Metabarcoding data (quality filtered chimera-free merged reads) from the two MiSeq runs needed to... more Metabarcoding data (quality filtered chimera-free merged reads) from the two MiSeq runs needed to generate the paper dataset
The potential of the 18S rRNA V9 metabarcoding approach for diet assessment was explored using Mi... more The potential of the 18S rRNA V9 metabarcoding approach for diet assessment was explored using MiSeq paired-end (PE; 2 × 150 bp) technology. To critically evaluate the method′s performance with degraded/digested DNA, the diets of two zooplanktivorous fish species from the Bay of Biscay, European sardine (Sardina pilchardus) and European sprat (Sprattus sprattus), were analysed. The taxonomic resolution and quantitative potential of the 18S V9 metabarcoding was first assessed both in silico and with mock and field plankton samples. Our method was capable of discriminating species within the reference database in a reliable way providing there was at least one variable position in the 18S V9 region. Furthermore, it successfully discriminated diet between both fish species, including habitat and diel differences among sardines, overcoming some of the limitations of traditional visual-based diet analysis methods. The high sensitivity and semi-quantitative nature of the 18S V9 metabarcoding approach was supported by both visual microscopy and qPCR-based results. This molecular approach provides an alternative cost and time effective tool for food-web analysis
"Puncher et al_Thunnus thynnus_384 SNPs with flanking sequences.txt" contains the flank... more "Puncher et al_Thunnus thynnus_384 SNPs with flanking sequences.txt" contains the flanking sequences of 384 SNPs discovered using reduced representation sequence libraries, 221 of which passed quality criteria and 95 that were used for population structure analysis
The Atlantic bluefin tuna is a highly migratory species emblematic of the challenges associated w... more 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 genome-wide 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=0.034). 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
A fine scale spatial resolution survey (3 15 nautical miles) was conducted during May 2004 in the... more A fine scale spatial resolution survey (3 15 nautical miles) was conducted during May 2004 in the Bay of Biscay (43.32–46.128N and 1.29–4.318W), to study the zooplankton commu-nity during the onset of spring stratification. Cluster analysis classified the 45 most abundant taxa into seven major groups. In the southern part of the surveyed area, a front separating neritic waters from eddies off the shelf delimited distinct zooplankton communities. On the northern side of the surveyed area, river plumes and the generation of internal waves over the shelf break were the main mesoscale structures determining the composition and abundance of the zooplankton assemblages. Canonical correspondence analysis (CCA) and generalized additive models (GAMs) were used to investigate the relationship between zooplankton species distribution and selected environmental variables (sea surface temperature and salinity along with water column stratification and fluor-escence pattern). Surface salinity and...
Natural mortality, due to predation, of juvenile plaice (Pleuronectes platessa) was estimated in ... more Natural mortality, due to predation, of juvenile plaice (Pleuronectes platessa) was estimated in a Scottish nursery ground (Tralee Beach) in the spring of 2009 by means of using a DNA based method (TaqMan real-time PCR based assay), targeting a plaice-specific cytochrome-b sequence, in the DNA extracted from potential predators' stomach contents. The assay was run in 1305 stomachs from a set of predators thought to be important sources of mortality of small juvenile plaice on inshore nursery grounds (including the brown shrimp, Crangon crangon, the shore crab, Carcinus maenas, and several fish species). Previous studies of predation on juvenile plaice have used visual examination of stomach contents but this is time-consuming and may underestimate true predation levels as remains may become unidentifiable due to maceration and digestion. Molecular based tools for detecting the presence of prey tissue in predator stomachs and scat are becoming increasingly used in marine ecology and provide an alternative or complementary approach to visual identification. In this sense, while 45.2 % of the brown shrimp's stomachs tested positive for plaice DNA after running the assay, only 5.7 % and 10.5 % did for, respectively, the shore crab and gobies. Results are discussed in relation to different biological and environmental variables.
The Atlantic bluefin tuna is a highly migratory species emblematic of the challenges associated w... more 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 genome-wide 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 Mediter...
Background: The introduction of NIS to estuaries and coastal embayment is of great concern. Comme... more Background: The introduction of NIS to estuaries and coastal embayment is of great concern. Commercial ships' ballast water discharge and the northwards progression of species due to the ongoing climate change arise as the main factors explaining the rising occurrence of NIS species in Northern Atlantic waters. In this regard, regular monitoring of the plankton communities is paramount as to be able to respond properly to this potential issue. Results: While monitoring the invasive copepod Acartia tonsa populations in the estuaries of Bilbao and Urdaibai (Basque country, Spain), we report here the Asian copepod Pseudodiaptomus marinus for the first time in the Iberian Peninsula waters. Individuals from both species were collected from July to October, 2013 for DNA sequencing of mitochondrial cytochrome c oxidase subunit I gene (MT-CO1). Phylogenetic analysis of MT-CO1 confirmed P. marinus identity. Conclusions: Phylogeographic distribution of A. tonsa haplotypes in Europe along with the Bilbao port traffic patterns suggested a secondary invasion from an European source to Basque estuaries. The successful establishment of the A. tonsa population and the appearance of Pseudodiaptomus marinus confirm the need for regular plankton monitoring of estuarine and port waters. This applies also to nearby systems as these populations could represent a source of future dispersal.
Metabarcoding (large-scale taxonomic identification of complex samples via analysis of one or few... more Metabarcoding (large-scale taxonomic identification of complex samples via analysis of one or few orthologous DNA regions, called barcodes) is revolutionizing analysis of biodiversity of marine zooplankton assemblages. Metabarcoding relies on high-throughput DNA sequencing (HTS) technologies, which yield millions of DNA sequences in parallel and allow large-scale analysis of environmental samples. Metabarcoding studies of marine zooplankton have used various regions of nuclear small-(18S) and large-subunit (28S) rRNA, which allow accurate classification of novel sequences and reliable amplification with consensus primers, but-due to their relatively conserved nature-may underestimate species diversity in a community. To discriminate species, more variable genes are needed. A limited number of metabarcoding studies have used mitochondrial cytochrome oxidase I (COI), which ensures detection of species-level diversity, but may require group-specific primers and thus result in inconsistent amplification success rates. Reference databases with sequences for accurately-identified species are critically needed to allow taxonomic designation of molecular operational taxonomic units (MOTU) and comparison with previous studies of zooplankton diversity. Potential and promising applications of metabarcoding include rapid detection of impacts of climate change, monitoring and assessment of ecosystem health, calculation of biotic indices, characterization of food webs and detection of introduced, non-indigenous species.
which increased the taxonomic assignment success from 23.7 to 50.5 %. When the communities were s... more which increased the taxonomic assignment success from 23.7 to 50.5 %. When the communities were studied along with environmental variables, similar spatial and temporal trends of taxonomic diversity were observed for metabarcoding and microscopic studies of zooplankton, but not for phytoplankton. This is most likely attributable to the lack of representative sequences for phytoplankton species in current databases. In addition, there was high correspondence in community composition when comparing abundances estimated from metabarcoding and microscopy, suggesting semiquantitative potential for metabarcoding. Furthermore, metabarcoding allowed the detection and identification of two non-indigenous species (NIS) found in the study area at abundances hardly detectable by microscopy. Overall, our results indicate that metabarcoding is a powerful approach with excellent possibilities for use in plankton monitoring, early detection of NIS and plankton biodiversity shifts.
The potential of the 18S rRNA V9 metabarcoding approach for diet assessment was explored using Mi... more The potential of the 18S rRNA V9 metabarcoding approach for diet assessment was explored using MiSeq paired-end (PE; 2 9 150 bp) technology. To critically evaluate the method 0 s performance with degraded/digested DNA, the diets of two zooplanktivorous fish species from the Bay of Biscay, European sardine (Sardina pilchardus) and European sprat (Sprattus sprattus), were analysed. The taxonomic resolution and quantitative potential of the 18S V9 metabarcoding was first assessed both in silico and with mock and field plankton samples. Our method was capable of discriminating species within the reference database in a reliable way providing there was at least one variable position in the 18S V9 region. Furthermore, it successfully discriminated diet between both fish species, including habitat and diel differences among sardines, overcoming some of the limitations of traditional visual-based diet analysis methods. The high sensitivity and semi-quantitative nature of the 18S V9 metabarcoding approach was supported by both visual microscopy and qPCR-based results. This molecular approach provides an alternative cost and time effective tool for food-web analysis.
Single nucleotide polymorphisms (SNPs) are the most abundant form of genetic variation and are hi... more Single nucleotide polymorphisms (SNPs) are the most abundant form of genetic variation and are highly adaptable to large-scale automated genotyping and population genetics studies. For nonmodel organisms, many SNP discovery projects are based on sequencing and assembly of a transcriptome and the calling of sequence variation in contigs. This paper develops a new method for avoiding intron/exon boundaries in genotyping primer design, based on the mapping of raw genome reads to a transcriptome assembly. Applied to a project in European anchovy SNP discovery, the attained conversion rate of 91.6% is the highest yet reported for a nonmodel teleost SNP discovery project.
Increased throughput in sequencing technologies has facilitated the acquisition of detailed genom... more Increased throughput in sequencing technologies has facilitated the acquisition of detailed genomic information in non-model species. The focus of this research was to discover and validate SNPs derived from the European anchovy (Engraulis encrasicolus) transcriptome, a species with no available reference genome, using next generation sequencing technologies. A cDNA library was constructed from four tissues of ten fish individuals corresponding to three populations of E. encrasicolus, and Roche 454 GS FLX Titanium sequencing yielded 19,367 contigs. Additionally, the European anchovy genome was sequenced for the same ten individuals using an Illumina HiSeq2000. Using a computational pipeline for combining transcriptome and genome information, a total of 18,994 SNPs met the necessary minor allele frequency and depth filters. A series of further stringent filters were applied to identify those SNPs likely to succeed in genotyping assays, and for filtering of those in potential duplicat...
Metabarcoding data (quality filtered chimera-free merged reads) from the two MiSeq runs needed to... more Metabarcoding data (quality filtered chimera-free merged reads) from the two MiSeq runs needed to generate the paper dataset
The potential of the 18S rRNA V9 metabarcoding approach for diet assessment was explored using Mi... more The potential of the 18S rRNA V9 metabarcoding approach for diet assessment was explored using MiSeq paired-end (PE; 2 × 150 bp) technology. To critically evaluate the method′s performance with degraded/digested DNA, the diets of two zooplanktivorous fish species from the Bay of Biscay, European sardine (Sardina pilchardus) and European sprat (Sprattus sprattus), were analysed. The taxonomic resolution and quantitative potential of the 18S V9 metabarcoding was first assessed both in silico and with mock and field plankton samples. Our method was capable of discriminating species within the reference database in a reliable way providing there was at least one variable position in the 18S V9 region. Furthermore, it successfully discriminated diet between both fish species, including habitat and diel differences among sardines, overcoming some of the limitations of traditional visual-based diet analysis methods. The high sensitivity and semi-quantitative nature of the 18S V9 metabarcoding approach was supported by both visual microscopy and qPCR-based results. This molecular approach provides an alternative cost and time effective tool for food-web analysis
"Puncher et al_Thunnus thynnus_384 SNPs with flanking sequences.txt" contains the flank... more "Puncher et al_Thunnus thynnus_384 SNPs with flanking sequences.txt" contains the flanking sequences of 384 SNPs discovered using reduced representation sequence libraries, 221 of which passed quality criteria and 95 that were used for population structure analysis
The Atlantic bluefin tuna is a highly migratory species emblematic of the challenges associated w... more 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 genome-wide 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=0.034). 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
A fine scale spatial resolution survey (3 15 nautical miles) was conducted during May 2004 in the... more A fine scale spatial resolution survey (3 15 nautical miles) was conducted during May 2004 in the Bay of Biscay (43.32–46.128N and 1.29–4.318W), to study the zooplankton commu-nity during the onset of spring stratification. Cluster analysis classified the 45 most abundant taxa into seven major groups. In the southern part of the surveyed area, a front separating neritic waters from eddies off the shelf delimited distinct zooplankton communities. On the northern side of the surveyed area, river plumes and the generation of internal waves over the shelf break were the main mesoscale structures determining the composition and abundance of the zooplankton assemblages. Canonical correspondence analysis (CCA) and generalized additive models (GAMs) were used to investigate the relationship between zooplankton species distribution and selected environmental variables (sea surface temperature and salinity along with water column stratification and fluor-escence pattern). Surface salinity and...
Natural mortality, due to predation, of juvenile plaice (Pleuronectes platessa) was estimated in ... more Natural mortality, due to predation, of juvenile plaice (Pleuronectes platessa) was estimated in a Scottish nursery ground (Tralee Beach) in the spring of 2009 by means of using a DNA based method (TaqMan real-time PCR based assay), targeting a plaice-specific cytochrome-b sequence, in the DNA extracted from potential predators' stomach contents. The assay was run in 1305 stomachs from a set of predators thought to be important sources of mortality of small juvenile plaice on inshore nursery grounds (including the brown shrimp, Crangon crangon, the shore crab, Carcinus maenas, and several fish species). Previous studies of predation on juvenile plaice have used visual examination of stomach contents but this is time-consuming and may underestimate true predation levels as remains may become unidentifiable due to maceration and digestion. Molecular based tools for detecting the presence of prey tissue in predator stomachs and scat are becoming increasingly used in marine ecology and provide an alternative or complementary approach to visual identification. In this sense, while 45.2 % of the brown shrimp's stomachs tested positive for plaice DNA after running the assay, only 5.7 % and 10.5 % did for, respectively, the shore crab and gobies. Results are discussed in relation to different biological and environmental variables.
The Atlantic bluefin tuna is a highly migratory species emblematic of the challenges associated w... more 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 genome-wide 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 Mediter...
Background: The introduction of NIS to estuaries and coastal embayment is of great concern. Comme... more Background: The introduction of NIS to estuaries and coastal embayment is of great concern. Commercial ships' ballast water discharge and the northwards progression of species due to the ongoing climate change arise as the main factors explaining the rising occurrence of NIS species in Northern Atlantic waters. In this regard, regular monitoring of the plankton communities is paramount as to be able to respond properly to this potential issue. Results: While monitoring the invasive copepod Acartia tonsa populations in the estuaries of Bilbao and Urdaibai (Basque country, Spain), we report here the Asian copepod Pseudodiaptomus marinus for the first time in the Iberian Peninsula waters. Individuals from both species were collected from July to October, 2013 for DNA sequencing of mitochondrial cytochrome c oxidase subunit I gene (MT-CO1). Phylogenetic analysis of MT-CO1 confirmed P. marinus identity. Conclusions: Phylogeographic distribution of A. tonsa haplotypes in Europe along with the Bilbao port traffic patterns suggested a secondary invasion from an European source to Basque estuaries. The successful establishment of the A. tonsa population and the appearance of Pseudodiaptomus marinus confirm the need for regular plankton monitoring of estuarine and port waters. This applies also to nearby systems as these populations could represent a source of future dispersal.
Metabarcoding (large-scale taxonomic identification of complex samples via analysis of one or few... more Metabarcoding (large-scale taxonomic identification of complex samples via analysis of one or few orthologous DNA regions, called barcodes) is revolutionizing analysis of biodiversity of marine zooplankton assemblages. Metabarcoding relies on high-throughput DNA sequencing (HTS) technologies, which yield millions of DNA sequences in parallel and allow large-scale analysis of environmental samples. Metabarcoding studies of marine zooplankton have used various regions of nuclear small-(18S) and large-subunit (28S) rRNA, which allow accurate classification of novel sequences and reliable amplification with consensus primers, but-due to their relatively conserved nature-may underestimate species diversity in a community. To discriminate species, more variable genes are needed. A limited number of metabarcoding studies have used mitochondrial cytochrome oxidase I (COI), which ensures detection of species-level diversity, but may require group-specific primers and thus result in inconsistent amplification success rates. Reference databases with sequences for accurately-identified species are critically needed to allow taxonomic designation of molecular operational taxonomic units (MOTU) and comparison with previous studies of zooplankton diversity. Potential and promising applications of metabarcoding include rapid detection of impacts of climate change, monitoring and assessment of ecosystem health, calculation of biotic indices, characterization of food webs and detection of introduced, non-indigenous species.
which increased the taxonomic assignment success from 23.7 to 50.5 %. When the communities were s... more which increased the taxonomic assignment success from 23.7 to 50.5 %. When the communities were studied along with environmental variables, similar spatial and temporal trends of taxonomic diversity were observed for metabarcoding and microscopic studies of zooplankton, but not for phytoplankton. This is most likely attributable to the lack of representative sequences for phytoplankton species in current databases. In addition, there was high correspondence in community composition when comparing abundances estimated from metabarcoding and microscopy, suggesting semiquantitative potential for metabarcoding. Furthermore, metabarcoding allowed the detection and identification of two non-indigenous species (NIS) found in the study area at abundances hardly detectable by microscopy. Overall, our results indicate that metabarcoding is a powerful approach with excellent possibilities for use in plankton monitoring, early detection of NIS and plankton biodiversity shifts.
The potential of the 18S rRNA V9 metabarcoding approach for diet assessment was explored using Mi... more The potential of the 18S rRNA V9 metabarcoding approach for diet assessment was explored using MiSeq paired-end (PE; 2 9 150 bp) technology. To critically evaluate the method 0 s performance with degraded/digested DNA, the diets of two zooplanktivorous fish species from the Bay of Biscay, European sardine (Sardina pilchardus) and European sprat (Sprattus sprattus), were analysed. The taxonomic resolution and quantitative potential of the 18S V9 metabarcoding was first assessed both in silico and with mock and field plankton samples. Our method was capable of discriminating species within the reference database in a reliable way providing there was at least one variable position in the 18S V9 region. Furthermore, it successfully discriminated diet between both fish species, including habitat and diel differences among sardines, overcoming some of the limitations of traditional visual-based diet analysis methods. The high sensitivity and semi-quantitative nature of the 18S V9 metabarcoding approach was supported by both visual microscopy and qPCR-based results. This molecular approach provides an alternative cost and time effective tool for food-web analysis.
Single nucleotide polymorphisms (SNPs) are the most abundant form of genetic variation and are hi... more Single nucleotide polymorphisms (SNPs) are the most abundant form of genetic variation and are highly adaptable to large-scale automated genotyping and population genetics studies. For nonmodel organisms, many SNP discovery projects are based on sequencing and assembly of a transcriptome and the calling of sequence variation in contigs. This paper develops a new method for avoiding intron/exon boundaries in genotyping primer design, based on the mapping of raw genome reads to a transcriptome assembly. Applied to a project in European anchovy SNP discovery, the attained conversion rate of 91.6% is the highest yet reported for a nonmodel teleost SNP discovery project.
Increased throughput in sequencing technologies has facilitated the acquisition of detailed genom... more Increased throughput in sequencing technologies has facilitated the acquisition of detailed genomic information in non-model species. The focus of this research was to discover and validate SNPs derived from the European anchovy (Engraulis encrasicolus) transcriptome, a species with no available reference genome, using next generation sequencing technologies. A cDNA library was constructed from four tissues of ten fish individuals corresponding to three populations of E. encrasicolus, and Roche 454 GS FLX Titanium sequencing yielded 19,367 contigs. Additionally, the European anchovy genome was sequenced for the same ten individuals using an Illumina HiSeq2000. Using a computational pipeline for combining transcriptome and genome information, a total of 18,994 SNPs met the necessary minor allele frequency and depth filters. A series of further stringent filters were applied to identify those SNPs likely to succeed in genotyping assays, and for filtering of those in potential duplicat...
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Papers by Aitor Albaina