The genome sequence of Rhynchonycteris naso, Peters, 1867 (Chiroptera, Emballonuridae, Rhynchonycteris)

We present a reference genome assembly from an individual male Rhynchonycteris naso (Chordata; Mammalia; Chiroptera; Emballonuridae). The genome sequence is 2.46 Gb in span. The majority of the assembly is scaffolded into 22 chromosomal pseudomolecules, with the Y sex chromosome assembled.


Introduction
Emballonurid bats are aerial insectivores.They are found in Africa and Indo-Malayan, Australian, Neotropical, and Holarctic regions.Although typically found in tropical forest regions, a few species have been found in semiarid and desert regions 5 .
The Emballonuridae family comprises two subfamilies: Taphozoinae and Emballonurinae.Emballonurinae consists of 14 genera and 55 species 6 .The genus Rhynchonycteris is within Emballonurinae and comprises a sole species: Rhynchonycteris naso (Rhynchonycteris is one of four monotypic genera in Emballonuridae) (Figure 1).
Rhynchonycteris naso, the proboscis bat, has been found in tropical regions in middle and south America from the south of Mexico to the north of Bolivia and center of Brazil 5 .Proboscis bats are found up to 1500 meters elevation, generally at less than 500 meters elevation, often in lowland tropical forest, close to water bodies 5 .They roost in an exposed manner on tree trunks or man-made structures in the vicinity of water 7 .Their grey and brown marbled coat makes them well camouflaged, they often look like tree bark (see Figure 2) or may be perceived as swaying leaves since they may form a single line along the tree's length (Figure 2A-B) and can be observed rocking back and forth 5,8 .They live in stable multi-malemulti-female groups of usually <40 individuals 7 .Male mating strategies are based on both direct female-defense and male territoriality 9,10 .A substantial proportion of males are philopatric 9 .
Rhynchonycteris naso has been commonly referred to as long-nosed and or sharp-nosed bat in reference to the nose protruding from the rest of the face.They are small bats 36-48 mm body size with added ~11-17 mm of tail length, the forearm length is ~36-40 mm and they weigh around 3-6 g 5 .Although the family Emballonuridae is sometimes referred to as the "sac-winged bats", R. naso lacks wing sacs 7 .R. naso is classified in the IUCN Red List as a species of Least Concern.
Rhynchonycteris naso hunts small dipterans (such as mosquitoes, flies and caddisflies) 5,7 .The echolocation calls of R. naso are CF-FM with the CF component around 100 kHz during search flight 11 .The echolocation call frequency is lowered to 67 kHz during prey capture to maintain the peripheral acoustic view 12 .This strategy is different from the other members of the Emballonuridae family which use a constant frequency throughout the whole pursuit sequence.

Genome sequence report
The genome was sequenced from a single male R. naso collected on March 9th 2019, from a tree near the river in Gamboa, Panama (GPS coordinates: 9.1135734185584, -79.82011865195433).A total of 42x-fold coverage in Figure 1.Position of Rhynchonycteris naso in the phylogeny of Emballonuridae.The bat Rhynchonycteris naso is the only species currently recognized in the genus Rhynchonycteris 13 .Rhynchonycteris naso belongs to the Subfamily Emballonurinae, which currently includes 14 genera and 55 species 6 .
Pacific Biosciences Hi-Fi long reads (contig N50 20 Mb) was generated after removal of all reads shorter than 10kb.Primary assembly contigs were scaffolded with chromosome conformation (Hi-C) data, which was also used to attain chromosomelevel phasing 14 .The final assembly has a total length of 2.455 Gb in 40 sequence scaffolds with a contig N50 of 86 Mbp scaffold N50 of 286 Mbp (Table 1).The assembly has a BUSCO 15 completeness of 95.3% using the laurasiatheria reference set.Chromosomal pseudomolecules in the genome assembly of Rhynchonycteris naso are shown in Table 2.

Methods
The R. naso specimen was a male individual collected during a field expedition in Gamboa, Panama.Rhynconycteris naso was first identified by the roost location (a group of R. naso bats were hanging from a tree trunk in a line formation close to shallow waters).Furthermore, the shape of the face with a protruding nose, the gray-brown fur and the two light colored lines on the back of these bats determined the identification of this species as described previously (e.g.9,16.After going on a boat by the river close to Gamboa, a roost was spotted on a tree near the water near a location previously investigated by locals under the supervision of the expert fieldworkers Mirjam Knörnschild and Martina Nagy.The bat was caught using a hand net and after confirmation of the sex it was placed in a fabric bag and taken to the laboratories at the Smithsonian Institute in Gamboa for tissue harvesting.Capture and sampling were done under the project proposal 2019-0301-2022 approved by the Smithsonian Tropical Research Institute and the STRI Animal Care and Use Committee (ACUC) and collection and export was conducted under the collecting field number issued by UNARGEN SC/A-3-19.All work was conducted with approval by the Panamanian Ministry of Environment (Mi Ambiente).Tissues were removed from the subject individual immediately following euthanasia and were flash-frozen in liquid nitrogen and stored in a freezer at -80°C until shipping on dry ice, maintaining the cold chain.
All efforts were made to minimize any suffering of the animal.The animal was subjected to minimal handling after capture, and it was held in a clean cloth bag after capture as per best practices for field containment of bats 17 .After species identification, the individual was euthanized humanely by experienced researchers while monitoring and prioritizing the reduction of stress and suffering of the animal.The animal was euthanized by overdose of isoflurane inhalation (Formula CHF 2 OCClHCF 3 , CAS number 26675-46-7; Manufacturer Piramal Critical Care, Supplier US Pharmacy Systems, Product code 5034-1FL-SOL-ORA).Euthanasia by isoflurane inhalation is a humane approved method that rapidly causes unconsciousness and eventually death upon inhalation.Bats euthanized by this method are rendered unconscious within seconds due to their high respiration rate, and death occurs within a minute or two with no significant suffering by the animal.The animal was tested for absence of breathing and reflexes.After breathing stops, isoflurane exposure was extended for one more minute.Confirmation of death was done immediately by decapitation.Tissue samples were dissected and immediately snap frozen using liquid nitrogen.A total of 21 samples were collected including brain, blood, liver, spleen, heart, lung, testes, muscle and kidney.All data were recorded and reported in accordance with the ARRIVE guidelines -see data availability section and Table 1.
DNA was extracted using Nanobind extraction from muscle tissue following the Circulomics Nanobind HMW DNA Extraction Protocol.Pacific Biosciences HiFi libraries were constructed according to the manufacturer's instructions.Hi-C data was generated using the Arima Hi-C+ High Coverage kit from the same muscle tissue sample.Sequencing was performed by the Genomic Operations DNA Pipelines at Paratus Sciences on Pacific Biosciences Sequel IIe (HiFi reads) and Illumina NextSeq 2000 (Hi-C) instruments.
Assembly was carried out following the Vertebrate Genome Project Galaxy pipeline v2.0 18 .A brief synopsis of the method is as follows: Genome size was estimated using GenomeScope2 19 .Hifiasm with Hi-C phasing was used for genome assembly (Cheng, Haoyu et al. 2021).The quality of the assembly was evaluated using Merqury 20 and BUSCO 21 .Scaffolding with Hi-C data (Rao, Huntley et al. 2014) was carried out with YaHS (Zhou, McCarthy et al. 2023).PretextView was implemented to generate a Hi-C contact map (Figure 3). Figure 4-Figure 6 were generated using BlobToolKit 22 .All bioinformatics software utilised for the R. naso analysis are depicted in Table 3.The Rhynchonycteris naso genome sequencing initiative is part of the Bat1K genome sequencing project.The genome assembly is released openly for reuse.Underlying data may be available for non-commercial research purposes upon request.Please email info@batbio.orgfor more information.
The genome assembly for Rhynchonycteris naso (proboscis bat) can be found in the European Nucleotide Archive and NCBI.
All raw sequence data and the assembly have been deposited in the ENA (PRJNA1076651, PRJNA1076652) and NCBI (raw data).Data accession identifiers are SAMN39947078.
Data accession identifiers are reported in Table 1.

Luciano Chaves Franco Filho
Instituto Evandro Chagas, Ananindeua, Brazil The Data Note presents a high-quality reference genome assembly for the proboscis bat, Rhynchonycteris naso, from the family Emballonuridae.The genome, covering around 2.46 Gb, is organized into 22 chromosomal pseudomolecules, including the Y chromosome.The assembly achieved a contig N50 of 86 Mb and a scaffold N50 of 287 Mb, demonstrating the completeness and accuracy of the genome sequence.This work contributes to the Bat1K project, aiming to sequence the genomes of all bat species, and provides valuable insights into the genetic makeup of R. naso, an insectivorous bat species found in tropical regions of Central and South America.

Wenhua Yu
Guangzhou University, Guangzhou, Guangdong, China Authors presented a exciting reference genome assembly from an individual male Rhynchonycteris naso.No doubt it will benefit the future genomic analyses on Emballonuridae and other.
However, I have 2 minor comments for its improvement: 1) The use of Rhynchonycteris naso Peters, 1867 is not correct.I think it should be Rhynchonycteris naso (Wied-Neuwied, 1820).Pls check "Mammal species of the World" for the further details.

Is the rationale for creating the dataset(s) clearly described? Yes
Are

Xiuguang Mao
School of Ecological and Environmental Sciences, East China Normal University, Shanghai, China This manuscript presents a high-quality chromosome-scale assembly for Rhynchonycteris naso and also provides detailed characteristics about this species in the Introduction section.

I have two minor comments:
1) In Figure 1, 'Rhynchonycteris' should be italic.
2) "The final assembly has a total length of 2.455 Gb in 40 sequence scaffolds with a contig N50 of 86 Mbp scaffold N50 of 286 Mbp (Table 1)".But in Table 1, the scaffold N50 length is 287 Mb.

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: Comparative genomics, genome assembly, evolutionary biology, speciation, bats 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 2 .
Figure 2. Proboscis bats, Rhynchonycteris naso Individuals of R. naso.(A-B) These bats roost in a line formation often on trees and near the water.They sometimes look like tree bark or lichen due to their grey and brown marbled coat and light stripes on their backs [Photos taken near a river close to Gamboa, Panama by Ine Alvarez van Tussenbroek].

Figure 3 .
Figure 3. Hi-C Contact Map of the Rhynchonycteris naso haplotype 1 assembly with 11 scaffolds, visualized using PretextView.Scaffolds below 10 Mb were removed for creating the Hi-C Contact Map.

Figure 4 .
Figure 4. Genome assembly metrics generated using blobtoolkit for the Rhynchonycteris naso genome assembly.The larger snail plot depicts scaffold statistics including N50 length (bright orange) and base composition (blue).The smaller plot shows BUSCO completeness in green.

Figure 6 .
Figure 6.Cumulative sequence plot generated for the Rhynchonycteris naso assembly using blobtoolkit.The grey line shows the cumulative length for all chromosomes/scaffolds in the assembly.Colored lines represent Phylum represented in the assembly.

Figure 5 .
Figure 5. GC coverage plot generated for the Rhynchonycteris naso assembly using blobtoolkit.Individual chromosomes and scaffolds are represented by each circle.The circles are sized in proportion to chromosome/scaffold length.Histograms show the sum length of chromosome/scaffold size along each axis.Color of circles indicate taxonomic hits of each Phylum represented in the assembly.

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.

have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
://doi.org/10.21956/wellcomeopenres.22103.r95139This 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.

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.

have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
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.