Title: Phylogenetic relationships among three species of long-eared Mytosis in British Columbia inferred from mitochondrial DNA sequences.

Please note: this report was copied from an obscure B.C. Ministry of Environment web site that no longer exists, therefore archiving this document on this server has made it possible for it to be still viewed, Rick.

Project Leader: Dave Nagorsen, Royal British Columbia Museum, 675 Belleville St., Victoria, BC, V8V 1X5,
G.C. van Zyll de Jong, Research Associate, Royal Ontario Museum

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Three morphologically similar species of long-eared bats of the genus Mytia occur in British Columbia: M. evotis, M. keenii and M. septentrionalis (van Zyll de Jong 1979 ), The dark coastal form of M. evotis closely resembles M. keenii and M. evotis, M.e. pacificus closely resembles M. keenii. The great similarity of these two taxa represents a problem in identification and also raises questions regarding the phylogenetic relationship of these bats.

Mytosis keenii, keen long-eared bat, is of special interest because it appears to be endemic in the coastal rain forest. Nothing is known of the biology of this species. Current field work, to determine the status of the species and to study its biology and ecology, is hampered because of difficulties in field identification. Myotis keenii and the west coast M. evotis are virtually identical in external appearance.

In a recent study (van Zyll de Jong and Nagorsen 1994), we have sought to clarify the systematic relationship of the two taxa using morphological data and we developed a discriminate function based on a few external measurements, that may be useful in the identification of live bats in the field. Although our study confirmed the distinctness of M. evotis and M. keenii, questions; regarding the phylogenetic relationships of the three long-eared bats in British Columbia persist. We propose that the phylogenetic relationships of these taxa can be elucidated further by the analysis of mitochondrial DNA sequences.

Description of the work to be done - the molecular data to falsify 1 & 2 will come from freshly obtained tissue samples of the three species of Myotis and from an outgroup taxon. Small skin biopsies can be collected without harming the bat. An initial replication of six specimens per species gives a requirement of 24 tissue samples for processing.

DNA extraction's. Small quantities of skin tissue will be digested in 10 mm Tris-HC1 (pH 8.0), 2mM EDTA, l0 mM NaCl, 1% SDS, 10 mg/ml DTT and 0.5 mg/ml proteinase K, overnight at 37 C with constant gentle mixing. The resulting solution is then extracted (2x) and chloroform/isoamyl ( lx ). The extract is then concentrated and desalted on a Centricon-30.

Amplification and sequencing. The extracted DNA is amplified in an amplification buffer in the presence of Tag (Thermus aquaticus) polymerase and universal primers for segments of the mitochondrial genome that evolve at different rates (Kocher et al 1989, Proc . Natl. Acad. Sci .USA 86:6196-6200 ) . single stranded mtDNA used for sequencing using dideoxynucleotide chain terminators. The products of sequencing are resolved by polyacrylamide electrophoresis and autoradiographed. The resulting sequences are then analyzed to reconstruct the phylogenetic relationships among the taxa using parsimony analysis including an appropriate outgroup.

Schedule -

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