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. 2025 Dec 23;29(2):114525. doi: 10.1016/j.isci.2025.114525

Male and female genomes of millipede (Trigoniulus corallinus) and identification of sex chromosomes

Yidan Chen 1, Wenyan Nong 1, Wai Lok So 1, Zhe Qu 1,6, Juan Diego Gaitan-Espitia 2, Zhen-peng Kai 3, William G Bendena 4, Kwok Fai Lau 5, Jerome HL Hui 1,7,∗
PMCID: PMC12857412  PMID: 41623493

Summary

Arthropods are arguably the most successful group of metazoans, comprising the majority of described animal species. Millipedes belong to the class Diplopoda of arthropods and are vital decomposers of litter and recycle nutrients in the soil ecosystems worldwide. The molecular mechanism of sex determination in millipedes remains unknown. Here, we sequenced and assembled two high-quality genomes of male and female rusty millipedes Trigoniulus corallinus with sizes of 546 Mb (scaffold N50 = 21.9 Mb) and 630 Mb (scaffold N50 = 20.7 Mb), respectively. Whole-genome resequencing was further carried out on 10 males and 10 females, and sequencing depth analyses showed 1 X chromosome and 2 X chromosomes on male and female genome assemblies, suggesting an XX/X0 system. Synteny analyses of T. corallinus with the centipede Strigamia acuminata showed little conservation between their X chromosomes, implying different selection pressures happened on sex chromosomes after their divergence from the myriapod ancestor. The survey of the sex chromosomes of a millipede species provides the first genomic evidence supporting an XX/X0 system in millipedes. It provides a foundational framework for future studies in myriapod genomics and contributes to a broader understanding of arthropod evolution.

Subject areas: Biological sciences, Zoology, genetics, Phylogenetics, Molecular biology

Graphical abstract

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Highlights

  • •

    The first male and female genomes of a millipede species

  • •

    Genomic evidence suggest XX/XO system in Trigoniulus corallinus

  • •

    Lack of synteny detected between millipede and centipede X chromosome

Biological sciences; Zoology; Genetics; Phylogenetics; Molecular biology

Introduction

The phylum Arthropoda comprises over 80% of all described animal species, including insects, crustaceans (e.g., shrimp and lobsters), chelicerates (e.g., scorpions, spiders, mites, ticks), and myriapods (e.g., millipedes and centipedes). The Myriapoda contains more than 16,000 terrestrial species, all grouped in a close evolutionary relationship with Pancrustacea (i.e., crustaceans and insects). Despite their ecological and evolutionary importance, compared to other arthropods, the myriapods are relatively understudied.

The Myriapoda comprises four classes of extant arthropods: the centipedes, millipedes, pauropods, and symphylans. Several genome sequences of centipedes and millipedes have been obtained, and these studies have provided important insights into the evolution of myriapods and arthropods.1,2,3,4 Regarding the sex determination system in myriapods, an earlier study in centipede Strigamia maritima genome identified a set of genomic scaffolds differentially represented in male and females, and a more recent genome assembly from an individual male centipede Strigamia acuminata suggested an XX/XY system in the centipedes.5,6 Knowledge of the millipede sex determination system primarily comes from karyotyping studies, which have suggested an XX/XY system for most investigated species.7,8,9 Additionally, XX/X0 and XX/XX0 systems have been proposed for few species within the order Spirostreptida (Table 1).

Table 1.

Cytological studies of millipede sex chromosome system

Taxon Sex chromosome Sex of samples Reference
Order: Glomerida
Glomeris connexa XX♀/XY♂ Male Warchałowska-Śliwa et al.10
Glomeris hexasticha XX♀/XY♂ Male and female Warchałowska-Śliwa et al.10
Order: Polydesmida
Chondromorpha mammifera XX♀/XY♂ Male Chowdaiah11
Polydesmus gracilis XX♀/XY♂ Male Achar12
Order: Pseudonannolenida
Pseudonannolene strinatii XX♀/XY♂ Not mentioned Campos and Fontanetti13
Pseudonannolene tocaiensis XX♀/XY♂ Male Fontanetti14
Order: Sphaerotheriida
Arthrosphaera bicolor XX♀/XY♂ Male Chowdaiah and Kanaka15
Arthrosphaera craspedota XX♀/XY♂ Male Ambarish et al.16
Arthrosphaera dalyi XX♀/XY♂ Male Chowdaiah and Kanaka15
Arthrosphaera davisoni XX♀/XY♂ Male Achar17
Arthrosphaera disticta XX♀/XY♂ Male Chowdaiah and Kanaka15
Arthrosphaera fumosa XX♀/XY♂ Male Nair Ambarish and Ramaiah Sridhar18
Arthrosphaera gracilis XX♀/XY♂ Male Chowdaiah and Kanaka15
Arthrosphaera hendersoni XX♀/XY♂ Male Chowdaiah and Kanaka15
Arthrosphaera lutescens XX♀/XY♂ Male Ambarish et al.16
Arthrosphaera magna XX♀/XY♂ Male Achar17
Arthrosphaera nitida XX♀/XY♂ Male Achar17
Arthrosphaera sp. XX♀/XY♂ Male Chowdaiah19
Arthrosphaera sp. XX♀/XY♂ Male Kadamannaya et al.20
Arthrosphaera sp. XX♀/XY♂ Male Achar17
Arthrosphaera zebraica XX♀/XY♂ Male Chowdaiah19
Order: Spirobolida
Aulacobolus excellens XX♀/XY♂ Male Achar21
Cingalobolus sp. XX♀/XY♂ Male Achar22
Rhinocricus sp. XX♀/XY♂ Male Fontanetti23
Trigoniulus sp. XX♀/XY♂ Male Achar22
Order: Spirostreptida
Alloporus araraquarensis XX♀/XY♂ Male and female de Godoy et al.8
Alloporus principes XX♀/XY♂ Male and female de Godoy et al.8
Carlogonus acifer XX♀/XY♂ Male Achar and Chowdaiah24
Carlogonus palmatus XX♀/XY♂ Male Achar22
Gonoplectus malayus XX♀/X0♂ Male Sharma and Handa25
Gymnostreptus olivaceus XX♀/XY♂ Male de Godoy et al.8
Harpurostreptus sp. XX♀/XY♂ Male Chowdaiah11
Ktenostreptus sp. XX♀/XY♂ Male Chowdaiah11
Phyllogonostreptus nigrolabiatus XX♀/XX0♂ Male Sharma and Handa25
Pseudonannolene halophil XX♀/XY♂ Male Fontanetti23
Pseudonannolene ophiulu XX♀/XY♂ Male Fontanetti23
Spirostreptus asthenes XX♀/XY♂ Male Achar26
Thyropygus alienus XX♀/XY♂ Male Achar22
Thyropygus sp. XX♀/XY♂ Male Chowdaiah11
Thyropygus sp. XX♀/XY♂ Male Achar22
Urostreptus atrobrunneus XX♀/XY♂ Male de Godoy et al.8
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Millipedes are usually described as having two pairs of jointed legs on most body segments. This traditional view may be an oversimplification, as developmental studies point instead to the “diplosegment” pattern emerging in evolution through decoupling of dorsal and ventral segmentation processes, not through segment fusion (Janssen et al. 2004). As indicated in their Latin name meaning, i.e., thousand feet, they remain the known animals with the greatest number of legs, with up to 1,306 legs recorded in female Eumillipes persephone.27 The Diplopoda contains >12,000 species making them the largest class of myriapods, and they were among the first animals colonizing land during the Silurian period. They can be found in the terrestrial habitats of all continents except Antarctica and play various important roles in the ecosystem such as decomposers on litter. In some cultures, millipedes have also been consumed as food, used in traditional medicine, or used in converting plant matter into compost. The rusty millipede Trigoniulus corallinus (Gervais, 1847), also known as amber or coral millipede, originates from Asia and has now become a cosmopolitan species found in the America as well.2 It was the first millipede species to have its genome sequenced.2,3 Additionally, it exhibits distinct morphological features that allow for the differentiation of sexes, making it a suitable model for investigation of sex determination systems in the millipedes.

In this study, we investigated the sex determination system of the rusty millipede T. corallinus, which is the first millipede species to have its genome sequenced,2,3,4 in order to better understand the evolution of millipede, myriapod, and arthropod sex determination systems.

Results and discussion

To date, myriapod genomes have primarily been determined from single individual of one sex.1,2,3,4,6,28,29 Sexes of millipede T. corallinus can be easily identified in adults, as males exhibit modified seventh-leg pairs that form gonopods, which are used for mating (Figure 1A). Here, using a combination of PacBio and Omni-C sequencing technologies, high-quality genomes of male and female T. corallinus have been obtained to understand the millipede sex determination system (Figures 1A, 1B, S1, and S2; Table S1). The final male and female T. corallinus genome assemblies were 546 and 630 Mb with scaffold N50 of 22 and 21 Mb, respectively. Comparison of the longest 15 scaffolds between the two genome assemblies, nevertheless, shows that the ones in the male genome (414 Mb) are larger than those of the females (397 Mb). As the female genome assembly has higher proportion of repeats than that of the male, we speculate that the difference in repeat sequence percentages could be a contributing factor to the differences in the male and female genome assemblies’ sizes (Figure S3). 93.8% and 92.4% complete Benchmarking Universal Single-Copy Ortholog (BUSCO) scores for arthropod genes were also revealed in the male and female genomes, respectively (Table 2). In addition, male and female haplotype-resolved genomes were also assembled with similar sizes and quality (Table 2). Comparison of COI gene sequences to published T. corallinus sequences also confirmed species identity (Figure S4). Repeat content contributes to around 70% of both genomes, and a total of 17,180 and 16,930 protein-coding genes were predicted from T. corallinus male and female genomes respectively, comparable to previous studies (Figures 1B, 1C, and S5).3,4

Figure 1.

Figure 1

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Rusty millipede Trigoniulus corallinus (Tco)

(A) Pictures showing the lateral side view of male (left) and female (right) T. corallinus.

(B) Distribution of genes and repeat sequence in Tco male and female genomes.

(C) Repeat content in each scaffold of Tco male (upper) and female (lower) genomes, y axis: percentage of each repeat/non-repeat content.

Table 2.

Statistics of Trigoniulus corallinus (Tco) assemblies

Genome Assembly size No. of scaffold Scaffold N50 No. of contigs Contig N50 BUSCO
Tco-Male 546,000,343 789 21,909,385 839 8,413,389 C: 94.2% [S: 93.8%, D: 0.4%]
F: 2.5%, M: 3.3%, n: 1,667, E: 5.9%
Tco-Male hap1 551,403,766 1,332 20,594,007 1,407 6,862,114 C: 93.9% [S: 93.6%, D: 0.4%]
F: 2.6%, M: 3.5%, n: 1,667, E: 5.9%
Tco-Male hap2 539,124,520 1,158 25,611,796 1,279 6,848,208 C: 91.8% [S: 91.5%, D: 0.4%]
F: 2.7%, M: 5.5%, n: 1,667, E: 5.7%
Tco-Female 629,611,431 386 20,721,897 447 6,970,027 C: 92.8% [S: 92.4%, D: 0.4%]
F: 2.6%, M: 4.6%, n: 1,667, E: 5.9%
Tco-Female hap1 473,745,732 417 19,948,754 481 6,471,690 C: 89.6% [S: 89.3%, D: 0.3%]
F: 2.8%, M: 7.7%, n: 1,667, E: 6.0%
Tco-Female hap2 574,469,078 438 21,871,028 547 5,407,862 C: 92.9% [S: 92.6%, D: 0.4%]
F: 2.6%, M: 4.4%, n: 1,667, E: 5.8%
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Tco-Male/Tco-Female, final version of T. corallinus male/female genome; hap1/hap2, haplotype-resolved genomes that are used for downstream analyses; C, complete BUSCOs; S, complete and single-copy BUSCOs; D, complete and duplicated BUSCOs; F, fragmented BUSCOs; M, missing BUSCOs; n, total BUSCO groups searched.

Based on cytological studies, most millipedes were suggested to contain an XX/XY system, while XX/X0 and XX/XX0 have been suggested for certain species in the order Spirostreptida (Table 2). T. corallinus belongs to the order Spirobolida, and cytological analyses suggested that Trigoniulus contains an XX/XY system.22 To identify the sex chromosomes of T. corallinus, we first aligned the male and female genomes to detect potential X and Y chromosomes with the rationale that either X or Y chromosomes would be expected in each male haplotype-resolved genome, while any Y chromosome sequence could not be aligned to the female genome (Figure 2). In these analyses, one scaffold (TcM_10/TcF_5) was detected in one of the male haplotype-resolved genomes (male haplotype 2, TcMH2_4), and also the female genome sequence, suggesting that this scaffold is the X chromosome. However, we could not detect evidence to support the existence of a Y chromosome based on these analyses. To eliminate the possibility of female heterogamety, female haplotype-resolved genomes were also aligned to male and female genomes, and no putative Z or W sex chromosomes were identified (Figures 3 and S6).

Figure 2.

Figure 2

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Syntenic analyses of male haplotype-resolved genomes

(A) Synteny between male T. corallinus haplotype-resolved genomes (Tco-M_hap1/hap2) and T. corallinus male genome (Tco-M).

(B) Synteny between T. corallinus male haplotype-resolved genomes (Tco-M_hap1/hap2) and T. corallinus female genome (Tco-F).

Figure 3.

Figure 3

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Syntenic analyses of female haplotype-resolved genomes

(A) Synteny between T. corallinus female haplotype-resolved genomes (Tco-F_hap1/hap2) and T. corallinus female genome (Tco-F).

(B) Synteny between T. corallinus female haplotype-resolved genomes (Tco-F_hap1/hap2) and T. corallinus male genome (Tco-M).

Given genome mis-assembly artifacts can potentially result in misidentification of sex chromosomes, we further re-sequenced 20 different individuals of T. corallinus (10 male and 10 female) to test sex chromosome identification by mapping the individual re-sequenced reads to both genome assemblies (Table S2). Again, we did not identify Z-, W-, or Y-linked scaffolds, while the TcM_10 and TcF_5 could be identified as X-linked scaffolds (Figures 4A–4D). Together, our analyses strongly suggest that millipede T. corallinus utilizes an XX/X0 sex system. However, we cannot completely eliminate the possibility of a very small or highly degenerate Y-linked chromosome based on current methods. It is worth mentioning that the understanding of millipede sex chromosomes in this study was based on re-sequencing of 10 males and 10 females, and future studies on individuals collected in other geographical regions will be useful in understanding the selection on autosomes and sex chromosomes.

Figure 4.

Figure 4

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Putative X-chromosome identification and analyses

(A and B) Number and percentage of X-linked windows in Tco (A) female and (B) male genomes; y axis: number of X-linked windows in each scaffold (blue), percentage of X-linked windows in each scaffold (orange).

(C and D) Number and percentage of Y-linked windows in Tco (C) female and (D) male genomes, y axis: number of Y-linked windows in each scaffold (blue), percentage of Y-linked windows in each scaffold (orange).

(E) Synteny between male T. corallinus (Tco-M) and millipede Helicorthomorpha holstii (Hho) genomes (upper), between Tco-M and centipede Strigamia acuminata (Sac) genomes (middle), and between Tco-M and centipede Thereuonema tuberculata (Ttu) genomes.

(F) Gene Ontology enrichment for genes on S. acuminata X chromosome (left) and Y chromosome (right).

(G) Expression of protein-coding genes on putative T. corallinus X chromosome TcM_10 (left) and TcF_5 (right), y axis: transcripts per million (TPM) of each gene.

With the first sequence obtained for a millipede X chromosome, a few important questions can be addressed. To understand the sex chromosome evolution within myriapods, we first investigated the T. corallinus X chromosome scaffolds TcM_10 and TcF_5. The two scaffolds contain only 50 and 42 predicted protein-coding genes, which have much lower gene density and higher repeat content than autosomal scaffolds in T. corallinus (Figure 1B; Table S3). Moreover, most of these protein-coding genes do not show sequence similarities to other genes determined by eggNOG and NCBI ClusteredNR database. As X and Y chromosomes have been reported for the centipede Strigamia acuminata genome,6 we also compared the T. corallinus X chromosome to S. acuminata sex chromosomes and revealed little synteny conservation between them (Figure S7). Unlike T. corallinus X chromosome, S. acuminata X and Y chromosomes contain 1,115 and 178 predicted protein-coding genes respectively, and gene ontology analyses suggested that several gene pathways are enriched (Figure 4F).

Given the availability of two male and two female transcriptomes of T. corallinus generated in previous studies,3,4 we investigated the expression level of the predicted coding genes on the T. corallinus X chromosome to test for the possibility of dosage compensation. The limitation of this analysis was that most of the genes did not show detectable expression in the datasets available; only 4 genes on TcF_5 and 7 genes on TcM_10 had detectable expression, and with low expression levels. We detected evidence for potential sex-biased expression of these genes (Figure 4G), but we caution that in-depth transcriptomic studies are required to draw meaningful conclusions concerning dosage compensation or male-/female-biased expression, as only a few lowly expressed genes were detected in this study.

Conventional understanding of the millipede sex system comes from cytological studies, with the present study providing the first genomic investigation and evidence for an XX/X0 system in a millipede. The situation contrasts with most millipedes, which were suggested or inferred to have the XX/XY system, including species in the order Spirobolida that T. corallinus belongs to. Arthropods comprise majority of extant living animals, including insects, crustaceans, chelicerates, and myriapods. The sex determination systems within these groups have evolved along diverse trajectories. For instance, some insects, such as dipterans (flies) use the XX/XY system, while lepidopterans (butterflies and moths) employ the ZW system. Additionally, occurrences of Y or W chromosome losses and regains have been documented in various insect taxa.30 Given that this study represents the first investigation of sex determination in a single millipede species, it provides valuable insights into the evolution of sex systems within different myriapod lineages. Here, we propose that T. corallinus possesses an XX/X0 sex determination system, based on genome assemblies and resequencing of individuals. This contrasts with previous findings suggesting that Trigoniulus genus utilizes an XX/XY system, as determined by karyotyping (Achar, 1984). Given the limited number of millipede genomes investigating sex determination, it is early or premature to definitively conclude whether the sex system in millipedes is subject to rapid turnover or evolutionary change, potentially varying across different lineages, species, or populations. It will be important to investigate whether the situation in T. corallinus represents a typical or a secondarily derived situation in millipedes and examine the mode of Y chromosome degeneration if the XX/X0 system is derived. Indeed, male heterogamety may have been subject to different selection pressures in millipede and centipede evolution since their divergence from a common myriapod ancestor. Additional male and female pair of millipede genomes would be needed to differentiate between different possibilities and scenarios and to better understand sex chromosome evolution in arthropods.

Limitations of the study

The understanding of millipede sex chromosomes in this study was based on re-sequencing of 10 males and 10 females; future studies on individuals collected in other geographical regions will be useful in understanding the selection on autosomes and sex chromosomes. In-depth transcriptomic studies will also be required to draw meaningful conclusions concerning dosage compensation or male-/female-biased expression, as only a few lowly expressed genes were detected in this study.

Resource availability

Lead contact

Requests for further information and resources should be directed to and will be fulfilled by the lead contact, Jerome Ho Lam Hui (jeromehui@cuhk.edu.hk).

Materials availability

This study did not generate new unique reagents.

Data and code availability

The data have been deposited to BioProject accession PRJNA1311309 and CUHK Research Data Repository (https://researchdata.cuhk.edu.hk/dataset.xhtml?persistentId=doi:10.48668/LWA9UV).

Acknowledgments

This work was funded by the Hong Kong Research Grant Council General Research Fund (14100919) and Collaborative Research Fund (C4015-20EF). Y.C. was funded by a studentship provided by The Chinese University of Hong Kong. We thank Peter Holland for comments on the manuscript.

Author contributions

Conceptualization, J.H.L.H.; investigation and formal analysis, Y.C., W.N., and J.H.L.H.; data curation, Y.C. and W.N.; visualization, Y.C.; writing – original draft, J.H.L.H. and Y.C.; writing – review & editing, all authors; supervision, J.H.L.H.

Declaration of interests

The authors declare no competing interests.

STAR★Methods

Key resources table

REAGENT or RESOURCE SOURCE IDENTIFIER
Biological samples
Trigoniulus corallinus Hong Kong –
Critical commercial assays
NucleoBond HMW DNA kit Macherey-Nagel –
Dovetail Omni-CTM kit Dovetail Genomics –
PureLink™ Genomic DNA Mini Kit Invitrogen™ –
Deposited data
T. corallinus PacBio sequencing data (male and female) This paper CUHK Research Data Repository (https://researchdata.cuhk.edu.hk/dataset.xhtml?persistentId=doi:10.48668/LWA9UV)
T. corallinus Omni-C sequencing data (male and female) This paper CUHK Research Data Repository (https://researchdata.cuhk.edu.hk/dataset.xhtml?persistentId=doi:10.48668/LWA9UV)
T. corallinus male and female genomes with annotations This paper CUHK Research Data Repository (https://researchdata.cuhk.edu.hk/dataset.xhtml?persistentId=doi:10.48668/LWA9UV)
T. corallinus male and female haplotype-resolved genomes This paper CUHK Research Data Repository (https://researchdata.cuhk.edu.hk/dataset.xhtml?persistentId=doi:10.48668/LWA9UV)
T. corallinus resequencing data (10 males and 10 females) This paper CUHK Research Data Repository (https://researchdata.cuhk.edu.hk/dataset.xhtml?persistentId=doi:10.48668/LWA9UV)
T. corallinus genome assembly Qu et al.3 NCBI: JAAFCE000000000
T. corallinus transcriptome sequencing data Qu et al.3 NCBI: PRJNA564195
Strigamia acuminata genome with annotations Edgecombe et al.6 Darwin Tree of Life Data Portal GCA_949358305.1
Helicorthomorpha holstii genome Qu et al.3 NCBI: JAAFCF000000000
Thereuonema tuberculata genome So et al.4 NCBI: JAFIDM000000000
Software and algorithms
hifiasm (version 0.19.8) Cheng et al.31 https://github.com/chhylp123/hifiasm
purge_dups (version 0.0.3) – https://github.com/dfguan/purge_dups
BWA (version 0.7.17) Li et al.32 https://github.com/lh3/bwa
pairtools (version 1.0.2) Open2C et al.33 https://github.com/open2c/pairtools
SAMtools (version 1.18) Li et al.34 https://github.com/samtools/samtools
YaHS (version 1.2a.2) Zhou et al.35 https://github.com/c-zhou/yahs
BUSCO software (version 5.8.2) Manni et al.36 https://gitlab.com/ezlab/busco
BLASTN (version 2.14.1) Altschul et al.37 https://blast.ncbi.nlm.nih.gov/Blast.cgi
Earl Gray TE annotation pipeline (version 5.0.0) Baril et al.38 https://github.com/TobyBaril/EarlGrey
RepeatMasker (version 4.1.5) – https://www.repeatmasker.org/RepeatMasker/
Trimmomatic (version 0.39) Bolger et al.39 https://github.com/usadellab/Trimmomatic
Kraken2 (version 2.0.8) Wood et al.40 https://github.com/DerrickWood/kraken2
Funannotate genome annotation pipeline (version 1.8.17) Palmer et al.41 https://github.com/nextgenusfs/funannotate
eggNOG (version 2.1.3) Cantalapiedra et al.42 https://github.com/eggnogdb/eggnog-mapper
NGenomeSyn (version 1.42) He et al.43 https://github.com/hewm2008/NGenomeSyn
minimap2 (version 2.24-r1122) Li et al.44 https://github.com/lh3/minimap2
picard toolkit (version 3.1.1) – https://broadinstitute.github.io/picard/
HISAT2 (version 2.2.1) Kim et al.45 https://daehwankimlab.github.io/hisat2/
FeatureCounts (version 2.0.6) Liao et al.46 https://subread.sourceforge.net/featureCounts.html
clusterProfiler (version 4.10.1) Wu et al.50 https://github.com/YuLab-SMU/clusterProfiler
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Experimental model and study participant details

In this study, Trigoniulus corallinus (12 male and 12 female) with mixed development stage were collected from the campus of The Chinese University of Hong Kong (CUHK) (10 males and 9 females), WWF Island House Conservation Studies Center Yuen Chau Tsai Island (1 female), KukPo (1 male), Tai O (1 female), and High Island Reservoir (East Dam) (1 male and 1 female). They were cultured in plastic boxes containing gardening soil, rotting leaves and woods. Distilled water was sprayed to the surface of the substratum from time to time to maintain the humidity of the soil, and apple slices were also provided as food.

Method details

Experimental model

In this study, male and female Trigoniulus corallinus used for sequencing were collected from the campus of The Chinese University of Hong Kong (CUHK), WWF Island House Conservation Studies Center Yuen Chau Tsai Island, KukPo, Tai O, and High Island Reservoir (East Dam). Animals were kept in plastic boxes containing gardening soil, rotting leaves, and woods as previously described.2,3,4 Distilled water was sprayed to the surface of the substratum from time to time to maintain the humidity of the soil, and apple slices were also provided as food.

Genome sequencing and assembly

Genomic DNA of male and female T. corallinus was extracted using the NucleoBond HMW DNA kit (Macherey-Nagel) following the manufacturer’s instructions. Quality of the extracted DNA was checked with Qubit fluorometer and pulsed-field gel electrophoresis. Qualified genomic DNA samples were further used for PacBio library preparation and sequencing at The Chinese University of Hong Kong. Omni-C libraries (Dovetail Genomics) were also constructed for male and female T. corallinus following the manufacturer’s instructions, and sent to Novogene (Hong Kong) for further sequencing on Illumina platform. To obtain male and female T. corallinus genomes as well as the haplotype-resolved genomes, de novo genome assembly was carried out with hifiasm (version 0.19.8)31 for PacBio high-fidelity (HiFi) reads, then purge_dups (version 0.0.3) (https://github.com/dfguan/purge_dups) was used to identify and remove haplotypic duplication. Draft genome assemblies were further scaffolded by Dovetail Omni-C reads. In brief, read pairs were firstly mapped to the genome by BWA (version 0.7.17)32 with the parameter 'mem -5SP -T0'. Then, pairtools (version 1.0.2)33 was applied to process pair-end sequence alignments with the parameter “parse --min-mapq 40 --walks-policy 5unique --max-inter-align-gap 30 --nproc-in 8 --nproc-out 8 --chroms-path”, “sort --nproc 100”, “dedup --nproc-in 8 --nproc-out 8 --mark-dups --output-stats” and “split --nproc-in 8 --nproc-out 8 --output-pairs --output-sam”, and sam file was converted to bam file by SAMtools (version 1.18).34 The result bam file was used as the input of YaHS (version 1.2a.2)35 to scaffold the genome assembly. Read depth of Dovetail Omni-C reads for each genome was obtained by “samtools coverage” and further normalized by the average read depth of the genome. For quantitative assessment of genome assembly completeness, BUSCO software (version 5.8.2)36 was applied with the parameter “-m genome -l arthropoda_odb12”. To confirm the species identify, the published T. corallinus mitochondrial cytochrome oxidase subunit I (COI) gene sequence2 was aligned to male and female T. corallinus genomes by BLASTN (version 2.14.1)37 with the parameter “-evalue 0.001”.

Repeat annotation

Repeat sequences were annotated in male and female T. corallinus genomes to obtain soft-masked genomes for gene model prediction, using Earl Gray TE annotation pipeline (version 5.0.0) (https://github.com/TobyBaril/EarlGrey)38 with parameter “-r arthropoda -d yes -e yes”. For RepeatMasker (version 4.1.5) (https://www.repeatmasker.org/RepeatMasker/) that was used in the Earl Gray TE annotation pipeline, Dfam (version 3.7)39 and RepBase (RepeatMaskerEdition-20181026)40 databases were used. To compare the repeat contents between millipede and centipede, the genome sequence of a centipede Strigamia acuminata was downloaded from Darwin Tree of Life Data Portal (https://portal.darwintreeoflife.org; accession GCA_949358305.1; Edgecombe et al. 2023), with Earl Gray TE annotation pipeline applied using the same parameters.

Gene model prediction and function annotation

T. corallinus transcriptome sequencing data from previous studies3,4 were used in gene model prediction for T. corallinus male and female genomes. Trimmomatic (version 0.39)41 was applied to process raw reads with parameters “ILLUMINACLIP:TruSeq3-PE.fa:2:30:10 SLIDINGWINDOW:4:5 LEADING:5 TRAILING:5 MINLEN:25”, followed by Kraken2 (version 2.0.8)42 for classifying contamination with the default parameter. To detect protein-coding genes in T. corallinus genomes, the Funannotate genome annotation pipeline (version 1.8.17)43 was run with the soft-masked male and female genomes and cleaned transcriptome sequencing reads as input files. In brief, transcriptome sequencing data was used to obtain de novo genome-guided transcriptome assembly using “funannotate train” with parameters “--stranded RF --max_intronlen 350000 --no_trimmomatic”, followed by “funannotate predict” to generate consensus gene models with parameters “--genemark_mode ET --busco_seed_species fly --optimize_augustus --busco_db arthropoda --organism other --max_intronlen 350000”. UniProtKb/SwissProt protein evidence from UniProt (https://www.uniprot.org) (2025/02/26) was used in the “funannotate predict” step, and “funannotate update” with parameters “--no_trimmomatic --stranded RF” was used to update predicted gene models. For gene function annotation for predicted genes, eggNOG (version 2.1.3)44 was used with parameters “-m diamond --evalue 0.001 --score 60 --pident 40 --query_cover 20 --subject_cover 20 --itype proteins --tax_scope auto --target_orthologs all --go_evidence non-electronic --pfam_realign none --report_orthologs --decorate_gff yes”.

Genome synteny analyses

To reveal the syntenic relationships between male and female T. corallinus genomes, NGenomeSyn (version 1.42)45 was applied. In brief, the GetTwoGenomeSyn.pl script in NGenomeSyn was used to obtain link files by minimap2 (version 2.24-r1122)46 with parameters “-MappingBin minimap2 -MinAlnLen 10000” for scaffolds that are longer than 10 Mb. NGenomeSyn was then used to visualise the syntenic relationships. For the centipede S. acuminata, GetTwoGenomeSyn.pl was run with parameters “-MappingBin minimap2 -MinAlnLen 100 -MappingPara -x asm20”.

Resequencing of male and female individuals, and sex chromosome identification

Genomic DNA of 10 male T. corallinus and 10 female T. corallinus were extracted using the PureLink Genomic DNA Mini Kit (Invitrogen) following the manufacturer’s instructions, with DNA eluted with nuclease-free water. DNA quality was checked with gel electrophoresis and sent to Novogene (Hong Kong) for sequencing on Illumina platform. Trimmomatic (version 0.39) was used to process raw reads with parameters “ILLUMINACLIP:TruSeq3-PE.fa:2:30:10 SLIDINGWINDOW:4:5 LEADING:5 TRAILING:5 MINLEN:25”, and Kraken2 (version 2.0.8) was used to remove contamination with default parameters. To reveal the putative sex chromosome in T. corallinus genomes, cleaned whole genome resequencing data of 10 males and 10 females were aligned to male and female T. corallinus genomes using BWA (version 0.7.17-r1188) with parameters “mem -M”. The result SAM files were then converted to BAM files and further sorted with SAMtools (version 1.18). MarkDuplicates in the picard toolkit (version 3.1.1) (https://broadinstitute.github.io/picard/) was then applied to remove PCR duplications. Read-depth was calculated in 5-kb windows with SAMtools (version 1.18) and normalised by the average read depth of each sample. Windows with female/male read depth ≥ 1.5 were defined as X-linked windows, while windows with female/male read depth ≤ 0.3 were defined as Y-linked windows.47 In addition, Z-linked and W-linked windows were also checked with male/female read depth ≥ 1.5 and ≤ 0.3, respectively. Scaffolds with more than 50% sex chromosome-linked windows were considered as putative sex chromosome.

Sex-biased gene expression analyses on X chromosome

Gene expression level for published transcriptomic data of 2 males and 2 females T. corallinus3 on the X chromosome was shown using HISAT2 (version 2.2.1)48 with default parameters. Resulting SAM files were further converted to BAM files and sorted with SAMtools (version 1.18), and MarkDuplicates program in the picard toolkit (version 1.18) (https://broadinstitute.github.io/picard/) was applied to remove PCR duplications. FeatureCounts (version 2.0.6)49 was further applied with parameter “-p”, and Transcripts Per Million (TPM) was calculated to measure gene expression level for different samples.

Quantification and statistical analysis

GO enrichment in this study was done by clusterProfiler (version 4.10.1)50 package in R (version 4.3.3).

Published: December 23, 2025

Footnotes

Supplemental information can be found online at https://doi.org/10.1016/j.isci.2025.114525.

Supplemental information

Document S1. Figures S1–S7 and Tables S1–S3
mmc1.pdf (1.6MB, pdf)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document S1. Figures S1–S7 and Tables S1–S3
mmc1.pdf (1.6MB, pdf)

Data Availability Statement

The data have been deposited to BioProject accession PRJNA1311309 and CUHK Research Data Repository (https://researchdata.cuhk.edu.hk/dataset.xhtml?persistentId=doi:10.48668/LWA9UV).

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