This allowed us to assess the ability of these corals to manipulate the chemical conditions at the site of calcification (Ωcf), and hence their potential to acclimate to changing Ωsw. We show that lifelong exposure to low Ωsw did not enable the corals to acclimate and reach similar Ωcf as corals grown under ambient conditions. The lower Ωcf at the site of calcification can explain a large proportion of the decreasing P. astreoides calcification rates at low Ωsw. The naturally elevated seawater dissolved inorganic carbon concentration at this study site shed light on how different carbonate chemistry parameters affect calcification conditions in corals.Ocean acidification is expected to negatively impact calcifying organisms, yet we lack understanding of their acclimation potential in the natural environment. Here we measured geochemical proxies (δ pCO2 conditions cannot fully adapt the chemistry of their internal calcifying fluid compared to corals growing in ambient conditions.Ocean acidification is expected to have a negative impact on calcifying organisms, however, our understanding of the acclimation potential of corals in their natural habit is currently limited. Here, the authors find that scleractinian corals living in high  Our understanding of the potential fate of corals in the face of changing pCO2 in the ocean is based primarily on controlled laboratory studies (e.g. refs. 5), mesocosm studies mimicking coral community composition8, alkalinisation versus carbon dioxide-enrichment studies in natural coral reef sites10, and a number of field studies with naturally reduced calcium carbonate saturation state (Ωarag)14. These efforts have provided strong evidence that the calcification rates of a large number of coral species investigated to date will decline in response to projected pCO215. However, some studies also report that certain coral species were able to maintain high calcification rates or even benefit from elevated pCO218, suggesting a high resilience potential of some coral species to changing carbonate chemistry19. Specifically, the ability of an organism to control the biomineralization process clearly determines its ecological and physiological success under reduced pH conditions14. The process of calcification in corals is linked to their ability to control the pH at the site of calcification (pHcf) by removing protons out of the calicoblastic space between the tissue and skeleton, where calcification takes place5. This enables corals to sustain pHcf well above seawater pH (pHSW)20. The physiological capacity of corals to control pHcf may alleviate the decline in coral growth and increase coral resilience to future climate change19. Knowledge about internal calcifying fluid pHcf in corals has been derived from a few direct measurements under the calcifying cell layer either using microsensors22 or pH-sensitive dyes23. These studies confirmed an elevated pHcf of between 0.4 and 2 pH units above ambient seawater in the calicoblastic space. Indirectly, boron isotopes (δ11B) of coral skeletons, which represent the pHcf of the calcifying solution, also suggest an elevated pHcf (e.g. refs. 26). Boron isotopes are more readily accessible compared to direct measurements and have the additional benefit that they integrate pHcf history over longer time periods27. Studies suggest that pHsw is an important driver affecting pHcf28. However, it was recently demonstrated that changes in seawater dissolved inorganic carbon (DIC) or total alkalinity (TA) can also affect pHcf regulation23. Using B/Ca as a proxy for internal carbonate ion concentration (CO32−cf), provided geochemical evidence that corals can also modulate and adjust the internal DIC (DICcf) concentration. Together—the potential to upregulate DICcf and pHcf—allows for higher carbonate ion concentrations at the site of calcification and hence a higher Ωcf that facilitates calcification30.Ocean acidification is projected to lead to negative effects on calcifying organisms, particularly tropical corals31). For instance, in situ flume experiments mimicking natural  fluctuating conditions coupled with future pCO2 conditions showed that corals from acidified treatments could maintain a constant calcification pHcf irrespective of changes in seawater pHsw27. The authors argued that the fluctuating conditions the corals were exposed to likely favour this strong control on internal conditions. That year long experiment, however, cannot tell whether corals can maintain such strong control when exposed to reduced mean seawater aragonite saturation state (Ωsw) for their entire life span. Corals living for their entire life under continuously low Ωsw and variable environmental conditions can be used to test whether corals can maintain pHcf homoeostasis over long time spans in their natural ecosystem with its complex biological interactions. Because many natural ocean acidification sites also show strongly fluctuating conditions33 these settings may be ideal for testing the relationship between environmental variability and acclimation potential of corals to low Ωsw34.Over the last decade, a growing body of literature has provided evidence that corals subjected to daily and seasonally fluctuating environmental conditions are able to exert a stronger control over their internal physiological attributes, potentially allowing them to better cope with future changes  in Puerto Morelos, Mexico11. These geochemical proxies (δ11B-derived estimates of pHcf and B/Ca derived estimates of CO32−cf) allowed us to infer carbonate chemistry conditions at the site of calcification, which provide valuable new insights into the internal calcification regulation mechanisms in corals exposed to persistent low Ωsw, as well as fluctuating carbonate chemistry conditions33. Our results, combined with bio-inorganic calcification models30, identified critical regulation mechanisms and the inability of corals to fully acclimate to these conditions and sufficiently elevate their Ωcf to sustain growth rates similar to the same species of corals growing at ambient Ωsw.Here we measured geochemical proxies in the upper most recently formed skeletal parts of P. astreoides: 5 cores collected from the centre of the low Ωsw ojos and 7 from control present-day Ωsw sites adjacent (within a few meters) to the ojos11. Porites astreoides, the species used in this study, represents one of only three calcifying coral species found growing within the discharge impacted area, while nine coral species are found nearby under ambient present-day Ωsw. Previous studies indicate that although the abundance of P. astreoides was not significantly reduced at the low Ωsw ojos, its growth rate  decreased significantly by 37% compared to the same species collected at control sites11. The control sites have a relatively consistent Ωsw (on average: 3.92 ± 0.03 sd) year round compared to the ojos where Ωsw is always <2 and ranges from 0.77 to 1.85 .We used 12 cores from the coral 11B in the 12 corals analysed ranged from 23.1‰ to 27.6‰, with slight but significantly lower values for corals affected by the ojo discharge where Ωsw was low . These δ11B values translate into pHcf that are slightly lower  in the corals from sites with Ωsw < 2 with an average internal pHcf of 8.46 (±0.03 sem) compared to 8.54 (±0.01 sem) at the control sites  compared to the difference in environmental seawater pHsw of ~0.54 pH units between the sites. Hence, compared to pHsw in their surrounding environment, corals at the ojo centres maintained a higher pH gradient between seawater and the calcifying fluid (∆pH) in comparison to corals at control sites .The δ11B. This ratio varied between 442 and 721 µmol mol−1 and did not significantly correspond to Ωsw . Using the δ11B and B/Ca skeletal proxies together to constrain the carbonate system at the site of calcification suggests an elevation of CO32−cf not only due to shifts in internal pHcf but also due to an increase in DICcf . This is mainly due to the higher than ambient DICsw .Changes in coral skeletal B/Ca were determined along with δ19, see the “Methods” section) to calculate average Ωcf and associated relative calcification rates using our proxy data. We used the combined pHcf and the B/Ca-derived DICcf concentration at the site of calcification for corals from both ojo and control sites. With this bio-inorganic model both Ωcf  and calcification rates  decreased with decreasing Ωsw and correlated well with the observed calcification response previously reported for these corals11 . The internal calcifying fluid parameters are clearly distinct between corals from the different field sites, strongly indicating a combined effect of DICcf and pHcf modulation on P. astreoides calcification performance  decreased only slightly along the ambient Ωsw gradient in which the analysed corals live, highlighting the strong control of Porites asteroids corals over the biomineralization process. Yet the combined change in pHcf and DICcf corroborate the observed decline in calcification rate along the environmental gradient  that are localised in the calicoblastic epithelium, as well as other, not yet identified acid–base relevant transporters35. Carbonate chemistry at the calcification site clearly differs between coral growing at the control and ojo locations. The difference explains 41% of the observed difference in calcification rate; however, it still leaves 59% of the variation in calcification rate unexplained , and emphasise the importance of deciphering internal calcium regulation38.In our study, we took advantage of the inherent conditions of this submarine springs system, including the strong environmental fluctuations and the fact that carbonate chemistry is controlled by saline groundwater discharge, allowing us to provide new facets on drivers of coral calcification in natural settings affected by ocean acidification. In the subsequent discussion we will outline the novel insights we derive from the observed internal carbonate chemistry conditions at this natural low Ωcf reflects the strong effect of intracellular biological processes on coral calcification and is manifested in skeletal isotopic composition. The control of pHcf represents one mechanism to counter external seawater conditions39. The boron isotopic-derived pHcf values we report are similar to those reported in other studies for Porites27. The sensitivity of pHcf to changes in the environmental pHsw, however, differed between the different studies, as the environments the corals originated from were distinct40. All studies observed that pHcf stays within a narrower range (8.2–8.6) compared to large changes in seawater pHsw. They all highlight the generally strong control corals exert on pHcf. Despite this capacity for regulation, however, the observed pHcf was lower at lower Ωsw (e.g. refs. 41). Irrespective of whether corals maintained high pHcf, the corals exposed to low Ωsw maintained a higher proton gradient at lower pHsw  did not lead to full acclimation11. It is likely that there is a critical Ωarag threshold beyond which corals are no longer able to fully compensate for external acid–base changes. Such a critical threshold has been observed for corals grown at a Papua New Guinea CO2 seep site, where pHcf homoeostasis was only found for pHsw of >7.8 and Ωsw of >2.3. Beyond that, pHcf could not reach the same values as those under control conditions, and likely the coral’s physiological limit to compensate for changes was reached40. This lack of ability to fully compensate for the lower pHsw may be responsible for the slight differences in pHcf observed in this study.The ability of organisms to modify pHHsw Fig. . A poten30) allowed us to further pinpoint potential mechanisms of how external seawater conditions affect internal calcifying conditions and ultimately skeletal growth. Calcification was once thought to be a passive diffusion process of seawater that brings external DIC to the site of calcification 45 and by active ion transporters46 that result in an elevation of pHcf, thereby facilitating precipitation. At our study sites, DICsw is significantly higher at the low Ωsw sites (in average 2790 µmol kg−1 compared to control average DICsw of 2050 µmol kg−1) allowing us to decipher the role of external DICsw in modulating calcification regulation processes. If corals modify internal DICcf by simply up-regulating DICcf from the external concentrations baseline, we would expect higher DICcf values for the ojo corals where DICsw is higher. Under such assumption the elevated DICcf compensates for the slightly lower pHcf effect on Ωcf and calcification rates would essentially be similar between sites , and this impacts Ωcf (more precisely CO32−) and calcification. A recent study under laboratory conditions with Stylophora pistillata23 observed that changes in DICsw concentration modulates internal pHcf regulation, with higher external DICcf facilitating higher internal pHcf, resulting in a clear correlation between seawater DICsw/H+sw and pHcf. Since DICsw at the ojos is significantly higher than at the control sites, one could expect this to compensate reduced pHcf up-regulation induced only due to changes in seawater pHsw. Yet we do not see a strong correlation between pHcf and seawater DICsw/H+sw, suggesting different drivers for Ωcf regulation in P. astreoides compared to those observed in S. pistillata23. Nevertheless, the change in pHcf and the limited ability to upregulate DICcf at the ojos corroborates the observed calcification rate decrease of corals at the ojos. However, these parameters may not be the only drivers for the decline in growth. Recent studies identified internal calcium (Ca2+cf) regulation as an additional player in coral calcification responses and emphasised that regulation of Ca2+cf can contribute to a corals’ resistance to future ocean changes48. In this sense, the good agreement of our model with the observed calcification response may imply that internal average steady-state calcium concentrations (Ca2+cf) at the ojos are lower by some proportion that is related to the pHcf changes, since our model based on pHcf and CO32−cf can explain only 41% of the observed calcification decline. This suggests a strong link between Ca2+cf and pHcf and supports the idea of a plasma-membrane Ca-ATPase  responsible for pHcf regulation. However, it is possible that pHcf and Ca2+cf were both regulated by additional and/or different ion transport mechanisms 51.The use of our dual geochemical proxy data to model coral growth  we see. Recent work provided the first evidence that coral symbionts  and host genotypes can jointly affect coral calcification rates55. Similarly, possible interactions with the microbiome  or changes in energy acquisition and allocation processes to overcome environmental gradients58 can affect coral growth. Environmental factors may also affect pHcf and DICcf explaining some of the observed differences between the ojo and ambient corals at our study site. Studies have shown that a decrease in pHcf and DICcf is associated with increasing temperature38, yet at our sites the temperatures at the ojos is actually lower, on average, than at control sites. Salinity might also influence regulation processes, yet the measured average values (32.2 psu) as well as the salinity range measured (26–36 psu)44 at the springs can be tolerated by corals and the long-term exposure to such conditions may have allowed them to develop mechanisms to better cope and adapt to this variable environment59. Overall, these environmental and biological parameters may be responsible for the observed internal conditions in the calcifying fluid but likely also affect rates of processes that ultimately affect calcification, and thus contribute to the unexplained component in our relationship between calcification and the geochemically derived DICcf and pHcf. Our geochemical model approach assumes steady-state equilibrium conditions; however, the rates of the various transport processes involved in regulating the chemistry of the calcifying fluid will ultimately dictate the calcification response60: these rates may differ between individual coral genotypes, further contributing to the offsets between the model output and observations.The present study also indicates that the acclimation process in different corals encompass some degree of flexibility in terms of the relative role of pH11B and B/Ca) to constrain calcifying fluid carbonate chemistry in P. astreoides corals that spent their entire life (decades) under acidified low Ωsw conditions. We found that at the pHcf for corals at the low Ωsw was slightly lower than at the ambient conditions indicating inability to achieve optimal calcification conditions. We also determined that pHcf and DICcf are independently regulated and corroborated the calcification response in P. astreoides at this site. The study provides new insights into calcification responses of P. astreoides under changing environmental conditions and sheds light on the potential of corals to acclimate62. Using the geochemical proxies in combination with the bio-inorganic model brought forward by McCulloch et al. 30, we could explain 41% of the variability in coral growth rates along a Ωsw gradient. The variability which is not explained indicates that additional physiological and environmental processes contribute to the control of calcification rates in natural environments. This provides promising new avenues towards studying acclimation and adaptation potential of long-lived marine invertebrates such as corals.In this study, we utilised a dual geochemical proxy approach . Five cores were drilled in close proximity to the low pH discharge and seven cores were drilled from control sites outside the ojos discharge influence (~2–5 m away). After collection, cores were dried at 50 °C before further analysis. Water chemistry was measured at the different sites (summarised in Supplementary Table 64) and used to calculate carbonate chemistry  concentration compared to the ambient conditions away from the spring influence. The corals at these ojos are constantly exposed to these discharging water  the corals at these sites were exposed to low Ωsw for their whole life span, potentially allowing enough time for acclimation. Moreover, it is quite likely that strong selection processes have resulted in successful colonisation of the ojos by a fraction of the coral population that is better adapted to low pHsw and high CO2.Cores from colonies of 66; ~430 ± 8 μM, with no difference between ojos and control) and a boron isotopic composition (δ11Bsw) of 39.15  for the control site and 38.85  for the low pH ojos. Boron isotopic samples were analysed on a Neptune multi-collector inductively coupled mass spectrometer at National Cheng Kung University, Taiwan, using the standard-sample-standard bracketing technique67. The boric acid standard IAEA-B-1 was used as the reference standard (e.g. 39.77‰) to determine the δ11B of the samples, reproducibility (±0.25‰).Water samples were also taken for seawater boron concentrations . The measurement procedure followed Fietzke et al.68 and Wall et al.40 with slight modifications. Specifically, we used Multiplier and Faraday cups simultaneously to collect data for B10 and B11 (both on multiplier) as well as C12 (Faraday cup). This allows us to derive B/C and δ11B from the same skeletal material. Similar to previous work the cones were cleaned on a regular basis (every 2–4 days). The tubes going from the ablation cell to the plasma torch were checked for material deposition and cleaned by high flow rates overnight and/or mobilisation of the debris by increased flow rates transporting it out of the tubes. Prior to each measurement session the standard and samples were pre-ablated to remove surface contaminations . A standard-sample-bracketing method was used. The data of one measurement session contained 5–6 brackets. Both C12 and the variation of the standard (NIST SMR610) for each session were used to check for instrument stability and contaminations. Sessions were repeated when the standard drift was higher than the internal reproducibility of the standards (2 SD of the session on the standards). Twenty individual laser tracks (25 × 500 µm) were placed as close as possible to the edge of the skeletal section ), far enough away to avoid ablation through the skeletal part. Yet COC areas may not have been completely avoided. To account for this we: (a) subsequently screened the individual tracks for abnormalities in C12 indicative of either ablation through the coral skeletal part  or increased organics and excluded this parts from analysis, and (b) aimed for 20 tracks of ∼25 × 500 × 20 µm on all individuals to have a representative δ11B dataset per individual. By this approach we expect to cover a representative sample set and minimise the natural intra-skeletal variability and cover similar proportions in each of the different corals . The accuracy of our δ11B measurements has been checked by repeated analyses of Porites coral standard Jcp-1 and NIST SRM610, measured against a pellet of primary boron standard NBS951 (boric acid)  and briefly polished (Struers DiaPor Dur 9 µm polishing suspension) in preparation for boron analysis using a Struers TegraPol-21 with TegraForce-5 head (Grinder and Polisher). The δ68. This yields one δ11B value per sample and session with an average precision of <1‰ (1 SD) for ∼1.7 µg of carbonate sample. A minimum of 15 and up to 20 values of δ11B spread over the core surface in the upper few mm of each coral colony  were measured to obtain a representative data set per sample. The data set reflects the high variability in δ11B for a single colony, and replicates were averaged afterwards to yield values that reflect the mean δ11B value, hence the mean internal calcification conditions (see below).The data reduction followed Fietzke et al.10B and 11B) have been collected using a pair of ion counters, while carbon (12C) had been determined using a Faraday cup. B/C data are based on the integrated boron intensities (10B + 11B) divided by the 12C intensity. The calibration (conversion from intensity ratios to concentration ratios) has been done using a natural L. pertusa coral sample covering a B concentration range of about 450–950 µmol/mol, which had been determined before using LA–ICP–MS relative to standard NIST-SRM610 using 43Ca as internal standard. This calibration procedure resulted in: B/C [µmol/mol] = 78,800 × B/C [cps/cps]; . We used stoichiometric ratio of C/Ca = 1 as approximation for natural carbonates and translated B/C ratios in B/Ca [µmol/mol] ratios.B/C elemental ratios have been determined simultaneously with the boron isotope ratios via LA–MC–ICP–MS. Boron isotope data  of 1.027269 and pK*B averaged for the two sites  to quantify the extent of the physiological control on the internal pHcf.and related to the seawater aragonite saturation state . Compared to the pHcf range (8.2–8.8) derived from individually measured skeletal δ11B values such changes are minor  was calculated following McCulloch et al.19 IpHRAC model:Calcification rate . The individual average temperature data for the different sites were used  was calculated from pHcf and DICcf using seacarb. We first followed recent approaches20 by setting DICcf equivalent to double DICsw19. In a second step an advanced bio-inorganic model used both geochemically determined calcification parameters to calculate Ωcf. For the seacarb (R) calculations we used the measured average salinity and temperature for the different sites. We assumed [Ca]2+ concentrations that equals seawater values to calculate Ωcf.For a sole pH-regulation-based model we used seawater DIC concentration DIC11, by converting them into relative rates and setting the control site as 1 (or 100%19).Both modelled calcification rates were plotted against the measured calcification ratest-test . To understand how well the internal calcification conditions  can explain measured changes in net calcification rate we applied simple linear models regressing model-derived relative growth as a function of internal Ωcf. Similarly, changes in geochemical proxies as well as internal conditions were regressed to decipher correlation between these parameters and potential driving forces explaining changes in net calcification rate along the natural environmental seawater Ωsw gradient20.Statistical analysis of the geochemical proxies and derived internal calcification conditions was performed by comparing the two treatment groups (control conditions vs. reduced aragonite saturation state at the centres of the ojos) using Welch’s Data analysis and visualisation was done with R Studio version 3.0.1 .Supplementary InformationPeer Review FileSource Data


For echinoderms with feeding larvae, metamorphic and post-settlement success may be highly dependent on larval nutrition and the accumulation of energetic lipids from the diet. In contrast to the sea urchins, starfish and brittle stars within the Phylum Echinodermata, sea cucumber metamorphosis does not involve formation of a juvenile rudiment, but instead there is a rearrangement of the entire larval body. Successful metamorphosis in sea cucumbers is often associated with the presence in the late auricularia stage of an evolutionary novelty, the hyaline spheres (HS), which form in the base of the larval arms. Known since the 1850s the function of these HS has remained enigmatic—suggestions include assistance with flotation, as an organizer for ciliary band formation during metamorphosis and as a nutrient store for metamorphosis.Australostichopus mollis. Neutral lipids derived from the phytoplankton diet are transported by secondary mesenchyme cells , likely as free fatty acids or lipoproteins, from the walls of the stomach and intestine through the blastocoel to the HS; here, they are converted to triacylglycerol with a higher saturated fatty acid content. During metamorphosis the HS decreased in size as the triacylglycerol was consumed and LTC again transported neutral lipids within the blastocoel.Here using multiple methodologies  we show definitively that the HS are used to store neutral lipids that fuel the process of metamorphosis in in A. mollis functions as a nutrient storage structure that separates lipid stores from the major morphogenic events that occur during the metamorphic transition from auricularia–doliolaria–pentactula (settled juvenile). The discovery of LTC within the blastocoel of sea cucumbers has implications for other invertebrate larvae with a gel-filled blastocoel and for our understanding of lipid use during metamorphosis in marine invertebrates.The HS  The echinoderms, basal members of the Deuterostomia, are well known in an evo-devo context because of the presence of morphologically distinct larval forms in each echinoderm class , 2. In sHolothuroids are the echinoderm class that most fully represents the ancestral type of indirect larval development: a feeding dipleurula-type larva (the auricularia) followed by a non-feeding doliolaria , 2, 8. MSuccessful metamorphosis in sea cucumbers is often associated with the presence in the late auricularia stage of enigmatic structures, the hyaline spheres (HS), which form in the larval arms during the feeding phase, increase in diameter in the late auricularia before metamorphosis into the doliolaria stage and then decrease in size during the pentactula and juvenile stage , 12–16. Mortensen  first hyAustralostichopus mollis we directly test the hypothesis of a nutritional function for the HS using lipid mapping, resin-section light microscopy and lipid and fatty acid analyses. Our previous work has shown that, like asteroids and echinoids, A. mollis use the neutral lipids TAG and FFA to build the larval body [Here, using auricularia larvae of the New Zealand sea cucumber val body , and we val body , 4, thesAustralostichopus mollis reach the late auricularia stage approximately 14 days after fertilization, defined as when the left somatocoel was about half the length of the stomach and there was no elongation of the axohydrocoel  a ~ 5-µm-diameter, slightly flattened fibroblast-type cell with a clear nucleus and blue-staining cytoplasm with variable degree and number of cell projections  were strongly concentrated in the stomach Fig. a. In lat4 days weEarly-stage auricularia developed two granular masses, similar to those described by Semon  and MortAt about 19 days post-fertilization, hyaline spheres (HS) started forming in the posterolateral lobes of the late auricularia larvae, just above the GM Figs. c, 3a. HSTowards 20 days post-fertilization, more HS began appearing sequentially in the lateral and anterior larval lobes, reaching a maximum number of 10 per larva and increasing in size as development proceeded Fig. a. In ligDuring transition from the late auricularia to the doliolaria (approximately 22–23 days) the continuous ciliary band broke to form three ciliary rings Fig. a. The ceAll the doliolaria observed in culture had the blastocoel filled with HS and LTC; the latter were more abundant distally from the digestive tract Fig. a, d. GraIn the recently settled early pentactula (27 days), blastocoelic fibres were tightly packed in the centre of the juvenile body, holding the HS close to the digestive system but allowing transit of different cell types within the fibre matrix Fig. c, d. LTCNile Red and LipidTOX™ staining suggests that the HS in auricularia are accumulating large amounts of a neutral lipid from the diet. Using Iatroscan thin-layer chromatography–flame ionization detection (TLC–FID) we found a significant increase in the neutral lipids triacylglycerol (TAG) and free fatty acid (FFA) over the period of HS accumulation in the auricularia , a significant decline in polyunsaturated fatty acids  and no change in monounsaturated fatty acids  when compared to whole larvae with HS  and with HS (21 days) showed a significant increase in saturated fatty acids  derived from the phytoplankton diet are transported by LTC from the walls of the stomach and intestine through the blastocoel to the HS  we have resolved the long-standing enigma of the function of the hyaline spheres in sea cucumber larvae, showing that HS have an important nutritional role in late larval development and during metamorphosis in  HS Fig. . This me HS Fig. . Nearly In addition to confirming a nutritional role of the HS we have shown the mechanism whereby nutrients are transported from the digestive system of auricularia larvae, a significant gap noted in previous studies , 15. We It is important to note that the BS/LTC, whose function is described here, have been morphologically identified in previous studies on sea cucumber development, for example the “vesiculated mesenchyme cells” in the histological sections of Balser et al. ; their F, 6, withThe movement of mesodermal cells within the blastocoel of larval echinoderms has been known since Metchnikoff  describeOphiocoma pumila where an “oily accumulation” forms within the posterior tip of the ophiopluteus beneath the ciliary band [Holothurians are, however, unusual among planktotrophic echinoderms in transporting lipid reserves obtained from the phytoplankton food, which typically accumulates in the stomach wall in echinoids , 6 and aary band . This spary band . It woulPatiriella pseudoexigua, the species with the greatest lipid load, lipids are also secreted into the embryonic blastocoel where they are stored for the peri-metamorphic period [A significant advantage of positioning the lipid stores in the periphery of the larva is that it does not interfere with the process of morphogenesis in sea cucumbers and ophiuroids with a vitellaria , 9. In ec period , 36. StrA. mollis are primarily TAG, which are accumulated from the phytoplankton diet during the feeding larval stage for metamorphosis, as in other echinoderms .–39.A. moms . As thems , but did not follow these cultures through to metamorphosis. Such experiments could prove valuable in assessing the generality of the observation of Smiley [Apostichopus californicus that metamorphosis can proceed without HS. Recent transcriptome studies on the auricularia–doliolaria metamorphic transition in the Japanese sea cucumber Apostichopus japonicus have also shown KEGG enrichment of over 80 genes involved in the “fat digestion and absorption” category [Future research needs to consider whether HS are a prerequisite for metamorphosis in f Smiley  in Apostcategory . DetaileFinally, the relationship between the LTC and other secondary mesenchyme cells within the blastocoel of the auricularia needs to be examined in more detail. Mesoderm-derived immune cells have been classified into four types in sea urchin larvae , 31, 44,A. mollis larvae are storing neutral lipids for use during metamorphosis, i.e. they have a nutritional function. We also describe a secondary mesenchyme cell, the LTC, which moves lipids from the digestive system to the hyaline spheres during late larval development. Although the hyaline sphere itself appears to be unique to sea cucumbers, we hypothesize that the LTC will be found more generally in invertebrate larvae with a gel-filled blastocoel, including other echinoderms and the hemichordate tornaria.Here we definitively show that the HS in Australostichopus mollis larvae were obtained from 4 L rearing tanks of 1-µm-filtered sea water (FSW) set-up with gentle aeration under constant light and temperature conditions (12:12-h light:dark cycle at 19 ± 0.5 °C) as outlined in detail in Peters-Didier and Sewell [Isochrysis galbana, Dunaliella tertiolecta and Chaetoceros muelleri  supplied at a final concentration of 3000 microalgae cells per ml twice daily. A. mollis larval samples were obtained at the auricularia pre-hyaline spheres (14 days), late auricularia with HS (20 days), doliolaria (22 days) and pentactula/juvenile (24–27 days) stages for microscopy and lipid analysis.d Sewell . Larvae −1) in autoclaved FSW, incubated in the dark for 30 min at room temperature and rinsed 3× with autoclaved FSW after gentle centrifugation . One part of Nile Red stock solution consisting of 1:1000 dilution of Nile Red in acetone was then added to 500 parts of autoclaved FSW containing larvae and incubated for 1 h at 4 °C in the dark with occasional gentle inversion. Samples were rinsed in autoclaved FSW, immobilized in circular wells (200 µm deep × 2 mm diameter) on a transparent non-fluorescent Perspex slide and photographed with a Nikon 500 digital sight-cooled colour camera on an epifluorescence microscope (Leica DMR upright) fitted with two long-pass filters: I3  and A .Larvae were stained with Hoechst 33342 nuclear stain (Life Technologies) followed by Nile Red (Sigma): 1 µl of 1000× Hoechst 33342 dye was added to 1 ml of larval suspension (~ 40 larvae ml−1), left in the dark at room temperature for 48 h and rinsed 3x with autoclaved FSW after gentle centrifugation . The larvae were then stained with Hoechst 33342 dye as outlined above, and then 2 µl of 1000X LipidTOX™ green neutral lipid stain was added to 1 ml of the larval suspension and incubated for 30 min. Larvae were visualized in a Andor revolution XD confocal microscope set with a iXonEM (Andor™) camera attached to a Nikon Eclipse-Ti inverted microscope. Three laser channels were used: blue , green  and red . Images were processed using Imaris 7 (Bitplane) software. Unstained larvae were used as controls for all observations.For confocal microscopy, larvae were stained with LipidTOX™ Red phospholipid stain (Life Technologies) followed by Hoechst 33342 nuclear staining (Life Technologies) and LipidTOX™ green neutral lipid staining (Life Technologies). Two microlitres of 1000× LipidTOX™ Red phospholipidosis detection reagent was added to 1 ml of larval suspension in autoclaved FSW  and stained using a polychromatic staining method consisting of methylene blue-azure II and basic fuchsine [® 5 (Life Science).Samples of sea cucumber larvae were fixed and embedded in epoxy resin following the glutaraldehyde and cacodylate buffer method in Byrne et al. . Semi-thfuchsine . Throughfuchsine . Semi-thN = 200 individuals) and pentactula/juveniles (N = 60). Traditional TLC was also conducted on lipid extracts (45) from auricularia pre-HS (14 days) and with HS . Extracts (3 µl) were applied to pre-activated (100 °C for 10 min) aluminium-backed TLC plates  alongside 1 µl aliquots (10 mg/ml) of the highly purified lipid standards phospholipid (PL: L-α-phosphatidylcholine), free sterol (ST: cholesterol), free fatty acid  and triacylglycerol . TLC plates were developed in a neutral solvent system containing hexane–diethyl ether–acetic acid  and visualized using iodine vapour. Sample bands of TAG and FFA were scraped off the TLC plate, the lipids re-extracted using the Bligh and Dyer [® Plus factory-coated glass slides (Menzel-Gläser). Nile Red staining was in FSW for 30 min at the same concentration used to stain live larvae. Re-extracted lipids were observed under fluorescence microscopy using an I3 filter  and photographed using a Nikon 500 digital sight-cooled colour camera and images analysed with AnalySIS® 5 (Life Science).Lipid extraction and analysis using Iatroscan thin-layer chromatography–flame ionization detection (TLC–FID) was performed as described in Peters-Didier and Sewell  on larvaand Dyer  method, A. mollis larvae with and without hyaline spheres (N = 3) using C19:0 and C23:0 as the internal standards. FAME were separated and quantified using a gas chromatograph (GC 7890 Agilent system) equipped with a mass spectrometry detector (MSD 5975c) as described in Zárate et al. [Fatty acid methyl esters (FAME) were prepared using the method of Lepage and Roy  from tote et al. . FAME pee et al. . FA abunStatistical analyses were conducted using one-way ANOVA with SAS 9.4 or when assumptions of ANOVA were violated by permutational analysis of variance  using the PERMANOVA + 1 package within PRIMER 7.0.13  on a Euclidean distance matrix.


TBC1D4 gene encoding a canonical NFκB binding site, and edited it using CRISPR-Cas9 to remove this site. We show that this editing reduces TBC1D4 expression, local chromatin accessibility and binding of the p65 component of NFκB. We then used CRISPR without genomic editing to guide p65 back to the edited locus, demonstrating that this re-targeting, occurring ~182 kb from the gene promoter, is enough to restore the function of the locus, supporting the central role of transcription factors mediating the effects of functional variants.Functional variants in the genome are usually identified by their association with local gene expression, DNA methylation or chromatin states. DNA sequence motif analysis and chromatin immunoprecipitation studies have provided indirect support for the hypothesis that functional variants alter transcription factor binding to exert their effects. In this study, we provide direct evidence that functional variants can alter transcription factor binding. We identify a multifunctional variant within the  TBC1D4 gene, encoding an NFκB binding site, can alter transcription factor binding, and use CRISPR-Cas9 to reveal localization of the transcription factor to be the regulator of chromatin accessibility and p65 binding and ultimately TBC1D4 expression.Functional variants have been proposed to alter transcription factor binding. Here, the authors provide direct evidence that functional variants within the  A locus influencing DNA methylation in cis is referred to as a methylation QTL (meQTL)5, while there are also examples of loci influencing cis chromatin states6.The variability in genomic sequences between individuals that is explored for disease associations includes loci that have obvious functional properties. Some of these loci change the coding sequences of genes, but a large proportion exist in the non-coding majority of the genome and are associated with changes in genomic function. The main functional property of the genome that has been explored for association with DNA sequence polymorphism is gene expression. Differences in the level of expression of a gene that depends on the allele present at a locus nearby defines that nearby locus as an expression quantitative trait locus, or eQTL10. By changing TF binding, it is furthermore assumed that gene expression, DNA methylation, and chromatin states are altered as consequences12. Supporting the model of DNA sequence polymorphism influencing TF binding as the primary mechanism for these downstream functional consequences, chromatin immunoprecipitation has shown differences between individuals13 or alleles15 at a locus for TF binding associated with sequence variation at that site. While this is strong evidence for the TF binding model, it remains an association and not a direct test of the hypothesis. As an alternative hypothesis, it is proposed that genetic polymorphism at functional genetic variants influences enhancer RNA structure, altering its ability to act as an aptamer to recruit proteins locally, preventing it from facilitating local looping of chromatin16.When studied at the sequence level, these functional variants can be described to contain binding sites for sequence-specific transcription factors (TFs), with the presence of a sequence variant within that site assumed to alter the binding of the TF at this locus18. These approaches use reagents that have sequence-specificity to target a pre-defined locus, including zinc finger domain proteins19, the TALE binding domain of transcription activator-like effector nucleases (TALENs)22, and, more recently, the CRISPR system with an inactivated nuclease18. With the idea that ‘epigenetic’ regulators are those influencing chromatin states and DNA methylation, a number of enzymes that modify these biochemical properties at a locus have been targeted, with varying efficacies and gene regulatory outcomes39.These observations contrast with the assumptions being made in a second current area of genomic research, the use of ‘epigenetic editing’ to modify regulatory events at specific loci in the genomeThere is, however, some lack of coherence between the ideas being pursued by those studying functional variants and those performing epigenetic editing. The former group would regard TFs as the primary mediators of events that include transcriptional, DNA methylation, and chromatin state changes as consequences. The premise of many ‘epigenetic editing’ approaches is that altering chromatin states and DNA methylation is sufficient to alter transcriptional regulation, ignoring any need for TFs to function locally. It remains to be seen whether the successes or failures of these two independent approaches will reveal insights into physiological transcriptional regulation, for example, whether chromatin modifications by themselves can allow transcription to occur in the absence of local TF binding.Here, we seek to test the model that TF binding mediates functional genomic outcomes at a polymorphic and regulatory locus that we identified in the human genome. We use CRISPR for targeted mutagenesis at a TF binding site overlapping this multifunctional variant, followed by the further use of CRISPR as a targeting system to deliver to the edited locus the TF that could no longer bind to the site—a form of epigenetic editing that assumes a primary role for the TF in regulating the locus. The results reveal insights into both the normal physiology of functional genomic variants as well as guidance for epigenetic editing strategies.40, on which deep (Illumina Platinum genomes) sequencing has been performed41, and for which lymphoblastoid cell lines (LCLs) are available from the Coriell Institute. By studying a family, we could identify effects mediated by variants that are rare in wider populations.Our primary goal was to identify a small set of polymorphic loci with very strong functional effects on gene expression, chromatin accessibility, and DNA methylation. We performed our studies on the CEPH/Utah Pedigree 1463, which includes 17 members in three generations . When samples were harvested as a batch, different combinations of samples were used for biomaterial extraction. All batch information was noted and used as metadata in exploration of the sources of variability in the data analyses. We noted that two samples (GM12877 and GM12893) grew at a slower rate than the other 15. We also checked carefully whether GM12889 was an outlier in any of the functional assays, as we have found this cell line to have a previously unrecognized mosaic copy number neutral loss of heterozygosity of distal chromosome 1143, but did not observe any distinctive results from this sample.To reduce technical variability, substantial care was taken with the assays based on cultured LCLs. A single batch of reagents was used for all cell cultures, and all genome-wide assays were performed on the same flask of cells  assays using our optimized Illumina HiSeq X-based assay44 to profile DNA methylation throughout the genomes of these cell lines.We performed a set of genome-wide functional assays on the 17 samples. Directional RNA-seq was used for gene expression profiling, small RNA-seq to test miRNA expression, and the assay for transposase-accessible chromatin (ATAC-seq) to map nucleosome-free, BHLF1 gene, which is expressed early in the lytic cycle of EBV infection and expressed in subclonal populations of LCLs45. DNA sequence variants found in these assays were compared with those from the Illumina Platinum Genomes annotations41 to confirm sample identities. The influences on our functional assays are summarized in principal component plots in Supplementary Fig. Our analytical studies started by testing for sources of variability of our results. One of the cell lines that were growing more slowly than the others, GM12893, in addition to batch-affected lines GM12880 and 12879 were also outliers in terms of their gene expression  in this family46. This allowed us to compare the target genes affected by eQTLs (eGenes) between our study and the prior analysis of this family. We found 21 eGenes in common between the studies (Supplementary Data p < 1.3 × 10−10) despite the prior study’s older haplotype annotation and their use of an mRNA-seq assay46 as opposed to our RNA-seq assay that was not solely dependent upon oligo-dT priming. We further validated the results from both studies by performing qRT-PCR on several eGenes and found that those discovered by both studies almost universally showed the predicted expression differences among the children (8 out of 9)  to allow sensitive identification of all miRNAs being expressed, pooling reads across all 17 samples for prediction purposes. We show there to be 632 known human miRNAs expressed in these samples, plus 13 from the EBV genome, and we identified 112 candidate novel human miRNAs. We confirmed one of these novel human miRNAs using stem-loop PCR . Analytically, we mapped ATAC-seq reads with the steps of extra stringency to exclude blacklisted regions of the human genomeHaving identified these individual eGenes and caSummits, we then narrowed the focus to define candidate multifunctional variants affecting both gene expression and chromatin accessibility. We identified 62 haplotype blocks containing both an eGene and a caSummit which overlaps a heterozygous SNP. We also filtered to require that there be co-variation of the informative haplotype and SNP. For example, if the peak is associated with one but not the other paternal haplotype, but the SNP variation is carried on the maternal haplotype, the variation in chromatin accessibility cannot be attributed to that particular SNP within the paternal haplotype. As a further filter, with the assumption that one allele being active and the other silenced should be associated with a pattern of intermediate DNA methylation locally, we chose the subset of loci in which CG dinucleotides with DNA methylation values of 20–80% were found within the peak. The overall filtering process is summarized in Fig. GSDME and TBC1D4 genes. We focused on the TBC1D4 multifunctional variant, located in a ~6 Mb haplotype on chromosome 13q  to encode a canonical NFκB binding site52, with the human-specific (HS) allele changing that binding site in 3 positions, alterations strongly predicted to disrupt NFκB binding. We therefore chose this as the locus for genomic editing experiments. TBC1D4 is a gene encoding a Rab-GTPase-activating protein that responds to insulin by dissociating itself from the glucose transporter 4 (GLUT4) protein, allowing the latter to migrate to the plasma membrane where it can act to transport glucose into the cell53. Mutations of TBC1D4 have been associated with type 2 diabetes mellitus55, indicating that the protein has a role to maintain normal glucose homeostasis.The list of high-confidence candidate multifunctional variants included sequences within the 13q Fig. , upper, o49 Fig. , lower. tes Fig. . The Geotes Fig. 50. UsingTBC1D4 but not the adjacent COMMD6 or UCHL3 genes within the same interaction domain  have higher levels of expression of ain Fig. . More deain Fig. . In the ned Fig. .Fig. 4ExAs there are no informative CG dinucleotides immediately adjacent to the multifunctional variant, we are unable to test haplotype-specific DNA methylation patterns. However, we observe that DNA methylation in immediately flanking CGs is lower in individuals with the AA haplotype Fig. .Fig. 8DNcis-regulatory locus show it to be specifically active in LCLs57  strategy for ‘scarless’ editing60. It has recently been highlighted that CRISPR-mediated genomic editing can be associated with mutations that can be large in size and potentially missed if the screen is solely focused on a small, targeted desired mutation61. We, therefore, added long-range PCR of the 4 kb DNA region flanking the targeted locus as well as reverse transcriptase PCR (RT-PCR) of overlapping 1 kb segments of the 6.4 kb TBC1D4 transcript which spans 197.4 kb on chromosome 13. Of the 5 clones found to have edits at the desired site, these PCR-based analyses revealed 2 clones to have large mutational events at the locus by testing DNA and RNA58. We, therefore, focused our analyses on 3 clones, 2 of which had the desired clean replacement of the Ancestral (AA) with the human-specific (HS) allele, and one with a 7 bp deletion spanning the NFκB binding site that appeared by PCR and densitometry to be hemizygous58, an even more deleterious pair of mutations for comparison  to test the effect of replacing the functional, NFκB-binding AA with the inactive HS allele or the cell line with the completely deleted NFκB binding site. We show the results in Fig. nes Fig. . We thennes Fig.  and the nes Fig.  at the eTBC1D4 activity >2-fold relative to a condition in which the same system without the p65 protein was introduced into the LCLs with the HS allele. However, we were concerned that these effects could be partially or wholly due to the increased amount of this NFκB subunit in the cell, binding to other cis-regulatory sites at this gene. We, therefore, employed two additional strategies. We developed a control system to target a known NFκB binding site at the OXTR gene62, validating targeting by testing the Cas9-induced mutation rate at this locus in LCLs  drove a need to identify ‘epigenetic’ marks such as DNase hypersensitivity, or ChIP-seq mapping of active chromatin regulators, demonstrating enrichment for overlap of these candidate regulatory loci with the lead SNPs identified in the GWAS, finding even greater enrichment for other SNPs in linkage disequilibrium65. To progress from these relatively large areas of the genome, containing many candidate functional variants, to the specific variants mediating the disease, the approach has generally been to identify functional variants within these regions. Typically these variants are defined by associating co-variation of transcription levels with sequence polymorphism in cis to that gene, but there are now studies that use DNA methylation66 or chromatin characteristics67 to help identify loci with transcriptional regulatory properties. The locus that we studied within the TBC1D4 gene was selected for its effects on local gene expression, chromatin accessibility and local changes in DNA methylation, as well as its strong predicted effect on the binding of the NFκB TF based on sequence characteristics. This confirms that it should be possible to use different types of functional genomic assays to arrive at the same functional variants, and that the use of multiple functional assays simultaneously67 may be especially valuable in prioritizing certain loci as being more likely to have a regulatory function. A functional variant, once identified, can be linked to variable gene expression as an eQTL, with the target eGene representing a potential insight into the mechanism of the disease, based on the properties of the protein encoded by that gene.The interest in identifying functional variants has arisen from associations implicating haplotypes in disease heritability68, especially apparent from the systematic studies of the GTEx Consortium69. The cis-regulatory landscape of different cell types is well known to be distinctive70, implying sequence-specific mechanisms for targeting post-translational modifications of histones, DNA methylation, and nucleosomal remodeling in different cell types. Candidates for mediating this sequence-specificity should include TFs , but not the enzymatic mediators of chromatin states or DNA modifications, which inherently lack the ability to recognize specific sequences. The model of a primary recognition of specific sequences by different TFs as part of the process of driving lineage commitment is well-established. The fact that some TF binding sites (TFBSs) are variable between individuals, therefore, leads to the idea that the same canonical cell type from individuals differing in their DNA sequences at TFBSs can confer distinctive properties on that differentiated cell between individuals, differences that could be reflected by altered or disease phenotypes.One concern has been that the post-GWAS surveys for sequence variants with regulatory functions may have been performed in a cell type irrelevant to the disease process being studied, prompting the search for functional variants in the cell type(s) assumed to mediate the phenotype of interest. That eQTLs can have a cell type-specific activity is increasingly apparent71 and for DNA methylation influencing the genomic binding of at least one TF, NRF172. There is clear evidence from in vitro studies for DNA methylation influencing TF binding, both negatively and positively73. The question therefore arises whether our targeting of the p65 transactivation domains to a specific locus, an example of epigenetic editing, is exerting its positive effects on TBC1D4 transcription by means of replicating the function of the TF no longer capable of binding to the allele lacking the NFκB motif, or whether it is instead creating a local environment conducive to TF binding in general. As the targeting of the CRISPR complex is mediated through a guide RNA that forms an RNA:DNA hybrid74, loci which we have shown to be mostly inactive in the human genome75, and prior studies have revealed NFκB to bind strongly preferentially to double-stranded DNA76, we believe that, if anything, the targeting of the locus by CRISPR would have an inhibitory effect on local TF binding. While formally unproven by our studies, the primary mechanism for activation of the locus would appear to be more likely to be by the restoration of p65 than by non-specific effects on local chromatin organization. The question, therefore, arises about epigenetic editing more broadly, whether targeting of a locus to induce what may be secondary consequences of local TF binding  is the optimal approach. If the TF (or group of TFs) that would normally bind locally and recruit the mediators of these activation marks is not expressed in that cell, will transcription still be promoted? The different viewpoints inherent to the epigenetic editing and functional genetic communities could end up, even inadvertently, prompting some valuable insights into transcriptional regulation.This train of logic implies a primary role for TFs, with secondary effects on chromatin states and DNA modifications. Such a viewpoint raises a challenge to the idea that epigenetic regulation of the genome, defined in terms of chromatin patterns and DNA methylation, acts instead in a primary role to define where TFs can bind within the genome. There is support for both possibilities, mouse embryonic stem cell studies showing an instructive role for TFs on DNA methylationTBC1D4 gene from a promoter located ~182 kb telomerically.In summary, this study follows up on a wealth of excellent association data indicating a primary role for TFs in mediating effects of sequence variability at functional genetic loci. We harnessed the genetic and epigenetic editing capabilities of the CRISPR-Cas9 system to provide a direct test of this hypothesis, by engineering a locus to fail to bind the cognate TF, then restoring its targeting locally by guiding the transactivation domains of the TF back to the locus using CRISPR, increasing the transcription of the 2 incubator and maintained between 2 × 105 and 8 × 105 cells/ml.Lymphoblastoid cells (LCLs) derived from the 17-member CEPH Pedigree 1463 were purchased from the Coriell Institute and cultured in RPMI 1640 medium, supplemented with 15% fetal bovine serum , 100 IU/ml penicillin, and 100 μg/ml streptomycin (Life Technologies). Cells were kept in suspension in tissue culture flasks  at 37 °C in a 5% CO42. For each cell line, we collected 5 × 105 cells from two different cultures, harvested during the exponential growth phase. The cells were pelleted at 500×g at 4 °C for 5 min and washed with 50 μl of cold PBS. Samples were then centrifuged again at 500×g at 4 °C for 5 min. Cells were lysed in ice-cold lysis buffer  and immediately spun at 500×g at 4 °C for 5 min. The pellet was then resuspended in 50 μl of the transposase reaction mixture (TD buffer supplemented with 100 nM transposase). Following a 30-min incubation at 37 °C, the samples were purified using the DNA Clean and Concentrator-5 purification kit (Zymo). After purification, the libraries were amplified and indexed by combining: 10 µl transposed DNA, 5 µl PCR Primer Cocktail , 15 µl Nextera PCR Master Mix , 5 µl each of Nextera i5 and i7 indexed amplification primers , and 10 µl of nuclease-free water. The PCR reaction was carried out using the following conditions: 98 °C, 30 s; a total of 10 cycles of 98 °C, 10 s; 63 °C, 30 s; 72 °C, 1 min; 72 °C, 5 min. Subsequently, the libraries were purified using Agencourt AMPure XP beads; large fragments were filtered by using 0.6× magnetic bead volume (relative to the PCR mixture volume) and then keeping the supernatant. Primer-dimer and short fragments were removed by collecting bead-associated DNA in a 1:1 (bead solution volume:mixture volume) mixture. The libraries were quantified by Qubit HS DNA kit  and their quality assessed by 2% agarose gel electrophoresis and Bioanalyzer High-Sensitivity DNA Assay. Libraries were sequenced on an Illumina HiSeq 2500 to obtain 100 bp paired-end reads.The assay for transposase-accessible chromatin (ATAC)-seq libraries were prepared similarly to Buenrostro et al.44. A total of 2 × 106 cells was harvested, pelleted at 2000×g for 10 min and frozen at −80 °C until extraction of genomic DNA. Thawed cells were resuspended in 10 mM Tris pH 8.0, 150 mM EDTA solution before the addition of SDS to a final concentration of 1% in a 250 μl volume. Protein was digested overnight at 50 °C with 25 μg of Proteinase K , followed by an hour incubation at 37 °C with 5 μg of RNAse A (Sigma). Extraction of genomic DNA was then achieved through two consecutive phenol:chloroform steps, selecting the aqueous phase. The DNA was concentrated and purified via ethanol precipitation. Libraries were then prepared using 100 ng of genomic DNA and an additional spike-in of 0.5 ng of unmethylated lambda DNA as previously described44. Libraries were 250 bp paired-end sequenced on the Illumina HiSeq X system.Whole-genome bisulphite sequencing (WGBS) libraries for the Illumina HiSeq X (XWGBS) were prepared from each cell line similar to the method that we have previously describedCell pellets were treated with QIAzol lysis reagent (Qiagen) and total RNA was isolated using the miRNAeasy kit (Qiagen) combined with on-column DNase (Qiagen) treatment according to the manufacturer’s instructions. All dirRNA-seq libraries were prepared by removing rRNA from 1 μg of total RNA and generating strand-sensitive reads with a KAPA Stranded RNA-Seq Kit with RiboErase (HMR), following manufacturer’s protocol (Kapa Biosystems). Total RNA was depleted of rRNA by hybridization of complementary DNA oligonucleotides, followed by treatment with RNase H and DNase. RNA was then fragmented using heat and magnesium and submitted to first strand cDNA synthesis using random priming. After second strand synthesis and incorporation of dUTP, the ds-cDNA was A-tailed, ligated with adapters, and the library was amplified with primers carrying appropriate adapter sequences. The libraries were 100 bp paired-end sequenced.Libraries were constructed using NEBNext Small RNA Library Prep Kit (NEB). Input total RNA was ligated with 3′ adapters, and the excess of 3′ adapter was hybridized with a reverse transcription primer prior to the ligation of the 5′ adapter. Ligated RNA was converted into cDNA with a first strand reverse transcriptase, followed by PCR amplification. Library size was then selected by loading the PCR product into a 3% agarose dye-free gel of a Pippin Prep system (Sage Science). The libraries were 100 bp single-end sequenced. The metrics for all of the library sequencing performed in this project are shown in Supplementary Data 20 and random hexamers as primers 20 were used). All qPCR primers were designed using the NCBI Primer-BLAST web interface77  using both oligo(dT)Stem-loop primers , decoy version 1 for GRCh38 (hs38d1), and the Epstein Barr Virus genome, was used throughout the analysis79. Uniquely mapped reads were retained using samtools v0.1.19, followed by removal of mitochondrial reads by picard-tools v1.9280. Finally, duplicate reads were removed using both picard-tools (MarkDuplicates.jar) and samtools (samtools rmdup). Read1 reads were shifted using bedtools2 v2.26.0, as previously performed42. Leveraging the cell type similarity among the samples, all of the shifted reads were concatenated for each of the two replicate batches. Peaks were called for each replicate using MACS2 v2.1.0 81. Irreproducible discovery rates (IDRs) were found for the overlapping peaks using the method previously described82, and peaks with an IDR of less than 0.05 were retained and subsequently filtered by consensus blacklisted and mitochondria homologous regions, creating a ‘master’ peak list Kallisto v0.42.5 85. Estimated counts for genes were determined by Sleuth86. Genes on the sex and EBV chromosomes were removed and filtered for those with an average count of at least 10 among the children. Genes whose expression levels were associated with parental haplotype allele inheritance were discovered as aforementioned for caSummits except with additional regressors for batch extraction date and quartiled mean EBV expression. eGenes were defined by an FDR of less than 0.4. The statistical significance of the overlap between our eGene set and the previous study’s set was assessed using GeneOverlap v. 1.16.0.Sequenced reads were pseudo-aligned to GENCODE v24 long noncoding RNAs and protein-coding genes, as well as EBV genes, using 87 using the STAR aligner v. 2.5.2b with several parameter modifications specific to miRNAs . The aligner soft clips the 5′ ends of reads in order to find proper alignments. As this could lead to erroneous alignments, soft clipped reads were removed using samtools as well as the secondary alignments of reads. The primary alignments of all family members were concatenated and passed through miRDeep* for novel miRNA detection88. Only 221 putatively novel miRNAs with more than 50 supporting reads and a miRDeep* score greater than 10 were retained. A final set of 112 novel miRNAs were obtained by further filtering against the following miRNA databases: Rfam database v.1289, GENCODE v2478, fRNAdb90, NONCODE 201691, and FANTOM92.Small RNA sequence reads were aligned to the custom hg38 reference genome and a miRBase v21 human miRNAs gene transcript filesubRead v 1.5.0 featureCounts on all reads uniquely mapped by STAR and a miRBase v21 gtf also containing the 221 putative novel miRNAs93. Only miRNAs with at least 5 counts in 4 children were retained and were subsequently normalized using the trimmed mean of M values method94. These expression levels were regressed by haplotypic inheritance as previously mentioned to find eQTL-miRNAs (e-miRNAs).Quantification of miRNAs was achieved by using 95.To test if e-miRNA expression was mediating the effect of genetic variation on associated eGenes, the expression levels of eGenes were regressed by shared haplotype block miRNA expression levels, as well as by extraction date and quartile of EBV expression. e-miRNAs whose expression levels significantly (FDR < 0.05) predicted the expression of eGenes were not found to target their respective associated genes by testing validated targets stored in the multiMiR database44. Only CpGs with at least ten reads defining its methylation status on either strand were retained. Cytosines with coverage in the 99.9th percentile were removed to mitigate potential PCR artifacts. Finally, only the CpGs for which at least three children had intermediate methylation levels (25–75%) were used for discovering functional variants.To define CpGs with intermediate methylation levels, sequence reads were aligned to the custom hg38 reference genome and methylated and unmethylated counts for CpG were calculated as we have previously performed46 were explored for their potential to disrupt TF motif using HaploReg51. Rare variants were those with a minor allele frequency less than 0.05 in all 1000 Genomes superpopulations96.As shown in Fig. 56 was downloaded from the NCBI GEO database accession GSE55105. The fastq files were aligned using the Burrows-Wheeler Aligner version 0.7.13 (bwa mem –M –t < N > hg38.idxbase < R1 FASTQ >)79 to our custom hg38 genome.The ChIP-seq dataset from Zhao et al.6 cells was resuspended RPMI 1640 medium with 1% formaldehyde and incubated at room temperature under shaking for 10 min. The cross-linking was quenched with 0.125 M glycine, and the suspension was pelleted by centrifuging at 800×g for 5 min at 4 °C, then washed with 4 ml of ice-cold PBS and pelleted again. The pellets were then resuspended in Farnham lysis buffer and spun down at 800×g for 5 min at 4 °C. Subsequently, the samples were processed in a Biorupter using the setting of ON/OFF (30 s/30 s) cycles for 10 min. An average shearing size of 200 bp was obtained for each sample. The sonicated mixture was then spun at 10,000×g for 15 min at 4 °C, and the supernatant was collected. Protein G Dynabeads (ThermoFisher) were added to a 2 ml tube containing 1 mL PBS/BSA, the magnetic beads were washed 3 times to a final volume of 200 μl of PBS/BSA, and 20 μg of anti-p65 antibody  was added to each tube. The antibody was coupled to the beads for 3 h at 4 °C. Antibody-coupled beads were then washed again 3 times with PBS/BSA and then added to the 1 ml chromatin preparation and incubated at 4 °C overnight. The beads containing the immuno-bound chromatin were collected using a magnet, followed by washing 5 times in cold LiCl wash buffer and then washing with 1 ml TE buffer. The supernatant was then discarded and the bead pellet was resuspended in 200 μl IP elution buffer and incubated at 65 °C for 1 h, with vortexing every 15 min to elute the immuno-bound chromatin from the beads. The samples were then spun at 20,000×g for 3 min at room temperature. The supernatant containing the immunoprecipitated DNA was incubated at 65 °C overnight to complete the reversal of the formaldehyde cross-linking and then purified using the DNA Clean and Concentrator-5 kit (Zymo Research). The purified DNA was used as a template for quantitative PCR performed with the Light Cycler 480 Syber Green Master mix (Roche), according to the manufacturer’s instructions. Enrichment of DNA at the TBC1D4 cis-regulatory site was compared to a negative control region located within a gene desert of chromosome 13  where GM12878 ChIP-seq showed no H3K4me1, H3K27ac or NFκB enrichment. Enrichment at the promoter of IκBα, a p65 binding site validated by EMD Millipore, was used to assess the quality of the immunoprecipitation.For each tested cell line , a total of 20 × 1097, a total of 5 × 104 cells were pelleted by centrifugation at 500×g for 5 min at 4 °C and resuspended in 50 μl of ATAC-Resuspension Buffer (RSB) containing 0.1% NP40, 0.1% Tween-20, and 0.01% digitonin and kept on ice for 3 min. The suspension was washed with RSB devoid of NP40 or digitonin, and nuclei were pelleted by centrifugation at 500×g for 10 min at 4 °C. Nuclei were resuspended in 50 μl of transposition mixture . The reaction mixture was incubated 37 °C for 30 min with 3 g orbital shaking. Subsequently, the sample was purified with the DNA Clean and Concentrator-5 kit (Zymo) and submitted to qPCR as described for quantitative ChIP. A locus within a gene desert of chromosome 13  was used as a negative control. The enrichment of accessible chromatin at the TBC1D4 locus was normalized to the promoter region of GIN1, which was found to be highly accessible yet demonstrated invariable signal strength among the children.As performed by to Corces et al.98 was double digested, then purified on agarose gel to obtain a linearized fragment containing both the sgRNA expression and the dCas9 sequences. P2A Reverse complement oligonucleotides were annealed and extended, while the EGFP sequence was amplified by PCR from plasmid pBI-MCS-EGFP (Addgene plasmid #16542)99 using the nucleotides and primers described in Supplementary Data 63 with sequence-specific primers . Plasmid pCAG-eCas9-GFP-U6-gRNA was a gift from Jizhong Zou (Addgene plasmid #79145) and was used in combination with an ssODN template  cells were transfected with 33.3 µg of CRISPR-Cas9 plasmids in addition to 0.4 nmol of ssODN template for editing. The same number of cells was transfected with 2 μg of GFP plasmid to provide GFP control cells, while negative control cells received transfection reagents only. Transfections were conducted with the Cell Line Nucleofactor Kit V (Lonza) according to the manufacturer’s instructions. After transfection, cells were suspended in medium and incubated overnight under cell culture conditions, then replaced with fresh medium. GFP-positive cells were sorted after 48 h following the transfection. Cells were pelleted, washed twice, and suspended in sorting buffer . Cell suspensions were submitted to cell analysis and sorting in a FACSAria II cytometer (BD Biosciences). FACS data were analyzed using FACSDiva software (Becton Dickinson) with gating of single cells using FSC/W and SSC/W, and gating of GFP-positive cells. GFP-positive cells were identified by determining the GFP gating cutoff for control GFP-negative cells, further details are included in Johnston et al.58.LCLs were passaged at 3.5 × 10g, and the supernatant was filtered through a 0.3 micron sterile filter prior to use. When subsequent T7EI assays or qRT-PCR analyses were to be performed, cells were sorted directly into QuickExtract DNA extraction solution or RNAlater, respectively.Single GFP-positive cells were sorted 48 h after transfection into individual wells of a 96-well plate, containing a mixture of fresh and conditioned medium (1:1), in which the FBS concentration was increased to 20%. The 96-well plate was incubated for two weeks under cell culture conditions, then the content of each well was transferred to a new 96-well plate with additional conditioned medium. Conditioned medium was obtained from GM12881 cells, cultured in 20% FBS RPMI 1640 for 24 h. The medium was removed without disturbing cells at the bottom of the flask, centrifuged at 792×TBC1D4 or the OXTR loci, genomic DNA was isolated from transfected and control cell pellets using QuickExtract DNA Extraction Solution (Epicentre) according to the manufacturer’s instructions. DNA was then concentrated by ethanol precipitation. The 1 kb region containing the gRNA targeted region was amplified with forward and reverse primers (Supplementary Data To verify indel efficiencies of gRNAs targeting CRISPR-Cas9 to the Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Supplementary Data 7Supplementary Data 8Reporting SummarySource Data


Coelopa frigida along a latitudinal cline in North America. We test if the inversion is present in North America and polymorphic, assess which environmental conditions modulate the inversion karyotype frequencies, and document the relationship between inversion karyotype and adult size. We sampled nearly 2000 flies from 20 populations along several environmental gradients to quantify associations of inversion frequencies to heterogeneous environmental variables. Genotyping and phenotyping showed a widespread and conserved inversion polymorphism between Europe and America. Variation in inversion frequency was significantly associated with environmental factors, with parallel patterns between continents, indicating that the inversion may play a role in local adaptation. The three karyotypes of the inversion are differently favoured across micro-habitats and represent life-history strategies likely to be maintained by the collective action of several mechanisms of balancing selection. Our study adds to the mounting evidence that inversions are facilitators of adaptation and enhance within-species diversity.Large chromosomal rearrangements are thought to facilitate adaptation to heterogeneous environments by limiting genomic recombination. Indeed, inversions have been implicated in adaptation along environmental clines and in ecotype specialization. Here, we combine classical ecological studies and population genetics to investigate an inversion polymorphism previously documented in Europe among natural populations of the seaweed fly  When The ability to undergo polygenic specialization and local adaptation is related to the amount of genetic exchange between populations, because gene flow mixes unfavourable immigrant alleles with resident alleles. Negative modifiers of recombination can play an important role by limiting allele shuffling in parts of the genome –9. ChromEmpirical evidence supporting a link between inversions and local adaptation comes from clines of inversion frequencies along environmental gradients ,17,18 orCoelopa frigida provides a suitable model to understand how inversions facilitate adaptation to heterogeneous environments because it carries a large inversion, with the two forms called α/β ) and αα being the rarest karyotype ([5–21%]). Yet, inversion and karyotype frequencies were significantly heterogeneous between populations (deviance = 75(α), 41(αα), 102(αβ), 123(ββ); d.f. = 19, p < 0.001; electronic supplementary material, figures S2–S3 and table S4).All American populations were polymorphic for the inversion a and disp < 0.001) translating into a mean excess of heterokaryotypes of 20%, due to a deficit of both homokaryotypes . When considering the 20 populations individually, eight populations showed significant deviation from HWE with heterozygotes in excess, eight showed a slight excess of heterozygotes (non-significant) whereas four populations were at HWE (b).Significant deviation from HWE was observed among all American populations a; electronic supplementary material, figure S4). The two gradients included a climatic north–south cline, along which covaried air temperature, sea temperature and precipitations, and a west–east cline, with lower sea salinity and higher tidal amplitude in the western part of St Lawrence Estuary. Sampled wrackbeds varied in the seaweed composition, generally dominated by either Laminariaceae or Fucaceae and whose proportions correlated significantly negatively. Zoosteraceae and plant debris were present in 6 out of 20 locations and represented less than 50% . The abundance of other seaweed species correlated positively with sea temperature (figure 3a). Abiotic characteristics of the wrackbed were split into two independent dimensions: wrackbed surface and a summary variable associating wrackbed depth, temperature and salinity.American populations spanned heterogeneous environments, whose variations could be described by two large-scale gradients and heterogeneity in local wrackbed characteristics . Inversion frequency was also marginally associated with north–south climatic variation, or the correlated presence of other seaweeds, which both explained an additional 3% of variance in alternative models . The increase in α frequency with higher proportions of Laminariaceae (versus Fucaceae) was underlined by higher proportions of αα (and to a lesser extent αβ) relatively to ββ. The increase of α frequency with the abundance of plant debris was related to higher proportions of αβ relatively to ββ. The association between α frequency and the north–south cline (climate/other seaweeds) was mostly due to higher proportions of ββ relatively to αβ karyotypes in the south. Local conditions of the wrackbed were again the best predictors of karyotype proportions with the depth/T° of the wrackbed or its composition  predicting 28–34% of variance while climate (or the correlated abundance of other seaweeds) improved alternative model fit by 3–6% . The positive association between Laminariaceae and αα proportions, as well as the positive association between other seaweeds/warmer air temperature and ββ proportions showed parallelism on the Scandinavian cline .More detailed analyses to investigate variations in karyotype composition underlying variations in rearrangement frequency showed that each environmental predictor was differentially associated with karyotype proportions (figure 3(d)a). For both sexes, αα was the largest karyotype and ββ the smallest, with heterokaryotypes being intermediates. Size also varied significantly among populations , with significant differences in 67% (128/190) pairwise comparisons between populations . The mean size of each karyotype and sex observed at a given sampling location were significantly correlated , suggesting that local conditions similarly affect size in the three karyotypes and both sexes.Size was significantly associated with the inversion karyotypes, more strikingly for males than for females a. For boF1,19 = 3.8, p = 0.06; electronic supplementary material, figure S7D), with larger flies observed in populations with higher αα frequency and smaller flies found in populations with higher αβ frequency.Size variation was not significantly associated with environment . Size variation, controlling for karyotype and sex, was marginally associated with variation in karyotype frequencies . Male–female size difference increased at high ββ frequencies (+100% (αα), +70% (αβ)), but decreased at high αα frequencies (−30%(αα); electronic supplementary material, figure S8).The mean size difference between male and female, a potential indicator of mating success , was conin males b. Male–f4.C. frigida revealed the presence of a conserved α/β inversion polymorphism, previously known in European populations [α/β inversion may contribute to local adaptation [Investigating North American natural populations of ulations . Our resaptation . Paralle(a)C. frigida, we identified these characteristics in three adjacent coding regions whose haplotypes were perfectly associated with the Adh allozyme marker of the α/β inversion. This shows that recombination is strongly reduced between the two rearrangements, and provides the first reliable SNP marker for genotyping the α/β inversion in C. frigida. Examining haplotype variations at the three regions further revealed that both haplotypes are found in Europe and America and that haplotype divergence is much stronger than intercontinental variations between populations 5000 km apart. Thus, although further genomic studies are needed to confirm whether this holds true along the inversion, our results point towards a conserved inversion haplotype block throughout the species range. European and American populations also display a similar relationship between inversion karyotype and adult wing size [α/β inversion in C. frigida represents a widespread polymorphism, with similar features conserved throughout the range of the species.As a result of the reduced recombination rate within the inversion, nucleotide sequences within the inversion are generally characterized by high LD and strong divergence between the different rearrangements ,25,61. Iing size  as well ing size ,40, thus(b)C. frigida α/β inversion frequencies covary in parallel with the climatic cline and wrackbed composition between both continents . Although not a hard proof, parallel patterns of genetic or phenotypic variation are considered to be strong indirect evidence for local adaptation shaped by natural selection [Large climatic gradients or heterogeneous habitats impose spatially variable selection which favours the evolution of differently adapted phenotypes, selected for local environmental conditions ,3. Inverelection .C. frigida European and American clines exhibit a slight increase of the β arrangement at southern locations. This frequency shifts may be a direct effect of increased air temperature, if ββ are less cold-tolerant or if their smaller size/shorter development time is an advantage when warmer temperatures speed up wrackbed decomposition. The latter hypothesis has been used to explain the latitudinal size cline in Drosophila, with a smaller size and faster development time being favourable when larval food resources are ephemeral at warmer temperatures [C. frigida, this hypothesis is also supported by the significantly higher frequencies of ββ in warm wrackbeds. The latitudinal cline of frequencies could also be an indirect effect due to the different kinds of seaweeds associated with the southern part of both clines.Along large-scale latitudinal climatic gradients, both eratures . In C. fC. frigida is subjugated to wrackbeds, however, occasional mass migratory flight have also been reported which could maintain regular migration between colonies, up to a few hundreds of kilometres [Admittedly, clinal patterns of variation in allele frequencies could result from isolation by distance without the need to invoke selection. This, however, would unlikely result in similar directionality of the clinal variation across the two continents. Moreover, in Scandinavia, neither population differentiation nor any pattern of isolation-by-distance was observed with SSCP neutral markers over 300 km, suggesting that when the habitat is continuous, population structure is weak . This malometres ,67. GiveC. frigida inversion frequencies were local wrackbed characteristics, such as the depth, temperature and composition of the wrackbed. The influence of the wrackbed composition is of particular interest considering its scale of heterogeneity. The global ratio of Laminariaceae/Fucaceae changes at an order of magnitude along a spatial scale of 100–200 km, a scale at which dispersal is expected [αα karyotypes , a result which is consistent with better survival of αα on Laminariaceae in the laboratory [ββ. The amount of resources available in each substrate may be one of the factors explaining the different karyotype proportions. In fact, in the laboratory, Laminariaceae sustain a greater viability and a larger size than Fucaceae [αα. In the wild populations investigated here, higher αα proportions are also found in populations with large flies , and insect size is generally a good indicator of larval growth conditions [Drosophila, possibly linked to host-plant chemical compounds or microbiome fauna [Moreover, it is noteworthy that, rather than large-scale gradients, the best predictors explaining variation in expected , and at expected . Furtherboratory . Mixed wFucaceae , which snditions . Adaptatme fauna .C. frigida karyotype frequencies at a regional scale also vary with time and at a finer scale (i.e. within a heterogeneous wrackbed or between neighbouring beaches). Wrackbed depth and temperature can be patchy; the deposition of plant debris is linked to nearby river or storm events. Further work is needed to test whether the association between ecological predictors and karyotypes proportions observed at a regional scale hold true at a finer scale. If each of the three karyotypes is differentially favoured in each micro-habitat, they may represent a form of specialization maintained by micro-spatially varying selection balanced by gene flow. Balancing selection between micro-niches has been proposed in Timema cristinae stick-insects, in which an inversion underlies a green morph and a dark morph, respectively, cryptic on leaves or stems of the same host-plant [Interestingly, ecological factors identified as good predictors of st-plant . With a (c)Anopheles mosquitoes [Mimulus guttatus [Apis mellifera bees [C. frigida is more modest as all populations are polymorphic and frequencies remain at an intermediate range. Such pattern is not unusual in the literature, with clinal variation of frequencies in D. melanogaster spanning between 20 and 40% [Our results associating inversion karyotype frequencies with latitudinal gradients and seaweed habitats mirrors the well-described clines of inversion frequencies established along eco-climatic gradients in squitoes , the mosguttatus  and alonera bees . Yet, inera bees . By cont and 40% ; yet it C. frigida, our data and the literature suggest a fitness advantage for heterokaryotypes, which is likely to be the main mechanism underlying the persistence of this widespread polymorphism across space and time [αβ is in excess on both continents and, in the laboratory higher αβ survival is higher than homokaryotypes survival [Philomachus pugnax [Solenopsis invicta, one homokaryotype is lethal because of the accumulation of repetitive elements and deleterious mutations [C. frigida, none of the homokaryotypes is lethal, but the parallel, repeatedly striking deficit of the homokaryotypes suggests the presence of moderately deleterious effects. This is supported by evidence for genic selection in a series of inter- and intra-population crosses [Heterozygote advantage is one the earliest explanation for the persistence of genetic polymorphisms in natural populations . In C. fand time . Indeed,survival . In the s pugnax . Reduceds pugnax . In fireutations . In the  crosses , and remαβ correlated with smaller sizes for all karyotypes/sexes, the latter relationship probably being mediated by density. In the laboratory, smaller size is linked to higher density of larva and density increases overdominance, with a 2.6-fold viability difference between heterokaryotypes and both homokaryotypes at high density but only 1.2-fold at low density [Variability in the excess of heterokaryotypes (0–60%) indicates that heterozygote advantage may be modulated by local biotic and abiotic conditions. For instance, higher proportions of  density . Why hetC. frigida progeny suggest an excess of disassortative mating relatively to inversion karyotype, which may contribute to heterokaryote excess [Ovis aries), one allele confers higher reproductive success while the alternative allele increases survival, resulting in increased overall fitness in the heterozygote [C. frigida [. Because larger adults have a higher fertility, αα (and αβ) have a sexual advantage over ββ, particularly in males [ββ (and αβ) an egg-to-adult viability advantage over αα [ββ proportions, our results show that αα (and αβ) males are not only rarer, facing less adult competition from large-size males, but they are also larger, possibly because of lower larval competition from similar karyotypes. Thus, α rearrangement could benefit from the ‘advantage of the rare', a form of frequency-dependent selection, which is frequently involved in protecting polymorphisms [αα proportions, the observed lower male–female size difference and the numerous same-size competitors are expected to reduce αα males sexual advantage. As such, previous studies on European populations along with our results raise the hypothesis that the three karyotypes found in C. frigida may represent alternative life-history strategies with different relative investment in the trade-off between growth and reproduction, and for which balanced polymorphism could be maintained by a form of negative frequency-dependence selection [Heterokaryotype advantage can also result from reproduction . Notablye excess . Polymorrozygote . This me frigida . Becausein males . Yet, la over αα ,59. Suchorphisms . By contelection , somethi5.α/β inversion polymorphism is conserved between Europe and North America. Significant associations between karyotype frequencies and environmental variables on both continents provide strong indirect evidence for the role of the α/β inversion in local adaptation. As such, C. frigida represents an excellent system to elucidate the multifarious evolutionary mechanisms involved in the maintenance of structural variants. Our data indicate that the three inversion karyotypes are differently favoured by ecological conditions, and raises the hypothesis that they may represent three alternative life-history strategies, particularly in males. This joins theoretical predictions and accumulating evidence that within-species diversification and specialization can be made possible by the genomic architecture of the inversion itself [C. frigida will focus on population genomics to investigate the contribution of drift and demographic factors, to assess the age of the inversion and to identify which loci are the targets of selection and how linkage increases (or decreases) fitness, depending on the selective process involved. Our analysis combined with the abundant life-history literature on C. frigida suggests that several balancing selection mechanisms  interact to maintain this polymorphism. Further modelling could help to disentangle the relative contributions of these processes in shaping geographical patterns of inversion frequencies and spatial associations. Interestingly, several recent studies also highlighted the combined effects of several balancing selection mechanisms on inversion polymorphism [Our study shows that the n itself . Future morphism ,28. This