The genome sequence of the European shag, Gulosus aristotelis (previously Phalacrocorax aristotelis) (Linnaeus, 1761)
Hannah M. Ravenswater, Fiona Greco, Sarah J. Burthe, Emma J. A. Cunningham, Xianzhao Kan, Nicolas J. Rawlence
TL;DR
This paper presents the genome sequence of the European shag, a bird species, including detailed annotations of its genes and chromosomes.
Contribution
The paper provides a high-quality genome assembly and gene annotation for the European shag.
Findings
The genome assembly spans 1,279.1 megabases and includes 36 chromosomal pseudomolecules.
Gene annotation identified 16,474 protein-coding genes using Ensembl.
The mitochondrial genome is 18.61 kilobases in length.
Abstract
We present a genome assembly from an individual female Gulosus aristotelis, previously known as Phalacrocorax aristotelis, (the European shag; Chordata; Aves; Pelecaniformes; Phalacrocoracidae). The genome sequence is 1,279.1 megabases in span. Most of the assembly is scaffolded into 36 chromosomal pseudomolecules, including the Z and W sex chromosomes. The mitochondrial genome has also been assembled and is 18.61 kilobases in length. Gene annotation of this assembly on Ensembl identified 16,474 protein coding genes.
Genes, proteins, chemicals, diseases, species, mutations and cell lines named across the full text — each resolved to its canonical identifier and authoritative record.
Click any figure to enlarge with its caption.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5| Project accession data | ||
|---|---|---|
| Assembly identifier | bGulAri2.1 | |
| Species |
| |
| Specimen | bGulAri2 | |
| NCBI taxonomy ID | 126867 | |
| BioProject | PRJEB57282 | |
| BioSample ID | SAMEA10059652 | |
| Isolate information | bGulAri2, female: blood sample (DNA, Hi-C and RNA sequencing) | |
| Assembly metrics
|
| |
| Consensus quality (QV) | 61.7 |
|
|
| 100.0% |
|
| BUSCO
| C:97.2%[S:96.6%,D:0.6%],F:0.5%,M:2.2%,n:8,338 |
|
| Percentage of assembly mapped to chromosomes | 96.21% |
|
| Sex chromosomes | ZW |
|
| Organelles | Mitochondrial genome: 18.61 kb |
|
| Raw data accessions | ||
| PacificBiosciences SEQUEL II | ERR10462081, ERR10462082 | |
| Hi-C Illumina | ERR10466815 | |
| PolyA RNA-Seq Illumina | ERR11606292 | |
| Genome assembly | ||
| Assembly accession | GCA_949628215.1 | |
|
| GCA_949628205.1 | |
| Span (Mb) | 1,279.1 | |
| Number of contigs | 575 | |
| Contig N50 length (Mb) | 11.4 | |
| Number of scaffolds | 352 | |
| Scaffold N50 length (Mb) | 78.9 | |
| Longest scaffold (Mb) | 219.24 | |
| Genome annotation | ||
| Number of protein-coding genes | 16,474 | |
| Number of non-coding genes | 1,001 | |
| Number of gene transcripts | 26,595 | |
| INSDC accession | Chromosome | Length (Mb) | GC% |
|---|---|---|---|
| 1 | 219.24 | 41.0 | |
| 2 | 168.56 | 40.5 | |
| 3 | 131.34 | 41.0 | |
| 4 | 84.76 | 40.5 | |
| 5 | 71.72 | 42.0 | |
| 6 | 63.33 | 43.0 | |
| 7 | 55.81 | 43.0 | |
| 8 | 46.83 | 43.5 | |
| 9 | 39.05 | 42.0 | |
| 10 | 26.91 | 44.5 | |
| 11 | 24.85 | 44.0 | |
| 12 | 24.68 | 43.5 | |
| 13 | 19.06 | 47.0 | |
| 14 | 17.93 | 42.0 | |
| 15 | 17.37 | 46.0 | |
| 16 | 14.99 | 47.5 | |
| 17 | 13.73 | 48.0 | |
| 18 | 13.32 | 46.5 | |
| 19 | 9.84 | 47.5 | |
| 20 | 9.08 | 51.0 | |
| 21 | 8.89 | 52.5 | |
| 22 | 8.36 | 49.0 | |
| 23 | 8.19 | 52.5 | |
| 24 | 7.62 | 50.5 | |
| 25 | 3.89 | 57.0 | |
| 26 | 3.57 | 58.5 | |
| 27 | 2.68 | 47.5 | |
| 28 | 1.64 | 59.0 | |
| 29 | 1.11 | 54.5 | |
| 30 | 0.71 | 62.0 | |
| 31 | 0.63 | 57.5 | |
| 32 | 0.33 | 63.0 | |
| 33 | 0.23 | 61.5 | |
| 34 | 0.23 | 59.5 | |
| W | 31.35 | 47.5 | |
| Z | 78.89 | 40.5 | |
| MT | 0.02 | 44.5 |
| Software tool | Version | Source |
|---|---|---|
| BlobToolKit | 4.1.7 |
|
| BUSCO | 5.3.2 |
|
| gEVAL | N/A |
|
| Hifiasm | 0.16.1 |
|
| HiGlass | 1.11.6 |
|
| Merqury | MerquryFK |
|
| MitoHiFi | 2 |
|
| PretextView | 0.2 |
|
| purge_dups | 1.2.3 |
|
| sanger-tol/genomenote | v1.0 |
|
| sanger-tol/readmapping | 1.1.0 |
|
| YaHS | yahs-1.1.91eebc2 |
|
- —Wellcome Trust
- —NERC
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Taxonomy
TopicsGenetic diversity and population structure · Identification and Quantification in Food · Genomics and Phylogenetic Studies
Species taxonomy
Eukaryota; Opisthokonta; Metazoa; Eumetazoa; Bilateria; Deuterostomia; Chordata; Craniata; Vertebrata; Gnathostomata; Teleostomi; Euteleostomi; Sarcopterygii; Dipnotetrapodomorpha; Tetrapoda; Amniota; Sauropsida; Sauria; Archelosauria; Archosauria; Dinosauria; Saurischia; Theropoda; Coelurosauria; Aves; Neognathae; Suliformes; Phalacrocoracidae; Phalacrocorax; Phalacrocorax aristotelis (Linnaeus, 1761) (NCBI:txid126867).
Background
Gulosus aristotelis (previously known as Phalacrocorax aristotelis) and commonly known as the European shag, is a large seabird species within the cormorant family, Phalacrocoridae ( Croxall, 1987; Thanou et al., 2017). European shags have dark-green plumage in adulthood but that can often appear black, a yellow gape, green eyes, black legs and feet and a crest during the breeding season ( Wanless & Harris, 1997). Juvenile plumage is lighter brown and present up to two years old ( Wanless & Harris, 1997). The species displays sexual dimorphism, with males weighing 1900 g on average, compared to 1600 g in females ( Daunt et al., 2001). Sex is typically identified using vocalisations in adults ( Snow, 1960) and molecular techniques in juveniles ( Thanou et al., 2013).
European Shags are benthic foot-propelled pursuit divers, with a partially wettable plumage that facilitates diving in shallow waters but requires individuals to return to shore each day to dry and roost ( Grémillet et al., 1998), increasing their vulnerability to inclement weather conditions. The species’ diet primarily consists of fish with some predation of crustations, with both temporal and spatial variability consumption of prey species ( Howells et al., 2017, Howells et al., 2018; Velando & Freire, 1999).
Gulosus aristotelis breeds colonially, creating nest sites on cliff ledges or cavities under rocks ( Velando & Freire, 2001; Wanless & Harris, 1997). European shags start breeding at 2 to 3 years ( Aebischer, 1986), with the lifespan of over 14 years on average ( Herborn et al., 2014), with the oldest individual recorded on the Isle of May, an important seabird breeding colony where shags have been intensively monitored since the 1970s, at 23 years-old ( Hall, 2004). Breeding occurs seasonally, with shags laying on average a clutch of three eggs, which hatch asynchronously after an approximately 35-day incubation period ( Granroth-Wilding et al., 2014; Snow, 1960). Both sexes provide parental care, in the form of incubation and provisioning until fledging of chicks at approximately 55 days ( Daunt et al., 2007; Snow, 1960). During the non-breeding season, European shags remain coastal but may migrate variable distances from their breeding location. For example, on the Isle of May in Scotland, approximately 50% of the population migrates during the winter, with half the population remaining resident at the breeding area and half migrating up and down the entire East coast of the UK and, more rarely, across the North Sea to the Netherlands ( Acker et al., 2023).
G.aristotelis has a range that covers most of the coastline of Europe, with high concentrations the Atlantic coast and Mediterranean, and smaller populations at its southern range limit on the coast of North Africa ( GBIF Secretariat, 2023). With a European breeding population estimated at 152,000 ( BirdLife International, 2021), European shags are listed as ‘least concern’ but decreasing on the IUCN red list ( BirdLife International, 2018), with threats including climate change and extreme weather events, changing prey availability, pollution and disease ( BirdLife International, 2018; Hicks et al., 2019). Similarly to other seabirds, European shags are ecologically important, not only in their role as marine predators but as key indicators of marine ecosystem health and environmental change ( Parsons et al., 2008; Piatt & Sydeman, 2007).
Long term monitoring programmes of seabird colonies, including European shags, such as that carried out on the Isle of May in Scotland (from which this specimen originates), are crucial in building a picture of the interacting processes that threaten seabirds. Therefore, the availability of this complete reference genome is a vital step in combining molecular tools with existing large life history datasets. For example, this genome will immediately aid the completion of an epigenetic clock, widening access to age data for birds that have not been individually marked as chicks, as well as the impact of stressors on biological aging where birds are of known age. This includes responses to infection and the genome sequence will be used to explore the molecular mechanisms of resistance and immunity against parasitism and disease, including long term chronic infections with parasites that are ubiquitous across individuals ( Granroth-Wilding et al., 2017), and species differences in response to recent Avian Influenza outbreaks.
The genome of the European shag, Gulosus aristotelis, was sequenced as part of the Darwin Tree of Life Project and the Vertebrate Genomes Project (VGP). Here we present a chromosomally complete genome sequence for Gulosus aristotelis, based on one female specimen from the Isle of May National Nature Reserve, Scotland.
Genome sequence report
The genome was sequenced from a blood sample taken from one female Gulosus aritotelis (previously Phalacrocorax aristotelis) ( Figure 1) temporarily caught under licence from Isle of May National Nature Reserve, Scotland, UK (56.19, –2.57). A total of 42-fold coverage in Pacific Biosciences single-molecule HiFi long reads was generated. Primary assembly contigs were scaffolded with chromosome conformation Hi-C data. Manual assembly curation corrected 52 missing joins or mis-joins, reducing the scaffold number by 9.49%, and decreasing the scaffold N50 by 6.93%.
Photograph of a Gulosus aristotelis (previously Phalacrocorax aristotelis) (bGulAri2) from same study population as the specimen used for genome sequencing.Photograph by Fiona Greco.
The final assembly has a total length of 1,279.1 Mb in 352 sequence scaffolds with a scaffold N50 of 78.9 Mb ( Table 1). The snail plot in Figure 2 provides a summary of the assembly statistics, while the distribution of assembly scaffolds on GC proportion and coverage is shown in Figure 3. The cumulative assembly plot in Figure 4 shows curves for subsets of scaffolds assigned to different phyla. Most (96.21%) of the assembly sequence was assigned to 36 chromosomal-level scaffolds, representing 34 autosomes and the Z and W sex chromosomes. Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size ( Figure 5; Table 2). While not fully phased, the assembly deposited is of one haplotype. Contigs corresponding to the second haplotype have also been deposited. The mitochondrial genome was also assembled and can be found as a contig within the multifasta file of the genome submission.
Table 1.: Genome data for Phalacrocorax aristotelis, bGulAri2.1.
Genome assembly of Gulosus aristotelis, (previously Phalacrocorax aristotelis), bGulAri2.1: metrics.The BlobToolKit Snailplot shows N50 metrics and BUSCO gene completeness. The main plot is divided into 1,000 size-ordered bins around the circumference with each bin representing 0.1% of the 1,279,134,750 bp assembly. The distribution of scaffold lengths is shown in dark grey with the plot radius scaled to the longest scaffold present in the assembly (219,240,020 bp, shown in red). Orange and pale-orange arcs show the N50 and N90 scaffold lengths (78,889,319 and 13,321,506 bp), respectively. The pale grey spiral shows the cumulative scaffold count on a log scale with white scale lines showing successive orders of magnitude. The blue and pale-blue area around the outside of the plot shows the distribution of GC, AT and N percentages in the same bins as the inner plot. A summary of complete, fragmented, duplicated and missing BUSCO genes in the aves_odb10 set is shown in the top right. An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/bGulAri2_1/dataset/bGulAri2_1/snail.
Genome assembly of Phalacrocorax aristotelis, bGulAri2.1: BlobToolKit GC-coverage plot.Scaffolds are coloured by phylum. Circles are sized in proportion to scaffold length. Histograms show the distribution of scaffold length sum along each axis. An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/bGulAri2_1/dataset/bGulAri2_1/blob.
Genome assembly of Gulosus aristotelis, bGulAri2.1: BlobToolKit cumulative sequence plot.The grey line shows cumulative length for all scaffolds. Coloured lines show cumulative lengths of scaffolds assigned to each phylum using the buscogenes taxrule. An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/bGulAri2_1/dataset/bGulAri2_1/cumulative.
Genome assembly of Gulosus aristotelis, bGulAri2.1: Hi-C contact map of the bGulAri2.1 assembly, visualised using HiGlass.Chromosomes are shown in order of size from left to right and top to bottom. An interactive version of this figure may be viewed at https://genome-note-higlass.tol.sanger.ac.uk/l/?d=AXw7sVdmRrWp1DFeFKZT-Q.
Table 2.: Chromosomal pseudomolecules in the genome assembly of Phalacrocorax aristotelis, bGulAri2.
The estimated Quality Value (QV) of the final assembly is 61.7 with k-mer completeness of 100.0%, and the assembly has a BUSCO v5.3.2 completeness of 97.2% (single = 96.6%, duplicated = 0.6%), using the aves_odb10 reference set ( n = 8,338).
Metadata for specimens, barcode results, spectra estimates, sequencing runs, contaminants and pre-curation assembly statistics are given at https://links.tol.sanger.ac.uk/species/126867.
Genome annotation report
The Gulosus aritotelis (previously Phalacrocorax aristotelis) genome assembly (GCA_949628215.1) was annotated at the European Bioinformatics Institute (EBI) using the Ensembl rapid annotation pipeline. The resulting annotation includes 26,595 transcribed mRNAs from 16,474 protein-coding and 1,001 non-coding genes ( Table 1; https://rapid.ensembl.org/Phalacrocorax_aristotelis_GCA_949628215.1/Info/Index).
Methods
Sample acquisition and nucleic acid extraction
A blood sample was taken from a female Gulosus aristotelis (previously Phalacrocorax aristotelis) (specimen ID SAN0001768, ToLID bGulAri2) from the Isle of May National Nature Reserve, Scotland, UK (latitude 56.19, longitude –2.57) on 2021-06-29. The species was identified by Hannah Ravenswater (University of Edinburgh) and, Fiona Greco (University of Edinburgh) and Sarah Burthe (UK Centre for Ecology & Hydrology). Capture occurred via crook at the nest site during late chick rearing, and the individual released to the same location. Blood sampling was conducted by appropriately trained personal license holders, acting under a UK Home Office Project License in accordance with the Animals (Scientific Procedures) Act 1986. The blood sample was collected via brachial venepuncture of the live specimen using a 25-gauge needle. 75 μl blood was placed in 500 μl 95% ethanol, and then frozen at –80 °C within 120 mins.
The workflow for high molecular weight (HMW) DNA extraction at the Wellcome Sanger Institute (WSI) includes a sequence of core procedures: sample preparation; sample homogenisation, DNA extraction, fragmentation, and clean-up. In sample preparation, the bGulAri2 sample was weighed and dissected on dry ice ( Jay et al., 2023). The blood sample was homogenised using a PowerMasher II tissue disruptor ( Denton et al., 2023a).
HMW DNA was extracted using the Nanobind whole blood protocol ( Pacific Biosciences et al., 2023). DNA was sheared into an average fragment size of 12–20 kb in a Megaruptor 3 system with speed setting 31 ( Bates et al., 2023). Sheared DNA was purified by solid-phase reversible immobilisation ( Oatley et al., 2023): in brief, the method employs a 1.8X ratio of AMPure PB beads to sample to eliminate shorter fragments and concentrate the DNA. The concentration of the sheared and purified DNA was assessed using a Nanodrop spectrophotometer and Qubit Fluorometer and Qubit dsDNA High Sensitivity Assay kit. Fragment size distribution was evaluated by running the sample on the FemtoPulse system.
RNA was extracted from a blood sample from bGulAri2 in the Tree of Life Laboratory at the WSI using the RNA Extraction: Automated MagMax™ mirVana protocol ( do Amaral et al., 2023). The RNA concentration was assessed using a Nanodrop spectrophotometer and a Qubit Fluorometer using the Qubit RNA Broad-Range Assay kit. Analysis of the integrity of the RNA was done using the Agilent RNA 6000 Pico Kit and Eukaryotic Total RNA assay.
Protocols developed by the WSI Tree of Life laboratory are publicly available on protocols.io ( Denton et al., 2023b).
Sequencing
Pacific Biosciences HiFi circular consensus DNA sequencing libraries were constructed according to the manufacturers’ instructions. Poly(A) RNA-Seq libraries were constructed using the NEB Ultra II RNA Library Prep kit. DNA and RNA sequencing was performed by the Scientific Operations core at the WSI on Pacific Biosciences SEQUEL II (HiFi) and Illumina NovaSeq 6000 (RNA-Seq) instruments. Hi-C data were also generated from a blood sample from bGulAri2 using the Arima2 kit and sequenced on the Illumina NovaSeq 6000 instrument.
Genome assembly, curation and evaluation
Assembly was carried out with Hifiasm ( Cheng et al., 2021) and haplotypic duplication was identified and removed with purge_dups ( Guan et al., 2020). The assembly was then scaffolded with Hi-C data ( Rao et al., 2014) using YaHS ( Zhou et al., 2023). The assembly was checked for contamination and corrected using the gEVAL system ( Chow et al., 2016) as described previously ( Howe et al., 2021). Manual curation was performed using gEVAL, HiGlass ( Kerpedjiev et al., 2018) and PretextView ( Harry, 2022). The mitochondrial genome was assembled using MitoHiFi ( Uliano-Silva et al., 2023), which runs MitoFinder ( Allio et al., 2020) or MITOS ( Bernt et al., 2013) and uses these annotations to select the final mitochondrial contig and to ensure the general quality of the sequence.
A Hi-C map for the final assembly was produced using bwa-mem2 ( Vasimuddin et al., 2019) in the Cooler file format ( Abdennur & Mirny, 2020). To assess the assembly metrics, the k-mer completeness and QV consensus quality values were calculated in Merqury ( Rhie et al., 2020). This work was done using Nextflow ( Di Tommaso et al., 2017) DSL2 pipelines “sanger-tol/readmapping” ( Surana et al., 2023a) and “sanger-tol/genomenote” ( Surana et al., 2023b). The genome was analysed within the BlobToolKit environment ( Challis et al., 2020) and BUSCO scores ( Manni et al., 2021; Simão et al., 2015) were calculated.
Table 3 contains a list of relevant software tool versions and sources.
Genome annotation
The Ensembl Genebuild annotation system ( Aken et al., 2016) at the EBI was used to generate annotation for the Gulosus aritotelis assembly (GCA_949628215.1). Annotation was created primarily through alignment of transcriptomic data to the genome, with gap filling via protein-to-genome alignments of a select set of proteins from UniProt ( UniProt Consortium, 2019).
Wellcome Sanger Institute – Legal and Governance
The materials that have contributed to this genome note have been supplied by a Darwin Tree of Life Partner. The submission of materials by a Darwin Tree of Life Partner is subject to the ‘Darwin Tree of Life Project Sampling Code of Practice’, which can be found in full on the Darwin Tree of Life website here. By agreeing with and signing up to the Sampling Code of Practice, the Darwin Tree of Life Partner agrees they will meet the legal and ethical requirements and standards set out within this document in respect of all samples acquired for, and supplied to, the Darwin Tree of Life Project.
Further, the Wellcome Sanger Institute employs a process whereby due diligence is carried out proportionate to the nature of the materials themselves, and the circumstances under which they have been/are to be collected and provided for use. The purpose of this is to address and mitigate any potential legal and/or ethical implications of receipt and use of the materials as part of the research project, and to ensure that in doing so we align with best practice wherever possible. The overarching areas of consideration are:
• Ethical review of provenance and sourcing of the material
• Legality of collection, transfer and use (national and international)
Each transfer of samples is further undertaken according to a Research Collaboration Agreement or Material Transfer Agreement entered into by the Darwin Tree of Life Partner, Genome Research Limited (operating as the Wellcome Sanger Institute), and in some circumstances other Darwin Tree of Life collaborators.
The reference list from the paper itself. Each links out to its DOI / PubMed record.
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- 2Acker P Daunt F Wanless S : Additive genetic and environmental variation interact to shape the dynamics of seasonal migration in a wild bird population. Evolution. 2023;77(10):2128–2143. 10.1093/evolut/qpad 111 37343301 · doi ↗ · pubmed ↗
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- 4Aken BL Ayling S Barrell D : The Ensembl gene annotation system. Database (Oxford). 2016;2016: baw 093. 10.1093/database/baw 093 27337980 PMC 4919035 · doi ↗ · pubmed ↗
- 5Allio R Schomaker-Bastos A Romiguier J : Mito Finder: Efficient automated large‐scale extraction of mitogenomic data in target enrichment phylogenomics. Mol Ecol Resour. 2020;20(4):892–905. 10.1111/1755-0998.13160 32243090 PMC 7497042 · doi ↗ · pubmed ↗
- 6Bates A Clayton-Lucey I Howard C : Sanger Tree of Life HMW DNA Fragmentation: Diagenode Megaruptor® 3 for LI Pac Bio. Protocols.Io. 2023. 10.17504/Protocols.Io.81wgbxzq 3lpk/V 1 · doi ↗
- 7Bernt M Donath A Jühling F : MITOS: Improved de novo metazoan mitochondrial genome annotation. Mol Phylogenet Evol. 2013;69(2):313–319. 10.1016/j.ympev.2012.08.023 22982435 · doi ↗ · pubmed ↗
- 8Bird Life International: Gulosus aristotelis. The IUCN Red List of Threatened Species. 2018.