The genome sequence of the Spruce Carpet Moth, Thera britannica (Turner, 1925)

We present a genome assembly from an individual male Thera britannica (the Spruce Carpet Moth; Arthropoda; Insecta; Lepidoptera; Geometridae). The genome sequence is 381 megabases in span. Most of the assembly is scaffolded into 19 chromosomal pseudomolecules, including the assembled Z sex chromosome. The mitochondrial genome has also been assembled and is 15.9 kilobases in length. Gene annotation of this assembly on Ensembl has identified 12,457 protein coding genes.


Background
The genus Thera, within the family Geometridae, contains several species of moth that are difficult to distinguish morphologically. Whether each named species is genetically distinct is controversial and much taxonomic confusion persists. Thera britannica, the Spruce Carpet Moth, was described by the Australian paediatrician and entomologist Alfred Jefferis Turner in 1925, and was named for an association with the British Isles. Despite its specific name, the moth is not endemic to Britain but is found widely across northern and central Europe where its larval food plants grow, primarily spruce Picea sp. and Douglas fir Pseudotsuga menziesii (UKMoths, no date;GBIF Secretariat, 2022). In the UK, the moth has two generations per year and is found throughout England, Wales, Northern Ireland and southern counties of Scotland. In Ireland, records are primarily from the south of the country. The number of records of T. britannica has increased hugely since 1970, presumably due to commercial planting of conifers providing new habitats for breeding (Randle et al., 2019).
The forewings of T. britannica are a grey-brown ground colour, traversed by a wide, deeply fluted median band of darker brown outlined in cream. Almost identical markings occur in another UK species, the grey pine carpet T. obeliscata, with subtle and inconsistent differences in colour; however, T. obeliscata has a distinct larval food plant, pine (Pinus sp.). It has been suggested that the shape of each antennal segment in males is a more reliable character to distinguish between the two species (Townsend et al., 2010). To further complicate matters, a third species T. variata is described from mainland Europe, and sometimes considered conspecific to T. britannica (South, 1961;Townsend et al., 2010). Preliminary analysis of CO1 DNA barcode data does not reveal clear monophyletic groups for these three moths, suggesting either that they diverged very recently from each other, or there is occasional interbreeding, or even that they are not distinct species (P.O. Mulhair & P.W.H.H. analysis at (BOLD, 2023)). In contrast, this analysis shows clearer distinction for T. juniperata, T. fermata, T. cognata and T. cupressata. The genome sequence reported here is from an individual with a CO1 DNA barcode closest to several previously reported T. britannica specimens.
A genome sequence for T. britannica will be useful finding variable genetic loci of use in resolving the taxonomy of this genus, and also for probing the genetic basis of adaptation to coniferous food plants.

Genome sequence report
The genome was sequenced from one male T. britannica specimen ( Figure 1) collected in Wytham Woods, UK (latitude 51.77, longitude -1.33). A total of 55-fold coverage in Pacific Biosciences single-molecule HiFi long reads and 117-fold coverage in 10X Genomics read clouds was generated. Primary assembly contigs were scaffolded with chromosome conformation Hi-C data. Manual assembly curation corrected 29 missing or mis-joins, and removed two haplotypic duplications, reducing the scaffold number by 4%.
The final assembly has a total length of 380.6 Mb in 48 sequence scaffolds with a scaffold N50 of 22.9 Mb (Table 1). Most (99.71%) of the assembly sequence was assigned to 19 chromosomal-level scaffolds, representing 18 autosomes and the Z sex chromosome. Chromosome-scale scaffolds confirmed by the Hi-C data have been named in order of size (Figure 2- Figure 5; Table 2). The assembly has a BUSCO v5.3.2 (Manni et al., 2021) completeness of 98.5% (single 98.1%, duplicated 0.5%) using the lepidoptera_odb10 reference set. While not fully phased, the assembly deposited is of one haplotype. Contigs corresponding to the second haplotype have also been deposited.

Sample acquisition and nucleic acid extraction
A male T. britannica specimen (ilTheBrit1) was collected in Wytham Woods, Oxfordshire (biological vice-county: Berkshire), UK (latitude 51.77, longitude -1.34) on 8 September 2020, using a light trap. The specimen was collected and identified by Douglas Boyes (University of Oxford) and snap-frozen on dry ice.
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI). The ilTheBrit1 sample was weighed and   dissected on dry ice with tissue set aside for Hi-C sequencing. Thorax tissue 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. Low molecular weight DNA was removed from a 20 ng aliquot of extracted DNA using 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.   Illumina NovaSeq 6000 (10X) instruments. Hi-C data were also generated from head tissue of ilTheBrit1 using the Arima v2 kit and sequenced on the Illumina NovaSeq 6000 instrument.

Genome assembly
Assembly was carried out with Hifiasm (Cheng et al., 2021) and haplotypic duplication was identified and removed with purge_dups (Guan et al., 2020). One round of polishing was performed by aligning 10X Genomics read data to the assembly with Long Ranger ALIGN, calling variants with FreeBayes (Garrison & Marth, 2012). The assembly was then scaffolded with Hi-C data (Rao et al., 2014) using YaHS (Zhou et al., 2023). The assembly was checked for as described previously (Howe et al., 2021). Manual curation was performed using HiGlass (Kerpedjiev et al., 2018) and Pretext (Harry, 2022). The mitochondrial genome was assembled using MitoHiFi   -Silva et al., 2022), which performed annotation using MitoFinder (Allio et al., 2020). The genome was analysed, and BUSCO scores were generated within the BlobToolKit environment (Challis et al., 2020). Table 3 contains a list of all software tool versions used, where appropriate.

Genome annotation
The Ensembl gene annotation system (Aken et al., 2016) was used to generate annotation for the T. britannica assembly (GCA_939531255.1). 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).