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. 2023 Feb 17;13(2):e9816. doi: 10.1002/ece3.9816

Accumulation of gene copy number variations during the early phase of free‐spawning abalone speciation

Shotaro Hirase 1,✉, Masashi Sekino 2, Motoyuki Hara 3, Kiyoshi Kikuchi 1
PMCID: PMC9936805  PMID: 36818538

Abstract

The genetic basis of speciation in free‐spawning marine invertebrates is poorly understood. Although gene copy number variations (GCNVs) and nucleotide variations possibly trigger the speciation of these organisms, empirical evidence for such a hypothesis is limited. In this study, we searched for genomic signatures of GCNVs that may contribute to the speciation of Western Pacific abalone species. Whole‐genome sequencing data suggested the existence of significant amounts of GCNVs in closely related abalones, Haliotis discus and H. madaka, in the early phase of speciation. In addition, the degree of interspecies genetic differentiation in the genes where GCNVs were estimated was higher than that in other genes, suggesting that nucleotide divergence also accumulates in the genes with GCNVs. GCNVs in some genes were also detected in other related abalone species, suggesting that these GCNVs are derived from both ancestral and de novo mutations. Our findings suggest that GCNVs have been accumulated in the early phase of free‐spawning abalone speciation.

Keywords: ecological speciation, marine invertebrates, marine speciation, Western Pacific abalones, whole‐genome sequencing

Although gene copy number variations (GCNVs) and nucleotide variations possibly trigger the speciation of free‐spawning marine invertebrates, empirical evidence for such a hypothesis is limited. Our findings suggest that the accumulation of GCNVs contributes to the speciation of free‐spawning marine invertebrates during the early phase.

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1. INTRODUCTION

A major goal in marine biology is understanding the genetic basis of speciation in free‐spawning marine invertebrates (Pogson, 2016). Genomic analyses based on single nucleotide polymorphism (SNP) loci have identified candidates for genetic variation that contributes to marine speciation (Hirase et al., 2021; Momigliano et al., 2017). However, a growing body of evidence suggests that gene copy number variations (GCNVs) also play an important role in the ecological divergence of various organisms (Castagnone‐Sereno et al., 2019; Hirase et al., 2014; Ishikawa et al., 2019; Pezer et al., 2015) including marine species (Dorant et al., 2020). Therefore, GCNVs, as well as nucleotide variations, may trigger speciation in the ocean; however, empirical evidence for these hypotheses is limited.

In the current study, we focus on GCNVs in the Western Pacific abalones, Haliotis discus, H. madaka, and H. gigantea (Ino, 1952). These three species are estimated to have diverged recently from North America and are genetically close to the North American abalones (Geiger & Groves, 1999; Hirase et al., 2021). Population genomic analyses of these species revealed that although the three species are genetically distinct, there is evidence of historical and ongoing gene flow among these species. The most closely related pair, H. discus and H. madaka (genome‐wide F ST = 0.007), appears to occupy the early stages of the speciation continuum after the initial divergence of H. gigantea (Hirase et al., 2021). In the current study, we searched for GCNVs between H. discus and H. madaka based on whole‐genome sequencing (WGS) data. Our results showed the possibility of GCNVs accumulating during their speciation event. To further investigate whether the candidate GCNVs were derived from de novo or standing genetic variations, we examined GCNVs in H. gigantea and North American abalones that are expected to be genetically close to the Western Pacific abalones.

2. MATERIALS AND METHODS

2.1. Alignment of whole‐genome sequencing data

WGS data from the Western Pacific (eight H. discus, eight H. madaka, and six H. gigantea) and North American abalone species (three H. rufescens) were used. These were generated using Illumina HiSeq X Ten with the 150‐bp paired‐end protocol in our previous study (Hirase et al., 2021; Table A1 in Appendix A). The WGS data of five other North American abalone species (two individuals per species) were downloaded from the NCBI SRA (SRR7958743−SRR7958752; Masonbrink et al., 2019). The paired‐end reads were cleaned and aligned to the H. discus  hannai genome sequence (Nam et al., 2017) as described by Hirase et al. (2021). PCR duplicates were removed from constructed BAM files using SAMtools ver. 1.9 markdup (Li et al., 2009).

2.2. Identification of gene copy number variations

We compared the number of mapped reads for each gene, which was predicted in a previous study (Hirase et al., 2021), between H. discus and H. madaka using BAM files (Table A1 in Appendix A), and identified candidate GCNVs by referring to Hirase et al. (2014). If the number of mapped reads was significantly larger in one species, the gene would have been duplicated or multiplied specifically in that species. By contrast, if the numbers were significantly smaller, the gene would have been deleted, or its copy number would have decreased. Briefly, the number of mapped reads that overlapped with predicted gene regions (i.e., any exonic or intronic region) was counted using the featureCounts function of Subread ver. 1.4.6 (Liao et al., 2014). We removed genes onto which no reads were mapped in at least one individual, because an insufficient number of mapped reads may result in the detection of false GCNVs. We then searched for candidate GCNVs by detecting genes that showed significant differences in normalized read numbers between H. discus and H. madaka using the edgeR software package (Robinson et al., 2010) with a false discovery rate (FDR) < 0.01. For normalization with edgeR, the total number of mapped reads across the genome of each individual was used. To confirm that the number of identified GCNVs was significantly larger than expected by chance, we calculated an empirical p‐value based on a permutation test. In this test, we randomly reallocated eight H. discus and eight H. madaka individuals into two groups 10,000 times, performed the above analyses for each generated dataset, and obtained the null distribution of the numbers of GCNVs. Gene functions were annotated using BLASTP searches against the NCBI nonredundant (nr) protein database. We conducted BLASTN searches for candidate GCNVs against four North American abalone genomes (E‐value < 0.0000001): H. fulgens (halful_medaka.final_.fasta; https://abalone.dbgenome.org/downloads), H. sorenseni (h.sorensenigenomepilon3.pilon_.fasta; https://abalone.dbgenome.org/downloads), H. rufescens (Halruf.fasta; https://abalone.dbgenome.org/downloads; Masonbrink et al., 2019), and H. cracherodii (GCA_022045235; Orland et al., 2022).

To compare genetic differentiation between H. discus and H. madaka within and outside GCNVs, we calculated F ST values for nonoverlapping 1 kb sliding windows based on SNP loci using VCFtools ver. 1.1.12 (Danecek et al., 2011). For this calculation, SNP information (a vcf file) that was obtained in Hirase et al. (2021) was used; In this study, SNPs were called for H. discus and H. madaka, H. gigantea using SAMtools mpileup (−Q 30) and bcftools, and filtered using VCFtools (‐‐minQ 20 ‐‐remove‐indels ‐‐maf 0.05 ‐‐max‐alleles 2 ‐‐minDP 6 ‐‐max‐missing‐count 1). This SNP information was used for the detection of three‐or‐more‐different allelic pairs later (see next section).

2.3. Detection of three‐or‐more‐different allelic pairs

Three‐or‐more‐different allelic pairs (≥3 allele sequences) within a gene could be evidence of the GCNVs because ≥3 allele sequences of a gene cannot occur in a diploid genome (Hirase et al., 2014). Therefore, we examined whether ≥3 allele sequences were observed in the candidate GCNVs between H. discus and H. madaka, which were detected by read‐depth‐based analyses. The ≥3 allele sequences of candidate GCNVs were also identified in H. gigantea. In this analysis, we enumerated every pair of SNP positions, for each of the identified candidate GCNVs, that were located within 100 bp (within‐read‐length single nucleotide variation: SNP position pairs) using BAM files, the vcf file, and a custom Perl script (deposited in Dryad). Briefly, the number of different nucleotide pairs for each of the within‐read‐length SNP position pairs, which were supported by multiple (≥2) reads (by taking into account sequencing error) was counted. Then, we selected candidate GCNVs that were supported at least by one ≥3 allele sequence in at least three individuals from either species (Figure A1 in Appendix A). The ≥3 allele sequences in candidate GCNVs were also identified in North American abalone species. SNPs of North American abalones were called separately from those of the Western Pacific abalones, using the same method mentioned above, and filtered using VCFtools (−‐minDP 10 ‐‐remove‐indels ‐‐max‐missing‐count 0).

3. RESULTS

Significant differences in the numbers of mapped reads between H. discus and H. madaka (FDR < 0.01) were observed for 627 genes (Figure 1a), and this number was significantly higher than expected by chance (p < .05). This suggests the accumulation of gene copy number differences between the two species. Among these genes, 328 genes were expected to have more copies in H. discus (called as HD‐increased GCNVs), and 299 genes were expected to have more copies in H. madaka (called as HM‐increased GCNVs). Next, we compared the sliding‐window F ST within genes, where GCNVs were estimated, with those of all genes. The level of genetic differentiation in the genes where GCNVs were estimated was higher than that in all genes (Wilcoxon rank sum test; p < .05; Figure 1b).

FIGURE 1.

FIGURE 1

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(a) MA plot showing the relationship between average concentration (logCPM) and fold‐change (logFC) across the genes. Each gene is represented by an open dot. Genes that showed significant differences in the number of mapped reads between Haliotis discus and H. madaka (FDR < 0.01) are colored in red. (b) Boxplot and half‐eye plot for sliding‐window F ST values within all genes and those where gene copy number variations were expected.

Among the 328 HD‐increased GCNVs and 299 HM‐increased GCNVs, we focused on the top 10 HD‐increased GCNVs (Table A2 in Appendix A) and the top 10 HM‐increased GCNVs (Table A3 in Appendix A ) and found that the top 10 HD‐increased GCNVs included the small heat shock protein 20 (sHSP20) gene (STRG15773). In the reference genome of H. discus hannai (Nam et al., 2017), two sHsp20 genes were annotated as STRG.15773 (HDSC00791:156806‐157861) and STRG.24666 (HDSC01558:56682‐58572), which were predicted in our previous study (Hirase et al., 2021). Of the two genes, we detected HD‐increased GCNVs only in STRG15773 but not in STRG.24666 (Figure 2). Additionally, our BLASTN search suggested that there was only one sHsp20 gene in the genome assemblies of the four North American abalones: H. fulgens, H. sorenseni, H. rufescens, and H. cracherodii.

FIGURE 2.

FIGURE 2

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Distributions of normalized read depth in two genes possibly encoding small heat shock protein 20 (sHSP), STRG.15773 and STRG.24666. STRG.15773 is an HD‐increased gene copy number variation gene that was supported by the difference in the number of mapped reads between eight Haliotis discus and eight H. madaka individuals. Each boxplot represents the normalized read depth and average normalized read depth, respectively, of the mapped reads per 200‐bp nonoverlapping window for eight H. discus and eight H. madaka individuals. For normalization, the number of mapped read in each region was divided by the total number of mapped reads across the genome and multiplied by 10 million. Gene models are shown at the bottom of each panel.

Among the candidate GCNVs detected, four HD‐increased GCNVs had ≥3 allele sequences in at least three H. discus individuals and none in H. madaka individuals (Figure 3 ). Similarly, four HM‐increased GCNVs were supported by ≥3 allele sequences in at least three H. madaka individuals and no H. discus individuals (Figure 3). Among the eight genes where ≥3 allele sequences were detected in either H. discus or H. madaka, five genes also had ≥3 allele sequences in H. gigantea (Figure 3). Additionally, two of these five genes also had ≥3 allele sequences in the North American abalones (Figure 3 ). Although there were ≥3 allele sequences in one HM‐increased GCNV, STRG.16819, in all North American abalones, our BLASTN searches detected duplications/multiplications of this gene in the genome assembly of H. sorenseni, but not in those of the three North American abalone species, H. fulgens, H. rufescens, and H. cracherodii.

FIGURE 3.

FIGURE 3

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Four HD‐increased and four HM‐increased gene copy number variations that showed ≥3 allele sequences in at least three individuals. Each column represents an individual of each species, and the number in the box indicates the number of regions where ≥3 allele sequences were observed as shown in Figure A1 in Appendix A. Highlighted boxes show individuals that had ≥3 allele sequences in each gene. The ≥3 allele sequences were also identified in Haliotis gigantea and the six North American abalone species (Red: H. rufescens; Black: H. cracherodii; Green: H. fulgens; Pink: H. corrugate; Pinto: H. kamtschatkana; White: H. sorenseni).

4. DISCUSSION

Gene copy number variations can cause organisms to inhabit new ecological niches (Ishikawa et al., 2019). We obtained genomic evidence of significant amounts of GCNVs between H. discus and H. madaka, which have different ecological niches and have recently speciated (Hirase et al., 2021). This result suggests that the accumulation of gene copy number differences is present in the early speciation stages of free‐spawning abalones. In addition, the degree of genetic differentiation in the genes where GCNVs were estimated was higher than that in other genes, consistent with previous observations that many CNVs were found in genes for which SNP‐based analyses detected signatures of positive selection (Feulner et al., 2013; Gokcumen et al., 2011; Hirase et al., 2014). This trend possibly suggests that nucleotide divergence also accumulates in the genes with GCNVs in abalones. Alternatively, there may be some biases in the F ST estimates in the genes with GCNVs because alignments of multiple copies to one reference genome can cause the increased SNP variations in these genes (Feulner et al., 2013). This trend needs to be examined in more detail in the future.

Among the 627 candidate GCNVs detected, those in eight genes were confirmed by detecting ≥3 allele sequences in either H. discus or H. madaka. The method based on ≥3 allele sequences is not suitable for the detection of recently generated gene duplications, but instead robustly detects GCNVs of genetically distant species, because it does not depend on the efficiency of read mapping onto the reference genome, unlike depth‐based analysis (Nijkamp et al., 2012). We found that six of the eight genes had ≥3 allele sequences in H. gigantea and/or the North American abalone species. These results suggest that GCNVs between H. discus and H. madaka, which have recently diverged, are derived from both de novo (Zarrei et al., 2018) and standing genetic variations (Feulner et al., 2013). Within one gene (STRG.16819), we detected ≥3 allele sequences in more genomic regions in all the North American abalones species than in H. discus and H. madaka, which is consistent with the expectation that nucleotide mutations between standing duplicated genes have accumulated in the ancestral North American abalone species.

Our findings may suggest future issues regarding the genome assembly of abalones. Although the ≥3 allele sequences in one HM‐increased GCNV, the STRG.16819 gene, was detected in all North American abalone species, our BLASTN searches detected duplications/multiplications of genes in the genome assembly of H. sorenseni, but not in those of three North American abalone species, H. fulgens, H. rufescens, and H. cracherodii. Given that the genome assembly of H. cracherodii is a chromosome‐level assembly based on PacBio HiFi long‐reads (Orland et al., 2022), our results may imply that these GCNVs would have resulted from long segmental duplications/multiplications of genomic regions, which are longer than the PacBio HiFi long‐reads and remain unresolved (Vollger et al., 2019).

HD‐increased GCNVs included one of two genes that encode sHSP20 in the H. discus hannai genome. HSPs are the primary mitigators of environmental stress in various organisms (Chen et al., 2018) including abalones (Farcy et al., 2007; Huang et al., 2014; Kyeong et al., 2020). Variations in the copy number of Hsp genes have been found in diverse organisms. For example, in Drosophila, it has been suggested that Hsp70 genes evolved into seven copies in thermotolerant species (Evgen'ev et al., 2004). Hsp genes comprise several families based on their molecular weights (Kampinga et al., 2009). Among these, sHsp genes are the smallest members of the Hsp superfamily. In H. discus, two sHsp genes, sHsp26 and sHsp20, have been reported. The expression of these genes occurs in multiple tissues and is strongly affected by environmental stress (Park et al., 2008; Wan et al., 2012). In particular, sHsp20 mRNA expression is rapidly elevated upon exposure to thermal, oxidative, and multiple toxic metal stresses (Wan et al., 2012), suggesting that this gene contributes to the adaptation of abalones to various environments. Since there was only one sHsp20 in the genomes of the four North American abalones, the sHsp20 gene duplication may have occurred after the speciation of Western Pacific abalone from the North American abalones (Geiger & Groves, 1999; Hirase et al., 2021), and that the copy number of STRG15773 increased specifically in H. discus. Previous gene expression analyses have indicated that the sHsp20 gene is likely involved in abalone defenses against extreme environmental stress (Wan et al., 2012). Compared with other Western Pacific abalones, H. discus inhabits shallow depth zones where environmental fluctuations are more intense because of the influx of freshwater and the effects of varying water temperatures (Sinex, 1994). Therefore, it is possible that sHsp20 is involved in the ecological adaptation of H. discus.

Among the eight GCNVs in which ≥3 allele sequences were detected, one HM‐increased GCNV was annotated as a mucin gene (Figure 3). Aquatic invertebrates protect the surfaces of their bodies, gills, and intestines with a mucus layer, which is composed of mucin glycoproteins (Bakshani et al., 2018). The mucus layer serves as an antimicrobial barrier and physical protective layer and has several physiological functions (Stabili, 2019). Copy number variations of mucin genes may affect the adaptive divergence between H. discus and H. madaka. The expression of this gene has been reported to respond to thermal stress in hybrids of H. discus hannai and H. gigantea (Xiao et al., 2021). Given that mucin genes belong to multigenic families (Desseyn et al., 1997), this finding is consistent with the idea that GCNVs of multigenic family genes are more likely to occur than those of single‐copy genes (Hirase et al., 2014; Nguyen et al., 2006).

This study provides the first empirical data showing GCNVs in the early phase of marine invertebrate speciation and suggests that GCNVs accumulate in the early phase of marine invertebrate speciation. In addition, some GCNVs were detected in ancestral species, suggesting that GCNVs are derived from both ancestral and de novo mutations.

AUTHOR CONTRIBUTIONS

Shotaro Hirase: Investigation (lead); methodology (lead); writing – original draft (lead). Masashi Sekino: Resources (supporting); writing – review and editing (supporting). Motoyuki Hara: Resources (lead). Kiyoshi Kikuchi: Supervision (lead); writing – review and editing (equal).

FUNDING INFORMATION

This work was supported by the Japan Society for the Promotion of Science (KAKENHI 21580240, 17K19280, 22H00377).

CONFLICT OF INTEREST STATEMENT

We declare no competing interests.

ACKNOWLEDGMENTS

The authors are grateful to members of the Kikuchi laboratory for their helpful comments on this research.

APPENDIX A.

TABLE A1.

Summary of BAM files that used for identifying gene copy number variations in abalones.

Sample Accession number Species Depth Coverage (≥5 depth) Mapped_read
HDD_Iwafune02 DRR276800 Haliotis discus discus 8.95 0.447 90131141
HDD_Iwafune04 DRR276801 Haliotis discus discus 9.29 0.456 92970560
HDD_Shibushi01 DRR276798 Haliotis discus discus 8.06 0.412 80852568
HDD_Shibushi02 DRR276799 Haliotis discus discus 8.95 0.441 88559647
HDD_Goto01 DRR276794 Haliotis discus discus 9.39 0.451 89424345
HDD_Goto02 DRR276795 Haliotis discus discus 10.31 0.470 98078183
HDD_Izu01 DRR276796 Haliotis discus discus 8.69 0.431 88093824
HDD_Izu02 DRR276797 Haliotis discus discus 9.07 0.444 89655629
HM_Misaki01 DRR276780 Haliotis madaka 9.30 0.446 88899019
HM_Misaki14 DRR276781 Haliotis madaka 9.92 0.459 92831843
HM_Misaki22 DRR276806 Haliotis madaka 11.12 0.480 101515775
HM_Misaki23 DRR276807 Haliotis madaka 10.69 0.475 97172750
HM_Mugi06 DRR276802 Haliotis madaka 9.41 0.448 96155304
HM_Mugi07 DRR276803 Haliotis madaka 10.04 0.459 99682383
HM_Mugi08 DRR276804 Haliotis madaka 10.72 0.474 96993674
HM_Mugi09 DRR276805 Haliotis madaka 10.58 0.471 97057005
HG_Tsukumi01 DRR276810 Haliotis gigantea 10.49 0.439 98095031
HG_Tsukumi02 DRR276811 Haliotis gigantea 9.44 0.421 86327844
HG_Sado01 DRR276808 Haliotis gigantea 9.19 0.415 84306984
HG_Sado03 DRR276809 Haliotis gigantea 9.61 0.424 85917618
HG_Goto01 DRR276812 Haliotis gigantea 10.13 0.433 94554293
HG_Goto02 DRR276813 Haliotis gigantea 11.15 0.449 99878405
HR08 DRR276814 Haliotis rufescens 10.24 0.384 85679394
HR09 DRR276815 Haliotis rufescens 11.40 0.399 97400854
HR10 DRR276816 Haliotis rufescens 10.27 0.382 89689486
Black01 SRR7958745 Haliotis cracherodii 16.07 0.300 96336284
Black02 SRR7958746 Haliotis cracherodii 11.31 0.277 66930259
Green01 SRR7958751 Haliotis fulgens 13.39 0.272 79977247
Green02 SRR7958752 Haliotis fulgens 12.88 0.269 77150186
Pink01 SRR7958747 Haliotis corrugata 12.00 0.272 69409947
Pink02 SRR7958748 Haliotis corrugata 15.08 0.286 88385778
Pinto01 SRR7958749 Haliotis kamtschatkana 11.18 0.286 66467241
Pinto02 SRR7958750 Haliotis kamtschatkana 15.19 0.307 93488007
White01 SRR7958743 Haliotis sorenseni 13.91 0.303 98281227
White02 SRR7958744 Haliotis sorenseni 16.19 0.309 101403398
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FIGURE A1.

FIGURE A1

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Schematic diagram of the method for identifying three‐or‐more‐different (≥3) allele sequences (haplotypes) in candidate gene copy number variation (GCNV) genes. The number of different allelic sequences was counted for each of the identified GCNVs by enumerating every pair of SNV positions that were located within the read length.

TABLE A2.

The number of mapped reads to top 10 HD‐increased gene copy number variations that divided by the number of the total mapped reads and their results of edgeR.

Gene Annotation (top hit in BLASTP search) Haliotis discus discus Haliotis madaka logFC logCPM p‐Value FDR
Iwafune02 Iwafune04 Goto01 Goto02 Izu01 Izu02 Shibushi01 Shibushi02 Mugi06 Mugi07 Mugi08 Mugi09 Misaki01 Misaki14 Misaki22 Misaki23
STRG.52479 no hit 547 658 607 615 560 531 493 575 126 125 141 142 111 124 131 131 −2.261073 1.9575352 1.68 E‐154 2.70 E‐150
STRG.412 no hit 1536 3129 2825 3458 3579 3044 2969 3111 631 570 532 460 482 561 512 646 −2.5385 4.2779636 1.48 E‐79 1.59 E‐75
STRG.40757 XP_046329565.1| suppressor of cytokine signaling 4‐like [Haliotis rufescens] 266 237 235 278 255 221 216 228 55 42 32 56 30 42 30 38 −2.679361 0.6583958 2.04 E‐78 1.64 E‐74
STRG.48051 XP_046330233.1| protein SMG7‐like isoform X3 94 42 93 80 89 82 57 94 6 2 5 2 2 4 3 2 −4.656724 −1.077323 1.78 E‐50 9.52 E‐47
STRG.76 XP_046574497.1|uncharacterized protein LOC124282527 [Haliotis rubra] 186 354 276 407 501 532 339 420 64 52 69 96 49 55 65 117 −2.523546 1.3167576 4.13 E‐35 1.47 E‐31
STRG.15773 AMX23358.1|Hsp20 [Haliotis discus hannai] 97 81 115 108 103 89 89 89 32 26 14 20 17 16 21 15 −2.363456 −0.599399 3.40 E‐34 9.93 E‐31
STRG.45657 XP_046577082.1| uncharacterized protein LOC124284989 isoform X2 [Haliotis rubra] 53 40 57 44 47 55 38 33 5 4 6 2 7 2 2 4 −3.584297 −1.762253 1.69 E‐33 4.51 E‐30
STRG.43991 XP_046342493.1|septin‐7‐like isoform X1 421 461 422 405 355 427 382 381 208 225 222 218 224 208 193 239 −1.013928 1.7741263 8.39 E‐30 2.07 E‐26
STRG.37869 XP_046343616.1| beta‐1,4‐glucuronyltransferase 1‐like [Haliotis rufescens] 48 87 69 87 72 82 80 69 10 6 4 18 14 6 8 2 −3.212914 −1.069843 1.70 E‐29 3.91 E‐26
STRG.58440 XP_046344022.1|histone‐lysine N‐methyltransferase EHMT2‐like isoform X3 [Haliotis rufescens] 374 343 300 548 378 437 239 384 198 141 156 133 146 147 119 165 −1.413937 1.5279937 3.22 E‐26 6.09 E‐23
Total mapped number 9 E+07 9.3 E+07 8.9 E+07 9.8 E+07 8.8 E+07 9 E+07 8.1 E+07 8.9 E+07 8.9 E+07 9.3 E+07 1 E+08 9.7 E+07 9.6 E+07 1 E+08 9.7 E+07 9.7 E+07
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TABLE A3.

The number of mapped reads to top 10 HM‐increased gene copy number variations that divided by the number of the total mapped reads and their results of edgeR.

Gene Annotation (top hit in BLASTP search) Haliotis discus discus Haliotis madaka logFC logCPM p‐Value FDR
Iwafune02 Iwafune04 Goto01 Goto02 Izu01 Izu02 Shibushi01 Shibushi02 Mugi06 Mugi07 Mugi08 Mugi09 Misaki01 Misaki14 Misaki22 Misaki23
STRG.7630 XP_046362257.1|sushi, von Willebrand factor type A, EGF, and pentraxin domain‐containing protein 1‐like isoform X1 [Haliotis rufescens] 408 400 382 422 345 365 329 396 2262 2781 3308 2790 2494 2658 2845 2775 2.7368 4.02998 1.17 E‐225 3.75 E‐221
STRG.936 XP_046378335.1| uncharacterized protein LOC124150379 [Haliotis rufescens] 1038 1106 1029 1262 961 817 990 1015 3220 2533 2948 2895 2685 2861 3153 2935 1.3909 4.37891 2.89 E‐62 1.86 E‐58
STRG.17419 XP_046378335.1| uncharacterized protein LOC124150379 [Haliotis rufescens] 166 233 178 359 185 149 182 158 625 628 1044 1083 712 786 1096 1081 2.028 2.51044 1.27 E‐37 5.82 E‐34
STRG.24061 XP_046344413.1| uncharacterized protein LOC124125179 [Haliotis rufescens] 187 177 155 163 155 153 168 127 417 431 469 487 375 415 516 659 1.4363 1.74836 1.65 E‐36 6.62 E‐33
STRG.32296 XP_046330173.1| telomere length regulation protein TEL2 homolog isoform X2 [Haliotis rufescens] 585 359 472 449 421 645 403 568 2204 2440 1637 2847 2786 1907 1393 1512 1.9967 3.7715 2.20 E‐34 7.07 E‐31
STRG.59252 XP_046339330.1|countin‐3‐like isoform X1 [Haliotis rufescens] 32 8 5 36 36 39 6 16 210 203 215 200 203 192 264 212 3.1475 0.32136 1.87 E‐28 4.00 E‐25
STRG.73643 XP_046351856.1|tyrosine‐protein phosphatase nonreceptor type 23‐like isoform X2 [Haliotis rufescens] 110 79 50 67 49 34 38 48 226 310 290 278 202 316 208 257 2.0302 0.77255 1.53 E‐26 3.07 E‐23
STRG.53292 XP_046373113.1|uncharacterized protein LOC124146723 [Haliotis rufescens] 2 3 6 4 6 8 8 11 44 43 54 66 53 59 69 38 3.0092 −1.578 1.88 E‐25 3.02 E‐22
STRG.45154 XP_046335256.1|fibroblast growth factor receptor 3‐like isoform X1 [Haliotis rufescens] 25 36 23 23 37 31 26 29 106 130 117 80 107 106 130 107 1.8233 −0.4006 3.64 E‐25 5.56 E‐22
STRG.61550 XP_046326147.1|procollagen‐lysine, 2‐oxoglutarate 5‐dioxygenase 1‐like isoform X2 [Haliotis rufescens] 143 137 156 169 148 158 142 165 331 296 397 269 332 372 408 328 1.0557 1.40385 4.04 E‐23 5.63 E‐20
Total mapped number 9 E+07 9.3 E+07 8.9 E+07 9.8 E+07 8.8 E+07 9 E+07 8.1 E+07 8.9 E+07 8.9 E+07 9.3 E+07 1 E+08 9.7 E+07 9.6 E+07 1 E+08 9.7 E+07 9.7 E+07
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Hirase, S. , Sekino, M. , Hara, M. , & Kikuchi, K. (2023). Accumulation of gene copy number variations during the early phase of free‐spawning abalone speciation. Ecology and Evolution, 13, e9816. 10.1002/ece3.9816

DATA AVAILABILITY STATEMENT

The WGS data used in this study are provided in Table A1 in Appendix A. The perl script for detecting ≥3 allele sequences is deposited in the Dryad Digital Repository: https://datadryad.org/stash/share/WGpUYJzAGlXloIlShuopLT65WheRf1ra1veScjvF7vg.

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Associated Data

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

Data Availability Statement

The WGS data used in this study are provided in Table A1 in Appendix A. The perl script for detecting ≥3 allele sequences is deposited in the Dryad Digital Repository: https://datadryad.org/stash/share/WGpUYJzAGlXloIlShuopLT65WheRf1ra1veScjvF7vg.

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