The specimens used in this study were collected using mist-nets in 2023 during field expeditions to three nature reserves in Yunnan, China (Fig. 1). M. magnater and M. pusillus were collected in April 2023 from the Huanglianshan National Nature Reserve, Luchun County, Yunnan, China (N22.85°, E102.19°, altitude 866 m). M. fuliginosus was collected from two different localities: the Cangshan Erhai National Nature Reserve in Dali Bai Autonomous Prefecture, Yunnan, China (25.57 °N, 100.14 °E, altitude 1912 m, September 2023) and Yuanyang Guanyinshan Provincial Nature Reserve of Yunnan, China (N23.01 °, E102.94 °, altitude 2401 m, October 2023) (Bates and Harrison 1997). Specimens were preserved by immersion in absolute ethanol. The specimens were deposited at the Kunming Natural History Museum of Zoology, Kunming Institute of Zoology, Chinese Academy of Sciences (KIZ, CAS). M. magnater collection numbers are KIZ20230213, KIZ20230270, KIZ20230274, KIZ20230276, KIZ20230307 and KIZ20230312. The specimen collection numbers of M. fuliginosus are KIZ20230869, KIZ20230871, KIZ20230921, KIZ20230922, KIZ20231102 and KIZ20231111. The collection numbers of M. pusillus are KIZ20230263, KIZ20230265, KIZ20230305, KIZ20230306, KIZ20230311 and KIZ20230314.

Figure 1.  

The collection sites of the specimens used in this study.

Five external morphologies: tail length (TL), head-body length (HB), forearm length (FA), tibia length (TIB) and ear length (EAR) were measured in the field (Bates and Harrison 1997). The skull specimen was stripped under experimental conditions and the attached muscle tissue on the skull was carefully removed to maintain the integrity of the skull structure. A digital vernier caliper was used to measure the skull, the measurement accuracy being 0.01 mm. In terms of skull morphology measurement, we selected seven features for measurement: greatest skull length, from the anterior rim of the alveolus of the first upper incisor to the most projecting point of the occipital region (GSKL); postorbital breadth, narrowest dorsal width posterior to the postorbital constriction of the cranium (POB); mastoid width, the greatest width across the mastoid region (MAW); mandible length, from the posterior-most point of the condyle to anterior-most alveoli of lower incisors (ML); width across upper canines, taken across the outer-most points of the crowns of the upper canines (C-C); width across the upper third molars, taken across the outer-most point of the crowns of the 3^rd upper molars (M3-M3); complete upper canine-molar tooth row, taken from the anterior-most point of the crown of the upper canine to the posterior-most point of the crown of 3^rd upper molar (C-M3). Skull measurements were based on bat studies of the genus Pteropus in India and Sri Lanka (Kusuminda et al. 2022). The measurements of bat wing bones are undertaken following Bates and Harrison (1997); mt: metacarpal, from the extremity of the carpus to the distal extremity of the metacarpal. Ph: phalanx of the metacarpal, taken from the proximal to the distal extremity of the phalanx. For example: 1ph3mt, refers to the first phalanx of the third metacarpal. 2ph3mt/4mt/1ph4mt/5mt/1ph5mt: as above. It is worth noting that we did not take in situ measurements of the hand skeleton of M. pusillus during the field survey. Therefore, the morphological characteristics of the wings of M. pusillus were measured by using specimens immersed in absolute ethanol. Since the morphology of the hand bones did not change, most of the characteristics were considered to be reliable and not much different from those when they were alive in the natural state. During the assessment, it was observed that the second phalanx of the wing exhibited significant curling, a condition not conducive to accurate measurement. Consequently, these specimens had to be manipulated into a straight configuration for measurement, an intervention that may introduce discrepancies between the measured data and the specimens' natural state. Due to these methodological limitations, the morphological data derived from the second phalanx were deemed unreliable for inclusion in our analysis. These measurements were conducted by a single individual using consistent equipment, thereby minimising the influence of human error on the data.

Data processing was undertaken using the IBM SPSS Statistics v. 27.0.1 non-parametric 1-sample K-S (Kolmogorov-Smirnov) test (Berger and Zhou 2014). Inspection of the morphological data obtained by our measurements showed that all morphological data conformed to a normal distribution. Analysis of variance (ANOVA) was used to determine whether there were significant differences in morphological parameters amongst species and principal component analysis (PCA) was performed on the morphological measurements of specimens.

Genomic DNA from bat muscle tissue preserved in anhydrous ethanol was extracted using the TSP202-200 Animal Genomic DNA Extraction Kit (Bejing Tsingke Biotech Co., Ltd., Chian). We used the primers Molcit-F (5'-AATGACATGAAAAATCACCGTTGT3', Ibáñez et al. (2006)) and Cytb-H (5'-CTTTTCTGGTTTACAAGACCAG-3', Weyeneth et al. (2008)) to amplify the complete mitochondrial cytochrome b (cyt-b) gene. Polymerase chain reaction (PCR) was performed in a 50 μl system. The system contained 45 μl of enzyme: Gold Mix (green), 2 μl of forward primer, 2 μl of reverse primer and 1 μl of DNA template. The PCR protocol required an initial denaturation at 94°C for 2 minutes, followed by five cycles of denaturation at 94°C for 30 seconds, annealing at 50°C for 30 seconds and extension at 72°C for 1 minute, followed by 35 additional cycles: denaturation at 94°C for 30 s, annealing at 55°C for 40 s, extension at 72°C for 1 min and final extension at 72°C for 10 min and renaturation at 4°C. Finally. purifed samples were sequenced by the ABI 3730XL DNA Analyzer (USA) at Beijing Tsingke Biotech Co., Ltd (China).

The evolutionary history was inferred by using the Maximum Likelihood method and Hasegawa-Kishino-Yano model (Hasegawa et al. 1985), which has the highest log likelihood (-7525.55). The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbour-Joining and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach and then selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences amongst sites (5 categories (+G, parameter = 0.4761)). The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 30.02% sites). This analysis involved 43 nucleotide sequences. There were a total of 1141 positions in the final dataset. Evolutionary analyses were conducted in MEGA11 (Tamura et al. 2021). We selected Cyt b sequences provided by other researchers from the National Center for Biotechnology Information (NCBI) database for comparative study. Amongst them, Kerivoula furva was used as the outgroup. The relevant situation is shown in the following table (Table 1).

The number of base substitutions per site from between sequences are shown (Suppl. material 1). Analyses were conducted using the Maximum Composite Likelihood model (Tamura et al. 2004). This analysis involved 21 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All ambiguous positions were removed for each sequence pair (pairwise deletion option). There were a total of 1141 positions in the final dataset. Evolutionary analyses were conducted in MEGA11 (Tamura et al. 2021). The relevant sequences of M. magnater, M. fuliginosus and M. pusillus used are detailed in Table 1.

Body and Fur

M. magnater is characterised by a slender body plan, featuring an oval head, rounded ears with distinct tragus and a well-developed thumb. The second phalanx of the third metacarpal (2ph3mt) is notably elongated, approaching approximately 90% of the forearm length (FA). The FA is robust and covered with dense fur on the ventral aspect, while the mouth area is devoid of villi. The hind limbs are well-developed, presenting five distinct toes and the genitalia are well-developed. The body of M. magnater is densely covered with downy hair, presenting an overall tan hue, while the villi tips are marginally golden brown. The colouration along individual villi remains largely uniform from the tail root to the head, without a pronounced gradient. Both the dorsal and ventral body surfaces, as well as the head to tail hair colouration, exhibit consistency, with no significant variation (Fig. 2).

Figure 2.  

Three specimens of Miniopterus used in this study. A Miniopterus magnater, specimen collection number: KIZ20230213; B Miniopterus fuliginosus, specimen collection number: KIZ20231111; C Miniopterus pusillus, specimen collection number: KIZ20230306, specimens preserved in anhydrous ethanol. 1 Dorsal view; 2 Ventral view.

Skull

In the dorsal view, the anterior maxilla presents a concave oval configuration, with the incisors exhibiting a distinct inward retraction. The sagittal suture is prominently defined. The canines are markedly prominent and the orbits are notably large. The zygomatic arches are characterised by a straight lateral profile, with robust anterior and posterior extremities and a more delicate mid-section. From a lateral perspective, the skull of M. magnater is elegantly slender, featuring a subtle concavity at the maxillary level and a pronounced sagittal suture. The occipital region of the skull is broadly oval, with a significant indentation observable on the parietal bones. The zygomatic processes are well-developed, with the anterior portion being broad and the posterior tapering to a narrower point. Relative to M. fuliginosus and M. pusillus, the skull of M. magnater is notably elongated and the sagittal suture is comparatively more developed (Fig. 3).

Figure 3.  

Photograph of the skulls, mandibles, specimens. A Miniopterus magnater, collection number: KIZ20230213; B Miniopterus fuliginosus, collection number: KIZ20231111; C Miniopterus pusillus, collection number: KIZ20230306. 1 dorsal view of the cranium; 2 lateral view of skull; 3 ventral view of skull; 4 lateral view of the mandible; 5 occlusal view of mandible.

Dentition

Dental formula of the M. magnater : 2.1.2.3/3.1.3.3 = 36. The base of the canines of the cranium protrudes outwards and the first premolar is depressed medially, canine developed. First premolar is small, not as high as the incisor, the height of first premolar is not half that of second premolar, second premolar is higher in height than the posterior teeth, the height of the molar almost the same. Well-developed canines in the lower jaw, the height of first premolar and second premolar is basically the same and third premolar is significantly higher than first premolar and second premolar, the height of the molars decreases sequentially from front to back (Fig. 3).

Morphological measurement data

The descriptive statistics, as delineated in Table 2, reveals pronounced distinctions between M. magnater, M. fuliginosus and M. pusillus. Amongst the trio of species evaluated, M. magnater emerges as the most substantial in stature. Owing to the morphological congruence observed on external examination, we measured some morphological data of the skull and wings. The cranial measurements for M. magnater align well with prior scholarly works (Hodgson 1835, Smith and Xie 2008). Variability is noted in the extremities of the measured range; however, the mean values, juxtaposed with the standard deviation of these metrics, indicate a higher degree of precision in the cranial measurements and suggest limited inter-individual variability within M. magnater.

By means of morphometric data, the specimens of the three species of the genus Miniopterus collected in this study are in general agreement with the morphometric data of other scholars (Hodgson 1835, Smith and Xie 2008, Saikia et al. 2020, Kusuminda et al. 2022). Thus, we have reason to believe that these specimens belong to three species of Miniopterus, M. magnater , M. fuliginosus and M. pusillus.

By analysis of skulls based on measurement data, M. magnater demonstrates significant morphological distinctions from both M. fuliginosus and M. pusillus, as evidenced by pronounced differences in GTL, POB, MAW, M3-M3, C-M3, C-C and ML, with statistical significance indicated by a P-value of less than 0.01. In the context of the wing skeleton, 3mt, 4mt and 5mt of M. magnater and M. fuliginosus do not exhibit significant variability (P > 0.05, with a minimum P-value of 0.257). However, 1ph3m and 1ph4mt display highly significant differences. 1ph5mt presents significant to extremely significant differences, as detailed in Table 3 and Suppl. material 2.

By performing principal component analysis on the morphometric data, we obtained three principal components. The eigenvalue of principal component 1 is 15.894 and Variance explained is 88.048%. The eigenvalue and Variance showed that the differences between component 2 and component 3 were relatively small and the cumulative variance contribution was 95.815%. Except for TL, E, 3mt, 4mt, 5mt and ML, which have relatively low loadings, the other eigen loadings are relatively high. Overall, there was less loss of original information, which was suitable for principal component analysis (Table 4).

The scatter plots, generated from the principal component analysis, distinctly delineate the three Miniopterus species, with no overlap observed between M. magnater, M. fuliginosus and M. pusillus. The data points exhibit a clear demarcation, forming three discrete groups (Fig. 4).

Figure 4.  

Scatterplots of the samples for principal component factors 1 vs. 2 and 1 vs. 3, respectively.

Phylogenetic analyses have confirmed that our cytochrome b (Cyt b) sequences, in conjunction with those from other researchers, form a monophyletic group with M. fuliginosus, M. magnater and M. pusillus. The sequences display no evidence of hybrid relationships amongst the three species. These phylogenetic outcomes are congruent with the findings from morphological studies, thereby reinforcing the validity of our species identifications (Fig. 5).

Figure 5.  

Phylogenetic tree of the genus Miniopterus, based on the cytb gene constructed using the Maximum Likelihood method, nodes with bootstrap value < 0.70 are not labelled. The percentage of trees in which the associated taxa clustered together is shown next to the branches，represented by blue circles, with larger graphs indicating higher values.

Using the genetic distance table, we can see that, within the populations of all three species, the differences in genetic distances between the sequences of the specimens collected in this study and those of other researchers are relatively small, with an average difference of approximately 0.75% for M. magnater, 1.09% for M. fuliginosus and 0.20% for M. pusillus . In terms of inter-population variation, the difference in genetic distance between M. magnater and M. fuliginosus was relatively small, with a mean difference of about 6.39%. The mean difference in genetic distance between M. magnater and M. pusillus was about 13.40%. The mean difference in genetic distance between M. fuliginosus and M. pusillus is about 14.02% (Suppl. material 1). The results of genetic distances indicate that the specimens we collected in Yunnan Province, China, are indeed M. magnater , M. fuliginosus and M. pusillus belonging to the genus Miniopterus.

Comparative analysis of coat colouration reveals marked differences on the body surface between M. magnater and its congeners, M. fuliginosus and M. pusillus. The latter two species display a predominantly grey-black coat, with more pronounced golden hues at the fur tips.

Comparative morphological assessments reveal significant differences in overall morphology and size between M. magnater and its congeners, M. fuliginosus and M. pusillus, while M. magnater and M. fuliginosus exhibit relatively minor variations in body size. Forearm length, body length and hind foot length are markedly longer in M. magnater than in M. fuliginosus. In contrast, there is a substantial disparity in body size when comparing M. magnater with the smaller M. pusillus.

Morphometric data analysis indicates significant differences in the mean values of tail length (TL), forearm length (FA), head-body length (HB), tibia length (TIB) and ear length (E) between populations of M. magnater and M. fuliginosus (Table 2). Intraspecific body size variations within M. magnater are also evident, as demonstrated by the standard deviation of the morphometric measurements.

There was no significant difference in the arrangement structure of the teeth between M. magnater and M. fuliginosus and M. pusillus. According to previous studies, the M3-M3 of M. magnater was greater than 7.3 mm, M3-M3 of M. fuliginosus less than 7.3 mm (Smith and Xie 2008) and all specimens of M. magnater and M. fuliginosus in this study conformed to this characteristic (Fig. 3).

In terms of skull differences, of the three species, the smallest skull of M. pusillus differed considerably from the other two, with no sagittal suture present in the skull. The size difference between M. magnater and M. fuliginosus was relatively small, with both having a distinct sagittal suture.