Abstract

Species taxonomy

Eukaryota; Metazoa; Eumetazoa; Bilateria; Deuterostomia; Chordata; Craniata; Vertebrata; Gnathostomata; Teleostomi; Euteleostomi; Sarcopterygii; Dipnotetrapodomorpha; Tetrapoda; Amniota; Mammalia; Theria; Eutheria; Boreoeutheria; Laurasiatheria; Chiroptera; Yangochiroptera; Molossidae; Tadarida; Tadarida brasiliensis, (I. Geoffroy Saint-Hilaire, 1824) (NCBI:txid9438; subordinal taxonomy updated per Teeling et al, ( Teeling et al., 2005).

Introduction

Tadarida brasiliensis, commonly known as the Mexican free-tailed bat or the Brazilian free-tailed bat, is medium-sized New World insectivorous bat. Belonging to the family Molossidae, Tadarida is one of 21 genera that comprise the 4 ^th largest family in Chiroptera ( Simmons, 2023). The genus of Tadarida contains 8 species, with T. brasiliensis being the only New World bat of the genus ( Figure 1). While some genera with Molossidae show support for monophyly, Tadarida does not show evidence for forming a monophyletic clade ( Agnarsson et al., 2011). Since T. brasiliensis is the only New World bat of this genus, a subgenus classification of Rhizomops has been previously proposed ( Legendre, 1984), but morphological and genetic evidence does not support this distinction ( Gregorin & Cirranello, 2016; Ammerman et al., 2012). Genetic evidence from four genes produces a clade specifically formed of T. brasiliensis, Tadarida aegyptiaca and Sauromys petrophilus ( Ammerman et al., 2012), but more recent analysis using morphological evidence does not support this clade ( Gregorin & Cirranello, 2016). The closest relative of T. brasiliensis is T. aegyptiaca, with T. brasiliensis showing a higher relation to Old World molossids with the last shared common ancestor with the New World clade being 29 mya ( Ammerman et al., 2012).

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Figure 1.

Position of Tadarida brasiliensis in the phylogeny of Family Molossidae.

Tadarida brasiliensis is one of 8 species currently recognized in the genus Tadarida (Rafinesque, 1814). Tadarida belongs to the subfamily Molossinae ( Gervais, 1856), which currently includes 20 genera and 132 species. FIgure created with BioRender.com.

T. brasiliensis is one of the most widely distributed mammals in the New World, and one of the most abundant bat species. According to the International Union for Conservation of Nature (IUCN), T. brasiliensis is listed as Least Concern ( Barquez et al., 2015). The geographic range of the species includes most of the United States, Mexico, Central America, and southwestern South America, including Greater and Lesser Antilles ( Wilkins, 1989). Due to the species’ large range, and proposed behavioral and morphological differences, T. brasiliensis was once thought to comprise up to nine subspecies ( Schwartz, 1955). However, the population structure from genetic studies and morphological evidence do not confirm any subspecies classification and support gene flow between mainland and island populations ( Morales et al., 2016; Morales et al., 2018). Instead, phenotypic differences are hypothesized to be correlated to climatic variation across regions and individuals are recommended to be categorized as migratory or non-migratory ( Morales et al., 2016). The large geographic range is most likely related to the species’ large dispersal capabilities.

One of the defining traits of T. brasiliensis is the “free tail”, where the tail extends beyond the uropatagium which is characteristic of the Molossidae family ( Figure 2A). T. brasiliensis is the smallest of the New World molossids with an adult weight range of 11–14 g, average total body length of 95 mm, and the average forearm length of 42 mm ( Schmidly, 2018). The species has a short velvety brown pelage, with long hairs on the feet that extend past the toes ( Wilkins, 1989). The snout is short with wrinkled lips. The ears, when pressed forward, do not extend past the snout and do not meet at the midline. The species is not strongly sexually dimorphic, but reproductively active males have an enlarged gular gland which is a sebaceous gland found on the suprasternal neck region ( Krutzsch et al., 2002). This gland shows seasonal functionality and during the breeding season secretes a thick, oily, odorous substance in reproductive adult males.

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Figure 2.

Tadarida brasiliensis A) Adult individual of the Mexican free-tailed bat, Tadarida brasiliensis. Note the tail extending beyond the uropatagium, short wrinkled snout, and hairs extending beyond toes. Photo courtesy of Brock and Sherri Fenton with permission, Windsor Cave, Jamaica. B) An echolocation pulse sequence emitted by Tadarida brasiliensis while foraging over a pond. This sequence begins with a typically shallow frequency-modulated search-phase pulse and ends with a terminal buzz.

T. brasiliensis is known for forming large colonies, with a single colony sometimes containing several million individuals. The densest populations of the species are found in Texas, where in the summer an estimated 95–104 million bats (primarily females forming maternity colonies) occupy a few select caves known as guano caves ( Schmidly, 2018). T. brasiliensis primarily roost in caves or man-made structures such as buildings or under bridges but have also been found in hollow trees in the southeastern US ( Wilkins, 1989).

This species is classified as a migratory bat with some of the longest recorded bat migrations. T. brasiliensis is estimated to have an annual migration as far as 1500 km ( Villa-R & Cockrum, 1962), traveling from central and southwestern United States southward into Mexico ( Cockrum, 1969). While shorter seasonal movements in temperate bats are not uncommon, mostly in response to unfavorable climate ( Popa-Lisseanu & Voigt, 2009), longer migrations as seen in T. brasiliensis are rarer in bats. While temperature changes could be triggering this movement, another potential reason for this annual trip is the seasonal availability and distribution of food resources, such as movements of migratory moths ( Russell et al., 2005). Even during nightly foraging trips, individuals may travel more than 160 km from their roost to a foraging site and back in one evening, maintaining horizontal flight speeds of up to 44 m/s ( Davis et al., 1962; McCracken et al., 2016). This high dispersal capability may be responsible for the lack of distinct population structure across the wide species range. However, there are differences in migratory behavior based on geographic location and sex. Previous banding experiments propose sedentary or non-migratory subpopulations in parts of the United States ( Cockrum, 1969), but no genetic differentiation has been found to account for this difference in migratory phenotype ( Russell et al., 2005). Also, females seem to travel further with more males forming resident populations in the winter or not traveling as far into Mexico ( Russell et al., 2005).

T. brasiliensis is an aerial insectivore that relies upon echolocation to navigate and forage for prey ( Figure 2B). In open spaces this species emits a shallow frequency-modulated echolocation pulse that descends from approximately 25 to 20 kHz over a 15–20 ms duration. When approaching obstacles or prey, pulse durations are progressively shortened to 2 ms while the starting frequency of the fundamental harmonic is concomitantly raised to approximately 50 kHz, with higher (2 ^nd and 3 ^rd) non-overlapping harmonics also becoming prominent in the signal ( Schwartz et al., 2007; Simmons et al., 1978). T. brasiliensis is known to forage at high altitudes, with recorded foraging activity on migratory noctuid moths ( Lepidoptera) occurring as high as 3000 m ( Williams et al., 1973). However, T. brasiliensis can also forage near ground and their diverse diet varies seasonally depending on species abundance and availability ( McCracken et al., 2021; Ross, 1961).

Previously, there has only been a short read genome assembly of T. brasiliensis available (GenBank accession: GCA_004025005.1) which was generated as a part of the Zoonomia Project ( Zoonomia, 2020). Notable sequencing projects utilizing this assembly consist of comparative genomics analyses of diet, visual system adaptations based on foraging style, immunity and metabolic adaptations, and longevity across several bat species ( Blumer et al. 2022; Davies et al., 2020; Fushan et al., 2015; Moreno Santillan et al., 2021; Potter et al., 2021). As for future applications with the new reference quality long read genome assembly reported herein, T. brasiliensis has previously been proposed as an ideal mammal model for the genetic and epigenetic basis of migration, mostly due to the widespread species range, abundant population, and variation of the phenotype ( Merlin & Liedvogel, 2019). Functional genomics projects should be fruitful in this species compared to other North American bats due to their high population levels and therefore less conservation pressure, as sample sizes are often a limiting factor. Although there is continued potential for comparative projects across multiple bat species, access to this genome assembly will hopefully encourage more work regarding the genetic basis for T. brasiliensis’ unique traits.

Genome sequence report

The genome was sequenced from a single male Tadarida brasiliensis collected from the Texas A&M campus of College Station, Brazos County, Texas, USA. A total of 39-fold coverage in Pacific Biosciences Hi-Fi long reads (contig N50 86 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 2.28 Gb in 147 sequence scaffolds with a scaffold N50 of 111 Mb ( Table 1). The majority, 98.44%, of the assembly sequence was assigned to 25 chromosomal-level scaffolds, representing 23 autosomes (numbered by sequence length, and the X and Y sex chromosomes). Chromosomal pseudomolecules in the genome assembly of Tadarida brasiliensis are shown in Table 2. The assembly has a BUSCO ( Simao et al., 2015) completeness of 96.3% using the laurasiatheria reference set. While not fully phased, the assembly deposited is of one haplotype.

Methods

The T. brasiliensis specimen was an adult male individual collected on the evening of October 15, 2018. The bat was caught by hand net as it left a roost located in a building on the Texas A&M campus in College Station, Brazos County, Texas, USA. Capture, handling, and sampling were approved by the local institutional Animal Care and Use Committee (Texas A&M University animal use protocol # 2017-0163D) and by Texas Parks and Wildlife scientific collecting permit SPR-1104-610.

Upon capture, a dichotomous key ( Schmidly, 2018) was utilized to confirm species identity. The bats were identified as T. brasiliensis based on specific morphological features such as forearm measures and other external features. Additionally, other molossids found in Texas ( Eumops perotis, Nyctinomops femorosaccus, Nyctinomops macrotis) are much larger compared to T. brasiliensis. At the capture location, there is limited range overlap with other molossids except for N. macrotis which is an uncommon species. However, these two species can be distinguished based on ear morphology as N. macrotis’ ears meet on the midline of the head whereas T. brasiliensis’ ears do not.

After collection from the field, the specimen was brought back to the laboratory and spent 5 months in captivity before tissue extraction and sample preparation. The animal was euthanized with pentobarbital overdose on February 28, 2019. Tissue samples collected were blood, brain (left hemisphere, cerebellum, front and back cortex, dorsal and ventral striatum), liver, heart (ventricle), left and right lung, spleen, left and right kidney, arm muscle, and left and right testicle. In total, 23 tissue samples were collected. All tissue samples were flash frozen in liquid nitrogen and stored in a -80°C freezer until shipment with the cold chain maintained. All data were recorded and reported in accordance with the ARRIVE guidelines ( Kilkenny et al., 2010) – see data availability section and Table 1.

Hi-C chromatin confirmation capture

Chromatin confirmation capturing was done making use of the ARIMA-Hi-C (Material Nr. A510008) and the Hi-C+ Kit (Material Nr. A410110) and followed the user guide for animal tissues (ARIMA-Hi-C kit, Document A160132 v01 and ARIMA-Hi-C 2.0 kit Document Nr: A160162 v00). In brief, circa 50 mg flash-frozen powdered tissue was crosslinked chemically. The crosslinked genomic DNA was digested with the restriction enzyme cocktail consisting of two and four restriction enzymes, respectively. The 5’-overhangs are filled in and labelled with biotin. Spatially proximal digested DNA ends were ligated and finally the ligated biotin containing fragments were enriched and went for Illumina library preparation, which followed the ARIMA user guide for Library preparation using the Kapa Hyper Prep kit (ARIMA Document Part Number A160139 v00). The barcoded Hi-C libraries run on a NovaSeq6000 with 2x 150 cycles.

Assembly was carried out following the Vertebrate Genome Project pipeline v2.0 ( Rhie et al., 2020) as follows. HiFi reads were created with ccs (v6.0.0). HiFiasm (v0.16.0) was used to create the initial contig set. Haplotypic duplication was identified and removed with purge dups (v1.2.5) ( Guan et al., 2020). The quality of the assembly was evaluated using Merqury ( Rhie et al., 2020) and BUSCO ( Manni et al., 2021). Scaffolding with 10X data was carried out with Scaff10X (commit bc3a0cb), Bionano data with Bionano Solve (v 3.6.1) and Hi-C data ( Rao et al., 2014) with SALSA2 (commit e6e3c77) ( Ghurye et al., 2019). HiGlass ( Kerpedjiev et al., 2018) was implemented to generate Hi-C contact maps and perform manual curation of scaffolds into chromosomes. Figure 3, Figure 4, Figure 5 & Figure 6 were generated using BlobToolKit ( Challis et al., 2020). Software utilized for T. brasiliensis analysis are depicted in Table 3.

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Figure 3.

Hi-C Contact Map of the T. brasiliensis assembly with 25 chromosomes, visualized using HiGlass.

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Figure 4.

Genome assembly metrics generated using blobtoolkit for the T. brasiliensis 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.

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Figure 5.

GC coverage plot generated for the T. brasiliensis 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.

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Figure 6.

Cumulative sequence plot generated for the T. brasiliensis 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.

Table 3.

Software tools used.

Software tool	Version	Source
bamUtil	1.0.15	https://genome.sph.umich.edu/wiki/BamUtil:_bam2FastQ
FastQC	0.11.9	https://www.bioinformatics.babraham.ac.uk/projects/fastqc/
MultiQC	1.13	https://github.com/ewels/MultiQC
Genomescope	2.0	https://github.com/tbenavi1/genomescope2.0
HiFiasm	0.16.0	https://github.com/chhylp123/hifiasm
purge_dups	1.2.5	https://github.com/dfguan/purge_dups
BUSCO	5.3.2	https://busco.ezlab.org/
Merqury	1.3	https://github.com/marbl/merqury
Assembly-stats	17.02	https://github.com/rjchallis/assembly-stats
Scaff10X	4.2	https://github.com/wtsi-hpag/Scaff10X
Bionano Solve	3.6.1	https://bionano.com/software-downloads/
Arima-HiC Mapping Pipeline	-	https://github.com/ArimaGenomics/mapping_pipeline
SALSA	2.2	https://github.com/marbl/SALSA
HiGlass	1.11.7	https://github.com/higlass/higlass
samtools	1.9	https://www.htslib.org/
BlobToolKit	3.2.7	https://github.com/blobtoolkit/blobtoolkit

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Acknowledgments

Notes

[version 1; peer review: 2 approved, 2 approved with reservations]

Funding Statement

SCV was supported by a UKRI Future Leaders Fellowship, (MR/T021985/1), an ERC Consolidator Grant (101001702; BATSPEAK), and a Max Planck Research Group awarded by the Max Planck Society. ECT is a Wellcome collaborator and the Irish Research Council Laureate Award IRCLA/2017/58 and Science Foundation Ireland Future Frontiers 19/FFP/6790. MS and CW were supported by the US Office of Naval Research (ONRN00014-17-1–2736).

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References