This meta-analysis and meta-regression of randomized clinical trials evaluates the association of magnitude of differential blood pressure reduction and recurrent stroke in patients with stroke or transient ischemic attack. How much better is more blood pressure reduction vs less blood pressure reduction for secondary stroke prevention?In this meta-analysis and meta-regression that included 10 randomized clinical trials comprising 40 710 patients with stroke or transient ischemic attack, the risk of recurrent stroke was 8.4% with more intensive blood pressure lowering vs 10.1% with less intensive or no blood pressure lowering, a statistically significant difference. The greater the amount of differential blood pressure reduction, the greater the amount of reduction risk of recurrent stroke.This study suggests that more intensive differential blood pressure–lowering therapy may be beneficial for secondary stroke prevention. The degree to which more intensive blood pressure reduction is better than less intensive for secondary stroke prevention has not been delineated.To perform a standard meta-analysis and a meta-regression of randomized clinical trials to evaluate the association of magnitude of differential blood pressure reduction and recurrent stroke in patients with stroke or transient ischemic attack (TIA).PubMed, Embase, the Cochrane Central Register of Controlled Trials, and ClinicalTrials.gov were searched from January 1, 1980, to June 30, 2022.Randomized clinical trials that compared more intensive vs less intensive blood pressure lowering and recorded the outcome of recurrent stroke in patients with stroke or TIA.PRISMA) reporting guideline was used for abstracting data and assessing data quality and validity. Risk ratio (RR) with 95% CI was used as a measure of the association of more intensive vs less intensive blood pressure lowering with primary and secondary outcomes. The univariate meta-regression analyses were conducted to evaluate a possible moderating effect of magnitude of differential systolic blood pressure (SBP) and diastolic blood pressure (DBP) reduction on the recurrent stroke and major cardiovascular events.The Preferred Reporting Items for Systematic Reviews and Meta-analyses  with stroke or TIA were included for analysis. The mean duration of follow-up was 2.8 years . Pooled results showed that more intensive treatment compared with less intensive was associated with a reduced risk of recurrent stroke in patients with stroke or TIA . Meta-regression showed that the magnitude of differential SBP and DBP reduction was associated with a lower risk of recurrent stroke in patients with stroke or TIA in a log-linear fashion . Similar results were found in the association between differential blood pressure lowering and major cardiovascular events.More intensive blood pressure–lowering therapy might be associated with a reduced risk of recurrent stroke and major cardiovascular events. These results might support the use of more intensive blood pressure reduction for secondary prevention in patients with stroke or TIA. Indeed, recurrent stroke has declined substantially over decades, with improved blood pressure control as a leading cause.3 Furthermore, some trials4 compared antihypertensive therapy with placebo, while other trials5 compared a lower systolic blood pressure (SBP) target with a higher SBP-lowering target. Pooling of data from these 2 types of trials is needed to clarify the association between degree of increased blood pressure lowering attained with therapy and secondary stroke prevention.However, most individual randomized clinical trials have not significantly showed that blood pressure–lowering therapy reduces recurrent stroke in patients with stroke or TIA. Relatively small magnitude of blood pressure reduction and relatively few participants and inadequate statistical power in single randomized clinical trials may have attenuated signals of efficacy.Therefore, we performed a systematic review and meta-analysis of all relevant randomized clinical trials to evaluate the association between magnitude of differential blood pressure reduction and risk of recurrent stroke and performed a meta-regression to clarify whether larger differential blood pressure reduction magnitude was monotonically associated with a lower recurrent stroke risk in patients with stroke or TIA.PRISMA) reporting guideline was used for abstracting data and validity of this meta-analysis.6 The protocol was registered with PROSPERO (CRD42022309056).The Preferred Reporting Items for Systematic Reviews and Meta-analyses  independently screened and identified potential trials, and discrepancies were resolved by discussion with a third investigator (M.L.).We searched PubMed, Embase, the Cochrane Central Register of Controlled Trials, and the clinical trial registry maintained at ClinicalTrials.gov from January 1, 1980, to June 30, 2022, using the following terms: stroke or cerebrovascular accident or brain vascular accident or cerebral infarct or cerebrovascular disorder or intracranial vascular disease or cerebrovascular disease or cerebrovascular occlusion or transient ischemic attack AND antihypertensive or blood pressure lowering or blood pressure reduction AND recurrent or secondary prevention or previous or prior or history. We restricted the search to studies in humans and randomized clinical trials and did not apply language restrictions. We also reviewed prior meta-analysesCriteria for inclusion of a study were as follows: (1) the study design was a randomized clinical trial; (2) all or an identifiable subset of participants had a history of stroke or TIA; (3) the study evaluated more intensive vs less intensive blood pressure–lowering therapy, including the following possible comparisons: antihypertensive drug(s) vs placebo and a lower blood pressure target vs a higher blood pressure target; (4) recurrent stroke was reported as an end point; (5) the magnitude of SBP reduction between more intensive and less intensive blood pressure lowering was reported; and (6) treatment duration was of at least 1 year.1; (2) the study adopted only nonpharmaceutical approach, such as salt restriction or exercise, in the active treatment arm; (3) the study compared one antihypertensive drug vs another antihypertensive drug; (4) achieved SBP was higher in more intensive blood pressure–lowering group than less intensive blood pressure–lowering group after treatment; (5) more than 10% of patients were enrolled within 3 days after stroke; or (6) more than 10% of patients had end-stage kidney disease because of differential hemodynamic vulnerability of this disease. We extracted characteristics of each trial, which included patient age, sex, baseline blood pressure, number of patients in more intensive and less intensive blood pressure–lowering groups, duration of follow-up, magnitude of differential blood pressure reduction between more intensive and less intensive blood pressure–lowering groups, and number of recurrent stroke events and other outcomes in more intensive and less intensive blood pressure–lowering groups. Two investigators (C.-Y.H. and M.L.) independently abstracted the data and any discrepant judgments were resolved by joint discussion and by referencing the original report.Criteria for exclusion of a study were as follows: (1) the study was published before 1980 because strategies for secondary stroke prevention, including antihypertensive drug use, were substantially different then11Because all of the included studies were randomized clinical trials, the risk of bias  of the included trials was assessed by the Cochrane risk-of-bias algorithm.The primary outcome was recurrent stroke. The lead secondary outcome was major cardiovascular events. Major cardiovascular events were defined as the composition of nonfatal stroke, nonfatal myocardial infarction, and death from cardiovascular causes. Additional secondary outcomes were recurrent ischemic stroke, hemorrhagic stroke, fatal or disabling stroke, myocardial infarction, death from cardiovascular causes, death from any cause, and heart failure.P values were from 2-sided tests, and results were deemed statistically significant at P < .05. Heterogeneity was assessed by a P value determined by the use of χ2 statistics and I2 statistics, and I2 values of 0% to 29%, 30% to 49%, 50% to 74%, and 75% to 100% represent not important, moderate, substantial, and considerable inconsistency, respectively.12The analysis plan was performed on an intention-to-treat basis. We computed the fixed-effects estimate based on the Mantel-Haenszel method. Risk ratio (RR) with 95% CI was used as a measure of the association of more intensive vs less intensive blood pressure lowering with the primary and secondary outcomes. To explore the association between magnitude of differential blood pressure reduction and risk of recurrent stroke, analyses were performed for thresholds of SBP reduction of 4 mm Hg or lower, 5 mm Hg or lower, more than 5 mm Hg, more than 7 mm Hg, and more than 11 mm Hg, as well as magnitude of differential diastolic blood pressure (DBP) reduction of 2 mm Hg or lower, 3 mm Hg or lower, more than 3 mm Hg, and more than 4 mm Hg between more intensive vs less intensive blood pressure–lowering groups. The univariate meta-regression analyses were conducted with the fixed-effects model to evaluate a possible moderating effect of magnitude of differential SBP and DBP reduction on the recurrent stroke and major cardiovascular events. All 13 A sensitivity test was conducted to identify any trial that might have exerted a disproportionate influence on the summary treatment effect on the primary outcome by removing each individual trial from the meta-analysis one at a time. Another sensitivity test was conducted by restricting analysis within trials with recurrent stroke being the primary outcome in the original trial design. Grading of Recommendations, Assessment, Development and Evaluations was used to evaluate summaries of evidence for the primary and secondary outcomes.15The trim-and-fill method to identify and correct for funnel plot asymmetry arising from publication bias was used.16 we also conducted a subgroup analysis for Asians vs non-Asians. The Cochrane Collaboration’s Review Manager Software Package version 5.4 (RevMan) and Stata/SE version 15.1 (StataCorp LLC) were used for this meta-analysis and mete-regression.Subgroup analyses of included trials were conducted according to different study characteristics: mean baseline SBP levels (≥150 mm Hg vs 140-149 mm Hg), mean achieved SBP levels in the more intensive and less intensive blood pressure–lowering groups (≥140 mm Hg vs 130 to <140 mm Hg vs <130 mm Hg), study duration (<3 years vs ≥3 years), sample size (<3000 vs ≥3000 patients), time interval from index stroke to randomization (within 6 months from stroke vs within 3-5 years from stroke), entry event (ischemic stroke vs hemorrhagic stroke), study design (antihypertensive drugs vs placebo and a lower blood pressure target vs a higher blood pressure target), definition of differential blood pressure reduction (mean difference throughout the studies vs other definitions), and antihypertensive drugs used in the more intensive treated arm (angiotensin-converting enzyme [ACE] inhibitors vs angiotensin receptor blockers vs β-blockers vs diuretics vs ACE inhibitors plus diuretics). Since the risk of recurrent ischemic and hemorrhagic stroke is greater in Asian populations with high blood pressure compared with several other groups around the world,22 The characteristics of the included trials are shown in 23 Overall, 40 710 patients  with stroke or TIA were enrolled. The mean duration of follow-up was 2.8 years . Among the 10 included trials, 6 compared antihypertensive drug(s) vs placebo or no antihypertensive therapy,20 and 4 compared a lower blood pressure target vs a higher blood pressure target.22 In the Perindopril Protection Against Recurrent Stroke Study (PROGRESS), 2 different active groups  were compared with placebo, and we analyzed separately perindopril alone vs placebo and perindopril plus indapamide vs placebo.4 Across all trials, the mean baseline SBP was 146 mm Hg and DBP was 85 mm Hg.We identified 21 full articles for detailed assessment, of which 11 did not meet the inclusion criteria; therefore, the final analysis included 10 randomized clinical trials ,17 1 adopted mean difference after 2 years,20 1 adopted difference at first 6 months of treatment,22 and 1 adopted difference at the last study visit.5 The mean SBP reduction was 6.7 mm Hg and the mean DBP reduction was 2.8 mm Hg between active treatment and comparator groups. The Cochrane risk-of-bias assessment for the included trials is summarized in eFigure 2 in The definition of differential blood pressure reduction varied among included trials. Among included trials, 5 adopted mean blood pressure difference between the 2 groups throughout the studyP < .001; number needed to treat [NNT] in 3 years, 58). There was considerable inconsistency among included trials   .2,3,4,5,P = .001)   A.2,3,4,5 = .003) B.2,3,4,121 or 5 mm Hg or lower 21 and associated with a greater effect magnitude when the amount of differential SBP reduction was more than 5 mm Hg ,22 more than 7 mm Hg ,22 and more than 11 mm Hg  NT = 14) .4,1921 or 3 mm Hg or lower 21 and associated with a greater effect magnitude when the amount of differential DBP reduction was more than 3 mm Hg 22 and more than 4 mm Hg  NT = 14) .4,19,22P < .001; NNT in 3 years, 61). There was substantial heterogeneity among included trials     A.2,3,4,5 = .048) B.2,3,4,1P < .001; NNT = 88). There was considerable inconsistency among included trials  . There was substantial heterogeneity among included trials  . Heterogeneity was not important among included trials  . Heterogeneity was not important among included trials  . Heterogeneity was not important among included trials  . Heterogeneity was not important among included trials  . Heterogeneity was not important among included trials        trials with patients enrolled within 3 to 5 years from stroke  than patients enrolled within 6 months from stroke  and the lead secondary outcome . In the forest plot of the recurrent stroke, the larger of the differential SBP reduction, the larger of the reduction of recurrent stroke was found visually. In the meta-regression analyses, magnitude of differential SBP and DBP reduction were significantly associated with a lower risk of recurrent stroke in patients with stroke or TIA. Therefore, heterogeneity might be primarily driven by the various magnitude of differential blood pressure lowering among included trials. Similar findings were noted in the major cardiovascular events.8 in several aspects. First, we excluded trials comparing one antihypertensive drug vs another antihypertensive drug 24 or achieved SBP higher in more intensive blood pressure–lowering group than less intensive blood pressure–lowering group after treatment.25 This approach was taken to be in line with the objective of our study. Second, the study by Katsanos et al8 only pooled data from trials with antihypertensive therapy vs placebo, whereas we pooled data from trials with antihypertensive therapy vs placebo and a lower blood pressure target vs a higher blood pressure target. The latter approach includes all relevant trials of blood pressure–lowering therapy for secondary stroke prevention. Third, the study by Katsanos et al8 adopted achieved blood pressure levels for meta-regression analyses, whereas this study adopted magnitude of differential blood pressure reduction for meta-regression analyses and showed that the larger the magnitude of differential blood pressure reduction, the larger the reduction of recurrent stroke and major cardiovascular events.The current study was distinct from the study by Katsanos et al27 It accords as well with studies in broader cardiovascular event qualifying event trials, including the Action to Control Cardiovascular Risk in Diabetes Blood Pressure (ACCORD BP) trial,28 the Systolic Blood Pressure Intervention Trial (SPRINT),30 and a trial of older Asian individuals with hypertension.31 Furthermore, physiologically, intensive (SBP target <120 mm Hg) compared with standard (SBP target <140 mm Hg) blood pressure–lowering therapy was associated with increased, not decreased, cerebral perfusion, in participants with a history of cardiovascular disease.32 Taken together, this study does not refute the current practice guidelines recommending blood pressure lowering to an SBP target of less than 130 mm Hg as a major target for patients with stroke or TIA.The current study is consonant with and extends prior investigations. In the current study, greater achieved differential blood pressure lowering was beneficial even among patients at the lower end of the blood pressure spectrum. The degree of separation between more intensive and less intensive SBP lowering was associated with a uniform relative risk reduction regardless of whether patients with less intensive treatment SBP levels of 140 mm Hg or higher, 130-139 mm Hg, or less than 130 mm Hg. This finding accords with analyses of intensive blood pressure lowering in stroke qualifying event trials, including PROGRESS and the Warfarin-Aspirin Symptomatic Intracranial Disease (WASID) trial.34 However, post hoc analyses of several trials showed that treating SBP to a target of less than 140 mm Hg in patients with severe intracranial stenosis might be beneficial.37 Since the risk-benefit profile of more aggressive blood pressure lowering is not consistent among studies, secondary prevention guidelines recommend a less aggressive SBP target (<140 mm Hg) in patients with 50% to 99% stenosis of a major intracranial artery.38Among patients with high-grade vertebrobasilar or other intracranial stenoses, an inverse association of SBP with recurrent stroke risk was found in some studies.40 These findings support a strategy of a moderate blood pressure–lowering target during the first 6 months following stroke or TIA followed by a more aggressive target chronically.Although this meta-analysis excluded trials with blood pressure–lowering therapy used in acute stage of stroke or TIA, the time interval from stroke to randomization still varied among included trials. A subgroup analysis suggested that more intensive compared with less intensive blood pressure–lowering therapy was associated with substantial risk reduction of recurrent stroke in trials with patients enrolled within 3 to 5 years from stroke or TIA but only associated with modest risk reduction of recurrent stroke in trials with patients enrolled within 6 months from stroke or TIA. This finding accords with observational studies that have suggested that aggressive blood pressure lowering might not be beneficial within the first 6 months following the index ischemic stroke event.41 and this meta-analysis showed that more intensive compared with less intensive blood pressure–lowering therapy was associated with a substantial relative risk reduction in hemorrhagic stroke . Also, the risk of recurrent ischemic and hemorrhagic stroke was greater in Asian population with high blood pressure compared with several other groups around the world.16 Therefore, it is conceivable that more intensive blood pressure–lowering treatment might be especially beneficial for Asian populations.A subgroup analysis also suggested that more intensive compared with less intensive blood pressure–lowering therapy was associated with a larger risk reduction of recurrent stroke in trials with study duration of 3 years or more vs less than 3 years. It is conceivable that a longer duration of high blood pressure levels may cause more profound damage of vessels thereby leading to a higher likelihood of recurrent stroke in patients with stroke or TIA. Also, we found that more intensive compared with less intensive blood pressure–lowering therapy was associated with a larger risk reduction of recurrent stroke in Asian populations than non-Asian populations. In general, the ratio of hemorrhagic stroke to ischemic stroke is higher in Asian than in non-Asian populations,22 and in such settings, there might be a higher chance that stroke events were not well recorded. Still, a sensitivity test restricting analysis within trials with recurrent stroke reported as the primary outcome obtained similar results. Third, although we excluded trials before 1980, the included trials reflect a long period of clinical practice during which concomitant treatments for stroke prevention further evolved.1 However, the consistency in benefit of blood pressure lowering suggests substantial benefit on top of a wide range of background therapies. Fourth, the individual studies mostly have wide CIs and many of the pooled results of secondary outcomes are driven by a single study with a large weight. Fifth, more than half of the participants are contributed by the Prevention Regimen for Effectively Avoiding Second Strokes (PRoFESS)2 and PROGRESS4 trials. Still, sensitivity testing removing either trial yielded pooled results similar to the overall pooled estimates of the primary outcome.There are several limitations to this study. First, although it was conceivable that some patients in the included trials had baseline SBP less than 140 mm Hg, none of included trials had mean baseline SBP less than 140 mm Hg. Granular analysis related to baseline BP levels could not be conducted because this study was a trial-level meta-analysis, rather than an individual, patient-level pooled analysis. Second, the purpose of some included trials was not to evaluate association between blood pressure lowering and risk of recurrent stroke,This meta-analysis and meta-regression of randomized clinical trials for blood pressure–lowering therapy found that more intensive compared with less intensive blood pressure–lowering therapy may be associated with a reduction of recurrent stroke; further, the larger the magnitude of differential blood pressure reduction, the larger the reduction of cerebrovascular events. More intensive blood pressure–lowering therapy may be also associated with a risk reduction of major cardiovascular events, ischemic stroke, hemorrhagic stroke, fatal or disabling stroke, and death from cardiovascular causes in patients with stroke or TIA. These results might support the use of more intensive blood pressure reduction for secondary stroke prevention chronically.


Pocillopora colonies collected from 32 sites on 11 islands across the Pacific Ocean to characterize host-photosymbiont fidelity and to investigate patterns of gene expression across a historical thermal gradient. We report high host-photosymbiont fidelity and show that coral and microalgal gene expression respond to different drivers. Differences in photosymbiotic association had only weak impacts on host gene expression, which was more strongly correlated with the historical thermal environment, whereas, photosymbiont gene expression was largely determined by microalgal lineage. Overall, our results reveal a three-tiered strategy of thermal acclimatization in Pocillopora underpinned by host-photosymbiont specificity, host transcriptomic plasticity, and differential photosymbiotic association under extreme warming.Heat waves are causing declines in coral reefs globally. Coral thermal responses depend on multiple, interacting drivers, such as past thermal exposure, endosymbiont community composition, and host genotype. This makes the understanding of their relative roles in adaptive and/or plastic responses crucial for anticipating impacts of future warming. Here, we extracted DNA and RNA from 102  Pocillopora corals have a three-tiered strategy of thermal acclimatization that is underpinned by host–photosymbiont specificity, host transcriptomic plasticity, and differential photosymbiotic associations under extreme warming.Using data from the Tara Pacific expedition to investigate symbiont fidelity and patterns of gene expression across a thermal gradient, this study shows that  Recent environmental perturbations, particularly rising sea surface temperatures and increasing frequency of extreme heating events , are disrupting coral-dinoflagellate photosymbioses worldwide, increasing the frequency and severity of coral bleaching events3. There is therefore growing concern about the potential for heating-driven local extirpation of coral species and severe reef loss in the current century. However, in some regions, corals exhibit higher than average heat tolerances4 or show the ability to rapidly respond to and recover from acute thermal challenges5. Repeated sublethal exposure to elevated sea surface temperatures can also drive positive acclimatization (phenotypic plasticity) and/or selection for thermotolerant genotypes . However, in other species, warm pre-conditioning had no or negative effects on holobiont performance during subsequent thermal challenges7. The capacity for thermal acclimatization and/or adaptation may therefore play an important role in determining potential winners and losers among coral species in the face of global change8. Determining whether acclimatization capacity can keep pace with projected environmental change and in which symbiotic assemblages, is therefore of paramount importance for predicting responses of the coral-dinoflagellate photosymbiosis under a warming climate.Coral reefs are ecologically and economically important ecosystems whose existence depends upon the mutualistic, photosymbiotic, association between certain Cnidarian hosts and their dinoflagellate symbionts. Breakdown of this association can lead to expulsion of algal symbionts  and ultimately result in coral mortality and reef loss11. The mechanisms underlying these capacities as well as their limits and extent among different coral holobiotypes and the relative roles of each symbiotic partner remain under intense investigation. In some cases, alteration of the dominant photosymbiont lineage, from thermally sensitive to thermally tolerant Symbiodiniaceae for example, confers a higher resistance of the coral holobiont to heat stress13. In other cases, thermal acclimatization is achieved through altered gene expression in the host and/or symbiont, including high basal expression  of certain stress response genes, resulting in increased holobiont thermal tolerance15. Coral holobiont thermal sensitivity is therefore dependent on an array of interacting drivers, including environmental history19, endosymbiont community composition23, and host genotype25. However, the relative role of each of these factors in determining holobiont expression in natura remains poorly resolved. A remaining challenge, therefore, is to better understand how environmental context affects the complex interplay between the host and symbiont and to what extent certain genotypes and or host/symbiont pairings might confer resilience or resistance to projected environmental change. Finally, acclimatization or adaptation of a coral holobiont at the local (reef) scale is often not representative of capacities at larger (ocean) scales and therefore does not allow for accurate global projections of the survival or decline of a species or of reef ecosystems15.Understanding how corals will respond to ongoing climate change requires prior knowledge regarding their capacities for thermal acclimatization  and adaptation15. The Tara Pacific expedition provides a unique opportunity to address these important questions because it allows for comparative analyses of differences in holobiont community composition and photosymbiotic regulation among environments and species across the tropical Pacific Ocean26.Given the variety of components involved in maintaining the coral-dinoflagellate mutualism against environmental perturbation, recent analyses have begun to adopt a more integrative and multidisciplinary approach to understanding the impacts of climate change on coral health. Integration of various analysis methods  is needed to draw a global picture of coral holobiont adaptation and acclimatization capacitiesPocillopora spp. and their associated photosymbionts alongside metagenomic sequences and environmental context data from 11 islands across the Pacific Ocean  across the Pacific Ocean.To this end, we investigated gene expression profiles of 102 colonies of ean Fig.  in orderPocillopora lineages exhibit a high degree of host–photosymbiont fidelity across environments with instances of symbiotic flexibility and/or breakdown associated with corals living on reefs which have historically experienced elevated sea surface temperatures (SSTs). We also describe how host and photosymbiont expression profiles respond to different drivers and discuss how these mechanisms may represent a three-tiered strategy of thermal acclimatization/adaptation in Pocillopora corals. Finally, we discuss the implications of this strategy for continued persistence of Pocillopora holobionts under current and projected future ocean warming.Here we show that multiple Pocillopora hosts were taxonomically identified using genome-wide SNPs clustering and coalescent (SVD quartet) analysis as described in ref. 27. They obtained five distinct genetic lineages (SVD1 to SVD5), which were further assigned to the species level based on a set of Pocillopora reference sequences from refs. 28 and the mtORF sequences from ref. 29. This resulted in the identification of 12 colonies as belonging to P. cf. effusa , 19 colonies to P. meandrina , 17 colonies to P. verrucosa , 33 colonies to P. grandis  and 21 colonies to a cryptic P. verrucosa species , hereafter referred to as SSH5_pver . The mtORF phylogeny and Pocillopora species name connections with previously published studies are reported in Supplementary Fig. 30, complementary analyses of skeletal morphology will be necessary to confirm these species names with the respective type specimen vouchers31. We used two different genetic markers (nuclear ITS2 sequences and plastid-encoded psbAncr sequences) alongside transcriptome-wide SNPs to identify Symbiodiniaceae lineages in each Pocillopora colony. The ITS2 sequence profiles revealed that 84 Pocillopora colonies hosted Cladocopium C1/C42-based profiles, 9 colonies hosted Durusdinium D1-based profiles, and 9 colonies contained both Cladocopium and Durusdinium in varying proportions . These ITS2 profiles are all associated with Durusdinium glynnii. To identify the different Cladocopium lineages, we aligned metatranscriptomic reads of Pocillopora colonies on the predicted genes of the C. goreaui genome32 and called the SNPs for each Pocillopora colony hosting only Cladocopium. We clustered the frequencies of 3712 biallelic SNPs distributed across 1354 transcripts and obtained an optimal number of five different clusters potentially corresponding to five different lineages hereafter named L1 to L5  recently sequenced in three Cladocopium species: C. goreaui, C. latusorum, and C. pacificum33. The psbAncr sequence robustly separated Cladocopium L1 from L2/L3 and L4/L5. Cladocopium L1 is most similar to C. goreaui, whereas L2/L3 are two different taxa within C. latusorum and L4/L5 are two different taxa within C. pacificum  was incoherent between the three methods. They were assigned to L3, L2, or L5 according to the SNP, ITS2 and psbAncr analyses, respectively. Given that the SNP analysis is based on a larger number of variants distributed across the entire transcriptome, in comparison to the short and multi-copy (ITS2) or plastid-encoded (psbAncr) marker sequences, we consider that these two Cladocopium populations belong to L3.ver Fig. . We alsoeages SVD to SVD5,Pocillopora species that we sampled  exhibited near perfect symbiotic fidelity—i.e., a one to one pairing of photosymbiont lineage and host—which persisted across all sampled environments  were always found in symbiosis with C. pacificum (L2). Similarly, P. meandrina colonies  always hosted C. latusorum (L5). Finally, P. verrucosa which was present in both the Central and Western Pacific, displayed near perfect symbiotic fidelity with C. pacificum (L3) across its range, with only one instance of a difference in photosymbiont association (one colony in Guam hosting D. glynnii) which we discuss, below. The P. meandrina/P. verrucosa hybrid on Niue island (I09S03C010) hosted C. pacificum (L3).Three nts Fig. . P. cf. Pocillopora species (P. grandis and SSH5_pver) suggest symbiont flexibility. Pocillopora SSH5_pver was the only lineage sampled on Rapa Nui and is in symbiosis with C. goreaui. SSH5_pver was also present on Ducie and Moorea islands in symbiosis with C. pacificum (L3). The two symbionts of SSH5_pver perfectly match to two genetic subclades of SSH5_pver  and hosted C. latusorum (L5) and/or D. glynnii. P. grandis was also present on Ducie and Gambier islands where it was found in association with C. latusorum (L4). Similarly, the two Cladocopium symbionts of P. grandis match two genetic subclades that are geographically separated.The two other ver Fig.  that arePocillopora with its Cladocopium symbionts, we performed a cophylogeny analysis by testing phylogenetic congruence using the procrustean approach34. The host–symbiont associations that contributed the most to the global phylogenetic congruence between Pocillopora and Cladocopium  were the dependence of P. grandis, P. meandrina, and P. verrucosa on C. latusorum L4, L5, and C. pacificum respectively  and SSH5_pver on C. goreaui and C. pacificum L3 in the central Pacific.To understand whether the observed host–symbiont fidelity is the result of long-term cospeciation of ely Fig. . ConversThe variation partitioning approach allowed us to identify genes whose expression levels were driven primarily by the sampling environment, the host genetic lineage, or the photosymbiont genotype (see “Methods”). For the host, among the 1821 genes with greater than 50% of their expression variation attributed to these three factors, 57% (1042 genes) were linked to the environment, 33% (606 genes) were associated with the host genetic lineage, and 9.5% (173 genes) were attributed to the photosymbiont genetic lineage Fig. . FunctioPocillopora host, genes with expression dependent on the sampled island were enriched in biological process ontologies related to lipid and carbohydrate metabolism , ion transmembrane transport (16 of 82 genes) including transmembrane transport of ammonium (3 of 11 genes), and calcium (9 of 64 genes), as well as regulation of DNA repair (9 of 102 genes) and autophagy , active transport of protons across membranes , and oligosaccharide processing  and xenobiotic transport (3 of 77 genes), DNA recombination (8 of 156 genes), and response to ionizing radiation  and aquaporin-like or neutral solute transporters  particularly of calcium (3 of 64 genes), nitric oxide biosynthesis (1 of 1 gene), and assembly of the mitochondrial respiratory chain complex IV  and gluconeogenesis . Thirteen calcium-transporter and calcium-dependent protein kinases that have been shown to play a role in the establishment of symbiosis35 are also dependent on the host lineage  of gene expression. For the host, the 11 PCs retained after a-score maximization in the DAPC explained 55.18% of total gene expression variance . In Cladocopium, when colonies were assigned to pre-defined groups based on the environment, host lineage or photosymbiont lineage, we were able to correctly reassign 89%, 99%, and 99% of colonies to their respective groups (Supplementary Data P < 0.001). Therefore, based on proportions of colonies correctly re-assigned to pre-defined groups, our DAPC results revealed a marginally stronger influence of the genotype (or the genotype of the symbiotic partner) than of the environment for both the host and the symbiont. However, in the host we observed a higher number of genes associated with the environment (4386 genes) than associated with the host and symbiont lineages  along DF2 Fig. . ColonieCladocopium, genes whose expression strongly distinguished photosymbionts from Rapa Nui from all others (DF1 contributing genes) were enriched in protein family (Pfam) domains related to chlorophyll A-B binding (35 genes) and ion transport . The results of these analyses are presented in Supplementary Data binding 3 genes anCladocopium photosymbiont, mean climatological SST associated data were the strongest contributors  was primarily driven by differences in degree heating week severity between sampling locations. Colonies from islands with low DHW values  clustered around positive CCA1 values, whereas colonies from islands with historically elevated SSTs  clustered around negative CCA1 values , we identified 10 environmental variables (or variable clusters) that best explained the dispersion among host gene expression profiles Fig. . Similarues Fig. . In contues Fig. . NegativCladocopium photosymbiont, historical SST was strongly negatively correlated with CCA1 , but not between Cladocopium- and Durusdinium-containing colonies . In addition, intra-island comparison of expression profiles between colonies in the Eastern Tropical Pacific revealed only a small number of genes that were differentially expressed between Cladocopium- and Durusdinium-containing hosts (Supplementary Data Durusdinium-containing colonies (FDR-adjusted P ≤ 0.05 and a LFC ≥ 2) on the three islands, respectively  on the three islands, respectively  differed between islands , and limited flexibility for two species (P. grandis and SSH5_pver). For P. grandis and SSH5_pver, their two Cladocopium symbionts match to two distinct subclades that are geographically separated (discussed below). P. grandis/P. meandrina associated with C. latusorum and P. verrucosa with C. pacificum in concordance with previous observations33. The association of SSH5_pver with C. goreaui or C. pacificum and P. cf. effusa with C. pacificum has not been previously reported.Strong fidelity between genetically distinct coral hosts and specific Symbiodiniaceae species has been described for a wide variety of reef-building species42 including those inhabiting some of the hottest reefs in the world44. Previous studies have suggested that this tendency for high symbiotic fidelity in corals, and within pocilloporids specifically, reflects an underlying cospeciation between the Pocillopora host and its photosymbiont33, which supports the persistence of distinct holobiotypes which maximize holobiont fitness in a given environment.Our findings, therefore, support the general consensus that fidelity to a single putative symbiont species is the dominant pattern in most scleractinian corals45. We detected two broad instances of symbiont flexibility among the sampled Pocillopora species including a differential association with D. glynnii among P. grandis hosts in the Eastern Tropical Pacific  and within a single colony of P. verrucosa on Guam. Pocillopora - Durusdinium associations have been reported on previously. For example, in the far Eastern Tropical Pacific, P. meandrina (mtORF type 1) and P. verrucosa (mtORF type 3) hosts are known to flexibly alter their symbiotic associations between certain lineages of Cladocopium and D. glynnii depending on local thermal conditions41. Similarly, on Guam, Pocillopora colonies hosting D. glynnii were shown to have better maintained photosynthetic productivity under acute heat stress than those hosting Cladocopium46. Because these associations appear to be stable and persistent in these locations across time, it can be argued that this photosymbiotic association is stable between pocilloporids and D. glynnii, particularly in the far Eastern Tropical Pacific where the association is common, may indicate a stable strategy for survival on these warm reefs as discussed below.Despite this overarching tendency towards symbiotic fidelity in corals, symbiotic associations are also sometimes flexible indicating a potential role for acclimatory phenotypic plasticity to modulate holobiotypes under certain conditions, particularly acute heat stressPocillopora species with two different Cladocopium lineages was always observed in geographically distant islands supporting the hypothesis of speciation driven by niche diversification within Cladocopium genera33. In addition, this flexibility of symbionts matches perfectly with two different subclades of each Pocillopora species. Therefore this apparent flexibility is probably stable and not due to recent changes in the environment.The association of one Pocillopora phylogeny is similar to the Cladocopium phylogeny suggesting that the symbiont speciation is driven by the host in agreement with the cospeciation hypothesis29. However, we observed two major breaks in this cospeciation not previously reported. In the first instance, the association of SSH5_pver with two genetically distant Cladocopium could be due to the relative isolation of SSH5_pver colonies from Rapa Nui and consequent adaptation to the unique local environmental conditions in this location . The presence of C. pacificum in P. cf. effusa in the central Pacific is also incoherent with the cospeciation pattern, but the reasons for this difference in symbiotic association remain to be studied.In general, the Pocillopora host, we identified a greater number of genes whose expression profiles were correlated with variation in the environment than with either the host- or symbiont-genetic lineages. However, both analyses also indicated that the most influential genes  had expression profiles that were linked to the host genetic lineage. This mixed result, suggests that the steady-state expression profiles we measured in Pocillopora hosts are jointly responsive to the host genetic lineage and to the local abiotic environment. Host gene expression therefore reflects the classical conception of the phenotype as an integrated expression of the genotype and the environment. The high proportion of colonies that were correctly re-assigned to their genetic lineage based on their expression profiles . However, assessing coral holobiont transcriptomic plasticity is complicated by the fact that many different drivers  may interact to determine expression profiles in natura. For the Pocillopora genes with expression dependent on the symbiont lineage were involved in several important biological processes including those potentially related to regulation of calcification, symbiont photosynthesis, and to the scavenging of reactive oxygen species. Among them, ATPase-coupled transmembrane transporters are known to play a role in host calcification49 and as carbon-concentrating mechanisms in marine photosymbioses thereby regulating Symbiodiniaceae photosynthesis53. These responses likely serve to maintain symbiosome homeostasis and regulate photosymbiont productivity across environments.Although they represented only a small fraction of the top variant genes, Cladocopium were most strongly correlated to their respective genetic lineages and comparatively weakly influenced by the environment. We suggest two, potentially complementary, explanations for the difference in environmental sensitivity between the host and the symbiont: (i) it reflects a primarily host-driven homeostatic regulation of the photosymbiont micro-environment such that environmental influences on symbiont expression are minimized, and/or (ii) that symbiont transcriptomic profiles reflect inherent differences in physiological regulation between microalgal species which outweighed environmental influences over the range of thermal habitats we investigated in this study.In contrast to the host, gene expression profiles within 55. For example, following a simulated heatwave event, Acropora aspera hosts exhibited a significantly more robust transcriptional response than their associated photosymbionts with strong upregulation of several host genes including heat shock proteins54. In addition, bleaching susceptibility of corals following an acute heatwave was found to be tied to a reduced capacity of the host to maintain intracellular acid–base homeostasis56. Finally, in photosymbiotic Aiptasia, disruption of host homeostasis occurred prior to symbiont photoinhibition and bleaching suggesting that it is the cnidarian host that first responds to environmental perturbation57. Further research is necessary to determine whether the relatively stable gene expression profiles we observed within Cladocopium lineages across environments are a result of strong regulatory control by the Pocillopora hosts and the degree to which these profiles may exhibit plasticity under acute thermal stress.Previous research has suggested that Symbiodiniaceae themselves often exhibit relatively invariant expression profiles in response to environmental changePocillopora corals, we did observe a significant response to the environment among host lineages. Constrained correspondence analysis of expression variation between islands indicated that the strongest environmental factor contributing to colony gene expression was historical SST, specifically degree heating week exposure. This is coherent with the primacy of temperature as an abiotic driver in ectothermic organisms generally58 and given its specific, disruptive, effects on regulatory homeostasis within scleractinian corals19. Colonies from different genetic lineages and from geographically distant locations nevertheless displayed similar expression profiles linked to thermal stress anomaly , an evolutionarily distinct class of enzymes that detoxify xenobiotic compounds and ameliorate oxidative stress60.In terms of coral host transcriptomic plasticity, among genes whose variation in expression was principally correlated with the local environment, we observed an upregulation of genes traditionally associated with the environmental stress response (ESR) in colonies present on historically warm reefs. Host lineages from warm islands showed elevated expression of Heat Shock Protein Family A (HSP70) Member 12A-like. This class of chaperones is known to be involved in response to thermal stress in scleractinian corals62. Such shifts can occur over long time periods as a result of competitive exclusion between photosymbiont lineages within a colony44, or over relatively short time scales in response to acute stress63. However these short-term changes are usually reversibly plastic, typically reverting when the stressful conditions cease64. Both mechanisms can facilitate acclimatization to the local thermal environment, as has recently been shown in photosymbiotic giant clams65. Although the Pocillopora lineages we sampled typically exhibited high symbiotic fidelity with specific Cladocopium lineages, this was not always the case.The coral holobiont is composed of a diverse community of organisms which potentially display differing sensitivities to warming. Shifts in the composition of the holobiont can therefore be as important as shifts in gene expression for permitting acclimatization to thermal challengesPocillopora lineages hosted different symbiont communities under different environmental contexts. In these instances, host colonies either exhibited a difference in association between specific Cladocopium photosymbionts in different environments or were dominated by Durusdinium glynnii photosymbionts. Interestingly, in all these cases, when a given coral host lineage was present on multiple islands that spanned a thermal gradient, we saw a preferential association with one of three photosymbionts  on the warmer island(s). In fact, with the exception of a single P. cf. effusa (SVD1) colony on Moorea which hosted C. pacificum L2 symbionts, we always observed a preferential association with C. pacificum L3 and C. latusorum L5 in Pocillopora colonies inhabiting islands with mean historical SSTs >26.8 °C. This observation suggests that whereas pocilloporid corals typically exhibit symbiotic fidelity, they are capable of a limited form of symbiotic flexibility involving preferential association with specific lineages of Cladocopium (and Durusdinium) depending on the local environment67. This difference in association may serve to maximize holobiont fitness under elevated SST and suggests a potential strategy for community-driven thermal acclimatization.In total, we observed 23 instances in which Pocillopora colonies which were either simultaneously hosting two photosymbiont genera or which contained Durusdinium glynnii symbionts as the dominant community member 71. Although the prevalence of Durusdinium in our data was not correlated with TSA frequency, it was strongly correlated with TSA magnitude suggesting the potential for photosymbiont shuffling/switching in these colonies in response to a recent or historical thermal challenge72. Alternatively, because Pocillopora meandrina symbionts are generally acquired via vertical transmission, with oocytes being seeded with the maternal symbiont community73, it is also possible that these Cladocopium- and Durusdinium-containing colonies represent true, evolutionarily stable, holobiotypes which persist across generations on reefs which experience frequent and/or severe thermal stress. However, whereas it has been suggested that some corals can increase their heat tolerance limits by +1.0–1.5 °C following a change in their symbiont community composition , repeated or prolonged exposure to high temperature often still results in colony mortality74. Thus, the degree to which the specific association between pocilloporid corals and Durusdinium under elevated SSTs confers an adaptive advantage under prolonged future warming requires further investigation.Finally, we also observed 14 ber Fig. . These cDurusdinium symbionts in Pocillopora colonies on warm reefs, especially in the far Eastern Tropical Pacific, this difference in association had very little effect on the host gene expression we measured in natura. Our data indicate that photosymbiont community composition  had only a weak, and inconsistent, impact on host expression and that host expression was driven more by the environment than by photosymbiont community composition. This stands in contrast to previous studies which have reported a significant role of the photosymbiont in modulating coral host gene expression75. For example, a recent study in Montastraea cavernosa reported that colonies containing Durusdinium glynnii symbionts displayed elevated expression of ESR genes even under ambient conditions relative to colonies containing Cladocopium symbionts22. These results suggest that photosymbiont genotype significantly alters host expression even prior to environmental perturbation.Despite the increased prevalence of M. cavernosa colonies up to 14% of expression variation between individuals could be attributed to the switch between dominant symbiont lineages22. The environment played a much more significant role in determining Pocillopora gene expression in our dataset with 24% of expression variation being explained by the sampling island. Additionally, we observed very little to no overlap in differentially expressed genes between Cladocopium- and Durusdinium-containing Pocillopora colonies across the three islands  showed reduced expression of ESR.Despite the weak influence of the dominant symbiont lineage on overall C. pacificum L3, C. latusorum L5, and D. glynnii) which appear to have been in preferential association with Pocillopora hosts inhabiting islands with historically elevated SSTs and/or TSAs. Although non-selective mechanisms could account for this pattern , these mechanisms seem unlikely in this study. The fact that we observed the same photosymbiont communities in colonies inhabiting reefs ca. 14,000 km apart across the Pacific Ocean  and under very similar environmental conditions  suggests that the high holobiont fidelity and specificity we observed among multiple lineages of Pocillopora corals is reflective of a process of cospeciation between Pocillopora hosts and their photosymbionts. Given the similar strong patterns of cophylogeny between Pocillopora and Cladocopium we observed and the limited environmental plasticity of photosymbiont expression across the Pacific Ocean, we interpret this strategy as one relying on host-mediated transcriptomic plasticity to buffer the holobiont from environmental change alongside preferential association with specific, thermotolerant, Cladocopium lineages under elevated SSTs. However, at extreme SSTs/TSAs even these heat-tolerant Cladocopium lineages are ultimately replaced by Durusdinium D1 photosymbionts suggesting there may be upper limits to the effectiveness of this strategy under warming. Whereas high levels of gene flow between populations across large spatial scales in both Pocillopora hosts and their photosymbionts29 has been suggested as an beneficial trait for permitting adaptation to previous climate shifts29, the degree to which this strategy of high fidelity and specificity between Pocillopora hosts and their photosymbionts may act to aid or hinder their adaptation to predicted future warming warrants further research.Thus, in our dataset, we observed three photosymbiont lineages  traits. In contrast, Cladocopium expression profiles were primarily driven by algal genotype and displayed low phenotypic plasticity across environmental gradients. Overall, our data suggest the existence of a two-tiered strategy underpinning thermal acclimatization in Pocillopora holobionts with strong selection for specific photosymbiont lineages  coupled with high host transcriptomic plasticity acting as an environmental buffer.Overall, we observed high symbiotic fidelity among Durusdinium glynnii was largely absent from Pocillopora colonies in the Central Pacific and rare in the West Pacific (only two colonies) in our dataset, previous studies have shown that D. glynnii frequently occurs in Pocillopora corals in many locations in the Pacific, particularly those with elevated SSTs (more than 31.5 °C) or which repeatedly experience extreme thermal events76. Taken together, our results and previous studies show that the presence of D. glynnii photosymbionts is a common strategy for Pocillopora corals to survive heat stress.We observed photosymbiont flexibility only in association with elevated SSTs in the far Western and far Eastern Pacific. Although the thermotolerant photosymbiont Our study has important implications for continued coral reef conservation and the prediction of coral responses to future ocean warming. Our examination of the mechanisms underpinning acclimatization among lineages across an environmental gradient and our identification of potential candidate loci under thermal expression selection provide useful baseline metrics for informing future manipulative experiments focused on physiological mechanisms underlying these observations. Investigating the extent to which different thermal acclimatization strategies, including both transcriptional and compositional changes, are employed among lineages will allow us to better forecast the survival of coral species and reefs under future ocean warming.Pocillopora spp. colonies from 32 reef sites across 11 islands  were sampled as part of the Tara Pacific expedition26. At each island, fragments of three coral colonies were collected from each of three reef sites yielding a total of ca. 9 colonies sampled per island  data were extracted from day and night satellite measurements of SST at 1 km spatial resolution from 2002 to the day of sampling as described in ref. 77 . DNA and RNA were quantified on a Qubit 2.0 fluorometer with Qubit dsDNA BR (Broad range) and HS (High sensitivity) Assays and Qubit RNA HS Assay , respectively. The quality of total RNA was checked by capillary electrophoresis on an Agilent Bioanalyzer using the RNA 6000 Pico LabChip kit  and the RIN was calculated. Eluted DNA and RNA were stored at −20 °C and −80 °C, respectively, prior to library construction.Coral fragments were processed to extract and isolate DNA/RNA as described in refConstruction of DNA libraries began by shearing to a mean size of 300 bp using a Covaris E210 instrument . Size profiles of sheared materials were visualized on an Agilent Bioanalyzer DNA High Sensitivity chip, and sheared DNA was then end-repaired, 3’-adenylated, and ligated to Illumina compatible adapters using the NEBNext DNA Modules Products  and NextFlex DNA barcodes (Bioo Scientific Corporation). After two consecutive cleanups using 1× AMPure XP bead, DNA was amplified using Kapa Hifi HotStart NGS library Amplification kits , followed by 0.6× AMPure XP bead purification. DNA libraries were stored at −20 °C until sequencing.RNA libraries were prepared following the TruSeq Stranded mRNA sample preparation protocol. Extracted RNA (1 µg) was subjected to poly-A+ selection using oligo(dT) beads, and the resulting mRNA was chemically fragmented under elevated temperature before conversion into single-stranded cDNA using random hexamer priming. Then, the second strand was generated to create double-stranded cDNA. Double-stranded cDNA was purified by 1.8× AMPure XP bead cleanups, 3’-adenylated, and ligated to TruSeq RNA barcoded adapters with 6 bases  or NEXTflex DNA barcoded adapters with 12 bases . After another 1× AMPure XP bead cleanup, the ligated product was amplified by 15 cycles of PCR and purified by a final 0.8× AMPure XP bead cleanup. RNA libraries were stored at −80 °C until sequencing.https://github.com/institut-de-genomique/fastxtend), as well as read pairs that mapped to the Enterobacteria phage PhiX174 genome (GenBank: NC_001422.1). For metatranscriptomic reads, read pairs that mapped to ribosomal sequences were removed using SortMeRNA v2.181. After this filtering, we obtained between 39 M and 104 M metatranscriptomic reads  and libraries were quantified by qPCR with the KAPA Library Quantification Kit for Illumina Libraries (Kapa Biosystems) on an MXPro instrument (Agilent Technologies). DNA  and RNA  libraries were sequenced using 151 bp paired-end read chemistry on a NovaSeq or HiSeq4000 Illumina sequencer . Basecalling and calculation of Phred quality scores (Q scores) were performed during sequencing by the Illumina Real Time Analysis (RTA) software. Illumina bcl2fastq Conversion software converted raw BCL files generated by RTA to fastq data. For both metagenomic and metatranscriptomic reads, we removed short (<30 bp length) and low-quality nucleotides (Q score <20), adapter/primer sequences with an in-house script based on Fastx-Toolkit software  and SYM-VAR-REV primers (5’-CGGGTTCWCTTGTYTGACTTCATGC-3’). Complete protocols for DNA amplification, library preparation and sequencing of ITS2 amplicons are available in ref. 27. Briefly, we identified a set of genome-wide single nucleotide polymorphisms (SNPs) from metagenomic reads mapped to the Pocillopora meandrina genomic reference84 using the Genome Analysis Toolkit tool 85. We followed a modified version of the best practices guide for variant discovery with GATK which included indexing of the genomic reference , followed by identification of realignment targets  and realignment around detected indels . Variants were called for each colony individually  and resulting variant call files (VCFs) were merged into island-specific, multi-sample, cohort files  before performing joint genotyping across all 11 islands  with polyploidy defined at 186. Joint analysis of multiple samples  is recommended for discovery of germline SNPs and indels as it provides information regarding population-wide variance across a cohort of multiple samples. This, in turn, allows for a higher degree of sensitivity to detect low-frequency variants, a clearer distinction between homozygous and missing sites, a greater ability to avoid false positives, and results in more accurate sample genotyping as described in the GATK technical documentation87. The resulting SNPs were filtered using VCFtools (v0.1.12)88 to include only biallelic sites with minor allele frequencies ≥0.05, quality scores ≥30, coverage (minimum read depth) ≥16, linkage disequilibrium (r2) ≥ 0.2, and no missing data across colonies. These curated SNPs were then used to identify divergent host lineages using a coalescent analysis performed on the individual colonies as described in ref. 27 using RaxML.We performed host lineage assignment as described in ref. Symbiodiniaceae taxa were present in each coral colony, we used ITS2 amplicons determined for each sample. ITS2 profiles were obtained using SymPortal82, which uses the intragenomic diversity of Symbiodiniaceae ITS2 to define so-called ITS2 type profiles based on consistent co-occurrence of intragenomic ITS2 variants across all samples.To determine which Pocillopora colonies hosting Cladocopium (84 colonies), we further investigated their population structure using single nucleotide polymorphism (SNP) distributions across their coding sequences. We excluded from these analysis colonies containing a large proportion (>25%) of a second ITS2 profile and potentially affecting the SNP calling. SNPs from the resulting 82 colonies were identified from metatranscriptomic reads previously aligned on the predicted coding sequences of the Cladocopium goreaui genome (see next paragraph) following the same protocol as described for the host above. SNPs were filtered using VCFtools (v0.1.12)88 to include only biallelic SNPs with a quality score ≥30 and a coverage ≥4. The frequencies of these SNPs were then clustered using the Hclust function of the stats package (v4.2.2) in R with the completeLinkage method. The optimal number of clusters was determined with the gap statistics method89 (fviz_nbclust function in the R package factoextra v1.0.7). We then compared the Bray-Curtis-derived pairwise distances (based on ITS2 type profiles) of the Cladocopium-containing Pocillopora colonies to the SNP-based clustering of Cladocopium populations. In general, we observed a high degree of overlap between the two identification methods across Cladocopium lineages. The ITS2 profile distances indicate that Cladocopium lineages which were undefined in the SNP clustering method belong to L5  from metagenomes to assign Cladocopium lineages to Cladocopium species previously identified33. For this analysis, metagenomic reads of the 82 Pocillopora corals containing Cladocopium symbionts were aligned simultaneously on three psbAncr sequences  with bwa-mem (v2.2.1)90. Full-length reads aligned with more than 90% of identity were kept. The reads aligned on the psbAncr reference sequence covered by the highest number of reads were selected to build a consensus sequence for each sample (samtools mpileup). We selected two psbAncr sequences for each Cladocopium clade identified in ref. 29 and aligned them with the 82 consensus sequences with clustalW (MegaX software)91. A Bayesian phylogeny of this alignment was generated using MrBayes (v3.2.7a)92 with the GTR substitution model (nst=6), gamma-distributed rate variation (rates=invgamma), the birth-death clock model (brlenspr=clock:birthdeath). The MCMC was run for 1,000,000 generations with sampling every 1000 steps and the first 25% was removed as burnin. The phylogeny was represented with R packages ape (v5.6.2) and ggtree (v3.6.2)93  to conduct a Procrustean analysis of cophylogeny (PACo)94. The PACo approach uses a distance-based global fit to quantify the topological congruence between two phylogenetic trees and identifies the associations contributing most strongly to the cophylogenetic structure29. For Cladocopium, we used the SNP frequency clustering described above. For Pocillopora, we used a subsetted phylogenetic tree generated from the SVD quartet phylogeny of Hume et al.27 generated by selecting a single representative for each of the five host lineages. The host and photosymbiont phylogenetic trees were aligned with the untangle function (step2side method) of the R package dendextend (v1.17.1)95.To test for phylogenetic congruence between Pocillopora meandrina coral host reference genome , and Symbiodiniaceae sequences (the CDS of the Cladocopium goreaui genome32 and a Durusdinium transcriptome96) using Burrows–Wheeler Transform Aligner  with the default settings90. Host- and symbiont-mapped reads were then sorted and processed using SAMtools v1.10.297 to generate respective bam files. A read was considered a host contig if its sequence aligned to the P. meandrina predicted coding sequence with ≥95% of sequence identity and with ≥50% of the sequence aligned. Reads aligned to Cladocopium goreaui coding sequences with a cutoff of ≥98% of sequence identity over ≥80% of the read length were retained as symbiont reads. Reads were further filtered to remove those in which more than 75% of the read length was low complexity or less than 30% was high complexity. Read counts were normalized as transcript per million (TPM). Tables of raw and normalized counts are available98.Metatranscriptomic reads  were separately aligned to predicted coding sequences (CDS) of the Cladocopium expression profiles we used three complementary approaches, namely (1) quantitative partitioning of gene expression variance into the fraction attributable to each putative driver using a linear mixed model approach (variance partitioning), (2) discriminant analysis of principal components (DAPC) to determine which drivers best distinguish between a priori sample groupings (islands and lineages) based on proportions of correct reassignment, and (3) constrained correspondence analysis informed by both historical and in natura environmental data to identify primary abiotic drivers of gene expression.To evaluate the relative influence of the environment and genotype on the coral host and 100 to partition the variance attributable to the environment, the host genotype, and the symbiont genotype in the Pocillopora and Cladocopium expression datasets . Prior to performing the variance partitioning analysis, a correction was applied to remove a bias due to the two different library preparation methods used for RNA sequencing following the procedure described in ref. 99. Three variables were defined as random effects: Pocillopora genetic lineage, Cladocopium genetic lineage, and the island of sampling. In order to account for any potential biases in our estimation of gene expression variance introduced by variancePartition’s random sub-sampling of colonies we repeated the variance partitioning analysis 100 times with a random removal of two samples during each repetition. Genes with a median explained variance >50% for one of the three selected variables across these 100 rounds, hereafter referred to as top variant genes, were then selected for functional enrichment analysis (1821 genes for Pocillopora and 2424 genes for Cladocopium).In the first approach, we used the variancePartition package in R (v 1.21.2)102 to determine which genes are best able to discriminate between pre-defined groups of samples  and to assess how well these groups could be distinguished from one another based on similarity among their expression profiles. The purpose of DAPC is to find the linear combinations of genes which maximize the differences between pre-specified groups while minimizing the within-group variance thereby allowing for the determination of which genes most strongly discriminate between the pre-defined groups. Coefficients of these genes are called loadings and higher loading scores indicate a stronger discriminatory ability. The linear combinations of these genes’ expression values are referred to as discriminant functions and serve to orient the groups in multi-dimensional space.We used the adegenet (v2.1.10) in RPocillopora and Cladocopium gene expression were then assessed by comparing the proportion of colonies that were correctly re-assigned to their pre-defined groups under each clustering model. A higher proportion of colonies correctly re-assigned to their pre-defined groups indicates a greater power to discriminate between divergent expression profiles and, therefore, a greater influence of that grouping factor on gene expression. The input for these analyses were the normalized expression data  for all genes with 10 or more counts in at least 90% of colonies with lineage assignations . We began each model run with an ordination analysis to extract Principal Components (PCs) of gene expression. We then computed a-scores to determine the optimal number of PCs to retain under each clustering model for subsequent cluster identification using discriminant analysis102. A-score optimization resulted in our selecting 11 PCs to retain for the host and photosymbiont . Group memberships were then independently predicted for the colonies based on DAPC scores.We assessed three discriminant analysis clustering models each for the host and algal symbiont with (1) the island of collection , (2) coral genetic lineage or photosymbiont genetic lineage as the grouping factor for the coral and photosymbiont, respectively, , and (3) the genetic lineage of the symbiont  as the pre-defined grouping factor. The relative strength of environmental-, genetic-, and symbiont impacts on The number of principal coordinates (PCs) and discriminant functions (DF) retained, the proportion of colonies correctly re-assigned to their a priori groups, and the overall proportion of gene expression variance explained by each factor  are summarized in Supplementary Data r2 ≥ 0.7 using the corclust function of the klaR package (v1.7.1) in R. From this, we identified clusters of correlated variables  between the host/symbiont expression data and the environmental data in order to visualize how expression data were structured with respect to potential environmental drivers. To avoid model overfitting we first identified environmental variables that were highly correlated with one another at an Pocillopora hosts containing Cladocopium and those containing Durusdinium in the far Eastern Tropical pacific . Analyses were performed separately for each of the three islands and also for all islands together. The input for each analysis were the host expression data for all genes with 10 or more counts in at least 90% of colonies from that island  or among the three islands together . For each analysis, these gene counts were normalized and transformed using a variance stabilizing transformation in DESeq2 (v1.28.1)103. Genes significantly differentially expressed (DEGs) between colonies containing Cladocopium and Durusdinium were then identified using DESeq2 with the following criteria: absolute log-fold change (LFC) ≥ 2 after application of ashr log fold-change shrinkage104 and FDR-adjusted P value ≤ 0.05. Because of the uneven sampling of C- and D-containing colonies between islands, the number of DEGs identified in each inter-island pairwise comparison was highly dependent on the number of colonies input in the DESeq2 analysis. To correct for this and allow for quantitative comparison of DEGs between islands we performed multiple differential expression analyses using two, randomly subsampled, colonies (the lowest sample number) for each category (C- vs D-containing) for each island. This resulted in a range of DEGs estimates from which the mean number of DEGs (±SD) were calculated.We assessed differential gene expression between Pocillopora genes were obtained from the genomic reference provided in ref.84. These annotations were obtained using the Interproscan tool for each coding sequence106. Gene Ontology (GO) annotations were attributed with the Interpro2GO correspondence table (v2020/06/13)105. Functional annotations of Cladocopium goreaui genes were recovered from the published genome32.Functional annotations 107 package in R. For top variant genes, the number of GO annotations assigned to genes within that interest group was compared to the number of annotations assigned to the rest of the dataset, to evaluate whether any ontologies were more highly represented within the module than expected by chance . Cladocopium functional enrichment analyses were performed using independent Fisher’s exact tests for each Pfam (protein family) domain between genes present in each gene-of-interest category  as compared to all expressed genes of Cladocopium. P values were corrected for multiple testing with the Benjamini & Hochberg method implemented in the p.adjust function in R. For each Pfam term, the number of annotations assigned to genes within an interest group  was compared to the number of annotations assigned to the genome, to evaluate whether any ontologies were more highly represented within the module than expected by chance (Fisher’s exact test).Functional enrichment among top variant (variance partitioning) and discriminant (DAPC) genes were analyzed in the host using the goseq (v1.40.0)Further information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1-19Reporting Summary


The SNP rs671 of Human aldehyde dehydrogenase (ALDH) is G-A transition at 1510th nucleotides, which is an important clinical indicator of alcoholic liver disease, digestive tract cancer and some drug efficiency. The commonly used genotyping assay of this polymorphism is relatively time-consuming and costly.This study develops a rapid and accurate one-step CRISPR/Cas12b assay to distinguish the G1510A polymorphism of human ALDH2 free of DNA amplification. The method we established requires only one step of adding 1 μl genomic DNA sample to premixed system, and waiting for the acquisition of fluorescent signal, taking approximate 30 min.This method provides a potential tool for more accurate and reliable nucleic acid detection with a single base difference and supports the relevant disease diagnosis and personalized medicine.The online version contains supplementary material available at 10.1186/s13008-023-00095-6. It provided a potential tool for more efficiency and reliable SNP detection method and supported the relevant disease diagnosis and personalized medicine.The commonly clinical used genotyping assay of single nucleotide polymorphism (SNP) is relatively time-consuming and costly. We developed a rapid and accurate one-step CRISPR/Cas12b assay to distinguish homozygote and heterozygote of SNP rs671 of human aldehyde dehydrogenase 2 (ALDH2) gene without any DNA amplification. This assay requires only one step of adding at least 10https://www.ncbi.nlm.nih.gov/snp/rs671). The transition of G to A of rs671 leads to substitution of Glu487 to Lys, that exhibits a serious loss of ALDH2 enzyme activity and is consequently closely related to alcoholic poisoning, alcoholic liver disease, digestive tract cancer, and other diseases [Human aldehyde dehydrogenase (ALDH) is a family of enzymes that catalyzes the oxidation of acetaldehyde and other aliphatic aldehydes. Among 19 identified ALDH isoenzymes, the mitochondrial isoenzyme ALDH2, which highly expressed in the liver and stomach, is one of the key enzymes in the human ethanol metabolism pathway [diseases .With the development of molecular biology, a large number of genotyping methods for SNP have been developed. For example, DNA sequencing, the golden-standard assay for genetic diagnosis, is often used but is also time-consuming and costly. Fluorescence probe PCR is the most widely used assay in clinical practice, however, it requires a long detection time and high sample quality . Other eRecent advances in the diagnostics field have highlighted the role of clustered regularly interspaced short palindromic repeats-associated (CRISPR/Cas) method as a promising candidate for rapid nucleic acid detection owing to its high reliability and specificity . SeveralThe principle of the CRISPR/Cas12b-mediated ALDH2 rs671 SNP genotyping system was illustrated in Fig. CRISPR/Cas12b nuclease requires a protospacer-adjacent motif (PAM) sequence TTN for double-strand DNA cleavage, and the PAM sequence should be located in an appropriate position . The dis4 copies of the human genomics DNA containing ALDH2 1510GG, 1510AA, and 1510GA were tested in the assay, respectively. There was a weak signal under 4 × 103 RFU for DNA sample containing homozygous AA, while fluorescence signal dramatically up to more than 20 × 103 RFU for DNA sample containing homozygous GG. For heterozygous GA, the increasing signal falls in between homozygous GG and AA (12 × 103). These results suggested that Cas12b-mediated assay could distinguish the three genotypes of ALDH2 rs641 . When the genome DNA was 103 copies, the method was also able to distinguish three genotypes of ALDH2 rs671 SNP  of this assay was also determined by tenfold gradient concentrations of human gDNA at 104 copies for each) including the three diploid genotypes of ALDH2 rs671 SNP. The CRISPR/Cas12b-assisted method showed a high level of concordance with the reference fluorescence probe qPCR method  were analyzed and evaluated. The reference fluorescence probe qPCR method was carried out in hospital with Human ALDH2 Gene Polymorphism Detection Kit  that had been extensively used for ALDH2 rs671 SNP genotyping in clinical practice. In a clinical setting, methods of DNA isolation and preparation, quality of reagents, instrumentation may differ and significantly affect fluorescence output, so we add standard plasmid controls (104 copies) of human genomic DNA extracted from clinical sample to a premixed system and waiting for a total of 30 min for the acquisition of fluorescence signal. We didn’t combine any amplification step before Cas12b-mediated genotyping, since copy number of the clinical DNA samples is much higher than the lowest sensitivity of the method. Cas12b could hardly tolerate any mismatch between sgRNA and target DNA sequences, endowing higher specificity and accuracy when used for DNA polymorphism detection. Compared with traditional methods, this method does not require a complicated experimental operation, complex instrument, or long-time course. We believe that the CRISPR/Cas12b genotyping assay will play an irreplaceable role in the gene diagnosis and accurate treatment of clinical diseases.In this study, we established an accurate, rapid, and convenient CRISPR/Cas12b-assisted method to distinguish three genotypes of ALDH2 rs671 SNP. This method only requires one step of adding 30–40 ng (equivalent to 10Additional file 1: Table S1. Sequences of sgRNA candidates in this study.


Drosophila melanogaster, much less is known about the origin and evolution of piCs in this or any other species. To investigate piC evolution, we use a population genomics approach to compare piC activity and sequence composition across 8 geographically distant strains of D. melanogaster with high quality long-read genome assemblies. We perform extensive annotations of ovary piCs and TE content in each strain and test predictions of two proposed models of piC evolution. The ‘de novo’ model posits that individual TE insertions can spontaneously attain the status of a small piC to generate piRNAs silencing the entire TE family. The ‘trap’ model envisions large and evolutionary stable genomic clusters where TEs tend to accumulate and serves as a long-term “memory” of ancient TE invasions and produce a great variety of piRNAs protecting against related TEs entering the genome. It remains unclear which model best describes the evolution of piCs. Our analysis uncovers extensive variation in piC activity across strains and signatures of rapid birth and death of piCs in natural populations. Most TE families inferred to be recently or currently active show an enrichment of strain-specific insertions into large piCs, consistent with the trap model. By contrast, only a small subset of active LTR retrotransposon families is enriched for the formation of strain-specific piCs, suggesting that these families have an inherent proclivity to form de novo piCs. Thus, our findings support aspects of both ‘de novo’ and ‘trap’ models of piC evolution. We propose that these two models represent two extreme stages along an evolutionary continuum, which begins with the emergence of piCs de novo from a few specific LTR retrotransposon insertions that subsequently expand by accretion of other TE insertions during evolution to form larger ‘trap’ clusters. Our study shows that piCs are evolutionarily labile and that TEs themselves are the major force driving the formation and evolution of piCs.Animal genomes are parasitized by a horde of transposable elements (TEs) whose mutagenic activity can have catastrophic consequences. The piRNA pathway is a conserved mechanism to repress TE activity in the germline via a specialized class of small RNAs associated with effector Piwi proteins called piwi-associated RNAs (piRNAs). piRNAs are produced from discrete genomic regions called piRNA clusters (piCs). While piCs are generally enriched for TE sequences and the molecular processes by which they are transcribed and regulated are relatively well understood in  Organisms have evolved many mechanisms to minimize the genomic instability caused by transposable element (TE) activity . In animflamenco, which is almost entirely composed of and dedicated to silencing Ty3/mdg4 (formerly known as gypsy) retroviral-like elements  loci and hundreds of smaller (<10 kb) loci  loci . It is akb) loci . The arckb) loci . Over tid piRNAs . The ‘de pathway .de novo’ models of piC evolution are not mutually exclusive, but each makes contrasting predictions on how the structure, composition and activity of piCs are expected to evolve in a population. In the ‘trap’ model, piCs are expected to be (1) fewer in number, larger (10–100 kb), and mainly peri-centromeric and sub-telomeric; (2) piCs are expected to be syntenically stable archive of sequences of active and inactive TE families, and (3) piCs should gradually undergo TE sequence turnover via internal sequence rearrangements such as insertions and deletions (INDELs). The latter process is likely to be a fundamental characteristic of ‘traps’, wherein insertions of young and active TE families would replace older insertions of inactive TE families. That is because there is a limit to the genomic space afforded to clusters due to the possibility of spreading of their silent chromatin over host genes  abundant but smaller in size (1–10 kb), broadly distributed genome-wide, 2) highly polymorphic in population and typically strain-specific, and comprised mainly of insertions from active TE families, and 3) exhibit wholescale insertions and deletions, leading to loss or gain of entire clusters. These properties would ensure that piRNA production is biased toward active TE families. Several independent studies have provided observations that de novo formation of canonical piCs can emerge in the laboratory from artificial transgenes , we manually identified and curated their coordinates by searching for the nearest annotated gene flanking the piCs in their respective genome assemblies . We found that even with relaxed mapping criteria (0.33X to 3X coverage and >70% identity) to the reference genome, only ~85–90% of all piCs from any given strain could be lifted over to the reference genome. Further inspection of piCs that failed liftover revealed that they were relatively small clusters (500 – 5000 bp) and entirely absent in the reference genome or were large clusters (25–200 kb) that had undergone extensive structural rearrangements and therefore could not be lifted over to the reference genome using the NCBI remapping tool. To recover piCs that were >25 kb in size, apparently active in multiple strains but highly structurally variable  to 906 (proTRAC) of piCs are active only in a single or a few strains confirming that each strain has a very unique piC landscape (restrictive and proTRAC methods separately or the combined master list (restrictive’ pipeline) are shared between two and three strains, respectively. All such piCs, shared by 3 strains or less are together termed as ‘rare’ piCs. Rare piCs are not only extremely abundant but also exhibit significant piRNA expression ranging from 20–100 RPM, which is comparable to previously described canonical piCs like 38C, 80EF and traffic jam 3’UTR  (42AB and 38C are still only active in 6 to 7 of the 8 strains (see below). Taken together, these results suggest that ovarian piCs are extremely labile and poorly conserved in activity across D. melanogaster strains.Next, we examined the relationship between the size of piC and their level of sharing across strains. First, we note that median piC length predicted from each library is highly similar, with median length ranging from 5.7 kb to 7.5 kb . We find2.2e-16) . In othe42AB and 82E, two dual-stranded piCs  we selected because it is also highly expressed in some strains but is euchromatic, overlapping the 5’ UTR of the corto gene. It is only identified as a piC in strains A1 and A7 in this study using the restrictive pipeline and piC expression profiles clearly show that piRNA expression across the 82E region is only detectable in these two strains  and 80F9 (right) are two peri-centromeric piCs on chr3L  between strains and is annotated as a piC in only 6 strains. piRNA coverage of Trypsin genes-associated piC (left) and eEF1alpha1 associated piC (right) in The  strains . Other t browser . Exampleon chr3L . 80EF, a‘trap’ and ‘de novo’ models of piC evolution. Under the ‘trap’ model, we expect that the rate of insertions, likely representing the ‘trapping’ of TE insertions within common piCs would be balanced by that of deletions as to stabilize the size of the piCs. Thus, we expect a similar frequency of insertions and deletions within large piCs acting as traps. By contrast, we predict that clusters born ‘de novo’ would be dominated by insertions, likely corresponding to recent transposition events. To systematically detect SVs genome-wide for each of the strains relative to the iso-1 reference strain . Polarization led to loss of ~55% of INDEL calls as the ancestral or derived state of the loci could not be determined due to conflicts in calls between the two outgroup species. After this filtering, 1183 insertions and 1873 deletions were retained for analysis, of which 30% of insertions and 20% of deletions overlapped with master-list piCs , namely insertions and deletions (INDELs), within the piC genomic regions. INDELs are predicted to affect piCs differently under the e strain , we mappist piCs .D. melanogaster strains . Meanwhile, deletions overlapping piCs have a similar length distribution than those non-overlapping piCs   . We also = 0.39) . Howeverde novo’ from recent TE insertions.Next, we tested whether piCs are enriched for INDELs relative to the rest of the genome. To do this, we compared the INDEL counts overlapping piCs for each of the cluster frequency categories with those expected based on 1000 sets of randomly shuffled INDELs. We found that deletions were significantly enriched in common piCs, but not in rare piCs . Insertide novo annotation of TEs in each of the strain genome assemblies. This was necessary because many TE consensus sequences present in the reference TE library for D. melanogaster (FlyBase release 2019_05) were discovered and curated more two decades ago using primarily the iso-1 and Oregon-R strains , LTR retrotransposons , Rolling Circle (RC) transposons, and cut-and-paste DNA transposons. We found that TE copies from all three LTR superfamilies were significantly younger in piCs than non-piC regions (Ty1/copia) and one DNA transposon superfamily (Tc1/mariner) and used terminal branch lengths to estimate their relative age (Ty1/copia LTR retrotransposons (n=135) overlapping piCs are significantly younger than non-overlapping ones, while Tc1/mariner (n=89) DNA transposons show no such bias    and the youngest G-element insertions according to terminal branch length do not overlap piCs  . In othe= 0.903) . Analyzil active , we founlap piCs . Taken tTo examine the role of recent transposition events in driving piC sequence composition, we established a set of non-reference TE insertions (absent from the reference genome) in each of the 8 strains using the raw long read data available for each. Briefly, we applied TLDR (a long-read TE insertion detection tool)  with a cTy1/copia superfamily member  . This any member  with 79 de novo’ piCs  shared by no more than 3 strains) and within common larger ‘trap’-like piCs . To increase statistical power for this analysis, we used all TE sequences annotated by RepeatMasker in each genome, instead of only non-reference insertions. For iso-1, we found that only 4 TE families are significantly enriched within young piCs. Interestingly, all 4 are LTR retrotransposon families of the Ty3/Mdg4 superfamily  and all except flea belong to the Mdg1 lineage  across piCs and their flanking non-piC regions for each piC frequency class (strain-specific or shared by 2–8 strains). We found that the divergence of TE insertions in rare piCs (shared by 3 or less strains) is markedly lower (3.5–5%) than in their flanking regions (10–15%) . In addiTo further test this idea, we examined how TE coverage within piCs and surrounding regions change as piCs become more common and presumably older . First, D. melanogaster, we used population genomics methods to characterize piC variation across eight inbred strains. A crucial asset was the availability of high-quality genome assemblies for these strains  that are shared across most or all strains, a characteristic that is congruent with the ‘trap’ model , A2 (b3841_paired), A4 (b1_3852), A7 (t7_paired), B3 (b3864_paired), B6 (t1_paired) and Oregon-R were a gift from Anthony Long (UCI). iso-1 reference strain (#2057) was obtained from Bloomington Drosophila Stock Center. All stocks were maintained on standard cornmeal medium at 22°C under a 12-hr day/night cycle.Small RNA libraries were constructed by size fractionation on urea-polyacrylamide gel electrophoresis as described in Reads were first trimmed of the adapter sequences and quality filtered using cutadapt (v3.4). Read lemakewindows  per window from bam files. Windows with piRNA expression of 2 RPM or more were merged if located within 10 kb of each other into piRNA expression domains. RPKM values for piRNA expression was calculated for such domains (ranged from 500 bp to 330 kb).Active piRNA cluster (piC) annotation was conducted independently for each replicate of each strain using a custom pipeline adapted from previously described methods . Genome ewindows  was usedewindows  and iso-ewindows  followedrestrictive mode of annotation - domains with at least 8 unique piRNAs per kb per million total piRNA reads were selected and then merged if interrupted by low mappability region of 10 kb or less. Similarly, for the permissive mode, domains with at least 2 piRNAs per kb per million were selected and then merged if interrupted by low mappability region of 15 kb or less. While the permissive mode had very relaxed parameters and likely produced many false positive predictions, the primary function of this mode was simply to provide unique piRNA support for the piRNAs detected from proTRAC method (see below), which utilizes multi-mapping piRNAs and predicts the majority of piRNAs in extremely low-mappability regions.piC annotation from merged domains was conducted in two modes - permissive and restrictive. First piRNA reads that uniquely map to selected domains were recovered and separated for each domain using samtools to quantify unique piRNA sequences per domain. Additionally, theoretical mappability scores of 0–1 for 25 nt reads was computed using GEM  for bookended 500 bp windows for each genome assembly . For the The same processed small RNA libraries as described above were used for alternative piC annotation using proTRAC-v2.4.4 . Specifirestrictive pipeline and proTRAC were lifted-over from each strain to iso-1. Remap parameters chosen for identification of all piCs with minimum alignment coverage of 0.3 and maximum expansion or contraction of 3X, allowing for clusters with strain-specific structural variation to be detected. Similarly, gene annotation from the reference genome was also lifted over to the DSPR genomes but with higher mapping stringency of minimum alignment coverage of 0.9.piCs annotated from the above methods for each strain were lifted-over to the iso-1 reference genome (Release 6) using NCBI remap (NCBI Genome Remapping Service). Briefly piC coordinates from the custom restrictive, proTRAC and master-list are provided in Recovery of complete piC regions primarily depends on expression and density of uniquely mapped piRNAs along the length of the cluster. Clusters identified independently from two different strains may differ in length due to natural variation in piRNA expression or density along the length of the cluster. To identify homologous clusters across strains despite changed boundaries due to structural variation in piCs, most heterochromatic piCs were examined in the IGV browser. Clusters resulting from merged bins across annotated protein-coding genes were unmerged into separate clusters. Any cluster partitioned into multiple smaller clusters due to lack of any uniquely mapping piRNAs for more than 20 kb were merged to recover the complete cluster. All strain genome assembly-match piC annotations from D. simulans (wxd1) and D. sechellia (sech25) were mapped to the D. melanogaster iso-1 release 6 (GCA_000001215.4) without the Y chromosome with minimap-2.1 map-pb --N3 and resulting sam file was converted to bam and sorted  could be determined were then filtered and their SV length, AF, and read support averaged from results of SV callers using bedtools poverlap (https://github.com/brentp/poverlap) with 1000 bootstraps of random shuffling. Mean expected overlap counts against piC coordinates were compared to expected overlap.Filtered SVs of insertions and deletions class were used for parsing for further analysis. The vast majority of SVs are expected to have extremely low frequency of <0.1, which reflects the general deleterious nature of SVs. Since raw long read data used from published studies were from pooled sequencing of ~200 flies for DSPR strains and 60–80 flies for iso-1, only SVs with intra-strain AF of 0.2 or more were considered, to enrich for germline SVs. SV enrichment analysis was carried out using de novo TE annotation was conducted using several computational tools. Summary of all major steps is presented in a flow-chart in To create a comprehensive and accurate representative TE library representing the TE insertions contained in the 8 strains, D. melanogaster TE library on respective genome assemblies of 8 strains was generated  trees were constructed for all defragmented iso-1 TE insertions for superfamilies and families reported in MA model . ML treeMA model .de novo TE insertion analyses. Reads (>1 kb) were mapped to iso-1 reference genome  using minimap2.1 default parameters and resulting sam file converted to bam file, sorted and indexed using samtools . TE insertion enrichment analyses were performed using TE_analysis_Shuffle-bed.pl script  annotations for each small RNA library per strain from restrictive, proTRAC, and master-List.Supplementary_Table_S4. piC population frequency for each pipeline after remap to iso-1 genome (liftOver).Supplementary_Table_S5. Collapsed, genotyped, filtered, and polarized structural variant calls.Supplementary_Table_S6. TE classification and activity determined from non-reference TE insertions.Supplementary_Table_S7. Filtered and collapsed TLDR non-reference TE insertion calls.