The genome sequence of the Mauritius kestrel, Falco punctatus (Temminck, 1821)

We present a genome assembly from an individual male Falco punctatus (the Mauritius kestrel; Chordata; Aves; Falconiformes; Falconidae). The genome sequence is 1,279.3 megabases in span. Most of the assembly is scaffolded into 23 chromosomal pseudomolecules, including the Z sex chromosome. The mitochondrial genome has also been assembled and is 17.34 kilobases in length.


Background
The Mauritius kestrel (Falco punctatus) (Figure 1A) is a small falcon that is endemic to the island of Mauritius in the Indian Ocean.It is found in forest habitats where it feeds on a range of live caught prey, including endemic geckos, small birds, large insects, and non-native reptiles.The Mauritius kestrel is a territorial species, typically forming monogamous pairs.The birds build their nests in nest boxes or natural cavities in trees and cliffs.Clutches (2 to 5 eggs) are laid from early September onwards, and the last fledglings have usually left the nest by the following January or February.Mauritius kestrels fledge at around 35 days old, achieve independence at around 85 days, and are capable of breeding at 1 year of age.The Mauritius kestrel is usually single brooded but will lay a replacement clutch if its eggs or chicks are lost relatively early in the breeding season.
Prior to the first human settlements in Mauritius in the 1600s, the Mauritius kestrel was widely distributed in forest habitats across the island.By the 1950s, it was restricted to the remote Black River Gorges region due mainly to extensive forest loss and the widespread application of organochlorine and organophosphate pesticides.By 1974, the population had been reduced to only four known wild individuals, making the Mauritius kestrel one of the rarest birds in the World at that time (Jones et al., 1995).Despite many conservationists believing the species was doomed to extinction, an intensive conservation programme began in the late 1970s and led to a significant recovery in kestrel numbers and distribution (Jones et al., 1995;Nicoll et al., 2021) (Figure 1B).This programme included the management of the remnant wild population through nest box provision, supplementary feeding, and predator control, together with the supplementation of numbers in the wild through captive breeding and release.In response, Mauritius kestrel numbers grew to 350-400 individuals by 2000, and the species was downlisted to Vulnerable on the IUCN Red List.Today, the Mauritius kestrel is found in three main populations, and consists of ~248 individuals.Globally, the recovery of the Mauritius kestrel population is regarded as an iconic conservation success story.These conservation activities have been accompanied by an ongoing, intensive monitoring programme (Nicoll et al., 2003).During each breeding season, attempts are made to find as many Mauritius kestrel nests as possible to collect data on the timing of egg laying, the number of eggs laid, and the number of chicks fledged.Prior to fledging, chicks are ringed with a unique combination of coloured leg rings and a numbered metal ring.This has enabled the identification of individual adults returning to breed in subsequent years, and over time has built up an incredibly detailed picture of the lives of individual birds as the populations have recovered.These data have generated a range of unique ecological insights (Cartwright et al., 2014;Nevoux et al., 2013;Nicoll et al., 2006;Taylor et al., 2021), as well as helping assess and manage ongoing and future risks to the recovering populations (Nicoll et al., 2021).
The extreme historical population collapse, and the more recent population decline, suggest that the long-term viability of the species might be at risk of a high realised load of harmful deleterious variation (Dussex et al., 2023).The extensive sample archive and deep ecological knowledge of the species makes it a model study system for conservation genomics.Currently, hundreds of whole genomes are being re-sequenced from historical (pre-1900), recent (1990-2000) and contemporary samples to elucidate the genomic consequence and long-term implications of the historic bottleneck and the more recent population size decline.This research efforts are part of a collaboration between several universities (University of Kent, UK, University of East Anglia, UK, Copenhagen University, Denmark), the Natural History Museum London, the Government of Mauritius' National Parks and Conservation Service (NPCS) and the Mauritian Wildlife Foundation (MWF -conservation NGO, Mauritius).The conservation monitoring and management of the Mauritius Kestrel is conducted by the MWF in collaboration with the NPCS with guidance from the university partners; recent conservation actions have also been implemented by Ebony Forest Reserve (conservation NGO).

Genome sequence report
The genome was sequenced from blood sampled from a Falco punctatus collected from Bambou Mountains, Mauritius (-20.39, 57.45).A total of 39-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 25 missing joins or mis-joins and removed one haplotypic duplication, reducing the scaffold number by 3.36%, and increasing the scaffold N50 by 0.44%.
The final assembly has a total length of 1,279.3Mb in 315 sequence scaffolds with a scaffold N50 of 92.4 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 (92.98%) of the assembly sequence was assigned to 23 chromosomal-level scaffolds, representing 22 autosomes and the Z sex chromosome.Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size (Figure 5; Table 2).Chromosome Z was assigned by synteny to Falco biarmicus (GCF_023638135.1).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
Blood sampling of Mauritius kestrels has been conducted by the MWF and university researchers.The Mauritius kestrel population has been blood sampled extensively during restoration; most Mauritius kestrel chicks produced annually were blood sampled, and there has been opportunistic sampling of adults.Chicks are blood sampled in the nest at around 20-days; adults are captured at nest sites for blood sampling.For developing the reference genome, three individuals were captured, and blood samples stored in absolute ethanol at -20°C on 2020-01-29.Blood from specimen with ID SAN11000045 (ToLID bFalPun1) was used for DNA and RNA sequencing, while blood from specimen ID SAN11000046 (ToLID bFalPun2) was used for Hi-C sequencing.
The workflow for high molecular weight (HMW) DNA extraction at the Wellcome Sanger Institute (WSI) Tree of Life Core Laboratory includes a sequence of core procedures: sample preparation; sample homogenisation, DNA extraction, fragmentation, and clean-up.In sample preparation, the bFalPun1 sample was kept on dry ice (Jay et al., 2023).For sample homogenisation, blood was cryogenically disrupted using the Covaris cryoPREP ® Automated Dry Pulverizer (Narváez-Gómez et al., 2023).HMW DNA was extracted using the manual Nucleated Blood Nanobind ® protocol (Denton et al., 2023a).DNA was sheared into an average fragment size of 12-20 kb in a Megaruptor 3 system with speed setting 31 (Bates et al., 2023).Sheared DNA was purified by solid-phase reversible immobilisation (Strickland et al., 2023).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 a bFalPun1 blood sample in the Tree of Life Laboratory at the WSI using the RNA Extraction: Automated MagMax™ mirVana protocol (do Amaral et al., 2023).The RNA concentration was assessed using a Nanodrop spectrophotometer and a Qubit Fluorometer using the Qubit RNA Broad-Range Assay kit.Analysis of the integrity of the RNA was done using the Agilent RNA 6000 Pico Kit and Eukaryotic Total RNA assay.
Protocols developed by the WSI Tree of Life laboratory are publicly available on protocols.io(Denton et al., 2023b).

Sequencing
Pacific Biosciences HiFi circular consensus DNA sequencing libraries were constructed according to the manufacturers' instructions.Poly(A) RNA-Seq libraries were constructed using the NEB Ultra II RNA Library Prep kit.DNA and RNA sequencing was performed by the Scientific Operations core at the WSI on Pacific Biosciences Sequel IIe (HiFi) and Illumina NovaSeq 6000 (RNA-Seq) instruments.Hi-C data were also generated from blood sampled from bFalPun2 using the Arima v2 kit.The Hi-C sequencing was performed using paired-end sequencing with a read length of 150 bp on the Illumina NovaSeq 6000 instrument.
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.
Table 3 contains a list of relevant software tool versions and sources.

Wellcome Sanger Institute -Legal and Governance
The materials that have contributed to this genome note have been supplied by a Tree of Life collaborator.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 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.

Final assembly evaluation
The final assembly was post-processed and evaluated with the three Nextflow (Di Tommaso et al   2023).The genome sequence is released openly for reuse.The Falco punctatus genome sequencing initiative is part of the Vertebrate Genomes Project.All raw sequence data and the assembly 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.population declines in recent history.Thus, I believe this genome will be an extremely valuable resource for future conservation genomics studies, including the ongoing genome re-sequencing efforts mentioned in the note.The methods of genome assembly and assessment are sound and reflect the methods used in many Tree of Life genome notes.I noticed that this report is missing methods and result for the genome annotation, which I think should be included and released with the main genome assembly.Other than that, I have no further comments and am looking forward to the forthcoming genomics research on this species.

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: genome assembly, conservation and evolutionary genomics, invasive species 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.The fall and rise of the Mauritius kestrel.(A) A Mauritius kestrel (Falco punctatus; photo credit Samantha Cartwright) (B) Demographic trajectory over time (bottleneck and recovery), the line represents the number of monitored pairs (territorial pairs) observed each year during the 6-month breeding season.The bars represent the number of captive-breed individuals released into the free-living population.

Figure 2 .
Figure 2. Genome assembly of Falco punctatus, bFalPun1.1: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 1,279,277,844 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 (127,704,412 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (92,385,878 and 8,824,250 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 aves_odb10 set is shown in the top right.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/Falco%20punctatus/dataset/CAUJKR01/snail.

Figure 3 .
Figure 3. Genome assembly of Falco punctatus, bFalPun1.1: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/Falco%20punctatus/dataset/CAUJKR01/blob.

Figure 4 .
Figure 4. Genome assembly of Falco punctatus, bFalPun1.1: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/Falco%20punctatus/dataset/CAUJKR01/cumulative.

Figure 5 .
Figure 5. Genome assembly of Falco punctatus, bFalPun1.1:Hi-C contact map of the bFalPun1.1 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=J1IV79vMQyOL7YmXGxz9LA.

Table 1 . Genome data for Falco punctatus, bFalPun1.1. Project accession data
Assembly metric benchmarks are adapted from column VGP-2020 of "Table 1: Proposed standards and metrics for defining genome assembly quality" from Rhie et al. (2021).