Bats examined in this study were captured from the Maoershan National Nature Reserve (25°48'N–25°58'N, 110°20'E–110°35'E), which covers an area of 170.09 km² of mountains with varied vegetation types. Although some areas at lower elevations have been transformed into bamboo forests, most of the reserve is undisturbed with primary forests, especially at higher elevations (Huang and Jiang 2002). Four Plecotus bats were captured from two sites (Fig. 1; 25°26'20"N, 110°53'32"E, 2002 m a.s.l. and 25°54'42"N, 110°27'14"E, 1708 m a.s.l.) with harp traps during a bat survey along an elevational gradient in June 2022. These bat specimens, designated GD-221656, GD-221657, GD-221658, and GD-221659, were preserved in anhydrous ethanol after all examinations were completed. These specimens are stored at the Guangdong Institute of Zoology.

Figure 1. 

Distribution of Plecotus bats in China and other regions. The map shows the southernmost regions, not the entire China, where Plecotus bats have been found.

Morphological measurements of bats were performed with electronic digital calipers according to Dai et al. (2020). Definitions of the measurements are as follows: FA, forearm length; T, tail length; HB, head and body length; Thsu, thumb length excluding claws; Thcu, thumb length including claws; Tib, tibia length; Hfsu, hindfoot length excluding claws; Hfcu, hindfoot length including claws; Trag, tragus length; E, ear length; STOTL, total length of the skull; CBL, condylobasal length; CCL, condylo-canine length; MAW, mastoid width; CM³L, maxillary toothrow length; CCW, width across upper canines; M³M³W, width across upper molars; CM₃L, mandibular tooth row length; ML, mandible length; UJH, lower jaw height; BCW, braincase width; BCH, braincase height; ZYW, zygomatic width; RL, rostral length; Bulla, diameter of tympanic bulla; IOW, interorbital width. The wing shape of each bat was recorded by tracing on paper, followed by a determination of wing loading and wingspan ratio using IMAGE J according to the method of Norberg and Rayner (1987). The criteria of Bininda-Emonds and Russell (1994) and Aldridge and Rautenbach (1987) were used for classification of wingspan ratio and wing loading as follows: wingspan ratio: low, 6.1–7.3; high, ≥7.3; wing loading: very low, ≤6.45 N/m²; low, 6.45–10.3 N/m²; high, ≥10.3 N/m².

Morphological measurements of six Plecotus species (Suppl. material 1: table S1) were used for the principal component analysis (PCA) using ‘prcomp’ function of the R package ‘stats’ (R Core Team 2021). The following 10 craniodental measurements were assessed by PCA: STOTL, CBL, MAW, CM³L, M³M³W, CM₃L, BCW, BCH, Bulla, and IOW (Suppl. material 1: table S2).

Echolocation calls of four bats were recorded using a handheld ultrasound detector (UltraSoundGate 116Hm, Avisoft Bioacoustis, Germany) when they were allowed to fly in a room of 5 × 5 × 2.5 m³ in size. Ultrasound spectrograms were generated using the 512-point Fast Fourier Transform (FFT) algorithm with 96.87% of the frequency overlapped with a Hanning window. A total of 30 pulses were arbitrarily selected from each bat for determination of start frequency, end frequency, frequency of maximum energy, and pulse duration using the Batsound software (Pettersson Elektronik AB, Uppsala, Sweden). The values were determined based on the second (highest energy) harmonic and statistically compared with those of the study from Vietnam (Dai et al. 2020) using ‘kruskal.test’ function of the R package ‘stats’ (R Core Team 2021) as the data were non-normally distributed as determined by the Shapiro-Wilk and normal Q-Q plot.

To further identify the bats, DNA was extracted from a small piece of the wing membrane of each bat, and polymerase chain reaction was performed to amplify a portion of the mitochondrial cytochrome b gene (Cyt b) using primers Cyt b-F (5′-TAG AAT ATC AGC TTT GGG TG-3′) and Cyt b-R (5′-AAA TCA CCG TTG TAC TTC AAC-3′) (Li et al. 2006). Each PCR was conducted in a volume of 50 μl containing 8 μl of genomic DNA, 2 μl each of primer F and R (10 mM each), 13 μl of water, and 25 μl of HiFi DNA polymerase master mix. PCR conditions were as follows: 5 min at 94 °C, followed by 10 cycles of 60 s at 94 °C, 30 s at 46 °C, and 62 s at 72 °C; 25 cycles of 60 s at 94 °C, 40 s at 50 °C (+0.3 °C/cycle), and 60 s at 72 °C; 35 cycles of 60 s at 94 °C, 40 s at 54 °C, 60 s at 72 °C, and final elongation for 10 min at 72 °C.

The obtained sequences were deposited in GenBank under the following accession numbers: OP425735 (GD-221657), OP425736 (GD-221659), and OP425737 (GD-221656). No sequences were obtained from bat GD-221658 because of a failure in DNA isolation. The sequences were aligned with those of 30 Cyt b genes (Table 4) from GenBank for phylogenetic analysis using MAFFT software (Katoh and Standley 2013). Selection of the best-fit nucleotide substitution model was performed by MODELFINDER (Kalyaanamoorthy et al. 2017), and the phylogenetic tree was constructed using the maximum-likelihood (ML) method in IQ-TREE with 5,000 ultrafast bootstraps (Nguyen et al. 2015).

In PCA, the percentages of explained variance of the first two principal components (PC1 and PC2) were 65.8% and 12.3%, respectively, with a cumulative percentage of 78.1% (Suppl. material 1: table S2). PC1 results were derived from all measurements except those of tympanic bullae (Bulla) and interorbital width (IOW), whereas results of PC2 were from analysis of Bulla and IOW (Table 1). PCA plots revealed that the four investigated bats were clustered with P. homochrous from Vietnam but were widely separated from other bats including P. ariel (Thomas, 1911), P. kozlovi (Bobrinski, 1926), P. ognevi (Kishida, 1927), P. strelkovi (Spitzenberger, 2006), and P. wardi (Thomas, 1911). This result suggests that these four bats are P. homochrous.

Morphologically, the bats have long, wide auricles, each with a prominent tragus (Fig. 3B). The length of each auricle is about the same as that of a forearm (Table 1). The bases of the two ears intersect at the forehead (Fig. 2A). Hairs on the ventral fur have a dark base with mixed grey and yellowish tips; those on the dorsal fur also have a dark base and are bicolored with brown tips (Fig. 2A, C). The facial fur is dark, and the skin is pink (Fig. 2B). The thumbs are very short (Fig. 2C). The wing membrane is attached to the base of toes, and there is a small, triangular protrusion at the base of the tail membrane near the heel (keeled calcar) (Fig. 2A). The dental formula is I 2/3, C 1/1, P 2/3, and M3/3. The first upper incisor is double pointed and higher than the second upper incisor. The second upper premolar is absent (Fig. 3B). The cranium is 16.02–16.43 mm in length and 8.13–8.27 mm in zygomatic width (Table 1), with a slight sagittal crest, which is the smallest among all Plecotus species. The bullae are medium-sized (diameter 4.20–4.43 mm). A concavity is present in the front of the dorsal side of the cranium (Fig. 3C). The orbital ridge is in the anterior part of the eye socket (Fig. 3A). All these morphological characteristics are identical to those of P. homochrous from Vietnam (Dai et al. 2020).

Figure 2. 

Pictures of Plecotus homochrous (GD-221656) examined in this study A left side B face C dorsal side.

Figure 3. 

Cranial morphology of Plecotus homochrous (GD-221656) A cranium in dorsal view B cranium in ventral view C cranium in left side view D mandible in dorsal view E mandible left side view.

Echolocation calls of the four bats are of frequency-modulation (FM) with multiple harmonics. The maximum energy of calls is mostly in the second harmonic (Fig. 4). Sound parameters of echolocation calls vary among the four individuals. Start frequency, end frequency, frequency of maximum energy, and pulse durations are 74.0 ± 2.8 kHz, 52.2 ± 1.9 kHz, 58.7 ± 0.6 kHz, and 1.5 ± 0.2 ms (Mean ± SD), respectively. There is no significant difference in start frequency, end frequency, and frequency of maximum energy between the P. homochrous bats from Vietnam and the four bats examined in this study (P values 0.16, 0.53, and 0.26) (Table 2). However, there is a significant difference in pulse duration (P value 0.01). The four bats also have a very low wing loading (5.68 ± 0.29 N/m²) and a low wingspan ratio (6.82 ± 0.70) (Table 3), indicative of slow and flexible flights.

Figure 4. 

Amplitude and spectrogram of echolocation calls of bats examined in this study.

The phylogenetic tree reveals two major clades. The first clade contains P. auritus, P. homochrous, P. kozlovi, P. macrobullaris, P. ognevi, and P. sacrimontis (i.e. P. auritus group). The second one includes P. austriacus, P. balensis, P. kolombatovici, and P. teneriffae (i.e. the P. austriacus group). Bats GD-221656, GD-221657, and GD-221659 are clustered with P. homochrous from Vietnam (Fig. 5).

Figure 5. 

Phylogenetic tree of bats constructed based on results from the maximum-likelihood (ML) analysis of Cyt b gene sequences. Numbers on ML tree nodes are ultrafast bootstrap (UFB) support values.