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    Breeding and migration performance metrics highlight challenges for White-naped Cranes

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    The emergence and development of behavioral individuality in clonal fish

    All animal care and experimental protocols complied with local and federal laws and guidelines and were approved by the appropriate governing body in Berlin, Germany, the Landesamt fur Gesundheit und Soziales (LaGeSo G-0224/20).Experimental breeding and designThe all-female Amazon molly (Poecilia formosa) is a naturally clonal, live-bearing fish species that gives birth to broods of genetically identical offspring. Like all unisexual vertebrates, Amazon mollies are the result of inter-specific hybridization44,45. As such, this ‘frozen hybrid’ has a heterozygous genome from its ancestral P. mexicana mother and P. latipinna father alleviating concerns about reduced genetic variation and the resulting inbreeding depression often associated with artificially selected isogenic animals. Additionally, despite their clonal nature, the Amazon’s genome shows no evidence of increased mutation accumulation, genomic decay or transposable element activity suggesting the genomes of these animals are evolving in similar ways as sexual species46. They reproduce through gynogenesis where the meiotic process is disrupted so that the eggs contain a full maternal genome. The egg must be fused with a sperm from one of their ancestral species to stimulate embryogenesis, but this paternal DNA is not incorporated into the egg. This provides the opportunity to control when reproduction occurs by controlling the females’ access to male sperm donors.We placed adult females, as potential mothers of experimental fish, in individual (5-gallon) breeding tanks with two Atlantic molly (P. mexicana) males for one week to act as sperm donors. Amazon mollies give birth to broods of generally ~8-30 individuals. A brood is born at once (i.e. all individuals are born within minutes of each other) and birth generally happens early in the day close to dawn. These parental fish were lab-bred and themselves sisters, so of the same age and lineage, and were kept at similar social densities and under standardized environmental conditions throughout their lives to further minimize potential variation in maternal experience. Each breeding tank contained an artificial plant as refuge and was checked frequently each day for the presence of offspring, especially during the morning hours when births are most likely. Newborn mollies were always found in the morning and then singly netted by trained animal caretakers, into individual experimental tanks where their behavior was automatically recorded for the next 70 days (see below). Moving the fish from the maternal tank to the experimental tanks was done in a standardized manner (i.e. individual fish were netted and placed into small dishes of water and then placed in the tracking tanks to limit exposure to the air) by the same caretakers to minimize variation in experience among individual fish. Altogether, eight mothers provided offspring that completed the entire 10-week experiment (Supplementary Table 1).Experimental tanks (27 x 27 cm), made of white Perspex, consisted of four equally sized compartments, and were evenly lit from below using 6500K-LEDs. Environmental conditions were highly standardized across tanks: all tanks were on the same 11:13 (L:D) light schedule, water depth was maintained at 10 cm depth, temperature was maintained at 25 ± 1 °C by a room air conditioning system, and fish received a standardized amount of powdered flake fish food (TetraMin™) twice daily. Opaque blinds surrounded the tanks to further limit outside disturbances. All experimental tanks were connected to the same filtration system where water could mix in the sump tank, allowing chemical cues to be shared across all experimental fish. Previous work has shown exposure to just chemical cues of conspecifics is sufficient in preventing the developmental of pathological behavior that could be associated with development in complete isolation14. We initially placed a total of 40 newborn individuals into the tracking tanks. At the end of the 10-week experiment, we were able to achieve complete tracking data on 26 individuals; camera malfunctions prevented data collection on four individuals, two individuals jumped into neighboring tanks causing the loss of data of all four individuals as we could not verify their identity; four newborn individuals escaped through holes in the water outlet of the tanks; and four individuals died as newborns. All results in the manuscript are on these 26 animals, though including data from all 40 (e.g. patterns of individual variation on the first day post birth) did not change the results or their interpretation (see Supplementary Table 2).Behavioral trackingWe developed a custom recording system using Raspberry Pi computers, which are an upcoming low-cost, highly adaptable solution for many applications in the biological sciences25. Specifically, we created a local network of Raspberry Pi 3B + ’s, each connected to a Raspberry Pi camera positioned exactly above an experimental tank, commanded by a lab computer, and connected to the server on the institute network (Supplementary Fig. 1). We programmed the Raspberry Pi’s using pirecorder26 to take timestamped photos every 3 s across the daily light period, each day, for 10 weeks, and store them automatically in dedicated, automatically named folders on the server. Image settings and resolution were thereby optimized to minimize file size while assuring image quality. After the experimental period, we created videos of all the recorded images of each fish of each day. These videos were subsequently tracked with the Biotracker software27, using background subtraction, providing the x, y coordinates of each fish in each frame. We then processed the data, including scaling and converting the coordinates to mm, and, for each frame, computed fish’s swimming speed (cm/s) and distance from the tank walls (cm). We then summarized these variables both on an hourly and daily basis to compute fish’s median swimming speed, inter-quartile range of swimming speeds, activity (proportion of time spent moving >0.5 cm/s), and median border distance. To quantify fish’s body size over time, we randomly selected five photos per week of each compartment, making sure the fish was away from the compartment walls and did not show strong body curvature, and then used ImageJ software to measure total body length (mm) from the tip of the snout to the end of the body. By averaging the measurements of the five images, we acquired one body size measurement per week.Error checkingWe collected up to 924,000 photos on each individual throughout the experimental period resulting in a total of over 24 million data points collected on our experimental animals (N = 26 individuals). To ensure that our tracking software accurately captured the behavior of our fish, we checked for potential tracking errors in two ways. First, we estimated overall error rates. To do this, we selected at random a starting frame from within a day; then we manually checked each of the subsequent 200 frames and identified whether an error was made (fish was not properly located by BioTracker) or not (fish was properly located) by visual inspection of the videos. We estimated the error rate as the number of errors divided by the total number of checked frames. The overall median error rate over the entire observation period was estimated to be 7%. Error rates increased earlier in the observation period when the fish were smaller (Supplementary Note I). As such, as a second step, we manually went through and corrected all frames for the very first day of tracking (i.e. day 1 post-birth) for all fish (~13,200 frames per individual) as this is a critical time period for one of our research questions. This ensured that the resulting behavioral data were completely accurate for this day. This manual correction allowed us the additional opportunity to compare how well our automatically tracked (i.e. not manually corrected) data performed compared to the manually corrected data. We found that the automatically tracked data re-created near identical estimates of among- and within-individual variance components and most importantly the among-individual correlation between the automatically tracked and manually corrected data was over 0.98 for our behavioral variables (Supplementary Note I). This strongly suggests that any errors introduced by our automated tracking software have minimal influence of our behavioral variables at best and do not affect our interpretation of the results.Statistical analysesWe used linear mixed, or hierarchical, models to partition the behavioral variation across different times periods into its among- and within-individual components. Throughout we focused our analysis on the 26 individuals for which we had complete data for the entire 10-week observation period to ensure comparable variation over time and across models.Our first question of interest was to test when individual differences in behavior first appeared over the course of the experiment. We started by investigating behavior on the first day post birth (Fig. 1A, Supplementary Table 2) and then planned to proceed in a day-by-day fashion until significant repeatability in behavior was apparent (Supplementary Table 3). We used hourly median swimming speed (11 observations for each of 26 individuals) as our response variable and included ‘hour’ and ‘total length (TL)’ as fixed effects and ‘individual’ was included as our random effect of interest. Including TL as a covariate allowed us to test whether behavior was related to an offspring’s body size on its first day of life. We set the first hour of the day as 0 and mean-centered TL as this would allow the among- (and within-) individual variance components to be estimated at these values (i.e. the earliest possible moment from when we could record behavior in the fish). We estimated the adjusted repeatability of median swimming speed as the variance attributable to individual identity over the total variance not explained by the fixed effects. We additionally estimated both marginal and conditional R-squared values which estimate the variance explained by the fixed effects only and the variance explained by the fixed and random effects combined, respectively. As our individual experimental fish came from different mothers, we first explored a number of different variance structures including random intercepts and slopes for both individual ID and maternal ID. This allowed us to test whether maternal identity explained variation in individual behavior. However, the most supported model included random intercepts and slopes for individual ID and not for mother ID, indicating that our methods to reduce variation among mothers were successful (Table 1). We used median swimming speed as our behavioral variable of interest throughout the main manuscript, as this behavior was tightly correlated with most of our other behavioral variables (Supplementary Fig. 2); though results using the other behavioral variables yielded the same interpretation (i.e. that significant individuality in (any) behavior was present on the very first day post-birth; Supplementary Table 2).Our second research question was to investigate how individual behavioral variance changed over the course of the entire observation period (70 days). Again, we first explored several different variance structures to test the importance of maternal identity and/or individual identity on behavioral variation. We found support for the inclusion of random slopes at the individual level, but not maternal level (Table 1). This indicates that levels of among- (and within-) individual variation may differ throughout the observation period. To investigate patterns of change in the variance components, we ran a series of models where we centered the observation covariate on different days. Individual intercepts are estimated when all covariates are set to zero, so this allowed us to ‘slice’ the data to estimate the among- and within-individual variance at different time points over the ten weeks. We ran 11 models as we chose to center the data every 7 days (first model was centered on observation 1; 11th model was centered on observation 70). The predicted individual intercepts (best linear unbiased predictors) and estimated variance components from each model are plotted in Fig. 3.We also closely investigated any potential influence of body size and/or growth rate differences on behavioral expression and individual behavioral variation in this entire 10-week data set. First, we estimated the repeatability of both weekly total length and weekly growth rates to determine if individuals consistently differed in these traits. Then, we ran a series of models with median weekly swimming speed as the response variable and included either weekly total length, weekly growth rate, and/or overall growth rate (estimated over the entire 10 weeks), as our fixed effects of interest. Each model also included the random effects of individual intercepts and slopes. Finally, because body size varies both among individuals (some individuals are on average larger than others) and within individuals (as they grow), we also performed within-individual centering of total length. In this fifth model, we included each individual’s average total length and their weekly deviation from their average length as the two fixed effects of interest. Individual identity and slopes were included as random effects. For all models, we estimated the variance explained by the fixed effects (marginal R2) and the fixed and random effects together (conditional R2). These results are reported in Table 2.For our third and final research question, we tested whether early-life behavior predicted later-life behavior. To test this, we estimated the among-individual correlation (including ‘individual ID’ as our random effect) in behavior using multivariate mixed models where the daily median swimming speeds in each week were the response variables (7 observations per week per individual; 10 weeks total; Fig. 4A). Then to investigate how the strength of these correlations may change over development, we used a linear model to test whether the correlation strength was predicted by the interaction between the first week included in the correlation and distance to the next week in the correlation (1, 2, 3, 4 or 5 weeks away in time; Fig. 4B).All models were performed using Markov Chain Monte Carlo estimation with the MCMCglmm package38 in R v3.6.139. We set our models to run 510,000 iterations with a 10,000 burn-in and thinning every 200 iterations. To ensure proper model mixing and convergence, we initially ran 5 independent chains and inspected posterior trace plots of parameter estimates (Supplementary Note II). In a preliminary analysis we tested three different prior settings (Supplementary Note II); results did not change with prior settings so we chose parameter-expanded priors for all models reported here as these are generally considered to be more robust. An R Markdown file with all the results presented here is included in Supplementary Note II.Reporting summaryFurther information on research design is available in the Nature Research Reporting Summary linked to this article. More

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    Phylogeny explains capture mortality of sharks and rays in pelagic longline fisheries: a global meta-analytic synthesis

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    The Blob marine heatwave transforms California kelp forest ecosystems

    The Santa Barbara Coastal Long Term Ecological Research program has monitored benthic communities in five kelp forests seasonally since 2008 using fixed transect diver surveys, and moored sensors at each reef have recorded bottom temperatures every 15 min. Blob-associated positive bottom temperature anomalies began in winter 2014 and persisted through autumn 2016 (Fig. 1a)18. Peak temperature anomalies occurred during the summer and autumn of 2014 and 2015 (Fig. 1a), and the average temperature anomaly in autumn 2015 was +3.1 °C, equivalent to an average daily temperature of 19.6 °C. In 2014 and 2015, 91 and 69% of autumn days, respectively, were classified as heatwave days as defined by Hobday et al.20. Seasonal chlorophyll-a concentration, a proxy for phytoplankton abundance, was obtained from satellite imagery at each of the five reefs over the 14-year period. The average chlorophyll-a concentration was anomalously low throughout the warming period, and exceptionally low during the springs of 2014 and 2015 (Fig. 1a), when upwelling-driven nutrient enrichment typically supports dense phytoplankton blooms.Fig. 1: Average seasonal bottom temperature anomaly, chlorophyll-a concentration anomaly, and percent cover and species richness of sessile invertebrates across five sites.The Blob, an anomalous warming period from spring of 2014 to winter of 2016, is highlighted in gray, coincident with (a) positive temperature anomalies (°C; solid line), negative chlorophyll-a anomalies (mg/m3; dashed line), and declines in (b) invertebrate cover (solid line) and species richness (number of unique species/taxa/80 contact points; dashed line). Seasons are denoted by Sp (Spring), Su (Summer), A (Autumn) and W (Winter).Full size imageMean sessile invertebrate cover averaged across all sites declined 71% during the Blob, reaching a 14-year minimum of 7% in autumn of 2015 (Fig. 1b and Supplementary Fig. 1). Species richness declined 69% during the same period (Fig. 1b and Supplementary Fig. 1). The responses of invertebrates to warming were not consistent across time even though the duration and intensity of warming was similar in 2014 and 2015, suggesting that extended periods of elevated seawater temperature were not solely responsible for the most severe loss of invertebrates. For ectotherms, increases in ambient seawater temperature should be met with increases in metabolic rate and food requirements to sustain metabolism21. Because of their sedentary lifestyle, sessile invertebrates cannot actively forage for food or seek spatial refuge from thermal extremes, and limitations in their planktonic food supply can result in metabolic stress over extended periods22,23. Anomalously low chlorophyll-a concentrations during the Blob (Fig. 1a), particularly in the spring of 2015, indicated that food limitation was a likely driver of invertebrate decline. Results from piecewise structural equation modeling (Fig. 2) that incorporated biological interactions with space competitors (understory macroalgae), predators (sea urchins), and foundation species (giant kelp) showed that the severity of warming had both a direct and indirect effect on the sessile invertebrate community. The proportion of heatwave days was a direct negative predictor of sessile invertebrate cover (−0.11) and species richness (−0.21). The proportion of heatwave days was an even stronger negative predictor of chlorophyll-a concentration (−0.26), yielding negative indirect effects on invertebrate cover (−0.07) and species richness (−0.05) due to the positive influence of chlorophyll-a concentration on sessile invertebrate cover (+0.26) and richness (+0.20).Fig. 2: Piecewise structural equation modeling (SEM) for sessile invertebrate cover and species richness.Arrows indicate directionality of effects on (a) invertebrate cover and (b) species richness. Red arrows show negative relationships; black arrows show positive relationships. R2 values are conditional R2. Arrow widths are proportional to effect sizes as measured by standardized regression coefficients (shown next to arrows). ***p  More

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    Droplet microfluidics-based high-throughput bacterial cultivation for validation of taxon pairs in microbial co-occurrence networks

    Conception of the workflow to demonstrate the microbial associations from co-occurrence networks with microbial cultivationMicrobial co-occurrence networks are composed of nodes and edges, which usually represent microbes and statistically significant associations between microbes, respectively. We hypothesized that the microbial associations could be validated if the topological properties of networks are simplified, and if the microbes representing the nodes can be cultivated. To test this hypothesis, we designed a workflow as shown in Fig. 1. A total of 12,096 wells from 126 96-well plates were inoculated with droplets of series diluted environmental samples, wells from each 96-well plate represented the same combination of given culture condition, sample type (plants, roots, and sediments) and dilution rate (from 10–1 to 10–7). After being cultivated at 30 °C for 10 days, 69 effective (Supplementary Table S3) plates with  > 30% wells showing microbial growth were retained for downstream microbial community analysis. Microbial DNA in each well was extracted, bar-coded, and sequenced for the inference of co-occurrence networks. The wells of plates showing high abundances of target Zotus were targeted for microbial isolations. Lastly, the cultivated microbial isolates were matched to Zotus in the network and used for demonstration of microbial interactions.Figure 1Overview of experimental demonstration of microbial interactions in co-occurrence networks. For detailed description, please refer to the method section.Full size imagePrevalent Zotu pairs in the co-occurrence networksDepending on the microbial density in samples, the 96-well plates harbored different numbers of wells with microbial growth. We obtained 65 96-well plates (6,091 wells) that were effective with microbial growth and data analysis for co-occurrence network reconstruction. After quality control and denoise, we obtained 130 Gbp sequence data. A total of 14,377 Zotus were annotated (Supplementary Table S4). There were 217 ± 94 (average ± standard deviation) prevalent Zotus, i.e., these Zotus appeared at frequencies ≥ 30% of wells in a given 96-well plate.Next, we analyzed Zotus compositions and abundances in each well of the 65 plates. Accordingly, we reconstructed 65 independent microbial co-occurrence networks and further retrieved the robust (Spearman’s |ρ| > 0.6 and P  More

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    Kinship dynamics may drive selection of age-related traits

    “This new study is inspired by some our earlier theoretical work applied to killer whales that suggests that age-related changes in relatedness are important for the evolution of menopause,” says Samuel Ellis, the first author of the study. “Reproduction can be thought of as a form of generalized harm as the birth of an offspring increases within-group competition for resources. Kinship dynamics — the ways in which local relatedness changes over an individual’s lifetime — are one way that menopause could be favored, because older females are more inclined to cease reproduction to not harm their group mates than younger females. Here we wanted to generalize this concept to both sexes, and to other species without menopause.” More

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    Viral metagenomics reveals persistent as well as dietary acquired viruses in Antarctic fur seals

    After massive parallel sequencing of the nucleic acids obtained from fur seal scats, a wide variety of invertebrate and vertebrate viral hosts assignations with low nucleotidic and amino-acidic identities were obtained, most of them corresponding to animal species not described before in Antarctica. These results make us reconsider the use of closed RefSeq databases for viral discovery, especially because the studied area was a remote geographical area where a high number of new viral species is expected to occur22.After repeating the analysis of the contigs obtained using BLASTn, a high number of miss-assignments was observed, corresponding almost entirely to contigs newly assigned as unclassified Eukaryotic Circular Rep-Encoding Single-Stranded DNA (CRESS-DNA) viral sequences. CRESS viruses have been detected ubiquitously in many different animals without any recognised role in the development of any disease23,24,25,26.These results are in accordance with the recent reporting of CRESS sequences also being ubiquitous in a wide variety of environments and at high proportions, including Antarctica, where they have been described to represent more than 50% of sequences obtained from glacier waters27.Viral-host distributionVirome studies in other Arctocephalus species from subantarctic and South American regions revealed a 5% of viral sequences with predominance of bacteriophages followed by viruses from the Parvoviridae family28. The methodology here applied provided an increase of 12–25% viral reads when probe-based Target Enrichment Sequencing (TES) was applied, that in comparison with Untargeted Viral Metagenomics (UVM) approaches conducted in these type of samples28 could be considered an optimal result.Most of the viral species detected in feces corresponded to unknown viruses, 83.59% from the total of sequences, followed by viruses that infect invertebrates, 8.75%, bacteriophages, 4.46%, and vertebrate viruses, 3.11% (Fig. 1).Figure 1Host distribution of viral assignations sequenced from fecal (A) and serum (B) samples collected from male A. gazella.Full size imageAs expected, when applying both targeted and untargeted sequencing methodologies, TES approach resulted in a recovery of many vertebrate viral assignations (Table 1) whereas untargeted sequencing enabled a better detection of viruses known to infect invertebrates (Table 2). To describe the complete A. gazella fecal virome, sequences obtained by both sequencing methodologies were considered all together, representing a total of 2.62 million reads.Table 1 Vertebrate viral assignations obtained from fecal samples sequencing from male A. gazella. Ranges of Genome coverage, nucleotide identity and aminoacidic identity are expressed in percentages.Full size tableTable 2 Invertebrate viral assignations obtained from fecal samples sequencing from male A. gazella. Colours represent the presence of each assignation in the processed pools. Ranges of Coverage, NT ID and AA ID are represented in percentages.Full size table
    A. gazella virusesFur seal picorna-like virusFur seal picorna-like virus was firstly described in a fecal sample obtained from A. gazella in King George Island in the South Shetland Islands, Antarctica by Krumbholz and co-workers16.In this study, we report a total of 19 contigs resulting after assembling 2671 reads obtained from 4/4 fecal pools analysed being the most prevalent virus described in this study. One of the contigs covered 96.91% of the fur seal picorna-like virus genome and presented a nucleotide homology of 99.38% with the reference strain described in 2017. The other contigs coverage ranged from 19.75 to 21.22% with a 45.92 to 90.5% nucleotide identity with reference strain NC_035110. Four contigs matching the ORF2 polyprotein are represented in Fig. 2 where differences among them and with the reference strain are showed.Figure 2Nucleotide alignment of ORF2 sequences from the A. gazella picorna-like contigs compared to the ORF2 from RefSeq NC_0351110. In consensus strain, position 1 represents position 6523 from RefSeqs genome.Full size imagePicornaviruses are known to cause a wide variety of diseases in vertebrate hosts, especially mammals29, but the role of Fur seal picorna-like virus in pathogenesis development is still unknown30. Many picornaviruses are transmitted horizontally via fecal–oral or airborne routes29. The fact that these sequences were detected in all the fecal pools obtained from animals with no evidence of disease may that suggest the virus may have a stable endemic relationship within that seal population.Torque teno pinniped virusLambdatorquevirus is a genus within the Anelloviridae family. The genus comprises 8 species named Torque teno pinniped virus 2 to 9 isolated from different pinniped species: A. gazella (Torque teno pinniped virus 6 and 7)17, Phoca vitulina (Torque teno pinniped virus 2, 3, 4)31, Zalophus californianus (Torque teno pinniped virus 5)32 and Leptonychotes weddellii (Torque teno pinniped 8 and 9)33.One contig with a nuleotide similarity of 95.12% against Torque teno pinniped virus 7 was obtained from one of the fecal pools. This virus had been described in these animals inhabiting Livingston Island in 2016, using rolling circle amplification and subsequent Sanger sequencing from buccal swabs17. However, sequences obtained in this study belong to partial ORF2 which is not the optimal genome region for typing purposes or phylogenetic analysis.These members of the Anelloviridae represent the more abundant viruses found in human, animals and environmental samples although their etiological role in any disease has not been clearly identified being considered a persistent virus ubiquitous to several different tissues34,35No Torque teno virus sequences were detected in serum samples which agree with what was observed for Zalophus californianus anellovirus prevalently detected in different tissues, like lung and liver, but not in blood samples. Interestingly, other known anelloviruses are typically found in blood or plasma samples32.MamastrovirusTwo of the fecal pools analyzed presented Mamastrovirus sequences. The presence of these viruses in humans and other mammals is widely known, as well as their involvement in gastroenteritis development36. The four contigs obtained (comprising 1008 sequences) showed homologies against reference genomes, ranging from 45.70% to 59.37% when compared at nucleotide level and 36.69% to 46.69% when compared at aminoacidic level. Phylogenetic analysis of partial OFR2 regions of these contigs indicate its closer similarity with sequences from California Sea Lion astroviruses, a virus that was determined as to be the most prevalent in fecal samples from these animals (Z. californianus)37. This finding suggests that these sequences may belong to a yet unknown virus like Z. californianus astrovirus and may indicate that such virus is prevalent in the sampled area (detected in 2/4 fecal pools studied) and the second more abundant virus (1008 reads) in the studied fecal samples (Fig. 3).Figure 3Phylogenetic consensus tree based on partial ORF2 sequences from the Mamastrovirus contigs sequenced from A. gazella scats (in bold). Bootstrap resampling with 1000 replicates.Full size imageAdeno associated virus 2Two of the studied fecal pools presented 138 sequences, forming 3 contigs with nucleotide identities ranging from 46.91 to 48.04% (Table 1), that matched adeno associated viruses previously described in Z. californianus, humans and other mammals with and unknow etiologic role (Fig. 4). The detected sequences probably correspond to fur seal adeno associated viruses never described before. The detection of these viruses is quite common in other mammals suggesting they could cause persistent infections in their hosts, but no etiological role has been attributed to them38.Figure 4Phylogenetic consensus tree of the Adeno-associated virus contigs sequenced from A. gazella scats (in bold). Bootstrap resampling with 1000 replicates.Full size imageNorovirusA norovirus contig was obtained in one of the four pools analyzed. Noroviruses are the most relevant non-bacterial gastroenteritis etiological agents in humans39, with its presence widely described in other mammals40. The contig detected in the fecal samples, represented the 4.43% of the viral genome, was in the VP1 region and comprised 56 reads with an identity  > 99% to California sea lion norovirus described by Teng and collaborators in 201841 (Fig. 5). Results obtained suggest these sequences belong to a putative new norovirus specie.Figure 5Phylogenetic consensus tree of the Norovirus contig sequenced from A. gazella scats (in bold). Bootstrap resampling with 1000 replicates.Full size imageViruses in serum samplesAll the viral sequences obtained from serum samples (970 reads) matched to CRESS-DNA viral sequences from unknown hosts.The fact that no other viruses were identified in serum samples suggests the animals tested were not under active viremia at the time of sample collection or it was not detectable by the applied methodology.Diet related virusesSeveral virus sequences similar to viruses known to have invertebrate animals as hosts were detected in fecal pools, mainly by UVM although some also by TES. These viruses are probably present in fur seal feces because of dietary habits although, since scats were collected from the ground nearby the animals, environmental cross-contamination should not be ruled out.Sequences with high coverage or similarities to any described virus are showed in Table 2.The high prevalence of virus sequences from crustaceans in the feces analyzed is hardly surprising because A. gazella inhabiting the Antarctic peninsula and the Atlantic sector of the Southern Ocean feed mostly on Antarctic krill Euphasia superba during the summer months42,43,44,45,46,47,48. Sequences from cephalopod viruses were also detected, although were much scarcer than those from crustaceans. This also agrees with current knowledge about the diet of A. gazella in the Atlantic sector of the Southern Ocean, where octopuses and squids are regularly consumed, although in low numbers44,45,46. It is worth noting than not cephalopod beak was recovered from the scats analyzed here48. Among all invertebrate viruses identified, some sequences present low identities with genomes from available databases, probably because Antarctica wildlife has been scarcely explored, forcing bioinformatic analysis to match them with the most similar viruses from these databases.No fish viruses were found in this study. Hard skeletal remains of fishes are often recovered from the scats of A. gazella from the Atlantic sector of the Southern Ocean42,43,44,45,46,47 and occurred indeed in the samples analysed here48, but stable isotope analysis of blood and whiskers revealed a negligible contribution of fish to the assimilate diet of juvenile and subadult male A. gazella49, which likely explain the absence of fish viruses in the samples analized here. Additionaly, no data on the virome present in the fish species regularly consumed by A. gazella has been published to our knowledge, with information limited to the bacteriome32, so even in case fish viruses were sequenced, it might not be correctly assigned to a fish host. Nevertheless, the methodology applied in this study had been successfully applied to the identification of the virome of Atlantic fishes50. Furthermore, Li and coworkers.37 and Wille and coworkers.22 also observed viral sequences probably corresponding to fish when analyzing the fecal virome of the California sea lions and Antarctic penguins.On the other hand, sequences highly similar to Coelho and Khabarov viral polymerases (greater than 98% of aminoacid identity), previously described in chinstrap penguins (Pygoscelis antarcticus) by Wille and coworkers22, were found in this study. The consumption of penguins by A. gazella during the summer months has been reported widely51,52,53,54,55, penguins feathers were reported from the scats analyzed in this study48 and stable isotope analysis of blood and whiskers revealed penguins as the second most relevant prey from juvenile and subadult male A. gazella in the population studied here49. This evidence is consistent with the presence of virus from chinstrap penguins in the samples analysed here. All in all, the study of fecal virome constitutes a very promising tool to explore the consumers’ diet. More