Stress Response to Climate Change and Postharvest Handling in Two Differently Pigmented Lettuce Genotypes: Impact on Alternaria alternata Invasion and Mycotoxin Production.

Many species of Alternaria are important pathogens that cause plant diseases and postharvest rots. They lead to significant economic losses in agriculture and affect human and animal health due to their capacity to produce mycotoxins. Therefore, it is necessary to study the factors that can result in an increase in A. alternata. In this study, we discuss the mechanism by which phenol content protects from A. alternata, since the red oak leaf cultivar (containing higher phenols) showed lower invasion than the green one, Batavia, and no mycotoxin production. A climate change scenario enhanced fungal growth in the most susceptible cultivar, green lettuce, likely because elevated temperature and CO2 levels decrease plant N content, modifying the C/N ratio. Finally, while the abundance of the fungi was maintained at similar levels after keeping the lettuces for four days at 4 °C, this postharvest handling triggered TeA and TEN mycotoxin synthesis, but only in the green cultivar. Therefore, the results demonstrated that invasion and mycotoxin production are cultivar- and temperature-dependent. Further research should be directed to search for resistant cultivars and effective postharvest strategies to reduce the toxicological risk and economic losses related to this fungus, which are expected to increase in a climate change scenario.

A short exposure to a semi-natural habitat alleviates the honey bee hive microbial imbalance caused by agricultural stress.

Honeybee health and the species' gut microbiota are interconnected. Also noteworthy are the multiple niches present within hives, each with distinct microbiotas and all coexisting, which we termed "apibiome". External stressors (e.g. anthropization) can compromise microbial balance and bee resilience. We hypothesised that (1) the bacterial communities of hives located in areas with different degrees of anthropization differ in composition, and (2) due to interactions between the multiple microbiomes within the apibiome, changes in the community of a niche would impact the bacteria present in other hive sections. We characterised the bacterial consortia of different niches (bee gut, bee bread, hive entrance and internal hive air) of 43 hives from 3 different environments (agricultural, semi-natural and natural) through 16S rRNA amplicon sequencing. Agricultural samples presented lower community evenness, depletion of beneficial bacteria, and increased recruitment of stress related pathways (predicted via PICRUSt2). The taxonomic and functional composition of gut and hive entrance followed an environmental gradient. Arsenophonus emerged as a possible indicator of anthropization, gradually decreasing in abundance from agriculture to the natural environment in multiple niches. Importantly, after 16 days of exposure to a semi-natural landscape hives showed intermediate profiles, suggesting alleviation of microbial dysbiosis through reduction of anthropization.

Novel strategies to improve chicken performance and welfare by unveiling host-microbiota interactions through hologenomics.

Fast optimisation of farming practices is essential to meet environmental sustainability challenges. Hologenomics, the joint study of the genomic features of animals and the microbial communities associated with them, opens new avenues to obtain in-depth knowledge on how host-microbiota interactions affect animal performance and welfare, and in doing so, improve the quality and sustainability of animal production. Here, we introduce the animal trials conducted with broiler chickens in the H2020 project HoloFood, and our strategy to implement hologenomic analyses in light of the initial results, which despite yielding negligible effects of tested feed additives, provide relevant information to understand how host genomic features, microbiota development dynamics and host-microbiota interactions shape animal welfare and performance. We report the most relevant results, propose hypotheses to explain the observed patterns, and outline how these questions will be addressed through the generation and analysis of animal-microbiota multi-omic data during the HoloFood project.

Population Structure and Diversity in European Honey Bees (Apismellifera L.)-An Empirical Comparison of Pool and Individual Whole-Genome Sequencing.

BACKGROUND: Whole-genome sequencing has become routine for population genetic studies. Sequencing of individuals provides maximal data but is rather expensive and fewer samples can be studied. In contrast, sequencing a pool of samples (pool-seq) can provide sufficient data, while presenting less of an economic challenge. Few studies have compared the two approaches to infer population genetic structure and diversity in real datasets. Here, we apply individual sequencing (ind-seq) and pool-seq to the study of Western honey bees (Apis mellifera). METHODS: We collected honey bee workers that belonged to 14 populations, including 13 subspecies, totaling 1347 colonies, who were individually (139 individuals) and pool-sequenced (14 pools). We compared allele frequencies, genetic diversity estimates, and population structure as inferred by the two approaches. RESULTS: Pool-seq and ind-seq revealed near identical population structure and genetic diversities, albeit at different costs. While pool-seq provides genome-wide polymorphism data at considerably lower costs, ind-seq can provide additional information, including the identification of population substructures, hybridization, or individual outliers. CONCLUSIONS: If costs are not the limiting factor, we recommend using ind-seq, as population genetic structure can be inferred similarly well, with the advantage gained from individual genetic information. Not least, it also significantly reduces the effort required for the collection of numerous samples and their further processing in the laboratory.

Recovering High-Quality Host Genomes from Gut Metagenomic Data through Genotype Imputation.

Metagenomic datasets of host-associated microbial communities often contain host DNA that is usually discarded because the amount of data is too low for accurate host genetic analyses. However, genotype imputation can be employed to reconstruct host genotypes if a reference panel is available. Here, the performance of a two-step strategy is tested to impute genotypes from four types of reference panels built using different strategies to low-depth host genome data (≈2× coverage) recovered from intestinal samples of two chicken genetic lines. First, imputation accuracy is evaluated in 12 samples for which both low- and high-depth sequencing data are available, obtaining high imputation accuracies for all tested panels (>0.90). Second, the impact of reference panel choice in population genetics statistics on 100 chickens is assessed, all four panels yielding comparable results. In light of the observations, the feasibility and application of the applied imputation strategy are discussed for different species with regard to the host DNA proportion, genomic diversity, and availability of a reference panel. This method enables leveraging insofar discarded host DNA to get insights into the genetic structure of host populations, and in doing so, facilitates the implementation of hologenomic approaches that jointly analyze host and microbial genomic data.

Transcriptomic dataset for Sardina pilchardus: Assembly, annotation, and expression of nine tissues.

European sardine or pilchard is a planktonic small pelagic fish present from the North Sea in Europe to the coast of Senegal in the North of Africa, and across the Mediterranean sea to the Black Sea. Ecologically, sardines are an intermediary link in the trophic network, preying on plankton and being predated by larger fishes, marine mammals, and seabirds. This species is of great nutritional and economic value as a cheap but rich source of protein and fat. It is either consumed directly by humans or fed as fishmeal for aquaculture and farm animals. Despite its importance in the food basket, little is known about the molecular mechanisms involved in protein and lipid synthesis in this species. We collected nine tissues of Sardina pilchardus and reconstructed the transcriptome. In all, 198,597 transcripts were obtained, from which 68,031 are protein-coding. Quality assessment of the transcriptome was performed by back-mapping reads to the transcriptome and by searching for Single Copy Orthologs. Additionally, Gene Ontology and KEGG annotations were retrieved for most of the protein-coding genes. Finally, each library was quantified in terms of Transcripts per Million to disclose their expression patterns.

Impact of dimethylpyrazole-based nitrification inhibitors on soil-borne bacteria.

Nitrogen (N) input from fertilizers modifies the properties of agricultural soils as well as bacterial community diversity, composition and relationships. This can lead to negative impacts such as the deterioration of system multifunctionality, whose maintenance is critical to normal nutrient cycling. Synthetic nitrification inhibitors (NIs) can be combined with fertilizers to improve the efficiency of N use by reducing N losses. However, analysis of their effects on non-target bacteria are scarce. This study aimed to analyze the effect of applying the NIs DMPP and DMPSA on the whole bacterial community. Through 16S rRNA amplicon sequencing we determined the differences between samples in terms of microbial diversity, composition and co-occurrence networks. The application of DMPP and DMPSA exerted little impact on the abundance of the dominant phyla. Nevertheless, several significant shifts were detected in bacterial diversity, co-occurrence networks, and the abundance of particular taxa, where soil water content played a key role. For instance, the application of NIs intensified the negative impact of N fertilization on bacterial diversity under high water-filled pore spaces (WFPS) (>64%), reducing community diversity, whereas alpha-diversity was not affected at low WFPS (<55%). Interestingly, despite NIs are known to inhibit ammonia monooxygenase (AMO) enzyme, both NIs almost exclusively inhibited Nitrosomonas genera among AMO holding nitrifiers. Thus, Nitrosomonas showed abundance reductions of up to 47% (DMPP) and 66% (DMPSA). Nonetheless, non-target bacterial abundances also shifted with NI application. Notably, DMPSA application partially alleviated the negative effect of fertilization on soil multifunctionality. A remarkable increase in populations related to system multifunctionality, such as Armatimonadetes (up to +21%), Cyanobacteria (up to +30%) and Fibrobacteres (up to +25%) was observed when DMPSA was applied. NI application substantially influenced microbial associations by decreasing the complexity of co-occurrence networks, decreasing the total edges and node connectivity, and increasing path distances.

Authoritative subspecies diagnosis tool for European honey bees based on ancestry informative SNPs.

BACKGROUND: With numerous endemic subspecies representing four of its five evolutionary lineages, Europe holds a large fraction of Apis mellifera genetic diversity. This diversity and the natural distribution range have been altered by anthropogenic factors. The conservation of this natural heritage relies on the availability of accurate tools for subspecies diagnosis. Based on pool-sequence data from 2145 worker bees representing 22 populations sampled across Europe, we employed two highly discriminative approaches (PCA and FST) to select the most informative SNPs for ancestry inference. RESULTS: Using a supervised machine learning (ML) approach and a set of 3896 genotyped individuals, we could show that the 4094 selected single nucleotide polymorphisms (SNPs) provide an accurate prediction of ancestry inference in European honey bees. The best ML model was Linear Support Vector Classifier (Linear SVC) which correctly assigned most individuals to one of the 14 subspecies or different genetic origins with a mean accuracy of 96.2% ± 0.8 SD. A total of 3.8% of test individuals were misclassified, most probably due to limited differentiation between the subspecies caused by close geographical proximity, or human interference of genetic integrity of reference subspecies, or a combination thereof. CONCLUSIONS: The diagnostic tool presented here will contribute to a sustainable conservation and support breeding activities in order to preserve the genetic heritage of European honey bees.

Response of Horticultural Soil Microbiota to Different Fertilization Practices.

Environmentally friendly agricultural production necessitates manipulation of microbe-plant interactions, requiring a better understanding of how farming practices influence soil microbiota. We studied the effect of conventional and organic treatment on soil bacterial richness, composition, and predicted functional potential. 16S rRNA sequencing was applied to soils from adjacent plots receiving either a synthetic or organic fertilizer, where two crops were grown within treatment, homogenizing for differences in soil properties, crop, and climate. Conventional fertilizer was associated with a decrease in soil pH, an accumulation of Ag, Mn, As, Fe, Co, Cd, and Ni; and an enrichment of ammonia oxidizers and xenobiotic compound degraders (e.g., Candidatus Nitrososphaera, Nitrospira, Bacillus, Pseudomonas). Soils receiving organic fertilization were enriched in Ti (crop biostimulant), N, and C cycling bacteria (denitrifiers, e.g., Azoarcus, Anaerolinea; methylotrophs, e.g., Methylocaldum, Methanosarcina), and disease-suppression (e.g., Myxococcales). Some predicted functions, such as glutathione metabolism, were slightly, but significantly enriched after a one-time manure application, suggesting the enhancement of sulfur regulation, nitrogen-fixing, and defense of environmental stressors. The study highlights that even a single application of organic fertilization is enough to originate a rapid shift in soil prokaryotes, responding to the differential substrate availability by promoting soil health, similar to recurrent applications.

Digging into the Genomic Past of Swiss Honey Bees by Whole-Genome Sequencing Museum Specimens.

Historical specimens in museum collections provide opportunities to gain insights into the genomic past. For the Western honey bee, Apis mellifera L., this is particularly important because its populations are currently under threat worldwide and have experienced many changes in management and environment over the last century. Using Swiss Apis mellifera mellifera as a case study, our research provides important insights into the genetic diversity of native honey bees prior to the industrial-scale introductions and trade of non-native stocks during the 20th century-the onset of intensive commercial breeding and the decline of wild honey bees following the arrival of Varroa destructor. We sequenced whole-genomes of 22 honey bees from the Natural History Museum in Bern collected in Switzerland, including the oldest A. mellifera sample ever sequenced. We identify both, a historic and a recent migrant, natural or human-mediated, which corroborates with the population history of honey bees in Switzerland. Contrary to what we expected, we find no evidence for a significant genetic bottleneck in Swiss honey bees, and find that genetic diversity is not only maintained, but even slightly increased, most probably due to modern apicultural practices. Finally, we identify signals of selection between historic and modern honey bee populations associated with genes enriched in functions linked to xenobiotics, suggesting a possible selective pressure from the increasing use and diversity of chemicals used in agriculture and apiculture over the last century.

EXFI: Exon and splice graph prediction without a reference genome.

For population genetic studies in nonmodel organisms, it is important to use every single source of genomic information. This paper presents EXFI, a Python pipeline that predicts the splice graph and exon sequences using an assembled transcriptome and raw whole-genome sequencing reads. The main algorithm uses Bloom filters to remove reads that are not part of the transcriptome, to predict the intron-exon boundaries, to then proceed to call exons from the assembly, and to generate the underlying splice graph. The results are returned in GFA1 format, which encodes both the predicted exon sequences and how they are connected to form transcripts. EXFI is written in Python, tested on Linux platforms, and the source code is available under the MIT License at https://github.com/jlanga/exfi.

Genetic structure of Iranian indigenous sheep breeds: insights for conservation.

Genetic structure and genetic diversity levels of indigenous Iranian sheep breeds are not clear, despite the interest this region has in itself as an important center for domestication of livestock. Early population genetic studies have reported high levels of diversity among Iranian sheep breeds until recently, when high admixture levels and genetic homogeneity have been detected. The rapid reduction of diversity observed in Iranian breeds might be due to an increasing trend of intensive crossbreeding practices or even total replacement of native breeds by highly specialized and productive ones. From a conservative perspective, this situation is highly concerning; thus, it might be wise to consider a conservation program in Iran to preserve the original genetic diversity in native sheep breeds. In this study, a total of 1065 animals with the purest morphological features representing 24 Iranian indigenous sheep breeds were sampled, corresponding to ancestral breed diversity. These samples were genotyped for 17 microsatellite loci in order to (1) determine the native ancestral diversity of Iranian breeds, (2) define the degree of genetic relationship among studied breeds, and (3) assess conservation priorities among defined groups. Our results showed no recent loss of diversity, but high genetic diversity levels for indigenous sheep breeds in Iran. Indeed, the analysis of conservation priorities pointed out the importance of 8 breeds for maintaining Iranian sheep breeds' maximum genetic diversity. Thus, under a genetic perspective, these 8 breeds should be the ones included into conservation programs for restocking endangered areas.

A novel transcriptome-derived SNPs array for tench (Tinca tinca L.).

Tench (Tinca tinca L.) has great economic potential due to its high rate of fecundity and long-life span. Population genetic studies based on allozymes, microsatellites, PCR-RFLP and sequence analysis of genes and DNA fragments have revealed the presence of Eastern and Western phylogroups. However, the lack of genomic resources for this species has complicated the development of genetic markers. In this study, the tench transcriptome and genome were sequenced by high-throughput sequencing. A total of 60,414 putative SNPs were identified in the tench transcriptome using a computational pipeline. A set of 96 SNPs was selected for validation and a total of 92 SNPs was validated, resulting in the highest conversion and validation rate for a non-model species obtained to date (95.83%). The validated SNPs were used to genotype 140 individuals belonging to two tench breeds (Tabor and Hungarian), showing low (FST = 0.0450) but significant (<0.0001) genetic differentiation between the two tench breeds. This implies that set of validated SNPs array can be used to distinguish the tench breeds and that it might be useful for studying a range of associations between DNA sequence and traits of importance. These genomic resources created for the tench will provide insight into population genetics, conservation fish stock management, and aquaculture.

Hygroregulation, a key ability for eusocial insects: Native Western European honeybees as a case study.

Sociality has brought many advantages to various hymenoptera species, including their ability of regulating physical factors in their nest (e.g., temperature). Although less studied, humidity is known to be important for egg, larval and pupal development, and also for nectar concentration. Two subspecies of Apis mellifera of the M evolutionary lineage were used as models to test the ability of a superorganism (i.e. honeybee colony) to regulate the humidity in its nest (i.e. "hygroregulation hypothesis") in four conservation centers: two in France (A. m. mellifera) and two in Portugal (A. m. iberiensis). We investigated the ability of both subspecies to regulate the humidity in hives daily, but also during the seasons for one complete year. Our data and statistical analysis demonstrated the capacity of the bees to regulate humidity in their hive, regardless of the day, season or subspecies. Furthermore, the study showed that humidity in beehives is stable even during winter, when brood is absent, and when temperature is known to be less stable in the beehives. These results suggest that humidity is important for honeybees at every life stage, maybe because of the 'imprint' of the evolutionary history of this hymenopteran lineage.

Association between combinations of genetic polymorphisms and epidemiopathogenic forms of bovine paratuberculosis.

Control of major mycobacterial diseases affecting livestock is a challenging issue that requires different approaches. The use of genetic markers for improving resistance to Mycobacterium avium subsp. paratuberculosis infection in cattle has been explored as a promising population strategy We performed paratuberculosis epidemiopathogenic phenotypic and genotypic characterization involving 24 SNPs in six candidate genes (NOD2, CD209, SLC11A1, SP110, TLR2 and TLR4) on 502 slaughtered Friesian cows. In the current study, we investigate whether recently proposed paratuberculosis (PTB) epidemiopathogenic (EP) forms (apparently free-AF, latent-LAT and patent-PAT) could be associated with some combination of these 24 SNPs. Best EP form grouping was obtained using a combination of 5 SNPs in four genes (CD209: rs210748127; SLC11A1: rs110090506; SP110: rs136859213 and rs110480812; and TLR2: rs41830058). These groups were defined according to the level of infection progression risk to patent epidemiopathogenic forms and showed the following distributions: LOWIN (low) with 39 (8%) cases (94.9% AF/5.1% LAT/0% PAT); LATIN (low) with 17 (3%) cases (5.9% AF/94.1% LAT/0% PAT); AVERIN (average) with 413 (82%) cases (52.1% AF/38.5% LAT/9.4% PAT) and PATIN (patent) with 33 (7%) cases (36.4% AF/24.2% LAT/39.4% PAT). Age of slaughter was significantly higher for LATIN (88.3 months) compared to AVERIN (65.3 months; p = 0.0007) and PATIN (59.1 months; p = 0.0004), and for LOWIN (73.9 months) compared to PATIN (p = 0.0233), and nearly significant compared to AVERIN (p = 0.0572) These results suggest that some selected genetic polymorphisms have a potential use as markers of PTB EP forms and thus add a new tool for the control of this widespread infection.

Genomic selection signatures in sheep from the Western Pyrenees.

BACKGROUND: The current large spectrum of sheep phenotypic diversity results from the combined product of sheep selection for different production traits such as wool, milk and meat, and its natural adaptation to new environments. In this study, we scanned the genome of 25 Sasi Ardi and 75 Latxa sheep from the Western Pyrenees for three types of regions under selection: (1) regions underlying local adaptation of Sasi Ardi semi-feral sheep, (2) regions related to a long traditional dairy selection pressure in Latxa sheep, and (3) regions experiencing the specific effect of the modern genetic improvement program established for the Latxa breed during the last three decades. RESULTS: Thirty-two selected candidate regions including 147 annotated genes were detected by using three statistical parameters: pooled heterozygosity H, Tajima's D, and Wright's fixation index Fst. For Sasi Ardi sheep, chromosomes Ovis aries (OAR)4, 6, and 22 showed the strongest signals and harbored several candidate genes related to energy metabolism and morphology (BBS9, ELOVL3 and LDB1), immunity (NFKB2), and reproduction (H2AFZ). The major genomic difference between Sasi Ardi and Latxa sheep was on OAR6, which is known to affect milk production, with highly selected regions around the ABCG2, SPP1, LAP3, NCAPG, LCORL, and MEPE genes in Latxa sheep. The effect of the modern genetic improvement program on Latxa sheep was also evident on OAR15, on which several olfactory genes are located. We also detected several genes involved in reproduction such as ESR1 and ZNF366 that were affected by this selection program. CONCLUSIONS: Natural and artificial selection have shaped the genome of both Sasi Ardi and Latxa sheep. Our results suggest that Sasi Ardi traits related to energy metabolism, morphological, reproductive, and immunological features have been under positive selection to adapt this semi-feral sheep to its particular environment. The highly selected Latxa sheep for dairy production showed clear signatures of selection in genomic regions related to milk production. Furthermore, our data indicate that the selection criteria applied in the modern genetic improvement program affect immunity and reproduction traits.

Spatial dynamics and mixing of bluefin tuna in the Atlantic Ocean and Mediterranean Sea revealed using next-generation sequencing.

The Atlantic bluefin tuna is a highly migratory species emblematic of the challenges associated with shared fisheries management. In an effort to resolve the species' stock dynamics, a genomewide search for spatially informative single nucleotide polymorphisms (SNPs) was undertaken, by way of sequencing reduced representation libraries. An allele frequency approach to SNP discovery was used, combining the data of 555 larvae and young-of-the-year (LYOY) into pools representing major geographical areas and mapping against a newly assembled genomic reference. From a set of 184,895 candidate loci, 384 were selected for validation using 167 LYOY. A highly discriminatory genotyping panel of 95 SNPs was ultimately developed by selecting loci with the most pronounced differences between western Atlantic and Mediterranean Sea LYOY. The panel was evaluated by genotyping a different set of LYOY (n = 326), and from these, 77.8% and 82.1% were correctly assigned to western Atlantic and Mediterranean Sea origins, respectively. The panel revealed temporally persistent differentiation among LYOY from the western Atlantic and Mediterranean Sea (FST  = 0.008, p = .034). The composition of six mixed feeding aggregations in the Atlantic Ocean and Mediterranean Sea was characterized using genotypes from medium (n = 184) and large (n = 48) adults, applying population assignment and mixture analyses. The results provide evidence of persistent population structuring across broad geographic areas and extensive mixing in the Atlantic Ocean, particularly in the mid-Atlantic Bight and Gulf of St. Lawrence. The genomic reference and genotyping tools presented here constitute novel resources useful for future research and conservation efforts.

Unraveling the environmental and anthropogenic drivers of bacterial community changes in the Estuary of Bilbao and its tributaries.

In this study, 16S rRNA gene sequencing was used to characterize the changes in taxonomic composition and environmental factors significantly influencing bacterial community structure across an annual cycle in the Estuary of Bilbao as well as its tributaries. In spite of this estuary being small and characterized by a short residence time, the environmental factors most highly correlated with the bacterial community mirrored those reported to govern larger estuaries, specifically salinity and temperature. Additionally, bacterial community changes in the estuary appeared to vary with precipitation. For example, an increase in freshwater bacteria (Comamonadaceae and Sphingobacteriaceae) was observed in high precipitation periods compared to the predominately marine-like bacteria (Rhodobacterales and Oceanospirillales) that were found in low precipitation periods. Notably, we observed a significantly higher relative abundance of Comamonadaceae than previously described in other estuaries. Furthermore, anthropic factors could have an impact on this particular estuary's bacterial community structure. For example, ecosystem changes related to the channelization of the estuary likely induced a low dissolved oxygen (DO) concentration, high temperature, and high chlorophyll concentration period in the inner euhaline water in summer (samples with salinity >30 ppt). Those samples were characterized by a high abundance of facultative anaerobes. For instance, OTUs classified as Cryomorphaceae and Candidatus Aquiluna rubra were negatively associated with DO concentration, while Oleiphilaceae was positively associated with DO concentration. Additionally, microorganisms related to biological treatment of wastewater (e.g Bdellovibrio and Zoogloea) were detected in the samples immediately downstream of the Bilbao Wastewater Treatment Plant (WWTP). There are several human activities planned in the region surrounding the Estuary of Bilbao (e.g. sediment draining, architectural changes, etc.) which will likely affect this ecosystem. Therefore, the addition of bacterial community profiling and diversity analysis into the estuary's ongoing monitoring program would provide a more comprehensive view of the ecological status of the Estuary of Bilbao.

Insights on the drivers of genetic divergence in the European anchovy.

Anchovies represent the largest world's marine fish catches and the current threats on their populations impose a sustainable exploitment based on sound scientific information. In the European anchovy (Engraulis encrasicolus), the existence of several populations has been proposed but a global view is missing. Using a multidisciplinary approach, here we assessed the divergence among different ecotypes and its possible causes. SNPs have revealed two functionally distinct ecotypes overlapping in the Central Mediterranean, with one ecotype confined near the river estuaries. The same SNPs outliers also segregated two distinct populations in the near Atlantic, despite their large spatial distance. In addition, while most studies suggested that adaptation to low salinity is key to divergence, here we show that the offshore ecotype has higher environmental tolerance and an opportunistic feeding behaviour, as assessed by the study of environmental conditions, anchovy diet and trophic levels, and passive egg dispersal. These results provide insights into the anchovy evolutionary history, stressing the importance of behaviour in shaping ecotypes.