Research ArticleEVOLUTIONARY BIOLOGY

Two different epigenetic information channels in wild three-spined sticklebacks are involved in salinity adaptation

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Science Advances  20 Mar 2020:
Vol. 6, no. 12, eaaz1138
DOI: 10.1126/sciadv.aaz1138
  • Fig. 1 Experimental space-for-time approach.

    We characterized DNA methylation profiles (via RRBS) and whole genomes [whole-genome sequencing (WGS)] of fish from three populations of wild-caught three-spined sticklebacks locally adapted to 6 (blue; n = 15), 20 (green; n = 16), and 33 (yellow; n = 15) PSU. We also bred and acclimated sticklebacks from the mid-salinity location (20 PSU) within one (“within-generational”) or over two (“transgenerational”) generations to decreased (6 PSU) or increased (33 PSU) salinity while maintaining a control group at its original salinity (n = 11 to 12 per group; see details in the figure). Differential methylation within and across generations was assessed and compared to natural populations locally adapted to the corresponding salinity, serving as the hypothetical future DNA methylation state to capture long-term adaptation processes.

  • Fig. 2 Graphical summary of the main results.

    We used the Baltic Sea salinity gradient to study the role of DNA methylation in local salinity adaptation and the response to salinity change in a space-for-time approach. To assess the potential future acclimatization and adaptation processes of the natural stickleback population from 20 PSU (KIE; green) to the predicted desalination (63), we compared differences in DNA methylation at CpG sites between wild-caught and laboratory-bred sticklebacks. Following the experiment timeline (bottom), we compared methylation levels of the experimental control group from 20 PSU to within- and transgenerational acclimation of 20 PSU sticklebacks to 6 PSU (DNA from left to right). The population locally adapted to 6 PSU serves as the hypothetical future state in which salinities will decrease (blue; DNA on the right). The three main results are written in the circles with schematically and horizontally corresponding DNA methylation changes. (i) Sixty-three percent of the DMS between the populations remained stable under experimental salinity change. (ii) The direction of experimental methylation change was dependent not only on the treatment but also on the degree of genetic differentiation between the populations [see Fig. 4 (A to D) for results]. (iii) Transgenerational salinity acclimation shifted DNA methylation patterns closer to the anticipated adaptive state found in the hypothetical future population [see Fig. 4 (E to H) for results]. For clarity, only one (6 PSU) of the two foreign salinity regimes tested (6 and 33 PSU) is shown. The results for the experimental fish acclimated to 33 PSU were very similar (see Fig. 1 for full experimental design and Fig. 4 for results).

  • Fig. 3 Gene Ontology terms for biological processes and molecular functions.

    Gene Ontology (GO) terms for biological processes and molecular functions under salinity increase (20 versus 33 PSU; yellow) and decrease (20 versus 6 PSU; blue) associated with pop-DMS are presented. The graph is split into GO terms associated with pop-DMS from natural stickleback populations across a salinity cline (wild) and their experimental inducibility (inducible and stable) in a two-generation acclimation experiment. The size of the circles refers to the number of genes of this term in the groups (in %), and the transparency refers to the false discovery rate–corrected P value (darker circles refer to a lower adjusted P value). This subset is filtered for GO terms including the following keywords: “channel,” “transport,” “water,” “chloride,” “potassium,” “homeostasis,” “ion-dependent,” “urine,” “ATP” (adenosine 5′-triphosphate), and “metabolic”; see fig. S2 for the full figure. cGMP, guanosine 3′,5′-monophosphate; cAMP, adenosine 3′,5′-monophosphate; G protein, heterotrimeric GTP-binding protein.

  • Fig. 4 The duration of acclimation (within-generational versus transgenerational) and level of genomic differentiation between populations influence DNA methylation at inducible sites.

    (A and B) Mean FST values for inducible pop-DMS (with a ± 5-kb window) under experimental salinity decrease (top; blue) and increase (bottom; yellow) that shifted methylation levels toward the values observed in either the field (expected) or the opposite direction (opposite). A randomization test (with 10,000 bootstraps) was performed for the difference between expected and opposite mean FST value (δ.mean.FST = expected mean FST – opposite mean FST) (C and D). Under the one-tailed hypothesis of increased genetic differentiation at opposite sites and an α of 0.05, the P value was calculated as values smaller than the true difference divided by 10,000 bootstraps. In (E to H), the y axis shows the percentage match between the within- and transgenerational acclimation groups in relation to the methylation differentiation level found in natural populations at inducible pop-DMS. This value was obtained by calculating the difference between the methylation change in the experiment (meth.diff.exp in %; control versus within-generational or control versus transgenerational) and the difference in methylation between natural populations (meth.diff.wild in %) as δ.meth.diff = 100 − (meth.diff.wild − meth.diff.exp). Mean values ± 95% confidence interval are shown for within- and transgenerational acclimation to decreased and increased salinity at expected and opposite inducible sites. Colors refer to the direction of DNA methylation change (hypomethylation or hypermethylation). Values closer to 100 indicate a shift in methylation pattern toward adaptive methylation levels found in natural populations, and asterisks indicate the significance level (***P ≤ 0.001 and **P ≤ 0.01) for the comparison between within- and transgenerational acclimation. “Main effect” refers to an effect of acclimation (within- or transgenerational), and “interaction effect” refers to an interaction of acclimation and methylation direction (hypo- or hypermethylation).

  • Fig. 5 Effects of salinity acclimation on fitness-correlated factors.

    For all five acclimation groups [control group (20 PSU), within-generational, and transgenerational acclimation to 6 or 33 PSU], survival rates in percent (A), standard length in centimeters (B), hepatosomatic index (C), and total weight in grams (D) are displayed. Letters indicate significant differences resulting from Tukey post hoc tests (table S3). HSI, hepatosomatic index.

  • Table 1 Differentially methylated genes across natural populations along a salinity cline.

    Genes derived from DNA methylation comparisons between natural populations associated with ≥10 pop-DMS [decreased salinity: KIE (20 PSU) versus NYN (6 PSU); increased salinity: KIE (20 PSU) versus SYL (33 PSU)]. Ensembl gene ID and name as well as the position on the chromosome are listed. The numbers refer to the numbers of DMS in the population comparison (wild). These DMS were classified into inducible, inconclusive, and stable sites according to their behavior in a two-generation salinity acclimation experiment with laboratory-bred sticklebacks from the mid-salinity population (20 PSU) exposed to experimental salinity increase or decrease (33 and 6 PSU, respectively). Furthermore, inducible sites were distinguished whether they matched methylation levels of the locally adapted population (expected) or not (opposite). Genes written in bold vary in both population comparisons. We used a Fisher’s exact test to assess whether pop-DMS associated to the same gene are correlated in their response to experimental salinity change (nonrandom distribution among the categories stable, inducible, and inconclusive) and reported corresponding P values. For a full table on all genes associated with one or more pop-DMS, see table S2 (A and B).

    Ensembl gene IDChromosomeStart
    position
    End
    position
    Gene
    name
    WildInducibleExpected
    inducible
    Opposite
    inducible
    StableInconclusiveFisher’s
    exact (P)
    Salinity decrease:
    ENSGACG00000008328Chr101286014412863850si:dkey-166 k12.1240009150.005
    ENSGACG00000019416Chr744518924453656HMX1 ortholog17000980.033
    ENSGACG00000013229Chr181532771715352321150003120.011
    ENSGACG00000017287Chr31345452713465167mmp16b120001200.001
    ENSGACG00000017584Chr31469081414694448CCNY1212120000.001
    ENSGACG00000018249Chr41214162512143011si:ch211-153b23.512110380.188
    ENSGACG00000008034Chr6936818793809411110100010.014
    ENSGACG00000009469Chr191665769173856egln2110001100.001
    ENSGACG00000004433Chr1721274572211376igsf21a1010100000.003
    ENSGACG00000007343Chr101066699510679875col9a210000640.227
    ENSGACG00000018407Chr41382833613837518Sncb10220530.848
    Salinity increase:
    ENSGACG00000020323Chr7170101601701117623000221<0.001
    ENSGACG00000013229Chr1815327717153523211510100140.125
    ENSGACG00000013359Chr111296088312968110sec14l1150001230.011
    ENSGACG00000019416Chr744518924453656HMX1 ortholog15330570.745
    ENSGACG00000002948Chr8218240221355ddx1014000680.077
    ENSGACG00000016350Chr143603545360492314101760.277
    ENSGACG00000006636Chr1847808934786820ZC3H12D130003100.034
    ENSGACG00000004667Chr1242734984286193tti1120001200.001
    ENSGACG00000015566Chr290430629051779casc4100001000.003

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/12/eaaz1138/DC1

    Fig. S1. Significant DMS throughout the genome for comparison between KIE versus NYN (20 versus 6 PSU; blue fish) and KIE versus SYL (20 versus 33 PSU; yellow fish).

    Fig. S2. GO terms for biological processes, cellular components, and molecular functions under salinity increase (20 versus 33 PSU; yellow) and decrease (20 versus 6 PSU; blue) associated with pop-DMS.

    Table S1. Relative distribution of DMS among genomic features.

    Table S2A. Differentially methylated genes between populations from KIE (20 PSU) and NYN (6 PSU).

    Table S2B. Differentially methylated genes between populations from KIE (20 PSU) and SYL (33 PSU).

    Table S3A. Tukey post hoc test results for survival rate.

    Table S3B. Tukey post hoc test results for SDL.

    Table S3C. Tukey post hoc test results for HSI.

    Table S3D. Tukey post hoc test results for total weight.

    Table S4. Summary statistics for whole-genome resequencing of wild-caught sticklebacks.

    Table S5A. Summary statistics for the RRBS of experimental fish.

    Table S5B. Summary statistics for the RRBS of wild-caught fish.

    Table S6. The number of DMS for each of the two pairwise population comparisons (pop-DMS).

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Significant DMS throughout the genome for comparison between KIE versus NYN (20 versus 6 PSU; blue fish) and KIE versus SYL (20 versus 33 PSU; yellow fish).
    • Fig. S2. GO terms for biological processes, cellular components, and molecular functions under salinity increase (20 versus 33 PSU; yellow) and decrease (20 versus 6 PSU; blue) associated with pop-DMS.
    • Table S1. Relative distribution of DMS among genomic features.
    • Table S2A. Differentially methylated genes between populations from KIE (20 PSU) and NYN (6 PSU).
    • Table S2B. Differentially methylated genes between populations from KIE (20 PSU) and SYL (33 PSU).
    • Table S3A. Tukey post hoc test results for survival rate.
    • Table S3B. Tukey post hoc test results for SDL.
    • Table S3C. Tukey post hoc test results for HSI.
    • Table S3D. Tukey post hoc test results for total weight.
    • Table S4. Summary statistics for whole-genome resequencing of wild-caught sticklebacks.
    • Table S5A. Summary statistics for the RRBS of experimental fish.
    • Table S5B. Summary statistics for the RRBS of wild-caught fish.
    • Table S6. The number of DMS for each of the two pairwise population comparisons (pop-DMS).

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