The genome sequence of Reeves’ muntjac, Muntiacus reevesi (Ogilby, 1839)

We present a genome assembly from an individual female Muntiacus reevesi (the Reeves’ muntjac; Chordata; Mammalia; Artiodactyla; Cervidae). The genome sequence is 2,656.2 megabases in span. Most of the assembly is scaffolded into 23 chromosomal pseudomolecules, including the X sex chromosome. The mitochondrial genome has also been assembled and is 16.35 kilobases in length.


Background
The Reeve's Muntjac or Chinese Muntjac Muntiacus reevesi reevesi is a small deer in the family Cervidae.They are 0.44 to 0.52 metres tall at the shoulder.Full grown males weigh 10 to 18 kg and females 9 to 16 kg (British Deer Society, 2024).Males have a distinctive pattern of facial stripes and small anthers that do not branch and usually curve inwards (British Deer Society, 2024).The Reeve's Muntjac is native to the forests, grassland and shrubland of south-east China but has been introduced into Europe: the first pair brought from China to London Zoo in 1838 being described as the type specimens (Chapman, 2021).Multiple other introductions followed, and Reeve's Muntjac were released in various counties including Bedfordshire, Oxfordshire, Northamptonshire, Kent, Devon, and on the Norfolk/Suffolk border.By 1994 this subspecies was present in over half of England and Wales, and has continued to spread (Chapman, 2021).It is also present in Northern Ireland and the Republic of Ireland (Chapman, 2021).This species' ecology means that the population can increase rapidly: males are fertile by the time they are nine months old and females, which can give birth at any time of year, are fertile at five to six months of age (Chapman, 2021).
Although numbers are declining in its native range, Reeve's Muntjac has been ranked at number 9 in a list of the "100 worst alien species in Europe" based on their impact on the native ecology (Nentwig et al., 2018).By overgrazing young coppice and the understorey of more mature woodland, Reeve's Muntjac may, with other species of deer, be contributing to habitat changes resulting in the decline of some bird species (Fuller et al., 2005;Gill & Fuller, 2007).They may be partly responsible for the possible increase in the range of the tick Ixodes ricinus (Gandy et al., 2023).Reeve's Muntjac cause damage to timber crops, allotments and gardens, and increasing numbers of road traffic accidents (British Deer Society, 2024).
We present a chromosomally complete genome sequence for a female Muntiacus reevesi reevesi, based on one specimen collected in Wytham Woods as part of the Darwin Tree of Life Project.

Genome sequence report
The genome was sequenced from adult female Muntiacus reevesi (Figure 1) collected from Wytham Woods, Oxfordshire, UK (51.77,.A total of 42-fold coverage in Pacific Biosciences single-molecule HiFi long reads was generated.
Primary assembly contigs were scaffolded with chromosome conformation Hi-C data.Manual assembly curation corrected 20 missing joins or mis-joins and removed one haplotypic duplication, reducing the scaffold number by 3.60%, and increasing the scaffold N50 by 0.77%.
The final assembly has a total length of 2,656.2Mb in 267 sequence scaffolds with a scaffold N50 of 114.5 Mb (Table 1).The snail plot in Figure 2 provides a summary of the assembly statistics, 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 (96.02%) of the assembly sequence was assigned to 23 chromosomal-level scaffolds, representing 22 autosomes and the X sex chromosome.Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size (Figure 5; Table 2).The X chromosome was identified based on synteny with Muntiacus reevesi (GCA_020226045.1).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.

Sample acquisition and nucleic acid extraction
The samples used in this study were taken from two Muntiacus reevesi individuals culled in Wytham Woods,       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.

Evaluation of final assembly
The final assembly was post-processed and evaluated with the three Nextflow (Di Tommaso et al The sanger-tol/blobtoolkit pipeline is a Nextflow port of the previous Snakemake Blobtoolkit pipeline (Challis et al., 2020).It aligns the PacBio reads with SAMtools and minimap2 (Li, 2018) and generates coverage tracks for regions of fixed size.In parallel, it queries the GoaT database (Challis et al., 2023) to identify all matching BUSCO lineages to run BUSCO (Manni et al., 2021).For the three domain-level BUSCO lineage, the pipeline aligns the BUSCO genes to the Uniprot Reference Proteomes database (Bateman et al., 2023) with DIAMOND (Buchfink et al., 2021) blastp.
The genome is also split into chunks according to the density of the BUSCO genes from the closest taxonomically lineage, and each chunk is aligned to the Uniprot Reference Proteomes database with DIAMOND blastx.Genome sequences that have no hit are then chunked with seqtk and aligned to the NT database with blastn (Altschul et al., 1990).All those outputs are combined with the blobtools suite into a blobdir for visualisation.
All three pipelines were developed using the nf-core tooling (Ewels et al., 2020), use MultiQC (Ewels et al., 2016), and make extensive use of the Conda package manager, the Bioconda initiative (Grüning et al., 2018) 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

Josephine Pemberton
The University of Edinburgh, Edinburgh, Scotland, UK This is a neat and succinct report of the work to assemble the genome of the Chinese or Reeve's muntjac, an introduced deer species to the UK.To the extent that I can judge the work has been thorough and extremely carefully QC'd prior to indexing.
All my specific comments relate to the Background section Para 1 end: In mature males there can be a small branch at the base of the antlers.
Suggest also mention that both sexes have tusks which are used by males in fights.
Para 2 end: Worth saying that individual mature females breed every 7 months and unlike in other deer species, males are fertile irrespective of the antler cycle (ref Chapman 2021).
Para 4 end: It would be worth mentioning that the related Indian muntjac (Muntiacus muntjak) is known for its extraordinarily low chromosome number (females 2N=6 and males 2N=7) and there is much interest in the chromosome fusions that have taken place en route to the Indian muntjac -see ref here: https://www.nature.com/articles/s42003-020-1096-9 .It seems to me this publication should be referred to regardless, given it describes an earlier assembly for reevesi (currently it is not cited) .As should any other previous genome sequences for this species.Is the rationale for creating the dataset(s) clearly described?
2. It would be helpful to have a little more context for the culling of the individuals used.The authors are clear about this work meeting ethical guidelines and things like that; but for those of us not familiar, a sentence or two about the context of this culling would helpful.For example, is there a regular cull of some individuals for population control, and these individuals were part of that process?Or, perhaps the collector just had a license for hunting, and the individuals were killed particularly for this work?Regardless, such context would help readers sleep easier regarding the situation within which these individuals were culled and these samples collected.Reviewer Expertise: Genomics 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 Muntiacus reevesi by Rufus46 (not the specimen used for genome sequencing).

Figure 2 .
Figure 2. Genome assembly of Muntiacus reevesi, mMunRee1.1:metrics.The BlobToolKit snail plot 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 2,656,184,892 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 (285,828,854 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (114,544,513 and 59,907,558 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 cetartiodactyla_odb10 set is shown in the top right.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/Muntiacus_reevesi/dataset/GCA_963930625.1/snail.

Figure 3 .
Figure 3. Genome assembly of Muntiacus reevesi, mMunRee1.1:BlobToolKit GC-coverage plot.Sequences are coloured by phylum.Circles are sized in proportion to sequence length.Histograms show the distribution of sequence length sum along each axis.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/Muntiacus_reevesi/dataset/GCA_963930625.1/blob.

Figure 4 .
Figure 4. Genome assembly of Muntiacus reevesi mMunRee1.1:BlobToolKit cumulative sequence plot.The grey line shows cumulative length for all sequences.Coloured lines show cumulative lengths of sequences assigned to each phylum using the buscogenes taxrule.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/Muntiacus_reevesi/dataset/GCA_963930625.1/cumulative.

Figure 5 .
Figure 5. Genome assembly of Muntiacus reevesi mMunRee1.1:Hi-C contact map of the mMunRee1.1 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=C-2GvvVfStOlDyKBbe-IFQ.

Figure 1 :
Figure 1: Legend could point out that the tusk is visible.
the rationale for creating the dataset(s) clearly described?No 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: Conservation genomics 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.Genome sequence report; Location coordinates no need to mention here as it is included in the methodology.Reduce repetitions.○ Methods; Mention the maturity level of the animal and voucher number of the sample if you stored at the museum.○ Include and specify how did you use RNA data in the genome analysis.○ 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.

Darwin Tree of Life Project Sampling Tree
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 '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.

have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
No competing interests were disclosed.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.