The genome sequence of the Large Brook Dun, Ecdyonurus torrentis (Kimmins, 1942)

We present a genome assembly from an individual female Ecdyonurus torrentis (the Large Brook Dun; Arthropoda; Insecta; Ephemeroptera; Heptageniidae). The genome sequence is 503.2 megabases in span. Most of the assembly is scaffolded into 11 chromosomal pseudomolecules, including the X sex chromosome. The mitochondrial genome has also been assembled and is 15.69 kilobases in length.


Background
Ecdyonurus torrentis (Figure 1) is a western Palearctic species found across central Europe from France to Ukraine.It is absent from Fennoscandia.It is found throughout Britain and Ireland, generally in northern and western areas, although there are scattered records from the south-east of England and the south of Ireland (GBIF Secretariat, 2023).
This species is considered a eurytherm and is typically found in the upper and middle reaches of watercourses (Buffagni et al., 2009).It is found at a range of altitudes, from upland streams to lowland watercourses.Larvae of E. torrentis are typically found in riffle areas of rivers and streams.They are usually found clinging to submerged stones, although they may swim if disturbed (Elliott et al., 1988).
Ecdyonurus torrentis is univoltine, overwintering as larvae and adults emerging between May and September (Elliott et al., 1988;Landa, 1968;Macan, 1957;Sowa, 1975;Sowa, 1979;Wise, 1980).In some years adults can be found as early as March.The flight period is often related to the altitude of the emergence site.In upstream reaches the flight period can last for up to three months, whereas at lower altitudes the flight period may be as short as one month (Wise, 1980).The larvae feed by either scraping algae from the substrate or gathering fine particulate organic matter from the sediment (Elliott et al., 1988).
The genome sequence for Ecdyonurus torrentis will aid in understanding the biology, physiology and ecology of the species and the relationship between this species and other Ecdyonurus species in Europe.

Genome sequence report
The genome was sequenced from one female Ecdyonurus torrentis collected from River Rye, Yorkshire, UK (54.21, -0.98).A total of 67-fold coverage in Pacific Biosciences single-molecule HiFi long reads and 76-fold coverage in 10X Genomics read clouds were generated.Primary assembly contigs were scaffolded with chromosome conformation Hi-C data.Manual assembly curation corrected 200 missing joins or mis-joins and removed 45 haplotypic duplications, reducing the assembly length by 1.77% and the scaffold number by 42.45%, and increasing the scaffold N50 by 1.06%.
The final assembly has a total length of 503.2 Mb in 121 sequence scaffolds with a scaffold N50 of 50.4 Mb (Table 1).The snailplot in Figure 2 summarises the assembly statistics is shown in Figure 2, 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 (97.25%) of the assembly sequence was assigned to 11 chromosomal-level scaffolds, representing 10 autosomes and the X sex chromosome.The X chromosome was identified by BUSCO synteny alignment to the curated male sample Siphlonurus alternatus (GCA_949825025.1).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.
Metadata for specimens, spectral estimates, sequencing runs, contaminants and pre-curation assembly statistics can be found at https://links.tol.sanger.ac.uk/species/2014018.

Sample acquisition and nucleic acid extraction
A female Ecdyonurus torrentis (specimen ID NHMUK014361706, ToLID ieEcdTorr1) was collected from River Rye, Yorkshire, UK (latitude 54.21, longitude -0.98) on 2019-05-06 using a kicknet.The specimen was collected and identified by Andrew Farr (independent researcher) and snap-frozen on dry ice.
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI).The ieEcdTorr1 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing.Posterior body tissue was cryogenically disrupted to a fine powder using a Covaris cryoPREP Automated Dry Pulveriser, receiving multiple impacts.High molecular weight  (HMW) DNA was extracted using the Qiagen MagAttract HMW DNA extraction kit.Low molecular weight DNA was removed from a 20 ng aliquot of extracted DNA using the 0.8X AMpure XP purification kit prior to 10X Chromium sequencing; a minimum of 50 ng DNA was submitted for 10X sequencing.HMW DNA was sheared into an average fragment size of 12-20 kb in a Megaruptor 3 system with speed setting 30.Sheared DNA was purified by solid-phase reversible immobilisation using AMPure PB beads with a 1.8X ratio of beads to sample to remove the shorter fragments and concentrate the DNA sample.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.

Sequencing
Pacific Biosciences HiFi circular consensus and 10X Genomics read cloud DNA sequencing libraries were constructed according to the manufacturers' instructions.DNA sequencing was performed by the Scientific Operations core at the WSI on Pacific Biosciences SEQUEL II (HiFi) and HiSeq X Ten (10X) instruments.Hi-C data were also generated from remaining tissue of ieEcdTorr1 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 Pretext (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.
Table 3 contains a list of relevant software tool versions and sources.

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

Is the rationale for creating the dataset(s) clearly described? Yes
Are the protocols appropriate and is the work technically sound?Yes

Are sufficient details of methods and materials provided to allow replication by others? Yes
Are the datasets clearly presented in a useable and accessible format?Yes Competing Interests: No competing interests were disclosed.
I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Isabel Almudi
University of Barcelona, Barcelona, Spain This data note presents the genome assembly of the mayfly Ecdyonurus torrentis from the Heptageniidae family of Ephemeroptera.Due to the relevance of this group for evolutionary and ecological studies, this genome assembly, and forthcoming Ephemeroptera genomes, will have great impact in these fields.
In order to generate this assembly, an adult female was collected and processed.The genome is 503,2 megabases assembled in 10 autosomes, one X chromosome and a genome.The quality of the assembly is excellent with high completeness as shown by BUSCO.Overall, the methods employed are adequate and clearly listed in the note.
Is the rationale for creating the dataset(s) clearly described?Yes Are the protocols appropriate and is the work technically sound?Yes

Are sufficient details of methods and materials provided to allow replication by others? Yes
Are the datasets clearly presented in a useable and accessible format?Yes Competing Interests: No competing interests were disclosed.
Reviewer Expertise: insect genomics, evolution & development I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Figure 1 .
Figure 1.Photograph of Ecdyonurus torrentis (not the specimen used for genome sequencing) by David Nicholls, Lea Meadows, 22 May 2015.

Figure 2 .
Figure 2. Genome assembly of Ecdyonurus torrentis, ieEcdTorr1.1: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 357,657,189 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 (90,518,639 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (65,719,635 and 55,360,379 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 diptera_odb10 set is shown in the top right.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/idCriRanu1.1/dataset/CATOTT01/snail.

Figure 5 .
Figure 5. Genome assembly of Ecdyonurus torrentis, ieEcdTorr1.1:Hi-C contact map of the ieEcdTorr1.1 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=GE7fh8rORWifzZkFK1tYag.

Table 2 . Chromosomal pseudomolecules in the genome assembly of Ecdyonurus torrentis, ieEcdTorr1. INSDC accession Chromosome Length (Mb) GC%
This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.