New records and an updated checklist of the genus Myotis (Chiroptera, Vespertilionidae) in Xizang, China

The mitochondrial cytochrome b (cyt b) gene was amplified by PCR using primers from Irwin et al. (1991) and Ruedi et al. (2013). Each 30 µL reaction contained: 13 µL of 2×Accurate Taq Premix (AG11009, Accurate Biotechnology Co., Ltd., China), 0.5 µL of each forward and reverse primer (10 µmol/L), 14 µL of RNase-free water, and 2 µL of DNA template (~20 ng/µL). Thermal cycling profile was: initial denaturation at 94 °C for 5 min; 37 cycles of 94 °C for 45 s, 46 °C for 30 s, and 72 °C for 60 s; and a final extension at 72 °C for 5 min.

Amplification products were verified by agarose gel electrophoresis and submitted to Sangon Biotech (Shanghai) for bidirectional Sanger sequencing. The 18 newly generated cyt b sequences from this study.Echolocation call recordingEcholocation calls of Myotis bats were recorded in flight state using an ultrasound detector (M500-384, Pettersson Elektronik AB, Sweden). Recordings were conducted in a quiet room (approximately 3 m×3 m×3 m).

Acoustic data were analyzed using BatSound v.4.0 (sampling frequency: 300 kHz, Fast Fourier Transform (FFT) size: 1024 points). For each species, more than 10 high-quality pulses were selected to measure three key parameters: peak frequency, pulse duration, and inter-pulse interval. Given that the Myotis calls typically consist of broadband frequency-modulated (FM) signals with multiple harmonics, where the first harmonic is the strongest and other harmonics (2nd–4th) decrease successively, only the first harmonic was analyzed in this study.