Data from: Shared and unique patterns of genomic differentiation and introgression between Japanese stickleback species across three sympatric sites
Data files
Jul 10, 2026 version files 115.55 MB
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OkinebeDatasetForDryad.zip
115.51 MB
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README.md
37.78 KB
Abstract
Hybridization and introgression frequently occur even between distantly related species. A central question in speciation research is which genomic regions act as barriers to gene flow and how genome-wide differentiation persists despite hybridization between partially reproductively isolated species. Ecological divergence is well known to promote genomic differentiation, especially during the early stages of speciation. However, the extent to which ecological divergence contributes to genomic divergence and the restriction of gene flow between more strongly isolated species, such as those exhibiting intrinsic hybrid incompatibilities, remains relatively unclear. One promising approach is to compare genomic differentiation and introgression patterns between sympatric sites of ecologically divergent distantly related species and those of ecologically similar species. Here, we identified a new sympatric site of threespine stickleback (Gasterosteus aculeatus) and Japan Sea stickleback (G. nipponicus) in the Okinebe River, Hokkaido, Japan. In this habitat, these two species differ in migratory life histories: G. nipponicus is anadromous (sea-run migratory), while G. aculeatus is resident. This contrasts with the other two previously studied sympatric sites, where both species are anadromous. We found that genomic differentiation in the Okinebe pair is maintained at high levels despite limited spatial isolation. Furthermore, the Okinebe pair had more genomic regions with high differentiation and fewer regions of introgression than the other sympatric pairs. These findings suggest that migratory differences may be able to contribute to the restriction of gene flow even between species with strong reproductive isolation. To better understand the role of ecological divergence in speciation, broader comparative studies across multiple sympatric species pairs with varying degrees of ecological differentiation and reproductive isolation are needed.
Dataset DOI: 10.5061/dryad.98sf7m0w7
Description of the data and file structure
Scripts and datasets used for the analyses in the study, “Repeated patterns of genetic differentiation and introgression between Japanese stickleback species across three sympatric sites”.
File: OkinebeDatasetForDryad.zip
Description:
The .zip file includes 19 folders corresponding to each figure, and one additional folder with population genetic analyses.
(1) Figure1:
Sympatric species in the Okinebe River system. (F) Admixture analysis of Gasterosteus sticklebacks with K = 2. Samples of Abashiri and Tokotan populations derived from Kakioka et al. (2020) were used as controls of pure species of G. nipponicus and G. aculeatus, respectively. Orange and blue indicate G. nipponicus and G. aculeatus, respectively. Grey and black arrowheads indicate BC1 and F1 hybrids, respectively (also see Figure S2).
Okinebe_Figure1.R: R scripts used for the analyses and figure generation in Figure 1.
Gasterosteus2013Admixture.txt: Output of admixture analysis of 2013 Gasterosteus sticklebacks used in Figure 1F.
Sample: Sample name.
Category1: Admixture proportion of G. nipponicus.
Category2: Admixture proportion of G. aculeatus.
Order: Order of samples used for visualization.
Gasterosteus2021Admixture.txt: Output of admixture analysis of 2021 Gasterosteus sticklebacks used in Figure 1F.
Sample: Sample name.
Category1: Admixture proportion of G. nipponicus.
Category2: Admixture proportion of G. aculeatus.
Order: Order of samples used for visualization.
Gasterosteus2022Admixture.txt: Output of admixture analysis of 2022 Gasterosteus sticklebacks used in Figure 1F.
Sample: Sample name.
Category1: Admixture proportion of G. nipponicus.
Category2: Admixture proportion of G. aculeatus.
Order: Order of samples used for visualization.
Gasterosteus2023Admixture.txt: Output of admixture analysis of 2023 Gasterosteus sticklebacks used in Figure 1F.
Sample: Sample name.
Category1: Admixture proportion of G. nipponicus.
Category2: Admixture proportion of G. aculeatus.
Order: Order of samples used for visualization.
(2) Figure2
Ecological and morphological divergence between sympatric species. (A) Stable isotope analysis of the three stickleback species collected in the Okinebe system. (B) Principal component (PC) analysis using morphological traits of Gasterosteus sticklebacks collected in the Okinebe system (see Figure S4C for loading of each PC). (C) PC analysis using morphological traits of Gasterosteus aculeatus collected in the Okinebe, Bekanbeushi and Tokotan systems (see Figure S4E for loading of each PC). Traits with high PC loadings are shown: DS1, first dorsal spine length; DS2, second dorsal spine length; GW, gape width; LPS, left pelvic spine length; SnL, snout length.
Okinebe_Figure2.R: R scripts used for the analyses and figure generation in Figure 2.
IsotopeAnalysis.txt: Dataset for isotope analysis used in Figure 2A.
Sample: Sample number.
Name: Sample name
N15:δ15N value (‰).
C13:δ13C value (‰).
Species: P (G. aculeatus), J (G. nipponicus), H (F1 hybrid), or B (BC1 hybrid).
Sex: F (Female) or M (Male).
Location: P (Pond) or R (River).
MorphologicalAnalysis.txt: Dataset for morphological analysis used in Figures 2B & 2C.
Sample: Sample name.
Species: P (G. aculeatus), J (G. nipponicus), H (F1 hybrid), or B (BC1 hybrid).
Sex: F (Female) or M (Male).
Group: Group used for visualization.
Location: P (Pond) or R (River).
Number: Sample number.
SL: Standard length (mm).
HL: Head length (mm).
BD: Body depth (mm).
JL: Jaw length (mm).
SnL: Snout length (mm).
GW: Gape width (mm).
ED: Eye diameter (mm).
DS1: First dorsal spine length (mm).
DS2: Second dorsal spine length (mm).
LPS: Left pelvic spine length (mm).
PGL: Pelvic girdle length (mm).
OkinebeMorphologyPCA.txt: PCA results used in Figure 2B.
Sample: Sample name.
PC1: PC1 of principal component analysis using morphological traits of Gasterosteus sticklebacks collected in the Okinebe system.
PC2: PC2 of principal component analysis using morphological traits of Gasterosteus sticklebacks collected in the Okinebe system.
Species: P (G. aculeatus), J (G. nipponicus), H (F1 hybrid), or B (BC1 hybrid).
Sex: F (Female) or M (Male).
aculeatusMorphologyPCA.txt: PCA results used in Figure 2C.
Sample: Sample name.
Species: P (G. aculeatus), J (G. nipponicus), H (F1 hybrid), or B (BC1 hybrid).
Sex: F (Female) or M (Male).
Location: P (Pond) or R (River).
PC1: PC1 of principal component analysis using morphological traits of Gasterosteus aculeatus collected in the Okinebe, Bekanbeushi and Tokotan systems.
PC2: PC2 of principal component analysis using morphological traits of Gasterosteus aculeatus collected in the Okinebe, Bekanbeushi and Tokotan systems.
(3) Figure3
Variation in reproductive stages between sympatric Gasterosteus species. (A) Three developmental stages of testis were identified based on flow cytometry data (upper panel) and histological sections (lower panel). Individuals 2021_Gas009, 2021_Gas010 and 2021_Gas002 are shown as representatives of Stage 1, Stage 2 and Stage 3, respectively. For data of other individuals, see Figures S5 and S6. (B) Comparison of standard length, plasma 11-ketotestosterone (11-KT) levels and plasma testosterone (T) levels among three gonad developmental stages.
Okinebe_Figure3.R: R scripts used for the analyses and figure generation in Figure 3.
Flowcyte1.txt: Dataset for flow cytometry analysis used in Figure 3A.
intensity: Fluorescence intensity measured by flow cytometry.
Testis: Histogram data of flow cytometry for control testis.
Fin: Histogram data of flow cytometry for control fin.
Okinebe2021_Gas002: Histogram data of flow cytometry for Okinebe2021_Gas002.
Okinebe2021_Gas008: Histogram data of flow cytometry for Okinebe2021_Gas008.
Okinebe2021_Gas009: Histogram data of flow cytometry for Okinebe2021_Gas009.
Okinebe2021_Gas015: Histogram data of flow cytometry for Okinebe2021_Gas015.
Okinebe2021_Gas018: Histogram data of flow cytometry for Okinebe2021_Gas018.
Okinebe2021_Gas019: Histogram data of flow cytometry for Okinebe2021_Gas019.
Okinebe2021_Gas022: Histogram data of flow cytometry for Okinebe2021_Gas022.
Okinebe2021_Gas023: Histogram data of flow cytometry for Okinebe2021_Gas023.
Okinebe2021_Gas024: Histogram data of flow cytometry for Okinebe2021_Gas024.
Okinebe2021_Gas027: Histogram data of flow cytometry for Okinebe2021_Gas027.
Okinebe2021_Gas029: Histogram data of flow cytometry for Okinebe2021_Gas029.
Okinebe2021_Gas033: Histogram data of flow cytometry for Okinebe2021_Gas033.
Okinebe2021_Gas034: Histogram data of flow cytometry for Okinebe2021_Gas034.
Okinebe2021_Gas035: Histogram data of flow cytometry for Okinebe2021_Gas035.
Okinebe2021_Gas036: Histogram data of flow cytometry for Okinebe2021_Gas036.
Okinebe2021_Gas038: Histogram data of flow cytometry for Okinebe2021_Gas038.
Okinebe2021_Gas039: Histogram data of flow cytometry for Okinebe2021_Gas039.
Okinebe2021_Gas045: Histogram data of flow cytometry for Okinebe2021_Gas045.
Okinebe2021_Gas081: Histogram data of flow cytometry for Okinebe2021_Gas081.
Flowcyte2.txt: Dataset for flow cytometry analysis used in Figure 3A.
intensity: Fluorescence intensity measured by flow cytometry.
Okinebe2021_Gas010: Histogram data of flow cytometry for Okinebe2021_Gas010.
Okinebe2021_Gas014: Histogram data of flow cytometry for Okinebe2021_Gas014.
aculeatusAndrogen.txt: Dataset for androgen levels used in Figure 3B.
Sample: Sample name.
Sex: F (Female) or M (Male).
Kidney: Kidney weight (mg).
Mature: Maturation stage.
KSI: Kidney Somatic Index.
SL: Standard length (mm).
Body: Body weight (g).
GSI: Gonad Somatic Index.
Testis: Testis weight (mg).
KT11: Concentration of plasma 11-KT (ng/mL).
T: Concentration of plasma T (ng/mL).
(4) Figure4
Comparison of FST between Gasterosteus nipponicus and Gasterosteus aculeatus across the three sympatric pairs and one allopatric pair. (A) Genome-wide FST distribution in each pair. The right histogram indicates the frequency distributions of FST. Orange, blue and black indicate high, intermediate and low FST regions, respectively. Black bars below each panel indicate regions where FST is smaller in the Okinebe pair than in other sympatric systems and used for phylogenetic analysis (see Figure S12). Red arrowheads below each panel indicate regions showing lower fd in the Okinebe than in the other sympatric systems and used for phylogenetic analysis (see Figure S14). (B) Pairwise comparison of FST between river systems. (C, D) Ven diagrams showing the overlaps of high-FST (C) and low-FST regions (D) among the comparisons. (E) Relationship between FST and recombination rate in the Okinebe system.
Okinebe_Figure4.R: R scripts used for the analyses and figure generation in Figure 4.
Fst_4pops.txt: FST calculated for every 10k window using WGS data.
chromosome: Chromosome.
window_pos_1: Start position of each genomic window.
window_pos_2: End position of each genomic window.
window: Genomic window name.
no_snps: Number of SNPs within each genomic window.
SNP: Median of each genomic window (Mb).
Oki_fst: FST between G. nipponicus and G. aculeatus in the Okinebe population.
Oki_State: HMM state inferred from FST values in the Okinebe population.
Toko_fst: FST between G. nipponicus and G. aculeatus in the Tokotan population.
Toko_State: HMM state inferred from FST values in the Tokotan population.
Bek_fst: FST between G. nipponicus and G. aculeatus in the Bekanbeushi population.
Bek_State: HMM state inferred from FST values in the Bekanbeushi population.
Allo_fst: FST between G. nipponicus and G. aculeatus in the allopatric population.
Allo_State: HMM state inferred from FST values in the allopatric population.
FstRecombinationRate.txt: Dataset for analysis used in Figure 4E.
number: Window number.
chromosome: Chromosome.
StartWindow: Start position of each genomic window.
MedianPoint: Median position of each genomic window.
cMMb: Recombination rate (cM/Mb).
cMMbPositive: Positive recombination rate (cM/Mb).
Name: Window name.
OkiFst: FST between G. nipponicus and G. aculeatus in the Okinebe population.
BekanFst: FST between G. nipponicus and G. aculeatus in the Bekanbeushi population.
TokoFst: FST between G. nipponicus and G. aculeatus in the Tokotan population.
AlloFst: FST between G. nipponicus and G. aculeatus in the allopatric population.
RecombinationRate_v5.csv: Recombination rate from Roesti et al. (2013) converted to version 5 genome of G. aculeatus.
number: Window number.
chr: Chromosome.
point: Start position of each genomic window.
cM: cM.
MedianPoint: Median position of each genomic window.
Length: Length of each genomic window (bp).
Mb: Length of each genomic window (Mbp).
cMVar: cM data from Roesti et al. (2013).
cMMb: Recombination rate (cM/Mb).
Figure4_FST_HMM folder
HMM_log10FST+1_3norm_Marques.R: Script from Marques et al. (2016) used for HMM analysis in Figures 4C & 4D.
Allopatric_fst_input_4pops.txt: Input data used for HMM analysis of FST in the allopatric system.
x: FST values.
Bekanbeushi_fst_input_4pops.txt: Input data used for HMM analysis of FST in the Bekanbeushi system.
x: FST values.
Okinebe_fst_input_4pops.txt: Input data used for HMM analysis of FST in the Okinebe system.
x: FST values.
Tokotan_fst_input_4pops.txt: Input data used for HMM analysis of FST in the Tokotan system.
x: FST values.
(5) Figure5
Comparison of fd across the three sympatric pairs and one allopatric pair. (A) Genome-wide fd distribution in each system. Orange dots indicate high fd (> 0.2) regions. Black bars below each panel indicate regions where FST is smaller in the Okinebe pair than in other sympatric systems and used for phylogenetic analysis (see Figure S12). Red arrowheads below each panel indicate regions showing lower fd in the Okinebe than in the other sympatric systems and used for phylogenetic analysis (see Figure S14). (B) A Venn diagram showing the overlaps of high fd windows among comparisons. (C) Comparison of FST between high fd and low fd genomic regions in the Okinebe pair. (D) Comparison of recombination rate between high fd and low fd genomic regions in the Okinebe pair. (E) Positive relationship between fd and recombination rate in the Okinebe system (ρ = 0.232 and p < 10−15).
Okinebe_Figure5.R: R scripts used for the analyses and figure generation in Figure 5.
Quebec_PO_Akkeshi_JS_Akkeshi_localFstats__50_25.txt: fd data of the Bekanbeushi system.
This file is a direct output from Dsuite.
Quebec_PO_Okinebe_JS_Okinebe_localFstats__50_25.txt: fd data of the Okinebe system.
This file is a direct output from Dsuite.
Quebec_PO_Tokotan_JS_Tokotan_localFstats__50_25.txt: fd data of the Tokotan system.
This file is a direct output from Dsuite.
Quebec_PugetSound_JS_Shinji_localFstats__50_25.txt: fd data of the allopatric population.
This file is a direct output from Dsuite.
fd_4pops.txt: fd data of 4 populations.
Chr: Chromosome.
Start: Start position of each genomic window.
End: End position of each genomic window.
Akkeshi: fd between G. nipponicus and G. aculeatus in the Bekanbeushi population.
Okinebe: fd between G. nipponicus and G. aculeatus in the Okinebe population.
Tokotan: fd between G. nipponicus and G. aculeatus in the Tokotan population.
Allopatric: fd between G. nipponicus and G. aculeatus in the allopatric population.
fdRecombinationRate_Okinebe.txt: fd and recombination rate in Okinebe system.
fd_Top: Rank of the fd value in descending order.
TOP%: Percentile rank from the top based on the fd value.
chr: Chromosome.
Median: Median position of each genomic window.
windowStart: Start position of each genomic window.
windowEnd: End position of each genomic window.
D: D values calculated by Dsuite.
f_d: f_d values calculated by Dsuite.
f_dM: f_dM values calculated by Dsuite.
d_f: d_f values calculated by Dsuite.
x: d_f values calculated by Dsuite after replacing all negative values with 0.
Length: Length of each genomic window (bp).
MedianPoint: Median position of each genomic window.
cMMb: Recombination rate (cM/Mb).
fstfd_Okinebe.txt: FST and fd in Okinebe system.
pop1: Population used in this analysis (JS_Okinebe).
pop2: Population used in this analysis (PO_Okinebe).
chromosome: Chromosome.
window_pos_1: Start position of each genomic window.
Name: Window name.
window_pos_2: End position of each genomic window.
avg_wc_fst: FST between G. nipponicus and G. aculeatus in the Okinebe population.
no_snps: Number of SNPs within each genomic window.
SNP: Median position of each genomic window (Mbp).
fd: High fd (1) or others (0).
(6) FigureS1
Analysis of Pungitius sticklebacks collected in the OkinebeRiver system. (A) Admixture analysis (K = 3) using ddRAD-seq data together with WGS data of three previously identified species: the brackish-water type, the freshwater type, and P. tymensis. We did not genotype Pungitius fish collected in the upstream region. (B) Histograms of the plate number and standard length of Pungitius sticklebacks collected at three sampling sites shown in Figure 1C. The gray colors indicate the completely plated morph, while the white colors indicate the partially plated morph. To count the lateral plates, ethanol-preserved samples collected in 2022 were stained with alizarin red to visualize bony structures, as described previously (Ishikawa et al. 2013). The lateral plate was counted under a stereoscopic microscope (S8APO; Leica Microsystems). The fish were also classified into partially plated and completely plated morphs, following Ishikawa et al. (2013): fish with more than two gaps of plates in the middle of the plate row were classified as the partially plated morph, while those with a continuous row of plates with only two or fewer gaps were classified as the completely plated morph. All analyzed Pungitius fish had keels, so no fish were classified into the low-plated morph. The ratios of partial vs. complete were 23:33, 57:60, and 27:35 in the river, pond, and upstream site, respectively, with no significant difference among sampling sites (Fisher’s exact test, P = 0.6031). The plate number also did not differ among the sampling sites (Figure S1B) (Kruskal-Wallistest, χ2 = 4.0221, df = 2, P = 0.1338).
Okinebe_FigureS1.R: R scripts used for the analyses and figure generation in Figure S1.
PungitiusAdmixture.txt: Output of admixture analysis of 2013 Pungitius sticklebacks used in Figure S1A.
Category1: Admixture proportion of brackish-water type.
Category2: Admixture proportion of freshwater type.
Category3: Admixture proportion of P. tymensis.
Group: Order used for visualization.
PungitiusPlate.txt: Dataset of plate number and standard length in Pungitius sticklebacks used in Figure S1B.
Name: Individual identifier.
Location: Collection site (Okinebe pond or Okinebe river).
PlateSum: Number of lateral plates.
SL: Standard length (mm).
Plate: Lateral plate phenotype (Complete or Partial).
(7) FigureS2
Triangle plots of Gasterosteus sticklebacks in the Okinebe River system with the hybrid index (the percentage of the genome derived from G. nipponicus) on the X-axis and the interspecific heterozygosity on the Y-axis. (A) Three hybrids collected in 2013. All fish collected in 2013 were used. (B) Three hybrids collected in 2021. As parental species, 10 G. aculeatus (Okinebe2021_Gas001-004, 006-011) and 10 G. nipponicus (Okinebe2021_Gas083-089, 091-093) were used.
Okinebe_FigureS2.R: R scripts used for the analyses and figure generation in Figure S2.
RAD2013GasFilt.vcf: SNP data used for the triangle plots in Figure S2A.
RAD2021GasFiltTriangle.vcf: SNP data used for the triangle plots in Figure S2B.
TrianglePlot_2013GasPop.txt: Population data used for the triangle plots in Figure S2A.
id: Individual identifier.
pop: Population. P: G. aculeatus, J: G. nipponicus, H: F1 hybrid, B: Backcross.
TrianglePlot_2021GasPop.txt: Population data used for the triangle plots in Figure S2B.
id: Individual identifier.
pop: Population. P: G. aculeatus, J: G. nipponicus, H: F1 hybrid, B: Backcross.
(8) FigureS3
Genome-wide FST distribution based on the ddRAD-seq data of Gasterosteus sticklebacks collected in 2021. Overall, 7,871 SNPs were used for the analysis. FST values were calculated for each 1,000 kb window.
Okinebe_FigureS3.R: R scripts used for the analyses and figure generation in Figure S3.
RAD2021Gas_RiverPvsPondP.windowed.weir.fst: FST calculated from ddRAD-seq data used in Figure S3A.
This file is a direct output from vcftools.
RAD2021Gas_PondJvsRiverJ.windowed.weir.fst: FST calculated from ddRAD-seq data used in Figure S3B.
This file is a direct output from vcftools.
RAD2021Gas_RiverJvsRiverP.windowed.weir.fst: FST calculated from ddRAD-seq data used in Figure S3C.
This file is a direct output from vcftools.
RAD2021Gas_PondJvsPondP.windowed.weir.fst: FST calculated from ddRAD-seq data used in Figure S3D.
This file is a direct output from vcftools.
(9) FigureS4
Ecological and morphological divergence among sympatric stickleback species in the Okinebe River system. (A) No substantial differences were found in the stable isotope ratios between individuals collected from the pond and the river. (B) Stable isotope analysis of lipid-eliminated samples showed qualitatively similar results to (A). (C) Loadings of morphological traits on PC1 and PC2 in the morphology PCA of the Okinebe fish shown in Figure 2B. (D) Variation of standard length between G. aculeatus (Gacu) and G. nipponicus (Gnip). (E) Loadings of morphological traits on PC1 and PC2 in the morphology PCA of G. aculeatus of the three river systems shown in Figure 2C. (F) Variation in standard length of G. aculeatus among the river systems.
Okinebe_FigureS4.R: R scripts used for the analyses and figure generation in Figure S4.
IsotopeAnalysis.txt: Dataset for isotope analysis used in Figure S4A.
Sample: Sample number.
Name: Sample name.
N15:δ15N value (‰).
C13:δ13C value (‰).
Species: P (G. aculeatus), J (G. nipponicus), H (F1 hybrid), or B (BC1 hybrid).
Sex: F (Female) or M (Male).
Location: P (Pond) or R (River).
LipidElimination.txt: Dataset for isotope analysis with lipid elimination used in Figure S4B.
Individual: Sample name.
N15:δ15N value (‰).
C13:δ13C value (‰).
Species: P (G. aculeatus), J (G. nipponicus).
Sex: F (Female) or M (Male).
Sample: Sample identifier.
Treatment: Treatment condition (before or after lipid elimination).
Okinebe20132023SLGW.txt: Dataset for standard length and gonad weight used in Figure S4D.
Sample: Sample name.
GW: Gonad weight (mg).
SL: Standard length (mm).
Species: P (G. aculeatus), J (G. nipponicus).
Sex: F (Female) or M (Male).
Group: Combined label indicating species, sex, and collection year.
Year: Collection year.
MorphologicalAnalysisForFigS4F.txt: Dataset for standard length of G. aculeatus used in Figure S4F.
Sample: Sample name.
Species: P (G. aculeatus), J (G. nipponicus).
Sex: F (Female) or M (Male).
Group: Combined label indicating species, sex, and collection site.
Location: Collection site.
Number: Sample identifier.
SL: Standard length (mm).
(10) FigureS5
Flow cytometry analyses. (B) Representative results of flow cytometry of a mature testis, which is mostly haploid (n), and a pectoral fin, which is diploid (2n). (C) Flow cytometry results and photos of all testes examined in this study.
Okinebe_FigureS5.R: R scripts used for the analyses and figure generation in Figure S5.
Flowcyte1.txt: Dataset for flow cytometry analysis used in Figure S5.
intensity: Fluorescence intensity measured by flow cytometry.
Testis: Histogram data of flow cytometry for control testis.
Fin: Histogram data of flow cytometry for control fin.
Okinebe2021_Gas002: Histogram data of flow cytometry for Okinebe2021_Gas002.
Okinebe2021_Gas008: Histogram data of flow cytometry for Okinebe2021_Gas008.
Okinebe2021_Gas009: Histogram data of flow cytometry for Okinebe2021_Gas009.
Okinebe2021_Gas015: Histogram data of flow cytometry for Okinebe2021_Gas015.
Okinebe2021_Gas018: Histogram data of flow cytometry for Okinebe2021_Gas018.
Okinebe2021_Gas019: Histogram data of flow cytometry for Okinebe2021_Gas019.
Okinebe2021_Gas022: Histogram data of flow cytometry for Okinebe2021_Gas022.
Okinebe2021_Gas023: Histogram data of flow cytometry for Okinebe2021_Gas023.
Okinebe2021_Gas024: Histogram data of flow cytometry for Okinebe2021_Gas024.
Okinebe2021_Gas027: Histogram data of flow cytometry for Okinebe2021_Gas027.
Okinebe2021_Gas029: Histogram data of flow cytometry for Okinebe2021_Gas029.
Okinebe2021_Gas033: Histogram data of flow cytometry for Okinebe2021_Gas033.
Okinebe2021_Gas034: Histogram data of flow cytometry for Okinebe2021_Gas034.
Okinebe2021_Gas035: Histogram data of flow cytometry for Okinebe2021_Gas035.
Okinebe2021_Gas036: Histogram data of flow cytometry for Okinebe2021_Gas036.
Okinebe2021_Gas038: Histogram data of flow cytometry for Okinebe2021_Gas038.
Okinebe2021_Gas039: Histogram data of flow cytometry for Okinebe2021_Gas039.
Okinebe2021_Gas045: Histogram data of flow cytometry for Okinebe2021_Gas045.
Okinebe2021_Gas081: Histogram data of flow cytometry for Okinebe2021_Gas081.
Flowcyte2.txt: Dataset for flow cytometry analysis used in Figure S5.
intensity: Fluorescence intensity measured by flow cytometry.
Okinebe2021_Gas010: Histogram data of flow cytometry for Okinebe2021_Gas010.
Okinebe2021_Gas014: Histogram data of flow cytometry for Okinebe2021_Gas014.
(11) FigureS7
Variation in gonad weight and standard length in G. aculeatus for males (A) and females (B). It should be noted that the gonad weights of samples in 2013 were measured from samples preserved in 99.5% ethanol, whereas the gonad weights of samples in 2023 were obtained immediately after euthanasia.
Okinebe_FigureS7.R: R scripts used for the analyses and figure generation in Figure S7.
Okinebe20132023SLGW.txt: Dataset for standard length and gonad weight used in Figure S7.
Sample: Sample name.
GW: Gonad weight (mg).
SL: Standard length (mm).
Species: P (G. aculeatus), J (G. nipponicus).
Sex: F (Female) or M (Male).
Group: Combined label indicating species, sex, and collection year.
Year: Collection year.
(12) FigureS8
Genome-wide FST distribution calculated from WGS data between G. nipponicus and G. aculeatus from the different river systems. FST values were calculated for each 10,000 bp window. The right histogram shows the frequency distribution across the genome.
Okinebe_FigureS8.R: R scripts used for the analyses and figure generation in Figure S8.
Okinebe_analysis_bcftools.filtered.win10k.v5_fst.txt: FST calculated for every 10k window using WGS data.
This file is a direct output from pixy.
(13) FigureS9
Genome-wide dXY (A) and π (B and C) distributions calculated from WGS data of Gasterosteus sticklebacks. dXY and π were calculated for each 10,000 bp window. The right histogram shows the frequency distribution across the genome. (D) Association between π and FST.
Okinebe_FigureS9.R: R scripts used for the analyses and figure generation in Figure S9.
Okinebe_analysis_bcftools.filtered.win10k.v5_fst.txt: FST calculated for every 10k window using WGS data.
This file is a direct output from pixy.
Okinebe_analysis_bcftools.filtered.win10k.v5_dxy.txt: dXY calculated for every 10k window using WGS data.
This file is a direct output from pixy.
Okinebe_analysis_bcftools.filtered.win10k.v5_pi.txt: Pi calculated for every 10k window using WGS data.
This file is a direct output from pixy.
(14) FigureS10
Relationships (A) between FST and recombination rate and (B) between fd and recombination rate in the allopatric pair.
Okinebe_FigureS10.R: R scripts used for the analyses and figure generation in Figure S10.
FstRecombinationRate.txt: FST and fd in the allopatric population.
number: Window number.
chromosome: Chromosome.
StartWindow: Start position of each genomic window.
MedianPoint: Median position of each genomic window.
cMMb: Recombination rate (cM/Mb).
cMMbPositive: Positive recombination rate (cM/Mb).
Name: Window name.
OkiFst: FST between G. nipponicus and G. aculeatus in the Okinebe population.
BekanFst: FST between G. nipponicus and G. aculeatus in the Bekanbeushi population.
TokoFst: FST between G. nipponicus and G. aculeatus in the Tokotan population.
AlloFst: FST between G. nipponicus and G. aculeatus in the allopatric population.
fdRecombinationRate_Allopatric.txt: fd and recombination rate in the allopatric population.
fd_Top: Rank of the fd value in descending order.
TOP%: Percentile rank from the top based on the fd value.
chr: Chromosome.
Median: Median position of each genomic window.
windowStart: Start position of each genomic window.
windowEnd: End position of each genomic window.
D: D values calculated by Dsuite.
f_d: f_d values calculated by Dsuite.
f_dM: f_dM values calculated by Dsuite.
d_f: d_f values calculated by Dsuite.
x: d_f values calculated by Dsuite after replacing all negative values with 0.
Length: Length of each genomic window (bp).
MedianPoint: Median position of each genomic window.
cMMb: Recombination rate (cM/Mb).
(15) FigureS11
FST of one river system was divided by FST of another river system, followed by log2 transformation. Arrowheads indicate regions used for phylogenetic analysis in Figure S12.
Okinebe_FigureS11.R: R scripts used for the analyses and figure generation in Figure S11.
Okinebe_analysis_bcftools.filtered.win50k.v5_fst.txt: FST calculated for every 50k window using WGS data.
This file is a direct output from pixy.
(16) FigureS12
Phylogenetic analysis of introgressed regions and their surrounding regions on Chr. 7 (A), Chr. 15 (B), and Chr. 21 (C and D). Genome sequences were phased, so there are two haplotypes for each fish. Arrowheads indicate the introgressed haplotype. Orange and blue labels indicate G. nipponicus and G. aculeatus, respectively. Bootstrap values of a branch separating haplotypes of two species are shown in red. Yellow arrowheads indicate haplotypes that are carried by a G. aculeatus individual, but cluster with G. nipponicus haplotypes.
chrVII_left_Okinebe.bed: Genomic position of used in Figure S12A.
chrom: Chromosome.
chromStart: Start position of genomic region.
chromEnd: End position of genomic region.
chrVII_target_Okinebe.bed: Genomic position of introgressed region in Chr.7 used in Figure S12A.
chrom: Chromosome.
chromStart: Start position of genomic region.
chromEnd: End position of genomic region.
chrXV_left_Okinebe.bed: Genomic position of used in Figure S12A.
chrom: Chromosome.
chromStart: Start position of genomic region.
chromEnd: End position of genomic region.
chrXV_right_Okinebe.bed: Genomic position of used in Figure S12B.
chrom: Chromosome.
chromStart: Start position of genomic region.
chromEnd: End position of genomic region.
chrXV_target_Okinebe.bed: Genomic position of introgressed region in Chr.15 used in Figure S12B.
chrom: Chromosome.
chromStart: Start position of genomic region.
chromEnd: End position of genomic region.
chrXXI_target1_left_Okinebe.bed: Genomic position of introgressed region in Chr.21 used in Figure S12C.
chrom: Chromosome.
chromStart: Start position of genomic region.
chromEnd: End position of genomic region.
chrXXI_target1_Okinebe.bed: Genomic position of introgressed region in Chr.21 used in Figure S12C.
chrom: Chromosome.
chromStart: Start position of genomic region
chromEnd: End position of genomic region
chrXXI_target1_right_Okinebe.bed: Genomic position of introgressed region in Chr.21 used in Figure S12C.
chrom: Chromosome
chromStart: Start position of genomic region.
chromEnd: End position of genomic region.
chrXXI_target2_left_Okinebe.bed: Genomic position of introgressed region in Chr.21 used in Figure S12D.
chrom: Chromosome.
chromStart: Start position of genomic region.
chromEnd: End position of genomic region.
chrXXI_target2_Okinebe.bed: Genomic position of introgressed region in Chr.21 used in Figure S12D.
chrom: Chromosome.
chromStart: Start position of genomic region.
chromEnd: End position of genomic region.
Okinebe_analysis_bcftools_filtered.v5.phased.imputemasked_chrVII_left.rmInvariants.phy.raxml.support.rename: Output of phylogenetic analysis using chrVII_left_Okinebe.bed.
Okinebe_analysis_bcftools_filtered.v5.phased.imputemasked_chrVII_target.rmInvariants.phy.raxml.support.rename: Output of phylogenetic analysis using chrVII_target_Okinebe.bed.
Okinebe_analysis_bcftools_filtered.v5.phased.imputemasked_chrXV_left.rmInvariants.phy.raxml.support.rename: Output of phylogenetic analysis using chrXV_left_Okinebe.bed.
Okinebe_analysis_bcftools_filtered.v5.phased.imputemasked_chrXV_right.rmInvariants.phy.raxml.support.rename: Output of phylogenetic analysis using chrXV_right_Okinebe.bed.
Okinebe_analysis_bcftools_filtered.v5.phased.imputemasked_chrXV_target.rmInvariants.phy.raxml.support.rename: Output of phylogenetic analysis using chrXV_target_Okinebe.bed.
Okinebe_analysis_bcftools_filtered.v5.phased.imputemasked_chrXXI_target1.rmInvariants.phy.raxml.support.rename:Output of phylogenetic analysis using chrXXI_target1_Okinebe.bed.
Okinebe_analysis_bcftools_filtered.v5.phased.imputemasked_chrXXI_target1_left.rmInvariants.phy.raxml.support.rename:Output of phylogenetic analysis using chrXXI_target1_left_Okinebe.bed.
Okinebe_analysis_bcftools_filtered.v5.phased.imputemasked_chrXXI_target1_right.rmInvariants.phy.raxml.support.rename: Output of phylogenetic analysis using chrXXI_target1_right_Okinebe.bed.
Okinebe_analysis_bcftools_filtered.v5.phased.imputemasked_chrXXI_target2.rmInvariants.phy.raxml.support.rename:Output of phylogenetic analysis using chrXXI_target2_Okinebe.bed.
Okinebe_analysis_bcftools_filtered.v5.phased.imputemasked_chrXXI_target2_left.rmInvariants.phy.raxml.support.rename: Output of phylogenetic analysis using chrXXI_target2_left_Okinebe.bed.
(17) FigureS13: fd of one river system was divided by fd of another river system, followed by log2 transformation. Because many fd values were zero, we added 1 to each value before division and then applied a log2 transformation. Black and red arrowheads indicate regions used for phylogenetic analysis in Figure S12 and S14.
Okinebe_FigureS13.R: R scripts used for the analyses and figure generation in Figure S13.
fd_4pops.txt: fd data of 4 populations.
Chr: Chromosome.
Start: Start position of each genomic window.
End: End position of each genomic window.
Akkeshi: fd between G. nipponicus and G. aculeatus in the Bekanbeushi population.
Okinebe: fd between G. nipponicus and G. aculeatus in the Okinebe population.
Tokotan: fd between G. nipponicus and G. aculeatus in the Tokotan population.
Allopatric: fd between G. nipponicus and G. aculeatus in the allopatric population.
(18) FigureS14
Phylogenetic analysis of introgressed regions on Chr. 14. Genome sequences were phased, so there are two haplotypes for each fish. Arrowheads indicate the introgressed haplotype. Orange and blue labels indicate G. nipponicus and G. aculeatus, respectively. Bootstrap values of a branch separating haplotypes of two species are shown in red. A yellow arrowhead indicates a haplotype that is carried by a G. aculeatus individual, but clusters with G. nipponicus haplotypes.
chrXIV_Okinebe.bed: Genomic position of used in Figure S14.
chrom: Chromosome.
chromStart: Start position of genomic region.
chromEnd: End position of genomic region.
Okinebe_analysis_bcftools_filtered.v5.phased.imputemasked_chrXIV_target.rmInvariants.phy.raxml.rename: Output of phylogenetic analysis using chrXIV_Okinebe.bed.
(19) TableS6
Model summary of GLM for stomach content analysis.
Okinebe_TableS6.R: R scripts used for the analyses in TableS6.
StomachJP.txt: Data for producing TableS6.
Number: Sequential sample number.
Species: aculeatus (G. aculeatus), nipponicus (G. nipponicus).
Ostracoda: Presence (1) or absence (0) of Ostracoda.
Amphipoda: Presence (1) or absence (0) of Amphipoda.
(20) PopulationGenetics
RAD2013Gas
AdmixtureRAD2013Gas.sh: Linux script for admixture analysis of 2013 Gasterosteus sticklebacks.
SampleListRAD2013Gas.txt: Sample list used for 1st SNP call.
RAD2013GasFiltThin_admixture.map: .map file used for admixture analysis of 2013 Gasterosteus sticklebacks.
RAD2013GasFiltThin_admixture.ped: .ped file used for admixture analysis of 2013 Gasterosteus sticklebacks.
RAD2013GasFilt.vcf: SNP data after filtering of 2013 Gasterosteus sticklebacks.
RAD2013Pun
AdmixtureRAD2013Pun.sh: Linux script for admixture analysis of Pungitius sticklebacks.
SampleListRAD2013Pun.txt: Sample list used for 1st SNP call.
SampleListbqsrRAD2013Pun.txt: Sample list used for 2nd SNP call.
RAD2013PunFiltThin_admixture.map: .map file used for admixture analysis of Pungitius sticklebacks.
RAD2013PunFiltThin_admixture.ped: .ped file used for admixture analysis of Pungitius sticklebacks.
RAD2013PunFilt.vcf: SNP data after filtering of Pungitius sticklebacks.
RAD2021Gas
AdmixtureRAD2021Gas.sh: Linux script for admixture analysis of 2021, 2022, 2023 Gasterosteus sticklebacks.
SampleListRAD2021Gas.txt: Sample list used for 1st SNP call.
SampleListbqsrRAD2021Gas.txt: Sample list used for 2nd SNP call.
RAD2021GasFiltThin_admixture.map: .map file used for admixture analysis of 2021, 2022, 2023 Gasterosteus sticklebacks.
RAD2021GasFiltThin_admixture.ped: .ped file used for admixture analysis of 2021, 2022, 2023 Gasterosteus sticklebacks.
RAD2021GasFilt.vcf: SNP data after filtering of 2021, 2022, 2023 Gasterosteus sticklebacks.
Oki2021_Pond_J.txt: List of G. nipponicus collected from Okinebe pond in 2021.
Oki2021_Pond_P.txt: List of G. aculeatus collected from Okinebe pond in 2021.
Oki2021_River_J.txt: List of G. nipponicus collected from Okinebe river in 2021.
Oki2021_River_P.txt: List of G. aculeatus collected from Okinebe river in 2021.
WGS2013Gas
1.fastq_processing
fastp_parallel.sh: Script for quality control on fastq files.
exec_fastp_parallel.sh: Script for parallel execution of quality control on fastq files.
2.mapping
bwa-mem2_parallel.sh: Script for mapping.
exec_bwa-mem2_parallel.sh: Script for parallel execution of mapping.
3.SNPcall
bcftools_call_parallel.sh: Script for SNP call.
exec_bcftools_call_parallel.sh: Script for parallel execution of SNP call.
vcf_filter_bcftools.sh: Script for filtering of vcf file.
bam_list.txt: List of bam files used for analysis.
Bekanbe_Okinebe_Tokotan.txt: List of sample names used for analysis.
4.pixy
pixy_command.sh: Script for FST, dXY & Pi calculation using pixy.
popmap.txt: Population list used for pixy analysis.
5.fd
run_dsuite.sh: Script for fd calculation using dsuite.
popmap.txt: POPMAP file used for dsuite analysis.
test_trios.txt: TRIO file used for dsuite analysis.
6.phylogeny
shapeit_phasing_parallel.sh: Script for phasing using shapeit.
exec_shapeit_phasing_parallel.sh: Script for parallel execution of phasing.
make_input_and_run_raxml.sh: Script for phylogenetic analysis using raxml.
keep_noWLD.txt: List used for analysis.
vcf2phylip_haplotype.py: script used for analysis.
Code/software
Scripts are included in the submitted datasets.
