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SPX gene and CRISPR/Cas9 knockoutUsing the SPX domain as a query to search in the P. tricornutum genome, we found a total of six genes that harbor an SPX domain (Supplementary Table 1), including Vpt1 and Vtc4 that were recently described by Dell’Aquila et al.21. Pfam analysis of the six identified sequences in P. tricornutum resulted in the identification of the SPX domain in these proteins, which shared several conserved sites with land plants’ SPX domains (Fig. 1a, b). Phylogenetic analyses further verified the high similarity of these sequences with other known SPX domain proteins (Supplementary Fig. 1). One of these genes possesses a SPX domain as the sole functional domain (named SPX, and its encoding gene named SPX gene, from here on) while the other five (including Vpt1 and Vtc4) contain at least one other domain. From our recently published transcriptome dataset27, we found that SPX, Vpt1, and Vtc4 genes were differentially expressed (log2 Fold Change > 1 and adjusted p value  More

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Our framework to predict unknown associations between known viruses and potential mammalian hosts or susceptible species comprised three distinct perspectives: viral, mammalian and network. Each perspective produced predictions from a unique vantage point (that of each virus, each mammal, and the network connecting them respectively). Subsequently, their results were consolidated via majority voting. This approach suggested that 20,832 (median, 90% CI = [2,736, 97,062], hereafter values in square brackets represent 90% CI) unknown associations potentially exist between our mammals and their known viruses, (18,920 [2,440, 91,517] in wild or semi-domesticated mammals). Number of unknown associations predicted by each perspective individually were as follows: mammalian only = 41,537 [4,275, 23,8971], viral only = 21,352 [2,536, 95,630], and network only = 76,081 [27,738, 20,5814]. Our results indicated a ~4.29-fold increase ([~1.43, ~16.33]) in virus-mammal associations (~4.89 [~1.5, ~19.81] in wild and semi-domesticated mammals).Additionally, we trained an independent pipeline including only the 3534 supported by evidence extracted from meta-data accompanying nucleotide sequences, as indexed in EID2 (55.82% of all associations – see Methods section and Supplementary Results 8). Our sequence-evidence pipeline indicated that 15,721 (median, 90% CI = [1,603, 88,553]) unknown associations could potentially exist (13,930 [1,298, 83,043] in wild or semi-domesticated mammals).In the following subsections we first illustrate the mechanism of our framework via an example, then further explore the predictive power of our approach for viruses and mammals.ExampleOur multi-perspective framework generates predictions for each known or unknown virus-mammal association (2,722,656 possible associations between 1,896 viruses and 1,436 terrestrial mammals). We highlight this functionality using two examples (Fig. 1). West Nile virus (WNV) a flavivirus with wide host range, and the bat Rousettus leschenaultia (order: Chiroptera). We first consider each of our perspectives separately, and then showcase how these perspectives are consolidated to produce final predictions.Fig. 1: Example showcasing final and intermediate predictions of West Nile Virus (WNV), and Rousettus leschenaultii.Panel A Top 60 predicted mammalian species susceptible to WNV. Mammals were ordered by mean probability of predictions derived from mammalian (all models), viral (WNV models) and network perspectives, and top 60 were selected. Circles represent the following information in order: 1) whether the association is known (documented in our sources) or not (potential or undocumented). Hosts are omitted for known associations. 2) Mean probability of the three perspectives (per association). 3) Median mammalian perspective probabilities of predicted associations. These probabilities are obtained from 3000 models (50 replicate models for each mammal), trained with viral features – SMOTE class balancing. 4) Median viral perspective probabilities of predicted associations (50 WNV replicate models trained with mammalian features – SMOTE class balancing). 5) Median network perspective probabilities of predicted associations (100 replicate models, balanced under-sampling). 6) Taxonomic order of predicted susceptible species. Orders are shortened as follows: Artiodactyla (Art), Carnivora (Crn), Chiroptera (Chp), primates (Prm), Rodentia (Rod), and Others (Oth). Panel B Top 50 predicted viruses of R. leschenaultii. Viruses were ordered by mean probability of predictions derived from mammalian (R. leschenaultii models), viral (all models) and network perspectives. Circles as per Panel A. Baltimore represents Baltimore classification. Panel C Median probability of predicted WNV-mammal associations in each of the three perspectives per mammalian order. Points represent susceptible species predicted by voting (at least two of the three perspectives – n = 137). Median ensemble probability is computed in each perspective (50 replicate models for each virus/mammal, 100 replicate network models). Predictions derived from each perspective at 0.5 probability cut-off. Supplementary Data 1 presents full WNV results. Panel D Median probability of virus-R. leschenaultii associations in the three perspectives per Baltimore group. Points represent susceptible species predicted by voting (at least two of the three perspectives – n = 64), predictions are derived as per panel C. Supplementary Data 2 lists full results for R. leschenaultii. Supplementary Fig. 7 illustrate the results when research effort into viruses and mammals is included in mammalian and viral perspectives, respectively.Full size image1) The mammalian perspective: our mammalian perspective models, trained with features expressing viral traits (Table 1), suggested a median of 90 [17, 410] unknown associations between WNV and terrestrial mammals could form when predicting virus-mammal associations based on viral features alone – a ~2.61-fold increase [~1.3, ~8.32]. Similarly, our results indicated that 64 [4, 331] new associations could form between our selected mammal (R. leschenaultia) and our viruses – a ~4.37-fold increase [~1.21, ~18.42] (Supplementary Results 4).Table 1 Viral traits & features used to build our mammalian models.Full size table(2) The viral perspective: our viral models, trained with features expressing mammalian traits (Table 2), indicated a median of 48 [0, 214] new hosts of WNV (~1.86- fold increase [~1, 4.82]). Results for our example mammal (R. leschenaultia) suggested 18 [3, 76], existing viruses could be found in this host (~1.95-fold increase [~1.16, ~5.00]) – Supplementary Results 5).Table 2 mammalian traits & features used to build our viral models.Full size table(3) The network perspective: Our network models indicated a median of 721 [448, 1,317] (~13.88 [9, 24.52] fold increase) unknown associations between WNV and terrestrial mammals, and that 246 [91, 336] existing viruses could be found in our selected host (R. leschenaultia), equivalent to a ~13.95 [~5.79, ~18.68] fold increase (Supplementary Results 6).Considering that each of the above perspectives approached the problem of predicting virus-mammal associations from a different angle, the agreement between these perspectives varied. In the case of WNV: mammalian and viral perspectives achieved 92.3% agreement [72.6%–98.5%]; mammals and network perspectives had 55.3% agreement [33.4%–69.5%]; and viruses and network had 52.9% agreement [19.8%–68.7%]. In the case of R. leschenaultia these numbers were as follows: 96.15% [82.44%, 99.58%], 87.24% [76.37%, 95.04%], and 87.61% [75.90%, 95.25%], respectively. The agreements between our perspectives across the 2,722,656 possible associations were as follows: 98.04% [90.36%, 99.73%] between mammalian and viral perspectives, 96.71% [88.62%, 98.92%] between mammalian and network perspectives, and 97.11% [91.57%, 98.95%] between viral and network perspectives.After voting, our framework suggested that a median of 117 [15, 509] new or undetected associations could be missing between WNV and terrestrial mammals (~3.45-fold increase [~1.3, ~12.2]). Similarly, our results indicated that R. leschenaultia could be susceptible to an additional 45 [5, 235] viruses that were not captured in our input (~1.37-fold increase [~1.26, ~13.37]). Figure 1 illustrates top predicted and detected associations for WNV (Supplementary Data 1) and R. leschenaultia (Supplementary Data 2). Supplementary Results 1 illustrate results with research effort into viruses, and mammals included as a predictor in our mammalian and viral perspective models, respectively. Predictions with and without research effort incorporated into models trained in these perspectives broadly agreed.Relative importance of viral featuresOur multi-perspective approach trained a suite of models for each mammalian species with two or more known viruses (n = 699, response variable = 1 if the virus is known to associate with the focal mammalian species, 0 otherwise). This enabled us to assess the relative importance (influence) of viral traits (Table 1) to each of our mammalian models. This in turn showcased variations of how these viral traits contribute to the models at the level of individual species (e.g. humans), and at an aggregated level (e.g. by order or domestication status). The results, highlighted in Fig. 2A, indicate that mean phylogenetic (median = 95.4% [75.6%, 100%]) and mean ecological (90.90% [43.50%, 100%]) distances between potential and known hosts of each virus were the top predictors of associations between the focal host and each of the input viruses. Maximum phylogenetic breadth was also important (74.7 0%, [16.60%, 100%]).Fig. 2: Results (viruses).Panel A Variable importance (relative contribution) of viral traits to mammalian perspective models. Variable importance is calculated for each constituent ensemble (n = 699) of our mammalian perspective (median of a suite of 50 replicate models, trained with viral features, with SMOTE sampling), and then aggregated (mean) per each reported group (columns). Panel B – Number of known and new mammalian species associated with each virus. Rabies lyssavirus was excluded from panel B to allow for better visualisation. Top 40 (by number of new hosts) are labelled. Species in bold have over 150 predicted hosts (Supplementary Data 3 lists details of these viruses including CI). Panel C Predicted number of viruses per species of wild and semi-domesticated mammals (group by mammalian order). Following orders (clockwise) are presented: Artiodactyla, Carnivora, Chiroptera, Perissodactyla, Primates, and Rodentia. Source of the silhouette graphics is PhyloPic.org. (Supplementary Data 4 lists aggregated results per mammalian order). Circles represent each mammalian species (with predicted viruses > 0), coloured by number of known viruses previously not associated with this species. Boxplots indicate median (centre), the 25th and 75th percentiles (bounds of box) and inter quantile range (whiskers) and are aggregated at the order level. Large red circles with error bars (90% CI) illustrate the median number of known viruses per species in each order. Number of species presented (n) is as follows: All = 1293 (Artiodactyla = 104, Carnivora = 177, Chiroptera = 548, Perissodactyla = 11, Primates = 171, and Rodentia = 282); Group I = 666 (94, 109, 156, 10, 160, 137); Group II = 371 (32, 120, 111, 1, 54, 53); Group III = 410 (87,62,123,9,51,78); Group IV = 739 (98, 102, 221, 9, 148, 161); Group V = 1129 (87, 173, 528, 8, 107, 226); Group VI = 358 (55, 64, 30, 6, 139, 64); and Group VII = 110 (3,2,53,1,43,8). Supplementary Fig. 8 presents results derived with research effort into mammalian hosts and viruses included in the constituent models trained in the viral and mammalian perspectives, respectively.Full size imageMammalian host rangeOur results suggested that the average mammalian host range of our viruses is 14.33 [4.78, 54.53] (average fold increase of ~3.18 [~1.23, ~9.86] in number of hosts detected per virus). Overall, RNA viruses had the average host range of 21.65 [7.01, 82.96] hosts (~4.00- fold increase [~1.34, ~14.15]). DNA viruses, on the other hand, had 7.85 [2.81, 29.47] hosts on average (~2.43 [~1.14, ~6.89] fold increase). Table 3 lists the results of our framework at Baltimore group level and selected family and transmission routes of our viruses. Figure 2 illustrates predicted mammalian host range of our viruses (Fig. 2B, Supplementary Data 3), and the increase in predicted number of viruses per species in species-rich mammalian orders of interest (Fig. 2C, Supplementary Data 4).Table 3 Predicted range of susceptible mammalian species of viruses per Baltimore group, family (top 15 families, ranked by fold increase) and transmission route.Full size tableRelative importance of mammalian featuresWe trained a suite of models for each virus species with two or more known mammalian hosts (n = 556, response variable = 1 if the mammal is known to associate with the focal virus species, 0 otherwise). This allowed us to calculate relative importance of mammalian traits (Table 2) to our viral models. We were also able to capture variations in how these features contribute to our viral models at various levels (e.g. Baltimore classification, or transmission route) as highlighted in Fig. 3A. Our results indicated that distances to known hosts of viruses were the top predictor of associations between the focal virus and our terrestrial mammals. The breakdown was: 1) mean phylogenetic distance – all viruses = 98.75% [93.01%, 100%], DNA = 99.48% [96.03%, 100%], RNA = [91.93%, 100%]; 2) mean ecological distance all viruses = 94.39% [71.86%, 100%], DNA = 96.36% [80.99%, 100%], RNA = [69.48%, 100%]. In addition, life-history traits significantly improved our models, in particular: longevity (all viruses = 60.9% [12.12%, 98.88%], DNA = 68.03% [11.22%, 99.69%], RNA = [13.55%, 96.37%]); body mass (all viruses = 62.92% [5.4%, 97.65%], DNA = 72.75% [18.49%, 100%], RNA = 57.45% [4.32%, 95.5%]); and reproductive traits (all viruses = 53.37% [5.67%, 95.99%]%, DNA = 59.46% [8.27%, 99.32%], RNA = 50.17% [4.85%, 92.17%]).Fig. 3: Results (Mammals).Panel A Variable importance (relative contribution) of mammalian traits to viral perspective models. Variable importance is calculated for each constituent model (n = 556) of our viral perspective (trained with mammalian features), and then aggregated (median) per each reported group (columns). Panel B Number of known and new viruses associated with each mammal. Labelled mammals are as follows: top 4 (by number of new viruses) for each of Artiodactyla, Carnivora, Chiroptera, Primates, Rodentia, and other orders. Species in bold have 100 or more predicted viruses (Supplementary Data 5). Panel C Top 18 genera (by number of predicted wild or semi-domesticated mammalian host species) in selected orders (Other indicated results for all orders not included in the first five circles). Each order figure comprises the following circles (from outside to inside): 1) Number of hosts predicted to have an association with viruses within the viral genus. 2) Number of hosts detected to have association. 3) Number of hosts predicted to harbour viral zoonoses (i.e. known or predicted to share at least one virus species with humans). 4) Number of hosts predicted to share viruses with domesticated mammals of economic significance (domesticated mammals in orders: Artiodactyla, Carnivora, Lagomorpha and Perissodactyla). 5) Baltimore classification of the selected genera (Supplementary Data 6). Supplementary Fig. 9 presents results derived with research effort into mammalian hosts and viruses included in the constituent models trained in the viral and mammalian perspectives, respectively.Full size imageWild and semi-domesticated susceptible mammalian hosts of virusesour framework indicated ~4.28 -fold increase [~1.2, ~14.64] of the number of virus species in wild and or semi-domesticated mammalian hosts (16.86 [4.95, 68.5] viruses on average per mammalian species). These results indicated an average of 13.45 [1.73, 65.04] unobserved virus species for each wild or semi-domesticated mammalian host (known viruses that are yet to be associated with these mammals). Our framework highlighted differences in the number of viruses predicted per order (Table 4). Figure 3 illustrates the predicted number of viruses in wild or semi-domesticated mammal by mammalian host range (Fig. 3B, Supplementary Data 5), and the top 18 virus genera (per number of host-virus associations) in selected orders (Fig. 3C, Supplementary Data 6). Supplementary Results 1 lists the results with the inclusion of research effort into mammalian species in our viral perspective models.Table 4 Predicted number of viruses per top 15 orders by fold increase in number of viruses predicted in wild or semi-domesticated mammalian hosts (per species).Full size tableNetwork perspective – Potential motifs
We quantified the topology of the network linking virus and mammal species by means of counts of potential motifs21. Figure 4 illustrates how potential motifs are captured in our network. Briefly, for each virus-mammal association for which we want to make predictions (n = 2,722,656, of which 6,331 are supported by our evidence, see methods section), we “force insert” this focal association into our network (Fig. 4A, B) and enumerate all instances of 3 (n = 2), 4 (n = 6), and 5-node (n = 20) potential motifs in which this association might feature if it actually existed21 (Fig. 4C visualises these different motifs). Following this process, a features-set is generated comprising the counts potential motifs for all included associations. Figure 4D illustrates the count of motifs (logged) grouped by mammalian order and virus Baltimore classification.Fig. 4: The network perspective – potential motifs (subgraphs) in our virus-host bipartite network.A The concept of potential motif. The association TBEV-P. leo is a forced insertion into the network prior to calculating motifs for the association. B Motifs space: networks represent 2 steps and 3 steps ego networks (union) of host (here P. leo) and virus (TBEV). 1, 2 and 3 step ego networks comprise the counting space for TBEV-P. leo potential motifs. Dark grey nodes represent viruses, light grey nodes represent hosts. Size of nodes is adjusted to represent overall number of hosts or viruses with known associations to the node. Red edges represent nodes reachable from the mammal (P. leo) in 1 or 2 steps (links). Blue edges represent nodes reachable from the virus (TBEV) with 1 or 2 steps (links). Humans and rabies virus were excluded from these networks. C 3, 4 and 5-node potential motifs in our virus-host bipartite network. Circles represent viruses and squares represent mammals. Red circles represent the focal virus (v), and blue squares represent the focal mammal (m) of the association v-m for which the motifs are being counted (dashed yellow line). This association has two states: either already known (documented in EID2), or unknown. Grey lines illustrate existing associations in our network. D Motifs counts. Heatmap illustrating distribution of motif-features (counts of potential motifs per each focal association) in our bipartite network, grouped by mammalian order and Baltimore classification. The counts are logged to allow for better visualisation. E Variable importance (relative contribution) of motif-features (variables) to our network perspective models (SVM-RW). Motifs (subgraphs) are coloured by the number of nodes (K = 3, 4, 5). Boxplots indicate median (centre), the 25th and 75th percentiles (bounds of box) and inter quantile range (whiskers). Points represent variable importance in individual runs (n = 100). Research effort into both viruses and mammals is included as independent variables in our network models (coloured in yellow).Full size imageRelative importance of network (motif) featuresFigure 4E illustrates that M4.1 was the most important feature in our network models: median = 100% [90.19%, 100%]. Followed by: M5.1 = 97.84% [89.19%, 99.93%], M5.7 = 98.8 97.22% [87.7%, 98.77%] and M4.6 = 96.75% [86.13%, 100%]. Research effort of viruses and mammals had relative importance = 90.26% [82.94%, 95.36%], 88.42% [78.38%, 94.87%] respectively. Overall, 5-node motif-features had median relative influence = 75.06% [1.21%, 98.14%]; whereas 3 and 4-node motif-features had relative influence = 71.69% [55.76%, 85.34%], and 61.06% [27.14%, 100%], respectively. Supplementary Fig. 29 illustrate the partial dependence of network perspective models on each of our network features.ValidationWe validated our framework in three ways: 1) against a held-out test set; 2) by systematically removing selected known viral-mammalian associations and attempting to predict them; and 3) against external data source, comprising viral-mammalian associations extracted using an exhaustive literature search targeting wild mammals and their viruses4,30.Our held-out test set comprised 15% of all data (randomly selected, n = 407,265; 954 known virus-mammal associations, see methods below). We removed this set from our network, computed network features (motifs), and trained constituent models in each perspective with the remainder data. We then estimated our framework performance metrics against the held-out test set. Our framework achieved overall AUC = 0.938 [0.862–0.959], F1-Score = 0.284 [0.464–0.124], and TSS = 0.876 [0.724–0.918], when trained without including research effort in its mammalian and viral perspectives. When research effort was included in these perspectives, performance metrics were as follows: AUC = 0.920 [0.823, 0.944], F1-Score = 0.272 [0.526, 0.093], and TSS = 0.840 [0.646, 0.888].The performance of our voting approach was better than any individual perspective, or combination of perspectives (Supplementary Tables 8–11). The most significant improvement was in F1-score, where individual perspectives scores were as follows: network = 0.104 [0.210–0.051], mammalian = 0.115 [0.009–0.064] (0.131 [0.284–0.035] with research effort), and viral = 0.181 [0.374–0.074] (0.196 [0.373–0.067]).Additionally, we conducted a systematic test to predict removed virus-mammal associations. In this test, we systematically removed one known virus-mammal association at a time from our framework, recalculated all inputs (including from network) and attempted to predict these removed associations. Our framework succeeded in predicting 90% of removed associations (90.70% for associations removed for viruses, 89.92% for associations removed from mammals, Supplementary Results 3).Finally, our framework predicted 84.02% [77.69%, 89.60%] of the externally obtained viral-mammalian associations (with detection quality  > 0) where both host and virus were included in our pipeline, and 77.82% [68.46%, 86.51%] (any detection quality). When including research effort in our mammalian and viral perspectives, these results were: 84.47% [78.15%, 89.60%], and 78.41% [68.83%, 86.37%], respectively. More

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Study siteThe experiment was conducted at Madingley Wood, Cambridgeshire, UK (0◦3.2´E, 52◦12.9´N) during summer 2018. Madingley Wood is an established field site with an ongoing long-term study of the blue tit and great tit populations. During the autumn and winter birds are caught from feeding stations using mist nets and they are fitted with British Trust of Ornithology (BTO) ID rings. Since 2012, blue tits and great tits have been fitted with RFID tags (BTO Special Methods permit to HMR), which enables collecting data remotely about their foraging behavior and social relationships. The study site has 90 nest boxes that are monitored annually during the breeding season. In 2018 chicks (n = 325) fledged successfully from 45 nest boxes (blue tits = 21, great tits = 24) and they were all ringed and fitted with RFID tags when they were approximately 10 days old. Because new juvenile flocks were arriving at our study site throughout the summer, we also conducted several mist-netting and ringing sessions in July and August to maintain a high proportion of blue tits and great tits ringed and RFID tagged for the experiments (on average 89%, see below). The study protocol was approved by the Animal Users Committee at the Department of Zoology, University of Cambridge.Food itemsWe investigated birds’ foraging choices by offering them colored almond flakes at bird feeders that were distributed throughout the wood. Before beginning the experiments, we allowed the birds to become familiar with the food items by providing plain ‘control’ almonds (plain and not colored) in paired feeders (1.5 m apart) at three locations (approximately 170 m from each other). The feeders were surrounded with metal cages to exclude larger birds, and we placed plastic buckets under the feeders to collect any spilled almonds and minimize birds’ opportunities to forage from the ground. We introduced the feeders at the beginning of June when the nestlings had fledged and were beginning to forage independently, and continued to provide these plain almonds in between our learning experiments (Fig. 3c).In the learning experiments, almond flakes were dyed with non-toxic food dye (Classikool Concentrated Droplet Food Colouring). We used three different color pairs: green (Leaf Green) and red (Bright Red), purple (Lavender Purple) and blue (Royal Blue), and orange (Satsuma Orange) and yellow (Dandelion Yellow). Almond flakes were dyed by soaking them for approximately 20 min in a solution of 900 ml of water and 30 ml of food dye and then left to air dry for 48 h. In the avoidance learning experiments, we made half of the almond flakes unpalatable by soaking them for one hour in 67% solution of chloroquine phosphate, following previously established methods from avoidance learning studies with birds in captivity14,23,24,25. The food dye was added to the solution during the last 20 min.Red and green are common colors used by aposematic, or cryptic prey, respectively9. Therefore, we investigated whether blue tits and great tits had initial color biases towards these colors before starting the main experiment. Because we did not want the birds in our study population to have any experience of the colors before the main experiment, this pilot study was conducted in Newbury, which is 130 km from our main study site. Birds were simultaneously presented with two feeders containing red and green almonds (both palatable) for 30 min and the number of almonds of each color taken by blue tits or great tits was recorded using binoculars. The position of the feeders was switched after 15 min to control for any preferences for feeder location, and the test was repeated on 9 different days. We did not find any evidence that birds had initial color preferences (t-test: t = 0, df = 15.69, p = 1). For the other two learning experiments, we chose color pairs that were available as a food dye and as different from red and green in the visible spectrum as possible to avoid generalization across experiments. These color pairs (blue/purple and yellow/orange) had similar contrast ratios as green and red, based on their RGB values (measured from photographs, see Supplementary Information). Although avian and human vision is different, the discriminability of colors is likely to be similar51, and rapid avoidance learning in each experiment shows that all colors were easily distinguishable. This was the main requirement for testing social information use, and subtle differences in color pair discriminability should only introduce noise to our data but not influence our conclusions.Learning experiments with colored almondsWe conducted three avoidance learning experiments with different color pairs throughout the summer: red/green, blue/purple, and yellow/orange (unpalatable/palatable). In addition, we conducted a reversal-learning experiment with the blue/purple color pair by making both colors palatable after birds had acquired avoidance to blue almonds. Each experiment followed a similar protocol, in which birds were presented with colored almonds at the same three feeding stations where they were previously offered plain almonds. Each feeding station had two feeders, where one contained the palatable color and the other contained the unpalatable color (except in the reversal learning test when both colors were palatable). We switched the side of the feeders every day to make sure that birds learned to associate palatability with an almond color and not a feeder position. The feeders were filled at least once a day (or more often if necessary) to make sure that birds always had access to both colors. We continued each avoidance learning experiment until >90% of all recorded visits were to the feeder with palatable almonds, indicating that most birds in the population had learned to discriminate the colors. This took 7 days in the red/green experiment and 8 days in the other two color pairs (blue/purple and yellow/orange). The reversal learning experiment was finished after 9 days when 50% of the visits were to the previously unpalatable color (blue), indicating that most birds had reversed their learned avoidance towards it.Recording visits to feedersWe monitored visits to all feeders using RFID antennas and data loggers (Francis Scientific Instruments, Ltd) that scanned birds’ unique RFID tag codes when they landed on a perch attached to the feeder. During the learning experiments, each day we also recorded videos from all three feeding stations (using Go Pro Hero Action Camera and Canon Legria HF R66 Camcorder). From the videos, we monitored the proportion of blue tits and great tits that did not have RFID tags and were therefore not recorded when visiting the feeders. We calculated the estimated RFID tag coverage for each day of the experiments by watching at least 100 visits to the feeders from the videos (divided equally among the three feeding stations) and recording whether blue tits and great tits had an RFID tag or not. We realized that the number of untagged individuals was very high (approximately 50% of all visiting birds) when we started the experiment with the first color pair (red/green; see Supplementary Fig. 3). We, therefore, stopped the experiment after two days and caught birds from the feeding stations with mist nets to fit RFID tags to new individuals. To maintain a high number of individuals RFID tagged for the other color pairs, we conducted a mist netting session a day before starting each experiment, as well as 4–5 days after it. We always switched the feeders back to containing plain almonds during mist-netting sessions to ensure that this would not interfere with the learning experiments. Apart from the first two days of the red/green experiment, the RFID tag coverage was on average 89% throughout the experiments (varying between 80 and 95%, Supplementary Fig. 3).Birds were recorded every time that they visited the feeders, i.e., landed on the RFID antenna. However, it is possible that birds did not take the almond during every visit. To get an estimate of how often birds landed on the antenna without taking the almond, and whether this differed between palatable and unpalatable colors, we analyzed the visits to the feeders from the video recordings. We watched videos from the five first days of each experiment (i.e., different color pairs) and analyzed 60 visits to each color (divided approximately equally among the three feeding stations). We recorded whether the feeding event happened (birds ate the almond at the feeder or flew away with it) or whether birds left the feeder without sampling the almond. Because the number of visits to the unpalatable feeder was low during the last days of the avoidance learning experiments, we decided not to analyze avoidance learning videos after day five (but recorded visits from all days of the reversal learning experiment). We found that in avoidance learning experiments birds started to ‘reject’ unpalatable almonds after two days, i.e., they sometimes landed on the feeder but flew away without taking the almond (see Supplementary Fig. 4a). This change was not observed at palatable feeders where birds continued to consume almonds at a similar rate as at the beginning of the experiment (Supplementary Fig. 4a). In reversal learning, the proportion of visits that did not include a feeding event did not differ between purple and blue almonds: birds showed similar hesitation towards both colors at the beginning of the experiment, but this wariness decreased when the experiment progressed, with birds taking the almond during most of their visits (Supplementary Fig. 4b).Statistical analyses and model validationForaging choices in learning experimentsWe first analyzed how birds’ foraging choices changed during the learning experiments using generalized linear mixed-effects models with a binomial error distribution. The number of times an individual visited each feeder on each day of the experiment was used as a bounded response variable, and this was explained by species (blue tit/great tit), individuals’ age (juvenile/adult), and day of the experiment (continuous variable), as well as bird identity as a random effect. When analyzing avoidance learning, initial exploration of data suggested that results were similar across all three experiments, so we combined the experiments in the same model. To investigate whether learning curves differed between the species or age groups, the day of the experiment was included as a second-order polynomial term, and we started model selections with models that included a three-way interaction between species, age, and day2. Best-fitting models were selected based on Akaike’s information criterion (see Supplementary Tables 1 and 2).Social networkTo investigate if birds used social information in their foraging choices, we first constructed a social network of the bird population based on their visits to feeders outside of the learning experiments, i.e., when birds were presented with plain almonds (in total 92 days, see Supplementary Information for the robustness of analysis to exclusion of network data before or after the experiment). We used only these data as individuals were likely to vary in their hesitation to visit novel colored almonds. We used a Gaussian mixture model to detect the clusters of visits (‘gathering events’) at the feeders52 and then calculated association strengths between individuals based on how often they were observed in the same group (gambit of the group approach). These associations (network edges) were calculated using the simple ratio index, SRI35.$$frac{x}{x+{y}_{{mathrm{A}}}+{y}_{{mathrm{B}}}+{y}_{{{mathrm{AB}},}}}$$
(2)
where x is the number of samples where individuals A and B co-occurred in the same group, yA is the number of samples where only individual A was seen, yB is the number of samples where only individual B was seen, and yAB is the number of samples where both A and B were observed in the same sample but not together. Network associations, therefore, estimated the probability that two individuals were in the same group at a given time, with the values scaled between 0 (never observed in the same group) and 1 (always observed in the same group).Social information use during avoidance learning: model descriptionIf social avoidance learning was occurring, then the more birds observed negative responses of others feeding on the unpalatable feeder, the less likely they would be to choose the unpalatable feeder themselves. Thus, we expected the probability of bird j choosing the unpalatable option at time t to decrease with ({R}_{-,j}left(tright)) (the real number of negative feeding events observed by j prior to time t). Likewise, if appetitive social learning was occurring, then the more birds observed positive responses of others feeding on the palatable feeder, the more likely they would be to choose the palatable feeder themselves (rather than the unpalatable feeder). So, we also expected the probability of j choosing the unpalatable option at time t to decrease as ({R}_{+,j}left(tright),)(the real number of positive events observed by j prior to time t) increased.However, we could not test for an effect of ({R}_{-,j}left(tright)) and ({R}_{+,j}left(tright)) directly, since birds often ate the almond away from the feeder, and therefore the real number of observed feeding events could not be measured. Instead, we aimed to test for a pattern following the social network that is consistent with these social learning processes. We reasoned that the probability that one individual i, observes a specific feeding event by another individual j, was proportional to the network connection between them, aij (probability they are in the same feeding group at a given time). Therefore, in each avoidance learning experiment (i.e., different color pair), we calculated the expected number of negative feeding events observed, prior to each choice (occurring at time t) as$${O}_{-,i}left(tright)={sum }_{j}{N}_{-,j}left(tright){a}_{{ij}},$$
(3)
where ({N}_{-,j}left(tright)) was the number of times j had visited unpalatable almonds prior to time t (i ≠ j), and summation is across all birds in the network, and likewise for the expected number of positive feeding events:$${O}_{+,i}left(tright)={sum }_{j}{N}_{+,j}left(tright){a}_{{ij}},$$
(4)
where ({N}_{+,j}left(tright)) was the number of times j had visited palatable almonds prior to time t (i ≠ j).We analyzed whether the expected observations of positive and/or negative feeding events of others influenced the foraging choices in the avoidance learning experiments using generalized linear mixed-effects models with a binomial error distribution. We used each choice (i.e., visit a feeder) as a binary response variable (1 = unpalatable chosen, 0 = palatable chosen), with the probability that unpalatable feeder is chosen on feeding event E given by ({p}_{E}={p}_{-,{i}(E)}left({t}_{E}right)), where i(E) is the individual that fed during event E and ({t}_{E}) is the time at which event E occurred. We then modeled the probability of i choosing the unpalatable option at time t as:$${p}_{-,i}left(tright)={rm{logit}}left(alpha +{beta }_{{rm{asoc}}+}{N}_{+,i}left(tright)+{beta }_{{rm{asoc}}-}{N}_{-,i}left(tright)+{beta }_{{rm{soc}}+}{O}_{+,i}left(tright)+{beta }_{s{rm{oc}}-}{O}_{-,i}left(tright)+{{{{rm{B}}}}}_{i}right),$$
(5)
where ({N}_{+,i}left(tright)) is the number of times a choosing individual had visited the palatable feeder (positive personal information), ({N}_{-,i}left(tright)) is the number of times a choosing individual had visited the unpalatable feeder (negative personal information), ({O}_{+,i}left(tright)) is the expected number of observed positive (positive social information) and ({O}_{-,i}left(tright)) observed negative feeding events (negative social information). Bird identity was included as a random effect, ({{rm{{B}}}}_{i}) (age and species were later added as variables, see below). Parameters ({beta }_{{rm{asoc}}+}) and ({beta }_{{rm{asoc}}-}) are the effects of asocial learning about the palatable and unpalatable foods, ({beta }_{{rm{soc}}+}) is the effect of social learning about the palatable food, and ({beta }_{{rm{soc}}-})is the effect of social avoidance learning about the unpalatable food. Estimation of these parameters, with associated Wald tests and confidence intervals, allowed us to make inferences about which effects were operating and the size of these effects. To aid model fitting we standardized all predictor variables and then back-transformed the effects to the original scale (see Supplementary Tables 3–5 for the model outputs). To assess the importance of asocial and social effects, we also ran separate models that excluded either asocial or social parameters and compared them to the initial model in Eq. (5) using Akaike’s information criterion (see Supplementary Table 6). However, in most cases, this reduced model fit significantly, and we, therefore, kept all parameters in the final models.Our approach took ({O}_{-,{i}}left(tright)) as a measure of ({R}_{-,j}left(tright)), and ({O}_{+,{i}}left(tright)) as a measure of ({R}_{+,j}left(tright))-, which we termed the ‘expected’ number of observations of each type. Strictly speaking, ({O}_{-,{i}}left(tright)) and ({O}_{+,{i}}left(tright)) were upper limits on the expected number of observations, assuming that birds observed all feeding events in the groups in which they were present, whereas only an unknown proportion of such events (({p}_{o})) was observed. Therefore, the real expected number of negative/positive observations would be (Eleft({R}_{-,j}left(tright)right)={p}_{o}{O}_{-,{i}}left(tright)) and (Eleft({R}_{+,j}left(tright)right)={p}_{o}{O}_{+,{i}}left(tright)) respectively. Thus, the coefficient, ({beta }_{{rm{soc}}-}), for the effect of ({O}_{-,{i}}left(tright)) could be interpreted as ({beta }_{s{rm{oc}}-}={p}_{o}acute{{beta }_{{rm{soc}}-}}) where (acute{{beta }_{s{rm{oc}}-}}) is the effect per observation. Note that since ({p}_{o}le 1), and(,{beta }_{{rm{soc}}-}=acute{{beta }_{s{rm{oc}}-}}{p}_{o}), ({beta }_{s{rm{oc}}-}) is more likely to underestimate than overestimate the effect per observation of a negative feeding event. An analogous argument applies to the coefficient, ({beta }_{{rm{soc}}+}), for the effect of ({O}_{+,{i}}left(tright)).Social information use during avoidance learning: extension to test for species effectsAfter fitting the initial model shown in Eq. (5), we further broke down the model to test whether individuals were more likely to learn socially by observing conspecifics than heterospecifics. This was done by splitting the expected number of observed positive and negative feeding events to observations of conspecifics (({O}_{+{rm{C}},i}left(tright)), ({O}_{-{rm{C}},i}left(tright))) and heterospecifics (({O}_{+{rm{H}},i}left(tright)), ({O}_{-{rm{H}},i}left(tright))), and including these in the model as separate explanatory variables thus:$${p}_{-,i}(t)={rm{logit}}left(begin{array}{c}alpha +{beta }_{{rm{asoc}}+}{N}_{+,i}(t)+{beta }_{{rm{asoc}}-}{N}_{-,i}(t)\ +{beta }_{{rm{soc}},+{rm{H}}}{O}_{+{rm{H}},i}(t)+{beta }_{{rm{soc}},-{rm{H}}}{O}_{-{rm{H}},i}(t)\ +{beta }_{{rm{soc}},+{rm{C}}}{O}_{+{rm{C}},i}(t)+{beta }_{{rm{soc}},-{rm{C}}}{O}_{-{rm{C}},i}(t) \ +{{{{rm{B}}}}}_{i}end{array}right)$$
(6a)
with ({beta }_{{rm{soc}},-{rm{H}}}) and ({beta }_{{rm{soc}},-{rm{C}}}) giving the effect of a negative observation of a heterospecific and conspecific, respectively, whereas ({beta }_{{rm{soc}},+{rm{H}}}) and ({beta }_{{rm{soc}},+{rm{C}}}) give the effect of positive observation of a heterospecific and conspecific, respectively. In general –/+ subscripts refer to negative/positive feeding events and C/H subscripts to feeding events by conspecifics/heterospecifics. By re-parameterizing the model thus:$${p}_{-,i}left(tright)={rm{logit}}left(begin{array}{c}alpha +{beta }_{{rm{asoc}}+}{N}_{+,i}left(tright)+{beta }_{{rm{asoc}}-}{N}_{-,i}left(tright) \ +{beta }_{{rm{soc}},{rm{H}}+}{O}_{+,i}left(tright)+{beta }_{{rm{soc}},{rm{H}}-}{O}_{-,i}left(tright)\ +left({beta }_{{rm{soc}},{rm{C}}+}-{beta }_{{rm{soc}},{rm{H}}+}right){O}_{+{rm{C}},i}left(tright)+left({beta }_{{rm{soc}},{rm{C}}-}-{beta }_{{rm{soc}},{rm{H}}-}right){O}_{-{rm{C}},i}left(tright)\ +{{{{rm{B}}}}}_{i}end{array}right)$$
(6b)
we were able to test for a difference between observations of negative feeds by conspecifics and heterospecifics (left({beta }_{{rm{soc}},{rm{C}}-}-{beta }_{{rm{soc}},{rm{H}}-}right)) and between observations of positive feeds by conspecifics and heterospecifics (left({beta }_{{rm{soc}},{rm{C}}+}-{beta }_{{rm{soc}},{rm{H}}+}right)).For all experiments there was no evidence for a difference between ({beta }_{{rm{soc}},-{rm{H}}}) and ({beta }_{{rm{soc}},-{rm{C}}}) (yellow/orange: Z = 0.803, p = 0.42; red/green: Z = 0.065, p = 0.95; blue/purple: Z = 1.113, p = 0.27). However, there was some evidence of a difference between ({beta }_{{rm{soc}},+{rm{H}}}) and ({beta }_{{rm{soc}},+{rm{C}}}) in two of the three experiments (yellow/orange: Z = 1.359, p = 0.17; red/green: Z = 1.417, p = 0.16; blue/purple: Z = 0.729, p = 0.47). Consequently, we reduced the model down to:$${p}_{-,i}left(tright)={rm{logit}}left(begin{array}{c}alpha +{beta }_{{rm{asoc}}+}{N}_{+,i}left(tright)+{beta }_{{rm{asoc}}-}{N}_{-,i}left(tright)\ +{beta }_{{rm{soc}},-}{O}_{-,i}left(tright)+{beta }_{{rm{soc}},+{rm{H}}}{O}_{+{rm{H}},i}left(tright)+{beta }_{{rm{soc}},+{rm{C}}}{O}_{+{rm{C}},i}left(tright)\ +{{{{rm{B}}}}}_{i}end{array}right)$$
(7)
for further analysis, i.e., with different effects for observations of conspecific/heterospecific positive feeds, but not of negative feeds. We did this for all color combinations (including blue/purple) to allow comparison across experiments (see Table 1). The R code used to run these models can be found in Supplementary data53 in ‘GLMM models Orange Yellow final.r’.Social information use during avoidance learning: simulations to test for a network effectNext, we tested whether the social effects we detected followed the social network. When using a network-based diffusion analysis (NBDA43), researchers can compare a network model with one in which the network has homogeneous connections among all individuals, but we found this to be unreliable for our model. Instead, we used a simulation approach to generate a null distribution for the null hypothesis of homogeneous social effects, taking the size of the social effects from the fitted models. We ran 1000 simulations (using the same procedure described above) for all social effects that were found to be significant in each avoidance learning model (each color pair; see Table 1). The total number of expected observations was kept equal, but we homogenized the observation effect across all birds by replacing the probability of bird i observing a feed by bird j, previously ({a}_{{ij}}), with ({sum }_{i}{a}_{{ij}}/n), where n is the number of birds in the experiment, (i.e., all birds had the same probability of observing each feeding event). The model was fitted to the simulated data each time to extract the Z value (Wald test statistic) of the social effect of interest. The distribution of these values was then used as a null distribution to test whether our observed social effect differed from the effects that did not follow the social network. To this end, we calculated the proportion of simulations that yielded a Z value as extreme or more extreme than that observed (judged by distance in either direction from the mean of the null distribution). The R code used to run these simulations can be found in Supplementary data53 in ‘Simulations to test if network effects follow network Orange Yellow.r’.Social information use during avoidance learning: extension to test for age effectsWe then aimed to test whether each of the three social effects detected differed based on the age class of the observed individual (adult versus juveniles). We, therefore, split the negative expected observations ({O}_{-,i}left(tright)) into the expected observations of adults ({O}_{-{rm{A}},i}left(tright)) and juveniles ({O}_{-{rm{J}},i}left(tright)), each with its associated coefficient in the model ({beta }_{{rm{soc}},-{rm{A}}}) and ({beta }_{{rm{soc}},-{rm{J}}}). Likewise, we split positive observations of conspecifics as ({O}_{+{rm{CA}},i}left(tright)) and ({O}_{+{rm{CJ}},i}left(tright)) and positive observations of heterospecifics as ({O}_{+{rm{HA}},i}left(tright)) and ({O}_{+{rm{HJ}},i}left(tright)) to give the model:$${p}_{-,i}left(tright)={rm{logit}}left(begin{array}{c}alpha +{beta }_{{rm{asoc}}+}{N}_{+,i}left(tright)+{beta }_{{rm{asoc}}-}{N}_{-,i}left(tright)\ +{beta }_{{rm{soc}},-{rm{A}}}{O}_{-{rm{A}},i}left(tright)+{beta }_{{rm{soc}},-{rm{J}}}{O}_{-{rm{J}},i}left(tright)\ +{beta }_{{rm{soc}},+{rm{HA}}}{O}_{+{rm{HA}},i}left(tright)+{beta }_{{rm{soc}},+{rm{HJ}}}{O}_{+{rm{HJ}},i}left(tright)\ +{beta }_{{rm{soc}},+{rm{CA}}}{O}_{+{rm{CA}},i}left(tright)+{beta }_{{rm{soc}},+{rm{CJ}}}{O}_{+{rm{CJ}},i}left(tright)\ +{{{{rm{B}}}}}_{i}end{array}right)$$
(8a)
As before, –/+ subscripts refer to negative/positive feeding events, C/H subscripts to feeding events by conspecifics/heterospecifics, and A/J subscripts to feeding events by adults/juveniles. We also fitted a re-parameterized version allowing us to test for a difference between expected observations of adults and observations of juveniles for each of the three social effects:$${p}_{-,i}left(tright)={rm{logit}}left(begin{array}{c}alpha +{beta }_{{rm{asoc}}+}{N}_{+,i}left(tright)+{beta }_{{rm{asoc}}-}{N}_{-,i}left(tright)\ +{beta }_{{rm{soc}},-{rm{J}}}{O}_{-,i}left(tright)+left({{beta }_{{rm{soc}},-{rm{A}}}-beta }_{{rm{soc}},-{rm{J}}}right){O}_{-{rm{A}},i}left(tright)\ +{beta }_{{rm{soc}},+{rm{H}}}{O}_{+{rm{H}},i}left(tright)+left({{beta }_{{rm{soc}},+{rm{HA}}}-beta }_{{rm{soc}},+{rm{HJ}}}right){O}_{+{rm{JA}},i}left(tright)\ +{beta }_{{rm{soc}},+{rm{C}}}{O}_{+{rm{C}},i}left(tright)+left({{beta }_{{rm{soc}},+{rm{CA}}}-beta }_{{rm{soc}},+{rm{CJ}}}right){O}_{+{rm{CA}},i}left(tright)\ +{{{{rm{B}}}}}_{i}end{array}right)$$
(8b)
The R code used to run these models can be found in Supplementary data53 in ‘GLMM models Orange Yellow final.r’. The main results of each model are presented in Table 2 and full model outputs in Supplementary Tables 3–5.Social information use during reversal learningTo investigate social information use during reversal learning, we used the order of acquisition diffusion analysis (OADA), a variant of NBDA43, which explores the order in which individuals acquire a behavioral trait44. The rate of social transmission between two individuals is assumed to be linearly proportional to their network connection, and the spread of trait acquisition is therefore predicted to follow the network patterns if individuals are using social information. We used NBDA to investigate whether the order of individuals’ first visit to the previously unpalatable blue almonds (mimics) followed the network. We fitted several different models that included (i) only asocial learning, (ii) social transmission of information following a homogeneous network (equal associations among all individuals), or (iii) social transmission of information following our observed network. Models that included social transmission were further divided into models with equal or different transmission rates from adults and juveniles, and from conspecifics and heterospecifics, by constructing separate networks for each adult/juvenile and conspecific/heterospecific combination. To investigate whether asocial or social learning rates differed between blue tits and great tits, we included species as an individual-level variable. We then compared different social transmission models that assumed that species differed in both asocial and social learning rates, only in asocial or only in social learning rates, or that they did not differ in either (see Table 3). The best-supported model was selected using a model-averaging approach with Akaike’s information criterion corrected for small sample sizes. All analyses were conducted with the software R.3.6.154, using lme455, asnipe56, and NBDA57 packages.Reporting summaryFurther information on research design is available in the Nature Research Reporting Summary linked to this article. More

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AnimalsTissue samples for expression analysis were collected on August 24 and September 9 2016 from a population of spring-spawning herring kept in captivity at University of Bergen; the rearing of captive herring was approved by the Norwegian national animal ethics committee (Forsøksdyrutvalget FOTS ID-5072). The tissue samples used for ATAC-sequencing were collected at Hästskär on June 19 2019 from wild spring-spawning Atlantic herring in the Baltic Sea, which do not require ethical permission.Genome scan and genetic diversity at the TSHR locusWe used a 2 × 2 contingency X2 test to estimate the extent of SNP allele frequency differences between seven spring- and seven autumn-spawning herring populations from the Northeast Atlantic (Supplementary Table 2), and thus, identify the major genomic regions associated with seasonal reproduction. The SNP allele frequencies were generated in a previous study16 and were derived from Pool-seq data. For the X2 test, we formed two groups, spring and autumn spawners, and summed the reads supporting the reference and the alternative alleles for the pools included in each group.To characterize genetic diversity at the TSHR locus, we calculated nucleotide diversity (π) and Tajima’s D for the same seven spring- and seven autumn-spawning Atlantic herring populations used in the genome scan (n = ~41–100 per pool) (Supplementary Table 2). The whole-genome re-sequence data of these pools were previously reported by Han et al.16 (for details of the pools used here see Supplementary Table 2). Unbiased nucleotide diversity π and Tajima’s D were calculated for each pool using the program PoPoolation 1.2.236, which accounts for the truncated allele frequency spectrum of pooled data. In brief, a pileup file of chromosome 15 was generated from each pool BAM file using samtools v.1.1037,38. Indels and 5 bp around indels were removed to exclude spurious SNPs due to misalignments around indels. To minimize biases in the π and Tajima’s D calculations, which are sensitive to sequencing errors and coverage fluctuations39, the coverage of each pileup file was subsampled without replacement to a uniform value based on a per-pool coverage distribution (the target coverage corresponded to the 5th percentile of the coverage frequency distribution, which was used as the minimum coverage allowed for an SNP to be included in the analysis) (Supplementary Table 2). We also calculated the diversity parameters but skipping the coverage subsampling step and obtained very similar results with both approaches (Supplementary Fig. 5), thus, we decided to keep working with the subsampled datasets as coverage subsampling is recommended by the software developers36. To exclude spurious SNPs associated with repetitive sequences and copy number variants, we applied a maximum coverage filter equivalent to the per-pool 99th percentile of the coverage frequency distribution (Supplementary Table 2). A minimum base quality of 20, a minimum mapping quality of 20, and a minor allele count of 2 were required to retain high quality SNPs for further analysis. Both, π and Tajima’s D statistics were calculated using a sliding window approach with a window size of 10 kb and a step size of 2 kb (the selected window-step combination offered a good genomic resolution while reducing the noise from single SNPs, after testing windows of 5, 10, 20, 40, 50, and 100 kb for non-overlapping and overlapping windows with a step size equivalent to 20% of the window size). Only windows with a coverage fraction ≥ 0.5 were included in the computations. In addition, we estimated the effective allele frequency difference, or delta allele frequency (dAF), between spring and autumn spawning groups at the TSHR locus using the formula dAF = abs(mean(spring pools)−mean(autumn pools)). For each of the diversity parameters, we assessed whether the mean differences between sets of SNPs within chr 15: 8.85–8.95 Mb (215 SNPs) and outside (214 635 SNPs) the TSHR region were statistically significant among spring- and autumn-spawning groups using a Wilcoxon test. Data postprocessing, statistical tests, and plotting were performed in the R environment40 (for specific parameters used in PoPoolation, see the associated code to this publication).Identification of the 5.2 kb structural variantSequences spanning the entire TSHR gene plus 10 kb upstream and downstream from two reference assemblies, ASM96633v115 and Ch_v2.0.218, were aligned using BLAST41 and the output was subsequently processed with a custom R script40. Repeats were annotated by CENSOR21 for the region harboring the 5.2 kb structural variant. To validate the structural variant, long-range PCR was performed with genomic DNA from two spring- and autumn-spawning Atlantic herring in a 20 μL reaction containing 0.8 mM dNTPs, 0.3 μM each of the forward and reverse 5.2kb-confirm primers (Supplementary Table 1) and 0.75 U PrimeSTAR GXL DNA Polymerase (TaKaRa) following the program: 95 °C for 3 min, 35 cycles of 98 °C for 10 s, 58 °C for 20 s and 68 °C for 2 min 30 s, and a final extension of 10 min at 68 °C.ATAC-seq analysisBSH and brain without BSH were dissected from two spring-spawning herring caught in the Baltic Sea and transported to the lab on dry ice, then kept at –80 °C before nuclei isolation. ATAC-seq libraries were constructed according to the Omni-ATAC protocol with minor modifications42. Briefly, tissue was homogenized in 2 ml homogenization buffer (5 mM CaCl2, 3 mM Mg(Ac)2, 10 mM Tris-HCl (pH = 7.8), 0.017 mM PMSF, 0.17 mM ß-mercaptoethanol, 320 mM Sucrose, 0.1 mM EDTA and 0.1% NP-40) with a Dounce homogenizer on ice. 400 μl suspension was transferred to a 2 ml tube for the density gradient centrifugation with different concentrations of Iodixanol solution. After centrifugation, a 200 μl fraction containing the nuclei band was collected, stained with Trypan blue and counted with a Countess II Automated Cell Counter (Thermo Fisher Scientific). An aliquot of 100,000–200,000 nuclei was used as input in a 50 μl transposition reaction containing 2 X TD buffer and 100 nM assembled Tn5 transposase for a 30-min incubation at 37 °C. Tagmented DNA was purified with a Zymo clean kit (Zymo Research). Purified DNA was used for an initial pre-amplification for 5 cycles, and the additional amplification cycle was determined by qPCR based on the “R vs Cycle Number” plot43. Amplified libraries were purified with a Zymo clean kit again, and library concentrations and qualities were evaluated using the 2200 TapeStation System (Agilent Technologies).ATAC-seq was performed with a MiniSeq High Output Kit (150 cycles) on a MiniSeq instrument (Illumina) and 7–9 million reads were generated for each ATAC-seq library. Quality control, trimming, mapping, and peak calling of the sequenced reads were conducted following the ENCODE ATAC-seq pipeline (https://www.encodeproject.org/atac-seq/). The trimmed reads were aligned to the Atlantic herring reference genome (Ch_v2.0.2)18 with Bowtie244 and the mapping rate was 85–95%. Duplicate reads, reads with low mapping quality and those aligned to the mitochondria genome were removed. The remaining reads (4–5 million) were subjected to peak calling by MACS245, where 22–32 K peaks were called. Sequenced library qualities were further evaluated by calculating the TSS enrichment score and checking the library complexity with the Non-Redundant Fraction (NRF) and PCR Bottlenecking Coefficients (PBC1 and 2). Finally, conserved peaks between two biological replicates were identified by evaluating the irreproducible discovery rate (IDR).Genotyping of six differentiated variants and haplotype analysisAll six genetic variants, including the 5.2 kb structural variant, two non-coding SNPs, two missense SNPs and the copy number variant of C-terminal 22aa repeat, were genotyped in 45 spring-, 67 autumn-spawning Atlantic herring and 13 Pacific herring. TaqMan Custom SNP assays were performed to genotype the four SNPs in 5 μl reactions with a template of 20 ng genomic DNA (ThermoFisher Scientific). Copy number of the C-terminal 22aa repeat was determined by the PCR product size generated with geno22aa primers. Genotyping of the 5.2 kb structural variant was performed in a PCR reaction containing two forward primers (geno5.2kb-1F and geno5.2kb-2F) and one reverse primer (geno5.2kb-R), which generated PCR products with different sizes between spring and autumn spawners. All the primers used for genotyping are listed in Supplementary Table 1.Tissue expression profiles by quantitative PCRTotal RNA was prepared from gonad, heart, spleen, kidney, gills, intestine, hypothalamus and saccus vasculosus (BSH), and brain without BSH (brain) of six adult spring-spawning Atlantic herring using RNeasy Mini Kit (Qiagen). RNA was then reverse transcribed into cDNA with a High-Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific). TaqMan Gene Expression assay (ThermoFisher Scientific) containing 0.3 μM primers and 0.25 μM TaqMan probe (Integrated DNA Technologies) was performed to compare the relative expression levels of TSHR among different tissues. qPCR with SYBR Green chemistry was used for TSHB and DIO2 in a 10 μl reaction of SYBR Green PCR Master Mix (ThermoFisher Scientific) and 0.3 μM primers, with a program composed of an initial denaturation for 10 min at 95 °C followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Ct values were first normalized to the housekeeping gene ACTIN, then the average expression for each gene in the gonad was assumed to be 1 for the subsequent calculation of the relative expression in other tissues.Plasmid constructsThe coding sequence for the herring single-chain TSH (scTSH) was designed following a strategy previously used for mammalian gonadotropins46 that contained an in-frame fusion of the cDNA sequences (5′–3′) of herring TSH beta subunit (NCBI: XM_012836756.1) and alpha subunit (NCBI: XM_012822755.1) linked by six histidines and then the C-terminal peptide of the hCG beta subunit. The designed sequence should generate a protein with a size of 30.6 kDa. Both scTSH and spring herring TSHR cDNA sequences were synthesized in vitro and cloned in the expression vector pcDNA3.1 by Genscript (Leiden, Netherlands). pcDNA3.1 plasmid expressing human TSHR was kindly provided by Drs. Gilbert Vassart and Sabine Costagliola (Université libre de Bruxelles, Belgium). Then, the spring herring TSHR and human TSHR plasmids were used as templates for site-directed mutagenesis to generate constructs coding for different mutant herring or human TSHRs. Plasmids for the dual-luciferase assay, including pGL4.29[luc2P/CRE/Hygro] containing cAMP response elements (CREs) to drive the transcription of luciferase gene luc2P and pRL-TK monitoring the transfection efficiency, were purchased from Promega. Five ng of each plasmid was used to transform the XL1-Blue competent cells (Agilent), plasmid DNA was subsequently extracted from 200 ml overnight culture of a single transformant clone using an EndoFree plasmid Maxi Kit (Qiagen).Cell cultureChinese hamster ovary (CHO) (ATCC CCL-61) and human embryonic kidney 293 (HEK293) (ATCC CRL-1573) cells were maintained in DMEM supplemented with 5% (CHO) or 10% FBS (HEK293), 100 U/ml penicillin, 100 μg/ml streptomycin and 292 μg/ml l-Glutamine (ThermoFisher Scientific) at 37 °C with 5% CO2. Epithelioma Papulosum Cyprini (EPC) cells (ATCC CRL-2872) were cultured in EMEM (Sigma) supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, 292 μg/ml l-Glutamine and 1 mM Sodium Pyruvate (ThermoFisher Scientific) at 26 °C with 5% CO2.Production of recombinant herring scTSHCHO cells were transfected with the scTSH expression plasmid using Lipofectamine 3000 (Invitrogen), stable clones were subsequently selected with 500 μg/ml G418 (Invitrogen) and screened for producing scTSH by western blot using a polyclonal antisera against the sea bass alpha subunit47. A positive clone was expanded in 225 cm2 cell culture flasks (Corning) in culture medium containing 5% FBS until confluence, then the cells were maintained in serum-free DMEM for hormone production for 7 days at 25 °C48. After 7 days, culture medium containing scTSH or without (negative control) was centrifuged at 15000 x g for 15 min and concentrated by ultrafiltration using Centricon Plus-70 / Ultracel PL-30 (Merck Millipore Ltd.). Then, western blotting was performed to confirm TSH production. Concentrated medium containing herring scTSH was denatured at 94 °C for 5 min in 0.1% SDS and 50 mM 2-mercaptoethanol, and then treated with 2.5 units of peptide-N-glycosidase F (Roche Diagnostics) at 37 °C for 2 h in 20 mM sodium phosphate with 0.5% Nonidet P-40, pH 7.5. All samples were run in 12% SDS-PAGE in the reducing condition and transferred to a PVDF membrane (Immobilon P; Millipore Corp.), then blocked overnight with 5% skimmed milk at 4 °C. After blocking, the membrane was incubated with polyclonal antisera against the sea bass alpha subunit (dilution 1:2000) for 90 min at room temperature, washed, and then further incubated with 1:25000 goat anti-rabbit immunoglobulin G (IgG) horseradish peroxidase conjugate (Bio-Rad Laboratories) for 60 min at room temperature. Immunodetection was performed by chemiluminescence with a Pierce ECL Plus Western Blotting Substrate kit (ThermoFisher Scientific).Cell surface expressionA Rhotag (MNGTEGPNFYVPFSNKTGVVYEE) was inserted at the N-terminus of herring TSHR for flow cytometry analysis of receptor cell surface expression. Anti-Rhotag polyclonal antibody was kindly provided by Drs. Gilbert Vassart and Sabine Costagliola (Université Libre de Bruxelles, Brussels, Belgium). PBS containing 1% BSA and 0.05% sodium azide was prepared as the flow cytometry (FCM) buffer for the washing and antibody incubation steps. 2.2 × 106 EPC cells were seeded in a 100 mm poly-d-Lysine-treated petri dish the day before transfection. Each dish was transfected with 10 μg TSHR or empty pcDNA3.1 expression plasmid using 20 μl jetPRIME transfection reagent in 500 μl jetPRIME transfection buffer (Polyplus transfection). Cells were harvested 24 h after transfection, then washed once in cold PBS and fixed in 2% PFA for 10 min at room temperature. After fixation, cells were washed three times with FCM buffer, then incubated with anti-Rhotag antibody or FCM buffer (negative control) for 1 h at room temperature. Cells were washed again with FCM buffer three times and stained with Alexa Fluor 488-labeled chicken anti-mouse IgG (H + L) antibody (1:200 dilution, ThermoFisher Scientific) or FCM buffer (negative control) for 45 min in the dark. After the fluorescent staining, cells were washed three times and resuspended in FCM buffer before analysis on a CytoFLEX instrument (Beckman Coulter). A minimum of 100,000 events was recorded for each sample, fluorescence intensities of negative control and cells transfected with empty pcDNA3.1 plasmid were used as the background for gating strategy. Cell surface expression was represented by the mean fluorescence intensity of the positively stained cell population.Dual-luciferase reporter assayEPC or HEK293 cells were plated in a 48-well plate at a density of 1 × 105 cells/well the day before transfection. A total of 250 ng plasmid mixture containing pGL4.29[luc2P/CRE/Hygro], TSHR expression plasmid (or empty pcDNA3.1) and pRL-TK with the ratio of 20:5:1 was prepared to transfect each well of cells using jetPRIME transfection reagent (Polyplus). Medium was replaced by fresh medium containing 10% FBS (TSH-induced condition) or serum-free medium (constitutive activity condition) 4 h after transfection. On day three, cells were treated with serum-free medium containing different dilutions of the concentrated scTSH medium for 4 h (TSH-induced condition) or directly subjected to the luminescence measurement without TSH induction (constitutive activity condition). Luminescence was measured using a Dual-Luciferase Reporter assay (Promega) on an Infinite M200 Microplate Reader (Tecan Group Ltd., Switzerland), and luciferase activity was represented as the ratio of firefly (pGL4.29[luc2P/CRE/Hygro]) to Renilla (pRL-TK) luminescence.5′-RACE to identify the herring DIO2 TSSTotal RNA was prepared from brain of a spring-spawning Atlantic herring using the RNeasy Mini Kit (Qiagen). Six μg of the isolated RNA was used for 5′-RACE with a FirstChoiceTM RLM-RACE Kit (ThermoFisher Scientific). One μl cDNA or Outer RACE PCR product was used as PCR template in a 20 μL reaction containing 0.8 mM dNTPs, 0.3 μM of each forward and reverse primer (Supplementary Table 1) and 0.75 U PrimeSTAR GXL DNA Polymerase (TaKaRa). Amplification was carried out with an initial denaturation of 3 min at 95 °C, followed by 35 cycles of 98 °C for 10 s, 58 °C for 20 s and 68 °C for 40 s, and a final extension of 10 min at 68 °C. The final 5′ RACE product was sequenced at Eurofins Genomics (Ebersberg, Germany).Sequence conservation analysisGenomic sequences covering the TSHR locus were extracted from Ensembl Genome Browser for Atlantic herring and 11 other fish species, including Amazon molly (Poecilia formosa), denticle herring (Denticeps clupeoides), goldfish (Carassius auratus), guppy (Poecilia reticulata), Neolamprologus brichardi, Japanese medaka (Oryzias latipes), northern pike (Esox lucius), orange clownfish (Amphiprion percula), spotted gar (Lepisosteus oculatus), three-spined stickleback (Gasterosteus aculeatus) and spotted green pufferfish (Tetraodon nigroviridis). The extracted sequences were firstly aligned using progressiveCactus49,50, and a subsequent alignment was generated using the hal2maf program from halTools51 with Atlantic herring assembly (Ch_v2.0.2)18 as the co-ordinate backbone. This alignment was used for the downstream phastCons score calculation by running phyloFit24 and phastCons25 from the PHAST package with default parameters. Peaks were called by grouping signals with a minimum phastCons score of 0.2 within 500 bp region.Structure modeling of human and herring TSHRsIn order to explore the possible interactions of the variant residues with other receptor interacting proteins and to study intramolecular interactions, we built a structural homology model for the herring TSHR (herrTSHR) complexed with herring TSH and Gs-protein. The TSHR hinge region that harbors the Q370H substitution and the C-terminus containing the 22aa repeat were excluded from the homology model due to the lack of structural templates for these regions. The homology model was constructed by using the following structural templates of evolutionarily related class A GPCRs: (i) the leucine-rich repeat domain (LRRD) complexed with hormone was modeled based on the solved FSHR LRRD – FSH complex structure (Protein Data Bank (PDB) ID: 4AY9)52,53, this part of model included herring TSHR Cys33 – Asn296 and fragments of the hinge region Gln297 – Thr312 and Ser393 – Ile421; (ii) the available structural complex of β2-adrenoreceptor with Gs-protein (PDB ID: 3SN6)54 was used as the template to model the seven-transmembrane helix domain (7TMD) of herring TSHR in the active conformation; (iii) the extracellular loop 2 (ECL2) was built by using the ECL2 of μ-opioid receptor (PDB ID: 6DDE)55. To prepare the template for herring TSHR modeling, the fused T4-lysozyme and bound ligand of β2-adrenoreceptor were deleted, the ECL1 and ECL3 loops were adjusted manually to the loop length of herring TSHR. Due to the lack of third intracellular loop (ICL3) in the β2-adrenoreceptor structure, amino acid residues of herring TSHR ICL3 were manually added to the template. Since herring TSHR does not have the TMH5 proline, which is highly conserved among all class A GPCRs and responsible for the helical kinks and bulges within this region56, we assumed a rather regular (stretched) helix conformation for the herring TSHR TMH5 and therefore replaced the kinked β2-adrenoreceptor TMH5 template with a regular α-helix. Moreover, the ECL2 template was substituted with μOR ECL2 structure because of its higher sequence similarity with herring TSHR in this region. Finally, amino acid residues of this chimeric 7TMD template and FSHR N-terminus were mutated to the corresponding spring herring TSHR residues and sequence of the heterodimeric FSH ligand was substituted by the herring TSH. All homology models were generated by using SYBYL-X 2.0 (Certara, NJ, US). The 7TMD structure was then fused with FSHR N-terminus at position 421. The assembled complex was subsequently optimized by the energy minimization under constrained backbone atoms (the AMBER F99 force field was used), followed by a 2 ns molecular dynamics simulation (MD) of the side chains. The entire TSHR complex was energetically minimized without any constraints until converging at a termination gradient of 0.05 kcal/mol*Å. Next, for autumn herrTSHR modeling, the spring TSHR sequence was substituted with autumn TSHR sequence. For humTSHR, the spring TSHR sequence was substituted with human TSHR, and the herring TSH ligand was replaced by the bovine TSH sequence. Both complex models were energetically minimized until converging at a termination gradient of 0.05 kcal/mol*Å.To investigate the microenvironment around the L471M mutation at TMH2 position 2.51, local short MD’s of 4 ns on Met4712.51 (spring herrTSHR), Leu471 (autumn herrTSHR) or Phe461 (humTSHR) and its surrounding amino acids were performed. During MD simulations, backbone atoms of the entire complexes as well as all side chains, except residues at positions 1.47, 1.51, 1.54, 2.48, 2.52, and 2.55 that form the hydrophobic patch around position 2.51, were constrained.Statistics and reproducibilityResults were presented as the mean + SD (standard deviation) calculated from at least four biological replicates for each experiment, and at least two independent experiments were conducted for each assay. Unpaired two-tailed Student’s t test was performed to calculate the P-values and means were judged as statistically significant when P ≤ 0.05.Reporting summaryFurther information on research design is available in the Nature Research Reporting Summary linked to this article. More