can be used toconstruct chromosome spanning assemblies.
The seed beetle Callosobruchus maculatus (Coleoptera; Bruchinae)
is a major agricultural pest on legume crops in arid regions of theworld, causing crop losses of up to 90% (Sallam 2013), and is an es­
tablished model system for studies in ecology and evolution (e.g.
Holmes et al. 2020; Arnqvist et al. 2022). The recent publication ofan annotated genome assembly of C. maculatus (Sayadi et al. 2019)
now provides opportunities for novel use of this model also in gen­etics and genomics.
Here, our aim was 2-fold. First, the current assembly of C. ma­
culatus is of high quality in terms of e.g. functional completenessand base accuracy but contains >15,000 contigs. We wereinterested in employing Hi-C to improve the contiguity by superscaffolding the current assembly, ideally into chromosomelevel scaffolds. This would, for example, enable detection of large
structural variants, analyses of linked selection, genomic land­
scapes of divergence, and comparative studies of genome collin­
earity and structural orthology. Second, we made a dedicatedeffort to improve the annotation of repetitive elements in the gen­ome of C. maculatus. The previous assembly is based on long-read
PacBio sequence data, and a run with RepeatModeler followed by
RepeatMasker identified a very high fraction of repeats in the as­sembly. However, more than half of these repeat sequences(54%) could not be attributed to any specific repeat class (Sayadi
et al. 2019). This likely reflects rapid sequence evolution of repeti­
tive elements in this group of insects, previously inferred from

Received on 16 June 2023; accepted on 30 October 2023
© The Author(s) 2023. Published by Oxford University Press on behalf of The Genetics Society of America.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which
permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

Downloaded from https://academic.oup.com/g3journal/article/14/2/jkad266/7471854 by Dennis Hejhal user on 21 May 2024

A chromosome-level assembly of the seed beetle
Callosobruchus maculatus genome with annotationof its repetitive elements

2 | G. Arnqvist et al.
Table 1. A comparison between the previous and the new genome assembly of C. maculatus.
New Hi-C assembly (CASHZR040000000.4)

1,007.82
15,778
2.07
1,183
6,491
212,245
26,5074,364
82.30%

1,012.33
10,661114.6275
472
98,582,428
39,277245
88.98%

85.4% (n = 1816)
4.8% (n = 103)
5.1% (n = 108)

85.7% (n = 1820)
4.2% (n = 90)
5% (n = 107)

Genome assembly size (Mbp)
No. of scaffolds/contigs
Maximum scaffold/contig length (Mbp)
Scaffold/contig L50
Scaffold/contig L90
Scaffold/contig N50 (bp)
Scaffold/contig N90 (bp)
Number of scaffolds/contigs >50 kbp:
% genome in scaffolds/contigs >50 kbp:
BUSCO assessment (n = 2124)
Complete BUSCOs (C)Duplicated BUSCOs (D)Fragmented BUSCOs (F)Completeness assessed with BUSCO v5.2.2, using the endopterygota_odb10 reference gene set.

studies of variation in genome size (Arnqvist et al. 2015; Boman andArnqvist 2023) and mitochondrial genomes (Sayadi et al. 2017). Our
efforts aimed at increasing our understanding of the apparently ra­pid evolution of tandem repeats and transposable elements (TEs)and at alleviating the general underrepresentation of wellannotated beetles in repeat databases used for repeat classification(Parisot et al. 2021).

Materials and methods
The genome of C. maculatus is ∼1.23 Gb (Arnqvist et al. 2015), andthe estimated repeat content of the genome is as high as 71%(Sayadi et al. 2019). The karyotype of the genome is 2n = 18 + XX/
XY, where the relative size of the 9 autosomes (% of the total hap­loid chromosomal length) ranges from 8.25 to 12.83, while the X is
7.69 and Y is a very small but distinct dot chromosome (Angus
et al. 2011). We used the annotated C. maculatus reference genome
assembly reported in Sayadi et al. (2019) as starting point forsuper-scaffolding and repeat annotation (GCA_900659725.1). In
that work, PacBio long-read sequences representing 32× genome
coverage with an average read length of 9.0 kb were assembled
using FALCON and subsequently error-corrected based on re­alignment of both PacBio (32×) and Illumina (125×) reads. This as­
sembly is 1.01 Gb in total size, with a contig N50 of 212 kb and thelongest contig spanning 2.1 Mb. Annotation of the assembly was
based on large amounts of transcriptome data, homology, andab initio prediction methods and identified 21,264 coding genes.
Analyses of conserved proteins sets showed a high fraction ofwell-assembled genes in the assembly (Sayadi et al. 2019). Yet, be­cause of the high repeat content of the genome, the assembly ishighly fragmented and contains >15,000 contigs.

Hi-C library preparation, sequencing, andassembly
We used a sample of live male C. maculatus from the isogenic ref­erence line SI4 for Hi-C sequencing. Beetles were killed by flash
freezing in liquid nitrogen and were then ground in batches into
a flour-like powder using a plastic pestle in an Eppendorf tube
on dry ice. The pestle and tube used were precooled in liquid nitro­
gen prior to grounding, and the tube contained some liquid nitro­gen, to ensure that the material remained frozen through theentire grinding, preparation, and transfer process. The sample
was then stored at −80°C.
The Hi-C library was prepared following the Arima Protocol“Arima-HiC_AnimalTissue_v00” (document number A160126

v00). In short, 400 mg of ground beetle material was crosslinked
in 2% formaldehyde for 20 min while rotating. The crosslinking
was stopped by addition of the Arima-kit provided by Stop
Solution. Before continuation, larger debris was allowed to sinkto the bottom and only material small enough to pipette wasused in subsequent reactions. Lysis was performed on crosslinked
tissue equivalent to ∼2 μg of chromatin. All steps of the Hi-C reac­tion were performed as described in the Arima protocol.
The chromatin was fragmented in an AFA Fiber Crimp-Cap
microTUBE using a COVARIS E220 with the following settings:
peak incident power 175 W, acoustic duty factor 10%, 200 cycles
per burst, and 50 s treatment time. The fragmented chromatin
was purified using AMPure XP beads and subjected to library prep­
aration following the “Arima-HiC Kit, Library Preparation using
Illumina TruSeq DNA PCR-Free Library Prep” protocol (document
number A160111 v01), and the Arima-kit reagents were supple­
mented by reagents of an Illumina TruSeq PCR-Free library prep­
aration kit. The library was amplified for 11 cycles using an
Illumina TruSeq DNA CD Index. The final library was analyzed
for fragment length distribution using an Agilent Fragment
Analyzer with a high-sensitivity NGS Fragment 1-6,000 bp kit
and for concentration using the Qubit high-sensitivity dsDNA
kit. Subsequently, the library was sequenced with a depth of∼800 million reads on a NovaSeq 6000 S4 flow cell with a read
length of 2 × 150 bp.
The Hi-C reads were preprocessed using Juicer (git-rev. 84f6957)(Durand et al. 2016) and a script provided by Arima Genomics
to generate ligation site positions in the input assembly(GCA_900659725.1) matching the sequence motifs “GATCGATC,”“GANTGATC,” “GANTANTC,” and “GATCANTC.” The resulting
list of valid Hi-C pairs was used as input for the scaffolder
3D-DNA (v. 180922). The resulting draft scaffolded assembly was
manually error-corrected and curated using the JBAT method pro­vided by the authors of 3D-DNA (Dudchenko et al. 2017).
In order to assess gene richness in major scaffolds and to iden­
tify scaffolds corresponding to the X chromosome, we mapped (1)all genes and (2) all putative X-linked genes identified in the origin­
al assembly from relative coverage in male and female samples(see Sayadi et al. 2019), using BWA-MEM (Li 2013), and assessed en­
richment among scaffolds in the new assembly.

Annotation liftover
The resulting Hi-C genome assembly was annotated by perform­
ing a coordinate conversion of the extant annotation [National
Center for Biotechnology Information (NCBI), accession

Downloaded from https://academic.oup.com/g3journal/article/14/2/jkad266/7471854 by Dennis Hejhal user on 21 May 2024

PacBio assembly (GCA_900659725.1)

Chromosome-level assembly of C. maculatus |3

PRJEB30475]. We performed the liftover using Liftoff (Shumate
and Salzberg 2021), using default parameters apart from a few
that were set to more stringent criteria (coverage >80%, sequence
identity >90%, distance scaling 5).

Annotation of repetitive elements
A repeat library was built by manually curating part of the output
of RepeatModeler open-1.0.11 (Smit et al. 2010). An initial repeat li­
brary was constructed using RepeatModeler and used to maskwith RepeatMasker (Smit and Hubley 2010). To improve the anno­tation, manual curation of the 38 most abundant repeats which
had already been classified by RepeatModeler as well as 89 of

the most abundant repeats out of the total 490 classified as “un­known” was performed through a “BLAST-extend-align-trim” ap­proach as previously described in Suh et al. (2018). Briefly,
for each repeat subfamily consensus sequence identified by
RepeatModeler, this approach consisted of (1) searching for copiesof the subfamily using BLASTN (Altschul et al. 1990), (2) selecting
the top 20 sequence hits, (3) extending their flanks, and (4) aligning
the extended sequences using MAFFT (Katoh et al. 2002). For man­ual curation, consensus sequences were constructed using
Advanced Consensus Maker (www.hiv.lanl.gov: Advanced
Consensus Maker, last accessed 2019) after trimming of the dis­cordant flanks. This new consensus was then classified using

Downloaded from https://academic.oup.com/g3journal/article/14/2/jkad266/7471854 by Dennis Hejhal user on 21 May 2024

Fig. 1. Hi-C contact map on the final C. maculatus assembly. Some 86% of the input assembly was arranged into 10 chromosome-length scaffolds.

4 | G. Arnqvist et al.

the CENSOR (Kohany et al. 2006) against the Repbase repeat data­base (Bao et al. 2015), NCBI’s Conserved Domain Database searchtool (Marchler-Bauer et al. 2017), and LAST alignment tool
(Kiełbasa et al. 2011). Based on homology to known repeats, target
site duplications (TSDs) in the alignment with flanks, presence ofconserved protein domains, and self-alignment, repeat classifica­tion was determined in line with previous studies (Wicker et al.
2007; Feschotte and Pritham 2007). The resulting partially curated
library was combined with a beetle-specific library from Repbase,created with RepeatMasker’s queryRepeatDatabase.pl script(-species coleoptera), and then used to mask the genome using
RepeatMasker.

Results and discussion
Hi-C assembly
After mapping to the input assembly, deduplicating and removing
short fragment reads, Juicer outputted 141 M valid Hi-C contacts
and additionally 154.7 M lower quality contacts [read-pairs below
the mapping quality (MAPQ) threshold]. The resulting scaffolded

assembly is 1.01 Gb in total size, which is on par with the predicted
genome size of 0.96 Gb (Arnqvist et al. 2015), and it shows a mas­sive improvement in scaffold length (see Table 1 for assembly sta­tistics). In the end, 86% of the assembly is contained within 10
well-supported chromosome-length scaffolds (Fig. 1). The relative
size and structure of the scaffolds is consistent with the 9 meta­
centric autosomes previously documented in C. maculatus using
cytogenetics (Angus et al. 2011) with the single considerably smal­ler scaffold corresponding to a substantial fraction of the meta­
centric X chromosome. The identity of the X chromosome
scaffold (23.3 Mb) was verified by the fact that 462 out of 658 puta­
tively X-linked genes (Sayadi et al. 2019) mapped to this scaffold.
We note that 81% of all genes and 83% of all coding sequences(CDSs) mapped to one of the 10 largest scaffolds. Gene density
was variable along scaffolds, but no scaffold showed a general en­
richment in gene content (Fig. 2).
All gene models in the liftover annotation carry additional at­tributes describing the coverage and identity statistics found dur­ing the liftover from the original annotation. A total of 36 genemodels (out of 21,264) did not map to the scaffolded assembly,

Downloaded from https://academic.oup.com/g3journal/article/14/2/jkad266/7471854 by Dennis Hejhal user on 21 May 2024

Fig. 2. A chromosomal ideogram of gene and repeat densities within the 10 longest scaffolds of the C. maculatus Hi-C assembly. Densities were calculated
in nonoverlapping 500 kbp bins. For scale, the length of the longest scaffold shown is 114.6 Mbp, and that of shortest is 23.3 Mbp.

Chromosome-level assembly of C. maculatus |5and a total of 744 gene models were partially mapped, using strin­
gent criteria.
Our effort illustrates the great utility of Hi-C sequencing forsuper-scaffolding of complex and large genomes. The new andimproved genome assembly of C. maculatus will no doubt aid in
the control of this widespread agricultural pest and should alsosignificantly increase the utility of this model species in future
genomic and genetic studies. For example, with chromosomelevel assemblies now being available for a growing number of bee­tle species (e.g. Herndon et al. 2020; Zhang et al. 2020; Chen et al.
2021; Keeling et al. 2022), future studies of shared synteny and gen­ome collinearity promises insights into the evolution of genome
structure in this large group of insects. Further, information on
physical colocalization of genes and contigs will enable analysesof the role of linkage and linked selection in evolutionary genomic
studies that utilize experimental evolution or artificial selection
and can provide detailed insights into the genomic landscape of
population and species divergence.

Repetitive elements
Both before and after automated classification and manual cur­ation of the most abundant repeats, the total masked repeat con­tent accounted for 70% percent of the assembly (Fig. 3). Throughour manual curation, we were able to classify 83 of the 89 mostcommon unclassified repeat subfamilies: 67 as DNA transposons,
7 as long interspersed nuclear elements (LINEs), 7 as LTR retro­transposons, and 2 as satellite DNA. This greatly reduced the pro­portion of repetitive elements which were unclassified from 31%of the assembly to 24%. Of all repeats with known classification
annotated by RepeatMasker, DNA transposons were the mostabundant, covering 18% of the genome, followed by LINEs (13%)
and LTR elements (2.3%).

Fig. 4. Dot plot of consensus sequences of the 13 newly identified large
nonautonomous Tc1-Mariner DNA transposons when aligned to eachother using LAST. The lengths of the consensus sequences range from 654
to 2,199 bp.

The most abundant superfamily was Tc1-Mariner, within theclass of DNA transposons, accounting for 9.75% of the genome.
A large portion of the annotated Tc1-Mariner elements (5.2% of
the genome, 41.7% of Tc1-Mariner elements) were of a group oflarge (1–2 kb) nonautonomous elements similar to miniature
inverted-repeat TEs (MITEs), possessing large terminal inverted

Downloaded from https://academic.oup.com/g3journal/article/14/2/jkad266/7471854 by Dennis Hejhal user on 21 May 2024

Fig. 3. Repeat landscape of the C. maculatus genome. The X-axis shows the Kimura 2-parameter distance of repeat copies to their respective consensus
sequence, with low values indicating that the repeat copy is more recent. The Y-axis shows the cumulative genome percentages of the repeats in each 1%bin of Kimura substitution level. The colored parts of the bars correspond to different classifications of repeats with gray being the unclassified repeats(“unknown”).

6 | G. Arnqvist et al.

Data availability
Preprocessing scripts are available at https://github.com/
ArimaGenomics/Scripts. Data, assembly, and annotation
files have been deposited at the European Nucleotide
Archive (ENA) under the BioProject PRJEB60338 as accession
CASHZR040000000. The repeat library has been deposited at the
Zenodo repository and is available at https://zenodo.org/record/
7994921. A file containing the genomic locations of repeats, aswell as the repeat family to which each identified repeat belongs,
has been deposited at Mendeley Data and is available at https://data.mendeley.com/datasets/6w6h63nw4s/2.

Acknowledgments
The authors thank Olga Dudchenko and Valentina Peona for ad­
vice on Hi-C scaffolding. Sequencing was performed by the
National Genomics Infrastructure at the Science for Life
Laboratory, Stockholm, financially supported by the Swedish
Research Council and the Knut and Alice Wallenberg
Foundation. The computations and data handling were enabled
by resources provided by the Swedish National Infrastructure

for Computing (SNIC) at UPPMAX partially funded by the
Swedish Research Council.

Funding
This work was supported by grants from the European Researchcouncil ERC (AdG-294333), the Swedish Research Council VR(621-2014-4523 and 2019-03611 to G.A.; 2016-05139 to Al.S), and
FORMAS (2018-00705 to G.A.; 2017-01597 to Al.S).

Conflicts of interest
The authors declare that they have no conflicts of interest.

Literature cited
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic localalignment search tool. J Mol Biol. 215(3):403–410. doi:10.1016/
S0022-2836(05)80360-2.
Angus RB, Dellow J, Winder C, Credland PF. 2011. Karyotype differ­ences among four species of Callosobruchus Pic (Coleoptera:
Bruchidae). J Stored Prod Res. 47(2):76–81. doi:10.1016/j.jspr.
2010.10.003.
Arnqvist G, Sayadi A, Immonen E, Hotzy C, Rankin D, Tuda M,
Hjelmen CE, Johnston JS. 2015. Genome size correlates with re­productive fitness in seed beetles. Proc R Soc B. 282(1815):
20151421. doi:10.1098/rspb.2015.1421.
Arnqvist G, Rönn J, Watson C, Goenaga J, Immonen E. 2022.
Concerted evolution of metabolic rate, economics of mating,ecology, and pace of life across seed beetles. Proc Natl Acad Sci
U S A. 119(33):e2205564119. doi:10.1073/pnas.2205564119.
Bao W, Kojima KK, Kohany O. 2015. Repbase Update, a database ofrepetitive elements in eukaryotic genomes. Mob DNA. 6(1):11.
doi:10.1186/s13100-015-0041-9.
Boman J, Arnqvist G. 2023. Larger genomes show improved bufferingof adult fitness against environmental stress in seed beetles. Biol
Lett. 19(1):20220450. doi:10.1098/rsbl.2022.0450.
Bouchemousse S, Falquet F, Müller-Schärer H. 2020. Genome assem­
bly of the ragweed leaf beetle: a step forward to better predict ra­
pid evolution of a weed biocontrol agent to environmental
novelties. Genome Biol Evol. 12(7):1167–1173. doi:10.1093/gbe/
evaa102.
Burton JN, Adey A, Patwardhan RP, Qiu R, Kitzman JO, Shendure J.
2013. Chromosome-scale scaffolding of de novo genome assem­
blies based on chromatin interactions. Nature Biotech. 31(12):1119–1125. doi:10.1038/nbt.2727.
Chen M, Mei Y, Chen X, Chen X, Xiao D, He K, Li Q, Wu M, Wang S,
Zhang F, et al. 2021. A chromosome-level assembly of the harle­quin ladybird Harmonia axyridis as a genomic resource to study
beetle and invasion biology. Mol Ecol Res. 21(4):1318–1332. doi:
10.1111/1755-0998.13342.
Coates BS, Walden KK, Lata D, Vellichirammal NN, Mitchell RF,
Andersson MN, McKay R, Lorenzen MD, Grubbs N, Wang Y-H,
et al. 2023. A draft Diabrotica virgifera virgifera genome: insights
into control and host plant adaption by a major maize pest insect.
BMC Genomics. 24(1):1–24. doi:10.1186/s12864-022-08990-y.
Cohen ZP, Brevik K, Chen YH, Hawthorne DJ, Weibel BD, SchovilleSD. 2021. Elevated rates of positive selection drive the evolutionof pestiferousness in the Colorado potato beetle (Leptinotarsa de­cemlineata, Say). Mol Ecol. 30(1):237–254. doi:10.1111/mec.15703.
Dudchenko O, Batra SS, Omer AD, Nyquist SK, Hoeger M, Durand NC,
Shamim MS, Machol I, Lander ES, Aiden AP, et al. 2017. De novo

Downloaded from https://academic.oup.com/g3journal/article/14/2/jkad266/7471854 by Dennis Hejhal user on 21 May 2024

repeats (TIRs), often being fully inverted and essentially forming
large palindromes (Fig. 4).
The genomes of a few other leaf beetles (Chrysomelidae) havenow been assembled, with larger assembly sizes not obviously
correlating to repeat content. Genomes of Leptinotarsa species
were found to harbor 27–34% repeats and are 512–643 Mb in size
(Cohen et al. 2021), Galerucella species 41–49% and 460–588 Mb(Yang et al. 2021), Ophraella communa 58.2% and 774 Mb(Bouchemousse et al. 2020), Gonioctena quinquepunctata 66% and1.73 Gb (Lukicheva et al. 2021), Diabrotica balteata 47% and1.61 Gb (King et al. 2023), Diabrotica virgifera 53% and 1.85 Gb(Coates et al. 2023), and Altica viridicyanea 63% and 865 Mb (Xueet al. 2021). However, different sequencing technologies wereused across these taxa, which will impact the portion of the repeat
content of the genomes sequenced, assembled, and annotated(Peona et al. 2021). The long-read data that forms the basis for
the C. maculatus assembly likely contributes to the higher repeat
content relative to assembly size compared with other leaf bee­tles. We note that the repeat content varied strikingly along scaf­
folds but no scaffold showed an obvious enrichment in repeats(Fig. 2).
A large portion of the repeat content of C. maculatus was
Tc1-Mariner DNA transposons. Of these, many were nonautono­
mous and palindromic, similar to MITE transposons in plants(Feng et al. 2002; Feschotte et al. 2002). However, the length of these
MITE-like Tc1-Mariner consensus sequences ranges up to 2.2 kb,
which is much larger than the typical size of MITEs (a few hundred
bp; Ye et al. 2016). The full extent of their impact on the overall
genome evolution, for example through their potential forcut-and-paste transposition, will need comparisons with otherspecies. However, it is clear that these large palindromic TEs
make up a significant portion of the C. maculatus repetitive land­
scape. In closing, we note that seed beetles may also provide fu­
ture insights into the possible role of repeats in adaptive
evolution, as genome size is associated with organismal function(Arnqvist et al. 2015; Boman and Arnqvist 2023) and because tran­
scripts related to DNA-mediated transposition show differential
abundance in experimental life history evolution lines in seedbeetles (Immonen et al. 2023).

Chromosome-level assembly of C. maculatus |Marchler-Bauer A, Bo Y, Han L, He J, Lanczycki CJ, Lu S, Chitsaz F,
Derbyshire MK, Geer RC, Gonzales NR, et al. 2017.
CDD/SPARCLE: functional classification of proteins via subfamily
domain architectures. Nucleic Acids Res. 45(D1):D200–D203. doi:
10.1093/nar/gkw1129.
Parisot N, Vargas-Chávez C, Goubert C, Baa-Puyoulet P, Balmand S,
Beranger L, Blanc C, Bonnamour A, Boulesteix M, Burlet N, et al.
2021. The transposable element-rich genome of the cereal pest
Sitophilus oryzae. BMC Biol. 19(1):1–28. doi:10.1186/s12915-021-01158-2.
Peona V, Blom MPK, Xu L, Burri R, Sullivan S, Bunikis I, Liachko I,
Haryoko T, Jønsson KA, Zhou Q, et al. 2021. Identifying the causes
and consequences of assembly gaps using a multiplatform gen­ome assembly of a bird-of-paradise. Mol Ecol Res. 21(1):263–286.
doi:10.1111/1755-0998.13252.
Sallam MN. 2013. Insect Damage: Damage on Post-Harvest. Roma,
Italy: FAO.
Sayadi A, Immonen E, Tellgren-Roth C, Arnqvist G. 2017. The evolu­tion of dark matter in the mitogenome of seed beetles. Genome
Biol Evol. 9(10):2697–2706. doi:10.1093/gbe/evx205.
Sayadi A, Martinez Barrio A, Immonen E, Dainat J, Berger D,
Tellgren-Roth C, Nystedt B, Arnqvist G. 2019. The genomic foot­print of sexual conflict. Nature Ecol Evol. 3(12):1725–1730. doi:
10.1038/s41559-019-1041-9.
Sedlazeck FJ, Lee H, Darby CA, Schatz MC. 2018. Piercing the dark
matter: bioinformatics of long-range sequencing and mapping.
Nat Rev Genet. 19(6):329–346. doi:10.1038/s41576-018-0003-4.
Shumate A, Salzberg SL. 2021. Liftoff: accurate mapping of gene annota­
tions. Bioinf. 37(12):1639–1643. doi:10.1093/bioinformatics/btaa1016.
Smit A, Hubley R. 2010. RepeatModeler open-1.0. http://www.
repeatmasker.org.
Smit A, Hubley R, Green P. 2010. RepeatMasker open-3.3.0. http://
www.repeatmasker.org.
Suh A, Smeds L, Ellegren H. 2018. Abundant recent activity ofretrovirus-like retrotransposons within and among flycatcher
species implies a rich source of structural variation in songbird
genomes. Mol Ecol. 27(1):99–111. doi:10.1111/mec.14439.
Wicker T, Sabot F, Hua-Van A, Bennetzen JL, Capy P, Chalhoub
B, Flavell A, Leroy P, Morgante M, Panaud O, et al. 2007.
A unified classification system for eukaryotic transposable
elements. Nature Rev Genet. 8(12):973–982. doi:10.1038/nrg2165.
Xue HJ, Niu YW, Segraves KA, Nie RE, Hao YJ, Zhang LL, Cheng X-C,
Zhang X-W, Li W-Z, Chen R-S, et al. 2021. The draft genome of thespecialist flea beetle Altica viridicyanea (Coleoptera: Chrysomelidae).
BMC Genom. 22(1):1–18. doi:10.1186/s12864-021-07558-6.
Yang X, Slotte T, Dainat J, Hambäck PA. 2021. Genome assemblies ofthree closely related leaf beetle species (Galerucella spp.). G3(Bethesda) Genes|Genomes|Genetics. 11(8):jkab214. doi:10.1093/
g3journal/jkab214.
Ye C, Ji G, Liang C. 2016. detectMITE: a novel approach to detect mini­
ature inverted repeat transposable elements in genomes. Sci Rep.
6(1):19688. doi:10.1038/srep19688.
Zhang L, Li S, Luo J, Du P, Wu L, Li Y, Zhu X, Wang L, Zhang S, Cui J.
2020. Chromosome-level genome assembly of the predator
Propylea japonica to understand its tolerance to insecticides andhigh temperatures. Mol Ecol Res. 20(1):292–307. doi:10.1111/
1755-0998.13100.

Editor: N. Whiteman

Downloaded from https://academic.oup.com/g3journal/article/14/2/jkad266/7471854 by Dennis Hejhal user on 21 May 2024

assembly of the Aedes aegypti genome using HiC yields
chromosome-length scaffolds. Science 356(6333):92–95. doi:10.
1126/science.aal3327.
Durand NC, Shamim MS, Machol I, Rao SS, Huntley MH, Lander ES,
Aiden EL. 2016. Juicer provides a one-click system for analyzing
loop-resolution HiC experiments. Cell Syst. 3(1):95–98. doi:10.
1016/j.cels.2016.07.002.
Feng Q, Zhang Y, Hao P, Wang S, Fu G, Huang Y, Li Y, Zhu J, Liu Y, Hu
X, et al. 2002. Sequence and analysis of rice chromosome 4.
Nature 420(6913):316–320. doi:10.1038/nature01183.
Feschotte C, Jiang N, Wessler SR. 2002. Plant transposable elements:
where genetics meets genomics. Nature Rev Genet. 3(5):329–341.
doi:10.1038/nrg793.
Feschotte C, Pritham EJ. 2007. DNA transposons and the evolution of
eukaryotic genomes. Ann Rev Genet. 41(1):331–368. doi:10.1146/
annurev.genet.40.110405.090448.
Herndon N, Shelton J, Gerischer L, Ioannidis P, Ninova M, Dönitz J,
Waterhouse RM, Liang C, Damm C, Siemanowski J, et al. 2020.
Enhanced genome assembly and a new official gene set for
Tribolium castaneum. BMC Genom. 21(1):1–13. doi:10.1186/
s12864-019-6394-6.
Holmes LA, Nelson WA, Dyck M, Lougheed SC. 2020. Enhancing theusefulness of artificial seeds in seed beetle model systems re­search. Methods Ecol Evol. 11(12):1701–1706. doi:10.1111/2041210X.13481.
Immonen E, Sayadi A, Stojković B, Savković U, D̵ ord̵ ević M,
Liljestrand-Rönn J, Wiberg RAW, Arnqvist G. 2023. Experimental
life history evolution results in sex-specific evolution of gene ex­pression in seed beetles. Genome Biol Evol. 15(1):evac177. doi:
10.1093/gbe/evac177.
Katoh K, Misawa K, Kuma K, Miyata T. 2002. MAFFT: a novel method for
rapid multiple sequence alignment based on fast Fourier transform.
Nucleic Acids Res. 30(14):3059–3066. doi:10.1093/nar/gkf436.
Keeling CI, Campbell EO, Batista PD, Shegelski VA, Trevoy SAL, Huber
DPW, Janes JK, Sperling FAH. 2022. Chromosome-level genome
assembly reveals genomic architecture of northern range expan­
sion in the mountain pine beetle, Dendroctonus ponderosae Hopkins(Coleoptera: Curculionidae). Mol Ecol Res. 22(3):1149–1167. doi:
10.1111/1755-0998.13528.
Kiełbasa SM, Wan R, Sato K, Horton P, Frith MC. 2011. Adaptive seeds
tame genomic sequence comparison. Genome Res. 21(3):487–493.
doi:10.1101/gr.113985.110.
King R, Buer B, Davies TGE, Ganko E, Guest M, Hassani-Pak K, Hughes
D, Raming K, Rawlings C, Williamson M, et al. 2023. The complete
genome assemblies of 19 insect pests of worldwide importance toagriculture. Pestic Biochem Phys. 191:105339. doi:10.1016/j.
pestbp.2023.105339.
Kohany O, Gentles AJ, Hankus L, Jurka J. 2006. Annotation, submis­
sion and screening of repetitive elements in Repbase:RepbaseSubmitter and Censor. BMC Bioinf. 7(1):474. doi:10.
1186/1471-2105-7-474.
Li H. 2013. Aligning sequence reads, clone sequences and assembly
contigs with BWA-MEM. arXiv:1303.3997. https://doi.org/10.
48550/arXiv.1303.3997.
Lukicheva S, Flot JF, Mardulyn P. 2021. Genome assembly of the coldtolerant leaf beetle Gonioctena quinquepunctata, an important re­source for studying its evolution and reproductive barriers be­
tween species. Genome Biol Evol. 13(7):evab134. doi:10.1093/
gbe/evab134.

7