The genome sequence of a stonefly, Nemurella pictetii Klapalek, 1900 [version 2; peer review: 2 approved, 1 approved with reservations]

We present a genome assembly from an individual male Nemurella pictetii (Arthropoda; Insecta; Plecoptera; Nemouridae). The genome sequence is 257 megabases in span. The majority of the assembly (99.79%) is scaffolded into 12 chromosomal pseudomolecules, with the X sex chromosome assembled. The X chromosome was found at half coverage, but no Y chromosome was found. The mitochondrial genome was assembled, and is 16.0 kb in length. the on the genome sequence of a stonefly, and information is this genome. The genome is high quality and will provide a new resource for an understudied


Background
Nemurella pictetii is a western palearctic species found from northern Spain to Siberia, and throughout the British Isles, although appears to be less common in Scotland. It is considered a eurytherm and can be found in a variety of habitats from rivers and streams to ponds and lakes. It does not appear to have any altitudinal preference; however, in Ireland it is more usually encountered in streams draining peaty soils usually at higher altitudes and in small headwater streams and seepages (Baars & Kelly-Quinn, 2006;Hynes, 1977). In Great Britain and Europe, larvae are known to occur in small upland lakes with stony shorelines and in vegetated ponds, wetlands and marshes (e.g. Lillehammer (1975) Nemurella pictetii is the sole representative of the genus Nemurella. The high-quality genome sequence described here is, to our knowledge, the first one reported for N. pictetii. This assembly, generated as part of the Darwin Tree of Life project, also represents a high-quality addition to an underrepresented taxon (Plecoptera) and wider group of insects (aquatic insects) (Hotaling et al., 2020). The genome sequence for N. pictetii will aid in understanding the biology, physiology and ecology of the species.

Genome sequence report
The genome was sequenced from one male N. pictetii ( Figure 1) collected from River Taff Fawr, Garwnant, Wales (latitude 51.8082, longitude -3.4449). A total of 68-fold coverage in Pacific Biosciences single-molecule long reads and 183-fold coverage in 10X Genomics read clouds were generated. Primary assembly contigs were scaffolded with chromosome conformation Hi-C data. Manual assembly curation corrected 62 missing/misjoins and removed 8 haplotypic duplications, reducing the assembly size by 2.35% and scaffold number by 65.52% and increasing the scaffold N50 by 8.35%.
The final assembly has a total length of 257 Mb in 20 sequence scaffolds with a scaffold N50 of 24.6 Mb ( Table 1). The majority of the assembly sequence (99.79%) was assigned to 12 chromosomal-level scaffolds, representing 11 autosomes (numbered by sequence length), and the X sex chromosome (Figure 1-Figure 4; Table 2). The sex of the specimen was

Amendments from Version 1
We thank the reviewer for his comments on the manuscript, and have made minor changes in response. We have also corrected some additional errors in Table 1, which listed incorrect BioSample and BioProject accession numbers.
Any further responses from the reviewers can be found at the end of the article       The genome sequence is released openly for reuse. The N. pictetii genome sequencing initiative is part of the Darwin Tree of Life (DToL) project. All raw sequence data and the assembly have been deposited in INSDC databases. The genome will be annotated and presented through the Ensembl pipeline at the European Bioinformatics Institute. Raw data and assembly accession identifiers are reported in Table 1. 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.

Manaaki Whenua Landcare Research, Lincoln, New Zealand
In the Background section, "although appears to be less common in Scotland" should be "although it appears to be less common in Scotland".
In the methods you should identify what life stage each of the individuals was -are these adults or juveniles, and if juvenile, which instar?
In stonefly genomics I have found issues with contamination from filamentous algae and diatoms in particular, which you appear not to have had issues with. When you say whole organism tissue what do you mean? Was the gut present? Was the outer cuticle present? My assumption is that you had an adult and dissected out the gut but it would be good to confirm.
Were any tissues stored for museum accession?
Was species ID confirmed through mitochondrial sequences?
Having an XO sex determination system appears relatively common in Plecoptera, as it appears to be in this species 1 . This should probably be made explicit.
I have no issues with the genome assembly and methods therein -it looks like a high quality assembly.

Are the protocols appropriate and is the work technically sound? Yes
Are sufficient details of methods and materials provided to allow replication by others? Partly Are the datasets clearly presented in a useable and accessible format? Yes © 2022 Hotaling S. 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.

Scott Hotaling
School of Biological Sciences, Washington State University, Pullman, WA, USA In this study, Macadam and colleagues report the assembly of a high-quality stonefly genomethe best to date for this entire order of insects. The study and methods contain no notable flaws and I see no issues in terms of its scientific robustness.

Background:
One missing component -placing this genome assembly in context for the group and insects as a whole. It fills a major niche for Plecoptera and adds a key species for aquatic insects broadly which are dramatically underrepresented (see my article Hotaling et al. 2020 1 ). These points can be highlighted for the reader. It might also be useful to note that the three previous stonefly genomes are exceptionally poor-quality and represent perhaps the lowest quality group of assemblies for any insect order (see my article Hotaling et al. 2021, Figure 1d 2 ). Adding these references is far from critical to the report's effectiveness. They would simply be "value added" for a future reader seeking broader context in terms of insect genomics.
○ Also there are double parentheses in the first paragraph of the section in the PDF, mid-way through. These may need correction.

Genome sequence report:
Described as being a female specimen in the first sentence but later as male. This should be confirmed/corrected.

Genome assembly:
There is a space missing in the PDF between " Table 3" and "contains" on the top of pg. 7.

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In this study, Macadam and colleagues report the assembly of a high-quality stonefly genome-the best to date for this entire order of insects.