Abstract:
The rufous horseshoe bat, Rhinolophus rouxii (family Rhinolophidae) has a wide distribution
and has been recorded from China, India, Myanmar, Nepal, Vietnam, and Sri Lanka. It is a
medium sized bat (11-13g), and shows enormous cryptic diversity. Recent research has shown
that R. rouxii contains many morphologically diverse, species complex and fur color as well as
the echolocation call frequency varies in different populations. We have used molecular
techniques for establishing the accurate identity of R. rouxii, recorded in one of the largest cave
populations in Sri Lanka. Bats were caught using a hand net and saliva samples (n=21) were
collected non-invasively from each bat using a sterile oral swab. Nucleic acids were extracted
from oral swab samples following the QIAmp Viral RNA Mini kit (extract both DNA and
RNA) protocol. Mitochondrial cytochrome b gene was amplified using cytochrome b, RrFP
and RrRP primers. PCR products were visualized using gel electrophoresis. Purified PCR
products were sequenced using Sanger sequencing. Sequence analyses were carried out using
the GeneiousPrime application. FASTA sequences of each bat obtained from sequencing were
trimmed and assembled to form the consensus sequences. Aligned sequences were compared
with sequences in the database at National Center for Biotechnology Information Support
Center (NCBI). Cytochrome b gene sequences of Rhinolophus rouxii bats were downloaded
from NCBI database and aligned with the consensus sequences using the Geneious alignment
tool. The phylogenetic tree was constructed using the Neighbor-Joining method. This study
identified the Rhinolophus species recorded in Wellawaya cave as Rhinolophus rouxii with an
average identity of 98.40%. Phylogenetic analysis showed it is closely related to the same
species identified in Karnataka district in India. This study reports the first molecular
confirmation carried out on Rhinolophus rouxii in Sri Lanka. This will provide the basis for a
genetic database for Sri Lankan bats.
