Abstract
We present a genome assembly from an individual male Crambus pascuella (White-banded Grass-moth; Arthropoda; Insecta; Lepidoptera; Crambidae). The assembly contains two haplotypes with total lengths of 792.17 megabases and 788.21 megabases. The haplotype 1 assembly and the haplotype 2 assembly each contain 30 chromosomal pseudomolecules, including the Z sex chromosome; these account for 98.92% and 99.36% of their respective sequences. The mitochondrial genome has also been assembled, with a length of 15.34 kilobases. This assembly was generated as part of the Darwin Tree of Life project, which produces genomes for eukaryotic species found in Britain and Ireland.
Keywords: Crambus pascuella, White-banded Grass-moth; Inlaid Grass-veneer, genome sequence, chromosomal, Lepidoptera
Species taxonomy
Eukaryota; Opisthokonta; Metazoa; Eumetazoa; Bilateria; Protostomia; Ecdysozoa; Panarthropoda; Arthropoda; Mandibulata; Pancrustacea; Altocrustacea; Allotriocarida; Hexapoda; Insecta; Dicondylia; Pterygota; Neoptera; Eumetabola; Endopterygota; Aparaglossata; Panorpida; Amphiesmenoptera; Lepidoptera; Glossata; Neolepidoptera; Heteroneura; Ditrysia; Obtectomera; Pyraloidea; Crambidae; Crambinae; Crambus; Crambus pascuella Linnaeus, 1758 (NCBI:txid989760)
Background
Crambus pascuella (Linnaeus, 1758), the White-banded Grass-moth, has yellowish-brown forewings with a conspicuous white longitudinal streak and a forewing length of 11–13 mm ( Sterling & Parsons, 2023). The species occurs in Europe, across Siberia to Japan, and in North America ( Ólafsson, 2009). In Britain and Ireland, it is common and widespread ( Sterling & Parsons, 2023).
The species inhabits damp grasslands, fens, wet heathland, marshes, moorland and damp woodland rides. Adults fly mainly from May to August, sometimes into September, with occasional records in early October. They are readily disturbed by day and are attracted to light. The larvae feed on deergrasses and sheep’s-fescue, possibly also other grasses and sedges, and live in a silken tube at the base of the foodplant ( Sterling & Parsons, 2023).
We present a chromosome-level genome sequence for Crambus pascuella, produced using the Tree of Life pipeline. The sequence is based on a specimen collected from Duxford, Cambridgeshire, England, UK (Figure 1). This assembly was generated as part of the Darwin Tree of Life Project, which aims to generate high-quality reference genomes for all named eukaryotic species in Britain and Ireland to support research, conservation, and the sustainable use of biodiversity ( Darwin Tree of Life Project Consortium, 2022).
Figure 1. Photograph of C. pascuella by Tony Morris.
Although numerous genomes have been sequenced for the family Crambidae, this assembly provides the first chromosome-level genome assembly for Crambus pascuella, enabling comparative analyses (data obtained via NCBI datasets, O’Leary et al., 2024). This is currently the only genome assembly available for this species. This assembly is the RefSeq reference assembly for this species.
Methods
Sample acquisition
The specimen used for genome sequencing was an adult male Crambus pascuella (specimen ID SAN00003315, ToLID ilCraPasc1; Figure 1). The same specimen was also used for Hi-C sequencing. It was collected from Duxford, Cambridgeshire, England, UK (latitude 52.095, longitude 0.1595) on 2023-06-14 by Charlotte Wright, who also identified it.
Nucleic acid extraction
High molecular weight (HMW) DNA extraction protocols developed by the Tree of Life Core Laboratory at the Wellcome Sanger Institute (WSI) are available on protocols.io ( Howard et al., 2025). Tissue from the ilCraPasc1 sample was weighed and triaged to select the appropriate extraction workflow. For library DTOL15016439, 3.0 mg of thorax tissue was homogenised by PowerMash using a PowerMasher II tissue disruptor. HMW DNA was extracted in the WSI Scientific Operations core using the Automated MagAttract v2 protocol. DNA was sheared into an average fragment size of 12–20 kb following the Megaruptor®3 for LI PacBio protocol. Sheared DNA was purified by automated SPRI (solid-phase reversible immobilisation).
The concentration of the sheared and purified DNA was assessed with a NanoDrop spectrophotometer and a Qubit Fluorometer using the Qubit dsDNA High Sensitivity Assay kit. Fragment size distribution was evaluated by running the sample on the Femto Pulse system. For this sample, final post-shearing DNA measurements included a Qubit concentration of 16.04 ng/μL and an average fragment size of 16.6 kb, yielding 753.88 ng of DNA. The 260/280 spectrophotometric ratio was 1.89, and the 260/230 ratio was 2.5. The Genomic Quality Number (GQN) was 7.7.
PacBio HiFi library preparation and sequencing
Library preparation and sequencing took place in the WSI Scientific Operations core. Library DTOL15016439 was prepared with the SMRTbell Prep Kit 3.0 (Pacific Biosciences) according to the manufacturer’s instructions. The kit includes reagents for end repair/A-tailing, adapter ligation, post-ligation SMRTbell bead clean-up, and nuclease treatment. Size selection and clean-up were performed using diluted AMPure PB beads (Pacific Biosciences). DNA concentration was quantified with a Qubit Fluorometer v4.0 (Thermo Fisher Scientific) and the Qubit 1X dsDNA HS assay kit. Final library fragment size was assessed with the Agilent Femto Pulse Automated Pulsed Field CE Instrument (Agilent Technologies) using the gDNA 55 kb BAC analysis kit.
The library was sequenced on a Revio instrument (Pacific Biosciences, California, USA). The prepared libraries selected for multiplexing were pooled based on genome size, library molarity and the intended plex level. Primers were annealed and polymerases were bound to generate circularised complexes following the manufacturer’s instructions. Complexes were purified using SMRTbell beads, diluted to the Revio loading concentration, and spiked with a Revio sequencing internal control. The pooled libraries were sequenced on a Revio 25M Plex SMRT cell in a 2-plex run. SMRT Link software (Pacific Biosciences) was used to configure and monitor the run and to perform primary and secondary data analysis.
Hi-C
Sample preparation and crosslinking
Hi-C data were generated from the ilCraPasc1 head sample using the Arima-HiC v2 kit (Arima Genomics). Following the manufacturer’s instructions, tissue was fixed and DNA crosslinked using TC buffer to a final formaldehyde concentration of 2%. The tissue was homogenised using the Diagnocine Power Masher-II. Crosslinked DNA was digested with a restriction enzyme master mix, biotinylated, and ligated. Clean-up was performed with SPRISelect beads before library preparation. DNA concentration was measured with the Qubit Fluorometer (Thermo Fisher Scientific) and Qubit HS Assay Kit. The biotinylation percentage was estimated using the Arima-HiC v2 QC beads.
Hi-C library preparation and sequencing
Biotinylated DNA constructs were fragmented using a Covaris E220 sonicator and size-selected to 400–600 bp using SPRISelect beads. DNA was enriched with Arima-HiC v2 kit Enrichment beads. End repair, A-tailing, and adapter ligation were carried out with the NEBNext Ultra II DNA Library Prep Kit (New England Biolabs), following a modified protocol where library preparation occurs while DNA remains bound to the Enrichment beads. Libraries were amplified with KAPA HiFi HotStart mix and a custom Unique Dual Index (UDI) barcode set (Integrated DNA Technologies). Depending on sample concentration and biotinylation percentage determined at the crosslinking stage, libraries were amplified with 10–16 PCR cycles. Post-PCR clean-up was performed with SPRISelect beads. Libraries were quantified using the AccuClear Ultra High Sensitivity dsDNA Standards Assay Kit (Biotium) and a FLUOstar Omega plate reader (BMG Labtech).
Prior to sequencing, libraries were normalised to 10 ng/μL. Normalised libraries were quantified again to create equimolar and/or weighted 2.8 nM pools. Pool concentrations were checked using the Agilent 4200 TapeStation (Agilent) with High Sensitivity D500 reagents before sequencing. Libraries were sequenced on the Illumina NovaSeq X, generating paired-end 150-bp reads.
Genome assembly
Prior to assembly of the PacBio HiFi reads, a database of k-mer counts ( k = 31) was generated from the filtered reads using FastK. The k-mer frequency distribution was analysed with GenomeScope (version 2.1.0) ( Ranallo-Benavidez et al., 2020), providing fitted point estimates of haploid genome size, heterozygosity and repeat content.
The HiFi reads were assembled using Hifiasm in Hi-C phasing mode ( Cheng et al., 2021, 2022), producing two haplotypes. Hi-C reads ( Rao et al. , 2014) were mapped to contigs using bwa-mem2 ( Vasimuddin et al., 2019). Contigs were further scaffolded with Hi-C data in YaHS ( Zhou et al., 2023), using the --break option for handling potential misassemblies. The scaffolded assemblies were evaluated using Gfastats ( Formenti et al. , 2022), BUSCO ( Manni et al., 2021) and MerquryFK ( Rhie et al., 2020).
The mitochondrial genome was assembled using MitoHiFi ( Uliano-Silva et al. , 2023). The MitoHiFi reference was the mitochondrial genome of Pseudargyria interruptella (NC_029751.1).
Assembly curation
The assembly was decontaminated using the Assembly Screen for Cobionts and Contaminants ( ASCC) pipeline. The flat files and maps for curation were generated with TreeVal. Manual curation was conducted primarily in PretextView and HiGlass ( Kerpedjiev et al. , 2018). Scaffolds were visually inspected and corrected as described by Howe et al. (2021). The two haplotype assemblies were combined for curation. Manual corrections included 25 breaks and 83 joins. This reduced the scaffold count by 11.9%. The curation process is described at sanger-tol/curation-resources. A Hi-C contact map of the haplotype 1 assembly was generated with PretextSnapshot.
Assembly quality assessment
The MerquryFK tool ( Rhie et al., 2020) was run in a Singularity container ( Kurtzer et al., 2017) to evaluate k-mer completeness and assembly quality for both haplotypes using the k-mer database ( k = 31) computed prior to genome assembly. The analysis outputs included assembly QV scores and completeness statistics.
The haplotype 1 assembly was assessed using BlobToolKit ( Challis et al., 2020). PacBio reads were mapped to the assembly to derive sequence coverage, and BUSCO ( Manni et al., 2021) was used to assess gene-set completeness. DIAMOND BLASTp searches of extracted BUSCO proteins and DIAMOND BLASTx searches of assembly sequences against UniProt Reference Proteomes ( Buchfink et al., 2021), followed by BLASTn searches of sequences without DIAMOND BLASTx hits against the NCBI nt database ( Altschul et al., 1990), supported taxonomic assignment. Coverage, sequence composition, taxonomic assignment and BUSCO results were integrated into a BlobDir for interactive inspection and visualisation. The snail and blob plots were rendered locally from this hosted BlobDir using BlobTk v0.8.3. The scaffold-length radial scale function for the snail plot was set to linear ( Challis & Blaxter, 2026). The blob plot used circular markers with area proportional to scaffold length ( Challis et al., 2020).
Ancestral linkage groups were painted along the chromosome-level scaffolds of the haplotype 1 assembly. Complete BUSCO genes (single-copy and duplicated) from the lepidoptera_odb10 lineage dataset were assigned to the 32 Lepidoptera Merian elements (MZ and M1–M31) described by Wright et al.. (2024) and plotted along chromosomes drawn to scale.
Genome sequence report
Sequence data
PacBio sequencing of the C. pascuella specimen generated 41.37 Gb (gigabases) from 3.68 million reads. These reads were used to assemble the genome. GenomeScope analysis using a p = 2 model gave a k-mer-based haploid genome size estimate of 777.10 Mb, with heterozygosity of 2.09% and repeat content of 46.39%; the full-model fit was 98.15% (Figure 2). The GenomeScope estimates guided expectations for the assembly. The NCBI assembly record reports 52× genome coverage. Hi-C sequencing produced 144.81 Gb from 479.50 million reads, which were used to scaffold the haplotype 1 assembly. Table 1 summarises the specimen and sequencing details.
Figure 2. GenomeScope model fitted to the k-mer frequency distribution.
The plot shows the observed and modelled k-mer spectra used to estimate haploid genome size, heterozygosity and repeat content from unassembled sequencing reads.
Table 1. Specimen and sequencing data for C. pascuella (BioProject PRJEB82173).
| Platform | PacBio HiFi | Hi-C |
|---|---|---|
| ToLID | ilCraPasc1 | ilCraPasc1 |
| Specimen ID | SAN00003315 | SAN00003315 |
| BioSample (source individual) | SAMEA115163332 | SAMEA115163332 |
| BioSample (tissue) | SAMEA115163470 | SAMEA115163469 |
| Tissue | thorax | head |
| Instrument | Revio | Illumina NovaSeq X |
| Library ID & run accession(s) | DTOL15016439: ERR13946464 | ERR13947484 |
| Read count total | 3.68 million reads | 479.50 million read pairs |
| Base count total | 41.37 Gb | 144.81 Gb |
Assembly statistics
The genome was assembled into two haplotypes using Hi-C phasing. Both the haplotype 1 and haplotype 2 assemblies were curated to chromosome level. The haplotype 1 assembly, which is proposed as the reference assembly for this species, has a total length of 792.17 Mb in 228 scaffolds (excluding organelle sequences), with 164 gaps, and its scaffold N50 is 27.65 Mb (Table 2). The scaffold auN for the haplotype 1 assembly is 28.3 Mb.
Table 2. Genome assembly statistics for C. pascuella .
| Genome assembly | Haplotype 1 | Haplotype 2 |
|---|---|---|
| Assembly name | ilCraPasc1.hap1.1 | ilCraPasc1.hap2.1 |
| Assembly accession | GCA_964341525.1 | GCA_964341515.1 |
| Assembly level | chromosome | chromosome |
| Span (Mb) | 792.17 | 788.21 |
| Number of chromosomes | 30 | 30 |
| Number of contigs | 392 | 308 |
| Contig N50 (Mb) | 8.28 | 7.88 |
| Number of scaffolds (excluding organelle sequences) | 228 | 132 |
| Scaffold N50 (Mb) | 27.65 | 27.97 |
| Scaffold auN (Mb) | 28.3 | 28.3 |
| Longest scaffold length (Mb) | 56.28 | 55.48 |
| Sex chromosomes | Z | Z |
| Organelles | Mitochondrial genome: 15.34 kb | - |
Most of the haplotype 1 assembly sequence (98.92%) was assigned to 30 chromosome-level scaffolds, representing 29 autosomes and the Z sex chromosome. These chromosome-level scaffolds, confirmed by Hi-C data, are named according to size (Figure 3; Table 3). Chromosome Z was identified through the detection of ancestral BUSCO genes. The exact order and orientation of the contigs on chromosome 23 (5,500 - 6,860 Kbp) are unknown.
Figure 3. Hi-C contact map of the C. pascuella haplotype 1 assembly.
Assembled chromosomes are shown in order of size and labelled along the axes, with a megabase scale shown below. The plot was generated using PretextSnapshot.
The mitochondrial genome was also assembled (length 15.34 kb, OZ205629.1). This sequence is included as a contig in the multifasta file of the haplotype 1 genome submission and as a standalone record.
Table 3. Chromosomal pseudomolecules in the haplotype 1 genome assembly of C. pascuella ilCraPasc1 (haplotype 2 also at chromosome level).
| INSDC accession | Molecule | Length (Mb) | GC% |
|---|---|---|---|
| OZ205600.1 | 1 | 37.64 | 37 |
| OZ205601.1 | 2 | 32.42 | 37 |
| OZ205602.1 | 3 | 31.87 | 37 |
| OZ205603.1 | 4 | 30.29 | 37 |
| OZ205604.1 | 5 | 30.17 | 37 |
| OZ205605.1 | 6 | 29.57 | 37 |
| OZ205606.1 | 7 | 29.13 | 37 |
| OZ205607.1 | 8 | 28.60 | 37 |
| OZ205608.1 | 9 | 28.53 | 37 |
| OZ205609.1 | 10 | 28.30 | 37 |
| OZ205610.1 | 11 | 28.21 | 37 |
| OZ205611.1 | 12 | 27.65 | 37.50 |
| OZ205612.1 | 13 | 27.31 | 37.50 |
| OZ205613.1 | 14 | 26.96 | 37 |
| OZ205614.1 | 15 | 26.63 | 37 |
| OZ205615.1 | 16 | 26.18 | 37 |
| OZ205616.1 | 17 | 25.30 | 37 |
| OZ205617.1 | 18 | 25.21 | 37.50 |
| OZ205618.1 | 19 | 24.30 | 37 |
| OZ205619.1 | 20 | 23.25 | 37.50 |
| OZ205620.1 | 21 | 22.76 | 37.50 |
| OZ205621.1 | 22 | 21.18 | 37.50 |
| OZ205622.1 | 23 | 20.08 | 37 |
| OZ205623.1 | 24 | 18.83 | 39 |
| OZ205624.1 | 25 | 18.05 | 37.50 |
| OZ205625.1 | 26 | 17.06 | 38 |
| OZ205626.1 | 27 | 15.85 | 37.50 |
| OZ205627.1 | 28 | 13.33 | 37.50 |
| OZ205628.1 | 29 | 12.69 | 37.50 |
| OZ205599.1 | Z | 56.28 | 37 |
Chromosome painting shows the distribution of BUSCO genes assigned to Lepidoptera Merian elements across the chromosome-level scaffolds of the haplotype 1 assembly (Figure 4).
Figure 4. Lepidoptera Merian elements painting of the haplotype 1 assembly of C. pascuella.
Chromosomes are drawn to scale, with coloured bars marking the positions of complete lepidoptera_odb10 BUSCO genes. Each BUSCO gene is coloured by its assignment to one of the 32 Merian elements (MZ and M1–M31).
Assembly quality metrics
For the haplotype 1 assembly, the estimated QV is 63.9, and for the haplotype 2 assembly, 64.9. When the two haplotypes are combined, the assembly achieves an estimated QV of 64.4. The k-mer completeness is 68.23% for the haplotype 1 assembly, 68.22% for the haplotype 2 assembly, and 99.42% for the combined haplotypes (Figure 5).
Figure 5. Evaluation of k-mer completeness using MerquryFK.
This plot illustrates the recovery of k‐mers from the original read data in the haplotype 1 assembly, the haplotype 2 assembly and the combined haplotypes. The horizontal axis represents k‐mer multiplicity, and the vertical axis shows the number of k‐mers. The black curve represents k‐mers that appear in the reads but are not assembled. The green curve corresponds to k‐mers shared by both haplotypes, and the red and blue curves show k‐mers found only in one of the haplotypes.
BUSCO analysis using the lepidoptera_odb10 reference set ( n = 5,286) identified 98.6% of the expected gene set (single = 98.2%, duplicated = 0.4%) in the haplotype 1 assembly. For the haplotype 2 assembly, BUSCO v.5.5.0 analysis identified 98.7% of the expected gene set (single = 98.1%, duplicated = 0.6%).
The snail plot in Figure 6 provides a summary of assembly contiguity, cumulative scaffold count, sequence composition and BUSCO completeness for the haplotype 1 assembly ( Challis & Blaxter, 2026). The relative sizes of the longest scaffold, scaffold N50 and N90, together with the dark-grey scaffold-length distribution, show how much of the assembly is contained in long scaffolds. The outer composition track shows variation in GC, AT and N content, while the central track shows the cumulative scaffold count and the BUSCO inset summarises gene-set completeness.
Figure 6. Assembly metrics for ilCraPasc1.hap1.1, the haplotype 1 assembly.
The BlobToolKit snail plot provides an overview of assembly metrics and BUSCO gene completeness. The circumference represents the length of the haplotype 1 assembly, and the main plot is divided into 1,000 bins around the circumference. The outermost blue tracks display the distribution of GC, AT, and N percentages across the bins. Scaffolds are arranged clockwise from longest to shortest and are depicted in dark grey. Scaffold length is shown on a linear radial scale, scaled to the longest scaffold. Red shading indicates the longest scaffold, and the deeper orange and pale orange shading represent the N50 and N90 lengths. The summary statistics include scaffold auN, the area under the scaffold Nx curve. The light grey spiral at the centre shows the cumulative scaffold count on a logarithmic scale. A summary of complete, fragmented, duplicated, and missing BUSCO genes in the lepidoptera_odb10 set is presented at the top right. The assembly can be explored interactively in the BlobToolKit viewer.
The blob plot in Figure 7 displays scaffolds from the haplotype 1 assembly by GC proportion and base coverage. Marker area is proportional to scaffold length, and colour indicates taxonomic assignment ( Challis et al., 2020).
Figure 7. BlobToolKit blob plot for ilCraPasc1.hap1.1, the haplotype 1 assembly.
The plot shows base coverage (vertical axis) and GC content (horizontal axis). Circles represent scaffolds; marker area is proportional to scaffold length, and colour indicates taxonomic assignment. The histograms along the axes display the total length of sequences distributed across different levels of coverage and GC content. The assembly can be explored interactively in the BlobToolKit viewer.
Table 4 lists the assembly metric benchmarks adapted from Rhie et al.. (2021) and the Earth BioGenome Project (EBP) Report on Assembly Standards. The three-part EBP metric, calculated for the haplotype 1 assembly, is 6.C.Q63.
Table 4. Earth BioGenome Project summary metrics for the C. pascuella assembly.
| Measure | Value | Benchmark |
|---|---|---|
| EBP summary (haplotype 1) | 6.C.Q63 | 6.C.Q40 |
| Contig N50 length | 8.28 Mb | > 1 Mb |
| Scaffold N50 length | 27.65 Mb | = chromosome N50 |
| Consensus quality (QV) | Haplotype 1: 63.9; haplotype 2: 64.9; combined: 64.4 | ≥ 40 |
| k-mer completeness | Haplotype 1: 68.23%; Haplotype 2: 68.22%; combined: 99.42% | > 90% |
| BUSCO | Haplotype 1: C:98.6% [S:98.2%, D:0.4%], F:0.3%, M:1.0%, n:5,286; Haplotype 2: C:98.7% [S:98.1%, D:0.6%], F:0.4%, M:0.9%, n:5,286 | S > 90%; D < 5% |
| Percentage of assembly assigned to chromosomes | 98.92% | ≥ 90% |
Notes: The EBP summary uses log10(Contig N50); chromosome-level (C) or log10(Scaffold N50); Q (Merqury QV). BUSCO v5.5.0 using the lepidoptera_odb10 lineage dataset: C=complete; S=single-copy; D=duplicated; F=fragmented; M=missing; n=orthologues.
Data availability
European Nucleotide Archive: Crambus pascuella (inlaid grass-veneer). Accession number PRJEB82173; https://identifiers.org/ena.embl/PRJEB82173. The genome sequence is released openly for reuse. The C. pascuella genome sequencing initiative is part of the Darwin Tree of Life Project ( PRJEB40665), the Sanger Institute Tree of Life Programme ( PRJEB43745) and Project Psyche ( PRJEB71705). All raw sequence data and the haplotype assemblies have been deposited in INSDC databases. The genome will be annotated using available RNA-Seq data and presented through the Ensembl pipeline at the European Bioinformatics Institute. Raw data and assembly accession identifiers are reported in Table 1 and Table 2.
Production code used in genome assembly at the WSI Tree of Life is available at https://github.com/sanger-tol. Table 5 lists software versions used in this study.
Table 5. Software 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. 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.
Funding Statement
This work was supported by Wellcome through core funding to the Wellcome Sanger Institute [220540, https://doi.org/10.35802/220540] and the Darwin Tree of Life Discretionary Award [218328, https://doi.org/10.35802/218328].
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
[version 1; peer review: 2 approved]
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