The genome sequence of the Northern Summer Mayfly, Siphlonurus alternatus (Say, 1824)

We present a genome assembly from an individual male Siphlonurus alternatus (the Northern Summer Mayfly; Arthropoda; Insecta; Ephemeroptera; Siphlonuridae). The genome sequence is 455.8 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 19.36 kilobases in length.


Background
Siphlonurus alternatus (Figure 1) is a Holarctic species found in Canada and the northern United States, and across Northern Europe, particularly Fennoscandia (GBIF Secretariat, 2023).In Britain and Ireland, it is a northern species with a highly localised distribution.It is anticipated that future surveys in south-west Scotland will turn up further records of this species (Macadam, 2019).This species is likely to be a eurytherm and is typically found in the middle reaches of watercourses (Buffagni et al., 2009).
It is found at a range of altitudes, from the foothills to lowland watercourses.Larvae of this species typically live in deep pools in rivers and streams, but can also be found in calcareous lakes (Bratton, 1990;Kimmins, 1932).The large nymphs are good swimmers and typically swim in short, darting bursts (Elliott et al., 1988).
Siphlonurus alternatus is univoltine, overwintering as eggs and emerging as adults between May and August (Elliott et al., 1988;Landa, 1968).Emergence of the adults typically takes place during daylight hours (Hirvenoja, 1964), and males of this species can be found swarming at dawn and dusk over light patches of substrate on the bed of the water body or floating plants such as water-lilies (Savolainen, 1978).The larvae feed by gathering or collecting fine particulate organic detritus from the sediment (Elliott et al., 1988).
The genome sequence for Siphlonurus alternatus will aid in understanding the biology, physiology and ecology of the species.

Genome sequence report
The genome was sequenced from one male Siphlonurus alternatus collected from Drumpail Burn,Scotland (54.92,.A total of 53-fold coverage in Pacific Biosciences singlemolecule HiFi long reads was generated.Primary assembly contigs were scaffolded with chromosome conformation Hi-C data.Manual assembly curation corrected 31 missing joins or misjoins and removed 10 haplotypic duplications, reducing the assembly length by 0.69% and the scaffold number by 12.9%, and increasing the scaffold N50 by 2.06%. The final assembly has a total length of 455.8 Mb in 107 sequence scaffolds with a scaffold N50 of 50.2 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 (97.87%) 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 homology to Sympetrum striolatum (GCA_947579665.1)(Crowley et al., 2023) and coverage.The Y chromosome could not be uniquely identified in the unlocalised scaffolds.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, spectra estimates, sequencing runs, contaminants and pre-curation assembly statistics can be found at https://links.tol.sanger.ac.uk/species/248243.

Sample acquisition and nucleic acid extraction
Specimens of Siphlonurus alternatus were collected from Drumpail Burn, Scotland, UK (latitude 54.92, longitude -4.77) on 2021-07-28.The specimens were collected and identified by Andrew Farr (independent researcher) and dry frozen at -80°C.One specimen (specimen ID NHMUK014543936, ToLID ieSipAlte2) was used for DNA sequencing and Hi-C data, and a second specimen (specimen ID NHMUK014543933, ToLID ieSipAlte3) was used for RNA sequencing.
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI).The ieSipAlte2 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing.Tissue from the whole organism was disrupted using a Nippi Powermasher fitted with a BioMasher pestle.High molecular weight (HMW) DNA was extracted using the Qiagen MagAttract HMW DNA extraction kit.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.RNA was extracted from whole organism tissue of ieSipAlte3 in the Tree of Life Laboratory at the WSI using TRIzol, according to the manufacturer's instructions.RNA was then eluted in 50 μl RNAse-free water and its concentration assessed using a Nanodrop spectrophotometer and Qubit Fluorometer using the Qubit RNA Broad-Range (BR) Assay kit.Analysis of the integrity of the RNA was done using Agilent RNA 6000 Pico Kit and Eukaryotic Total RNA assay.

Sequencing
Pacific Biosciences HiFi circular consensus DNA sequencing libraries were constructed according to the manufacturers'  (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   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)  "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.
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.
In the subheading "Genome sequence report" In the first line authors have mentioned "The genome was sequenced from one male Siphlonurus alternatus……….But authors have mentioned under the subheading "Sample acquisition and nucleic acid extraction" first line Specimens of Siphlonurus alternatus were collected from ……… Moreover authors have mentioned in the abstract "We present a genome assembly from an individual male Siphlonurus alternatus………………..There is a little confusion here because a specimen was used for DNA extraction and another specimen was used for RNA extraction.
Page no six first paragraph third line "DNA and RNA sequencing was performed".The sentence can be changed as DNA and RNA sequencing were performed.
Above all, I confirm that the manuscript meets the necessary scientific standard and is suitable for indexing"

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: Phylogenetic analysis of Noctuoidea moths 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.
Reviewer Report 14 May 2024 https://doi.org/10.21956/wellcomeopenres.22335.r80531 © 2024 Veale A. 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.

Andrew J. Veale
Manaaki Whenua Landcare Research, Lincoln, New Zealand The assembly looks excellent and the data note is fine.As per one of the previous reviewers, while RNA sequencing is mentioned in the methods it isn't mentioned in the results or discussion.I assume it was used for annotation, but annotation was also not mentioned in the results or discussion?
Was the second individual used for the RNA also male?Also, what tissues were used for the transcriptomes?
Mentioning that this was a male (I believe?), and that mayflies have a male XY female XX sex determination may be useful for those that do not know this.
Other than that it is fine.

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, however I have significant reservations, as outlined above.

Jerome H. L Hui
The Chinese University of Hong Kong, Hong Kong, Hong Kong Farr and colleagues report the genome sequence of mayfly Siphlonurus alternatus (Say, 1824).
Early record of this species in Britain could date back to 1913 (Macadam 2019, The Glasgow Naturalist).Molecular data of this species are scarce prior to this report, and are mainly confined to COI sequences deposited to the NCBI database.Therefore, this new genome resource will be useful for further studies, ranging from revealing their population structures, ecology, understanding their evolution with other insects, as well as studying the impact of climate change on them.
This genome resource is excellent from the summary statistics, with high BUSCO numbers, high sequence continuity, and majority of sequences contained on 11 pseudochromosomes (plus mitochondrion).All in all, this is another valuable contribution carried out by the Darwin Tree of Life Consortium.

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 The article shows the high-quality reference genome of an insect.The asm procedure is standard dtol.Many of the points raised by rev1 are available in the link provided in the last sentence of the Genome sequence report section: "Metadata for specimens, spectra estimates, sequencing runs, contaminants and pre-curation assembly statistics can be found at https://links.tol.sanger.ac.uk/species/248243".No need to add those in the article.The results are above the recommended EBP metrics, and the HiC contact map looks clean after curation.I think the information available is appropriate for this type of article, but in the same direction as commented by rev1, the article is missing a sentence or two expanding on the objectives and applications.
Finally, the article lacks information about the use of the RNA-Seq dataset generated from the specimen ieSipAlte3 (is it a male, like ieSipAlte2?).
The assembly looks very good, the article is appropriate for this data.

Is the rationale for creating the dataset(s) clearly described? Partly
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 This data note describes a high quality chromosomal assembly of the Northern Summer Mayfly, Siphlonurus alternatus, using the most advanced sequencing and assembly methods.The genome data are available at NCBI BioProject, and data is accessible (as checked for some of the chromosomes).

Comments:
I miss in the background more description on the rationale for generating this assembly.The only reference to that is the last sentence "The genome sequence for Siphlonurus alternatus will aid in understanding the biology, physiology and ecology of the species.".If I'm not mistaken, this is the first genome available for this species, this could be mentioned too.
RNA-seq is also generated for this species, however, this is not mentioned in the Results section.It would be helpful to describe how the RNA-seq was used or will be used (as mentioned in the Data availability will be used for gene annotation, but this could be mentioned earlier).

Small comments and typos:
-First sentence of the Background: "northern United States": Northern should be capitalized.
-Siphlonurus alternatus could be shortened to S. alternatus after the first mention in each section.
-In the first sentence of the Genome sequence report, the year of collection could be added (after e.g. the coordinates of sampling).
-Provide the N50 statistics after the manual assembly curation in "A total of 53-fold coverage in ... was generated (N50=)".
-Fix the sentence "The snailplot in Figure 2 summarises the assembly statistics is shown in Figure 2".
-Figure 2: Why it reads "Dataset: CATKWE01" in the bottom left?-Figures 2-4 in the PDF version are too big and take a lot of space of the page.
-"bwa-mem" is missing from Table 3 -For reproducibility of the assembly, I miss the options/parameters used in the programs and tools.This could be specified in Table 3.I also suggest sharing scripts used through e.g.GitHub.
Is the rationale for creating the dataset(s) clearly described?Partly 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: bioinformatics, population genetics, epigenetics, transposable elements, adaptation 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, however I have significant reservations, as outlined above.

Figure 1 .
Figure 1.Photograph of the Siphlonurus alternatus specimen used for RNA sequencing.

Figure 2 .
Figure 2. Genome assembly of Siphlonurus alternatus, ieSipAlte2.1: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 455,861,235 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 (70,536,023 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (50,231,148 and 22,999,722 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 insecta_odb10 set is shown in the top right.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/Siphlonurus%20alternatus/dataset/CATKWE01/snail.

Figure 5 .
Figure 5. Genome assembly of Siphlonurus alternatus, ieSipAlte2.1:Hi-C contact map of the ieSipAlte2.1 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=MXAHm5u1RPiiYHsY51vQdQ.

Table 3
contains a list of relevant software tool versions and sources.Wellcome Sanger Institute -Legal and GovernanceThe 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.

Table 2 . Chromosomal pseudomolecules in the genome assembly of Siphlonurus alternatus, ieSipAlte2. 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.