The genome sequence of Molossus nigricans (Chiroptera, Molossidae; Miller, 1902)

We present a genome assembly from an individual male Molossus nigricans (Chordata; Mammalia; Chiroptera; Molossidae). The genome sequence is 2.41 gigabases in span. The majority of the assembly is scaffolded into 24 chromosomal pseudomolecules, with the X sex chromosome assembled.


Species taxonomy Introduction
Molossid bats are swift aerial insectivores that are distributed throughout the world. As shown in Figure 1, they comprise two subfamilies, the South American endemic Tomopeatinae and the cosmopolitan Molossinae 1 , the latter consisting of 21 genera and 131 species 2 . Within this group, the genus Molossus comprises 15 species distributed broadly across the Neotropics 2,3 . Molossus nigricans, one of the largest species of Molossus, is found in Central America from southern Mexico, Belize, and Guatemala to Panama 2,4-6 . Adults of this species come in one of two color morphs, either black or red; a red individual is shown in Figure 2A and an individual with black pelage is shown in Figure 2B. Until recently M. nigricans was considered to be a subspecies of M. rufus 7 , but Loureiro et al. 3,4 demonstrated that it represents a distinct species.
The echolocation calls of M. nigricans are frequency modulated (FM) in the short first step of the call while the rest of the call is constant frequency (CF) 8 . The 20-35 kHz frequency range for molossid bats is apparently related to foraging strategies, allowing these species greater success in their open space foraging habitats 9 . Typically, molossid bats feed in open areas at relatively high altitudes where their relatively   low-frequency echolocation calls maximize response times and allow for better detection of a wide range of potential prey of different sizes 9,10 . In Belize, M. nigricans has a diverse diet including mostly beetles and hemipterans 11 . The species M. nigricans is not currently classified in the IUCN Red List of Threatened Species; the species complex from which it was split, Molossus rufus, is classified as Least Concern 12 .
The genus Molossus is morphologically conservative, and the level of genetic divergence is also low among species, which has masked recognition of the actual species diversity in the genus. Recently Loureiro et al. 3 Figure 1).

Genome sequence report
The genome was sequenced from a single male M. nigricans (field number BZ-404, catalog number AMNH:Mammalogy:280920) collected from the Lamanai Archaeological Reserve, Orange Walk District, Belize on 12 November 2021. A total of 41-fold coverage in Pacific Biosciences Hi-Fi long reads (contig N50 21 Mb) was generated after removal of all reads shorter than 10kb. Primary assembly contigs were scaffolded with chromosome confirmation Hi-C data. The final assembly has a total length of 2.41 Gb in 146 sequence scaffolds with a scaffold N50 of 81.9 Mb ( Table 1). The majority, 79.45%, of the assembly sequence was assigned to 24 chromosomal-level scaffolds, representing 23 autosomes (numbered by sequence length, and the X sex chromosome). Chromosomal pseudomolecules in the genome assembly of Molossus nigricans are shown in Table 2. The assembly has a BUSCO 13 completeness of 96.1% using the laurasiatheria reference set. While not fully phased, the assembly deposited is of one haplotype.

Methods
The M. nigricans specimen was a male individual of black pelage collected on an American Museum of Natural History (AMNH) field expedition at the Lamanai Archaeological Reserve in the Orange Walk District of Belize. The individual sampled was identified as M. nigricans based on morphometrics (e.g., forearm length, body mass) and morphological traits (e.g., fur color pattern) described by Loureiro et al. 4 ." The bat was caught in a 30 x 100 ft macro mist net 14 set in the High Temple Plaza at Lamanai (17.76736 N, 88.65270 W), an area known to be near a roost of this species. All efforts were made to minimize any distress or suffering by the animal. The individual sampled 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. After species identification, the individual was euthanized humanely the same night it was captured. The animal was identified as M. nigricans based on morphometrics and morphological traits described by Loureiro et al. 4 . The animal was euthanized by isoflurane inhalation, 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. Capture and sampling were conducted under Belize Forest Department Permit FD/WL/1/21 (16) and Belize Institute of Archaeology Permit IA/S/5/6/21(01), and samples were exported under Belize Forest Department permit FD/WL/7/22(08). All work was conducted with approval by the AMNH Institutional Animal Care and Use Committee (AMNHIACUC-20191212) 15 . All data were recorded and reported in accordance with the ARRIVE guidelines 16 -see data availability section and Table 1. Tissues were removed from the subject individual immediately following euthanasia and were flash-frozen in a liquid nitrogen dry shipper, with the cold chain maintained from field to museum to laboratory. 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 pipeline v2.0 17 with a few modifications as follows. An initial QC analysis was performed on the raw BAM file using FastQC. BAM files were converted to fastq format and merged for downstream processing. Genome size was estimated using GenomeScope2 18 . HiCanu was used for genome assembly 19 . Haplotypic duplication was identified and removed with purge dups 20 . The quality of the assembly was evaluated using Merqury 17 and BUSCO 21 . Scaffolding with Hi-C data 22 was carried out with SALSA2 23 HiGlass 24 was implemented to generate Hi-C contact maps. Figure 2- Figure 6 were generated using BlobToolKit 25 . Software utilised for the Molossus nigricans genome analyses are depicted in Table 3.

Linelle Ann Lacson Abueg
The Vertebrate Genome Laboratory, The Rockefeller University, New York, NY, USA The authors provide an appropriate and well-detailed description of their new M. nigricans genome. It might be interesting to note if other bats from the M. rufus species complex are also in the sampling area where this individual was found. Nonetheless, this genome will be a useful resource for those studying the genomics of molossid bats.

Comments:
in Genome Sequence Report: "While not fully phased, the assembly deposited is of one haplotype." I don't think the "one haplotype" part can be asserted here given that it is a pseudohaplotype assembly. Haplotype switches are still possible. ○ Table 2: is there a citation (karyotype?) for the chromosome number for M. nigricans mentioned in the caption, or is this referring to the observed scaffolds? ○ in Methods, these sentences: "The animal was identified as [...] within a minute or two with no significant suffering by the animal." are just re-stating the previous sentences starting with "The individual sampled was identified as [...] humanely the same night it was captured." but with more detail about the euthanasia. Maybe keep the second set of sentences, adding in the mist net information from the first set. ○ in Methods: "Vertebrate Genome Project" should be "Vertebrate Genomes Project". Spell out "QC" before using acronym. There is a period missing between "Scaffolding with Hi-C data" part and "HiGlass" part.  in Data Availability: "Underlining data" should be "underlying data" ○ Is the rationale for creating the dataset(s) clearly described?

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

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