Bats Out of Africa: Disentangling the Systematic Position and Biogeography of Bats in Cabo Verde

Cabo Verde Archipelago presents one of the largest knowledge gaps in the distribution and taxonomy of bats in the world. Old works indicated that there are five species classified as European taxa. We have conducted an integrative taxonomy to revise the systematic position and distribution of Cabo Verdean bats with molecular, morphological, and ecological data, to test their native or exotic origin, and infer possible colonization patterns based on fieldwork and museum samples. Results showed that Cabo Verde Hypsugo is closely related to those from the Canary Islands, in which the taxonomic status is under debate, presenting unique mitochondrial and nuclear haplotypes. We also expanded the distribution of Taphozous nudiventris for Fogo Island through pellets and acoustic identification, showed unique haplotypes for this species, and that Miniopterus schreibersii shared a haplotype with European, North African, and Western Asian specimens. The morphological and acoustic identification of Cabo Verdean specimens was challenging because of the lack of modern morphological descriptions and similarity of echolocation calls within the same genus. More studies are definitely needed to access the systematic of bat species in the archipelago, but this work is the first step for the establishment of conservation actions of the probable only native Cabo Verdean mammals.


Ancient DNA extraction protocol
The samples were cut in small pieces and about 50 mg was added to a 2.0 ml eppendorf tube together with 1 ml of extraction buffer (745 μL H2O; 9 ml EDTA 0.5 M (pH=8.0); 250 μL Proteinase K 10 mg/ml; 5 μL Tween 20) they were left overnight at 37°C in the incubator. In the next day, the samples were spun for 2 min at maximum speed and the supernatant was transferred to a 50 ml falcon tube containing 10 ml Binding Buffer (23.88 g Guanidine hydrochloride; 30 ml H2O; 50 ml Isopropanol; 25 μL Tween 20) and 400 μL 3M sodium acetate. A MinElute spin column was separated for each sample and an extension reservoir of a V-spin column was forced into the opening of the MinElute tube. The extension reservoir/MinElute assembly was removed from the collection tube and placed into a 50 ml falcon tube.
The solution with Binding buffer mixture was transferred into the extension reservoir, then it was centrifuged for 4 min at 1500 rpm and then for 2 min at 1500 rpm. The extension reservoir/spin column assembly was placed back into the collection tube and a dry spin was performed for 1 min at 6000 rpm in a centrifuge, then the flow-through was discarded. After that, 750 μL PE buffer was added and the samples were centrifuged at 6000 rpm for 30 s and the flow-through was discarded, this last step was repeated. After that, the spin column was turned and a dry spin was performed for 1 min at 13000 rpm, the spin column was then transferred into a 1.5 ml tube with the cap ripped off. Next, 25 μL TET buffer (~49.4 ml H2O; 100 μL EDTA 0.5 M (pH=8.0); 500 μL Tris-HCl 1 M (pH=8.0); 25 μL Tween 20) was added on top of the silica membrane and left standing for 5 min before centrifugation at 13000 rpm during 30 s. After that, more 25 μL of TET was added and the centrifugation was repeated. The 50 μL of the extraction was transferred to a fresh tube labeled with sample code.

DNA extraction with silica protocol
The feces were preserved in ethanol 96% and frozen. In order to proceed the DNA extraction, the samples were left in the greenhouse overnight to evaporate de ethanol. The entire process was done with filter tips and a negative control was prepared for the set of samples. The first step was adding 20 ml of PBS solution (6.08 g KH2PO4; 8.62 g NaCl and 1000 ml H2O) in a 50 ml falcon with the scat (entire pellet). The material was wash vigorously with a Pasteur pipette to promote the epithelial cells elution. About 15 ml of the supernatant was transferred to a 15 ml falcon and centrifuged at 4000 rpm during 20 min. After that, the supernatant was discarded and 2 ml of buffer L6 (147.8 g Guanidine Thiocyanate; 25 ml Tris-HCl 1 M (pH=6.4); 10 ml EDTA 0.1 M (pH=8.0); 3.25 ml Tríton X-100 and 250 ml H2O) was added to the pellet and the solution was vortexed. Then, the samples were incubated overnight at room temperature on constant agitation.
In the next day, they were centrifuged at 4000 rpm at 15 min, the supernatant was transferred to a new 15 ml falcon, 250 μL of silica solution was added and vortexed during 2 min to promote the bond of DNA molecules with silica solution. Then, the solution was centrifuged during 2 min at 4000 rpm. After that, the supernatant was discarded and 2.5 ml of buffer L2 (350 g Guanidine Thiocyanate; 59.2 ml Tris-HCl (pH=6.4); 23.7 ml EDTA (pH=8.0) and 592 ml H2O) was added to each sample and vortexed until the pellet dissolution. The tubes were centrifuged at 4000 rpm during 2 min, this last step was then repeated in order to clean the DNA solution. Then, 4 ml of ethanol 80% was added to dissolve the entire pellet and the samples were centrifuged again at 4000 rpm during 2 min.
The tubes were left in the greenhouse at 60°C to dry the pellet and remove all the ethanol. The DNA elution was made with 500 μL of ultra-pure water, the material was vortexed to dissolve the pellet and incubated at room temperature. After that, the solution was centrifuged at 4000 rpm during 10 min, the supernatant was recovered and transferred to a new eppendorf 1.5 ml. In order to remove all silica residuals, the samples were centrifuged at 8000 rpm during 1 min. The supernatant was then transferred to a column and centrifuged at 13000 rpm during 10 min to purify the DNA. The fluid was discarded, 500 μL of ultra-pure water was added to the column and centrifuged at 13000 rpm during 15 min. Then, the final step was adding 120 μL of ultra-pure water to a new tube, invert the column and centrifuge at 14000 rpm during 1 min. For the Silica Solution preparation, 6 g of Silica was dissolved in 50 ml of distilled water. The solution was incubated at room temperature wrapped in aluminium foil to avoid light during 24 hours to promote silica precipitation. The supernatant was discarded and 50 ml of distilled water was added, the solution was vortexed until the dissolution of the silica pellet. Then it was incubated at room temperature covered by aluminium foil during 5 hours to promote de silica precipitation. The supernatant was discarded (about 44 ml) and 60 μL of HCL 10 M (37%) was added.