Horseshoe Bat Species Research Articles

Identification of the same polyomavirus species in different African horseshoe bat species is indicative of short-range host-switching events

Polyomaviruses (PyVs) are considered to be highly host-specific in different mammalian species, with no well-supported evidence for host-switching events. We examined the species diversity and host specificity of PyVs in horseshoe bats (Rhinolophus spp.), a broadly distributed and highly speciose mammalian genus. We annotated six PyV genomes, comprising four new PyV species, based on pairwise identity within the large T antigen (LTAg) coding region. Phylogenetic comparisons revealed two instances of highly related PyV species, one in each of the Alphapolyomavirus and Betapolyomavirus genera, present in different horseshoe bat host species (Rhinolophus blasii and R. simulator), suggestive of short-range host-switching events. The two pairs of Rhinolophus PyVs in different horseshoe bat host species were 99.9 and 88.8 % identical with each other over their respective LTAg coding sequences and thus constitute the same virus species. To corroborate the species identification of the bat hosts, we analysed mitochondrial cytb and a large nuclear intron dataset derived from six independent and neutrally evolving loci for bat taxa of interest. Bayesian estimates of the ages of the most recent common ancestors suggested that the near-identical and more distantly related PyV species diverged approximately 9.1E4 (5E3-2.8E5) and 9.9E6 (4E6-18E6) years before the present, respectively, in contrast to the divergence times of the bat host species: 12.4E6 (10.4E6-15.4E6). Our findings provide evidence that short-range host-switching of PyVs is possible in horseshoe bats, suggesting that PyV transmission between closely related mammalian species can occur.

Environmental correlates of geographic divergence in a phenotypic trait: A case study using bat echolocation.

Divergence in phenotypic traits may arise from the interaction of different evolutionary forces, including different kinds of selection (e.g., ecological), genetic drift, and phenotypic plasticity. Sensory systems play an important role in survival and reproduction, and divergent selection on such systems may result in lineage diversification. Such diversification could be largely influenced by selection in different environments as a result of isolation by environment (IbE). We investigated this process using geographic variation in the resting echolocation frequency of the horseshoe bat species, Rhinolophus damarensis, as a test case. Bats were sampled along a latitudinal gradient ranging from 16°S to 32°S in the arid western half of southern Africa. We measured body size and peak resting frequencies (RF) from handheld individual bats. Three hypotheses for the divergence in RF were tested: (1) James’ Rule, (2) IbE, and (3) genetic drift through isolation by distance (IbD) to isolate the effects of body size, local climatic conditions, and geographic distance, respectively, on the resting frequency of R. damarensis. Our results did not support genetic drift because there was no correlation between RF variation and geographic distance. Our results also did not support James' Rule because there was no significant relationship between (1) geographic distances and RF, (2) body size and RF, or (3) body size and climatic variables. Instead, we found support for IbE in the form of a correlation between RF and both region and annual mean temperature, suggesting that RF variation may be the result of environmental discontinuities. The environmental discontinuities coincided with previously reported genetic divergence. Climatic gradients in conjunction with environmental discontinuities could lead to local adaptation in sensory signals and directed dispersal such that gene flow is restricted, allowing lineages to diverge. However, our study cannot exclude the role of processes like phenotypic plasticity in phenotypic variation.

Morphometric Variability in Horseshoe Bat Species, Rhinolophus Ferrumequinum and Rhinolophus lepidus Captured from District Malakand, Khyber Pakhtunkhwa, Pakistan

Morphometric Variability in Horseshoe Bat Species, Rhinolophus Ferrumequinum and Rhinolophus lepidus Captured from District Malakand, Khyber Pakhtunkhwa, Pakistan

Sensory Drive Mediated by Climatic Gradients Partially Explains Divergence in Acoustic Signals in Two Horseshoe Bat Species, Rhinolophus swinnyi and Rhinolophus simulator

Geographic variation can be an indicator of still poorly understood evolutionary processes such as adaptation and drift. Sensory systems used in communication play a key role in mate choice and species recognition. Habitat-mediated (i.e. adaptive) differences in communication signals may therefore lead to diversification. We investigated geographic variation in echolocation calls of African horseshoe bats, Rhinolophus simulator and R. swinnyi in the context of two adaptive hypotheses: 1) James’ Rule and 2) the Sensory Drive Hypothesis. According to James’ Rule body-size should vary in response to relative humidity and temperature so that divergence in call frequency may therefore be the result of climate-mediated variation in body size because of the correlation between body size and call frequency. The Sensory Drive Hypothesis proposes that call frequency is a response to climate-induced differences in atmospheric attenuation and predicts that increases in atmospheric attenuation selects for calls of lower frequency. We measured the morphology and resting call frequency (RF) of 111 R. simulator and 126 R. swinnyi individuals across their distributional range to test the above hypotheses. Contrary to the prediction of James’ Rule, divergence in body size could not explain the variation in RF. Instead, acoustic divergence in RF was best predicted by latitude, geography and climate-induced differences in atmospheric attenuation, as predicted by the Sensory Drive Hypothesis. Although variation in RF was strongly influenced by temperature and humidity, other climatic variables (associated with latitude and altitude) as well as drift (as suggested by a positive correlation between call variation and geographic distance, especially in R. simulator) may also play an important role.

Repetitive transpositions of mitochondrial DNA sequences to the nucleus during the radiation of horseshoe bats (Rhinolophus, Chiroptera)

Transposition of mitochondrial DNA into the nucleus, which gives rise to nuclear mitochondrial DNAs (NUMTs), has been well documented in eukaryotes. However, very few studies have assessed the frequency of these transpositions during the evolutionary history of a specific taxonomic group. Here we used the horseshoe bats (Rhinolophus) as a case study to determine the frequency and relative timing of nuclear transfers of mitochondrial control region sequences. For this, phylogenetic and coalescent analyzes were performed on NUMTs and authentic mtDNA sequences generated from eight horseshoe bat species. Our results suggest at least three independent transpositions, including two ancient and one more recent, during the evolutionary history of Rhinolophus. The two ancient transpositions are represented by the NUMT-1 and -2 clades, with each clade consisting of NUMTs from almost all studied species but originating from different portions of the mtDNA genome. Furthermore, estimates of the most recent common ancestor for each clade corresponded to the time of the initial diversification of this genus. The recent transposition is represented by NUMT-3, which was discovered only in a specific subgroup of Rhinolophus and exhibited a close relationship to its mitochondrial counterpart. Our similarity searches of mtDNA in the R. ferrumequinum genome confirmed the presence of NUMT-1 and NUMT-2 clade sequences and, for the first time, assessed the extent of NUMTs in a bat genome. To our knowledge, this is the first study to report on the frequency of transpositions of mtDNA occurring before the common ancestry of a genus.

Bat origin of human coronaviruses.

Bats have been recognized as the natural reservoirs of a large variety of viruses. Special attention has been paid to bat coronaviruses as the two emerging coronaviruses which have caused unexpected human disease outbreaks in the 21st century, Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Middle East Respiratory Syndrome Coronavirus (MERS-CoV), are suggested to be originated from bats. Various species of horseshoe bats in China have been found to harbor genetically diverse SARS-like coronaviruses. Some strains are highly similar to SARS-CoV even in the spike protein and are able to use the same receptor as SARS-CoV for cell entry. On the other hand, diverse coronaviruses phylogenetically related to MERS-CoV have been discovered worldwide in a wide range of bat species, some of which can be classified to the same coronavirus species as MERS-CoV. Coronaviruses genetically related to human coronavirus 229E and NL63 have been detected in bats as well. Moreover, intermediate hosts are believed to play an important role in the transmission and emergence of these coronaviruses from bats to humans. Understanding the bat origin of human coronaviruses is helpful for the prediction and prevention of another pandemic emergence in the future.

The use of museum samples for large-scale sequence capture: a study of congeneric horseshoe bats (family Rhinolophidae)

Museums hold most of the world's most valuable biological specimens and tissues collected, including type material that is often decades or even centuries old. Unfortunately, traditional museum collection and storage methods were not designed to preserve the nucleic acids held within the material, often reducing its potential viability and value for many genetic applications. High-throughput sequencing technologies and associated applications offer new opportunities for obtaining sequence data from museum samples. In particular, target sequence capture offers a promising approach for recovering large numbers of orthologous loci from relatively small amounts of starting material. In the present study, we test the utility of target sequence capture for obtaining data from museum-held material from a speciose mammalian genus: the horseshoe bats (Rhinolophidae: Chiroptera). We designed a ‘bait’ for capturing > 3600 genes and applied this to 10 species of horseshoe bat that had been collected between 93 and 7 years ago and preserved using a range of methods. We found that the mean recovery rate per species was approximately 89% of target genes with partial sequence coverage, ranging from 3024 to 3186 genes recovered. On average, we recovered 1206 genes with ≥ 90% sequence coverage, per species. Our findings provide good support for the application of large-scale bait capture across congeneric species spanning approximately 15 Myr of evolution. On the other hand, we observed no clear association between the success of capture and the phylogenetic distance from the bait model, although sample sizes precluded a formal test.

Acoustic identification and taxonomic remarks of Hipposiderids and Rhinolophids (Chiroptera: Hipposideridae, Rhinolophidae) in Tam Dao national park, Northeastern Nietnam

Hipposiderids and Rhinolophids are referred to all taxa of the two families Hipposideridae and Rhinolophidae, respectively. Their echolocation signals are diagnostic by a dominant “constant frequency” (CF) segment. Each echolocation signal of leaf-nosed bat species consists of two segments (“constant-frequency” and “terminal frequency-modulated”) while that of horseshoe bat species comprises three segments (“initial frequency-modulated”, “constant-frequency” and “terminal frequency-modulated”). Between 2008 and 2014, a series of field surveys was conducted in Tam Dao National Park (TDNP) with particular emphases on taxonomy and echolocation of CF bat species. Results from the surveys indicated that TDNP is a home to three species of Hipposideridae (Hipposideros armiger, H. larvatus and H. pomona) and five species of Rhinolophidae (Rhinolophus affinis, R. luctus, R. macrotis, R. pearsonii and R. pusillus). During the present study, R. luctus was only detected in the field based on a poor echolocation call while all seven remaining species were captured. The CF of the second harmonic and morphological features of each species are clearly distinguished from the respective ones of the remaining species. This paper provides the external and acoustic diagnoses of each species for identification in the field to strengthen survey achievements, monitoring and conservation of CF bats in the park in coming time.

Listening carefully: increased perceptual acuity for species discrimination in multispecies signalling assemblages

Communication is a fundamental component of evolutionary change because of its role in mate choice and sexual selection. Acoustic signals are a vital element of animal communication and sympatric species may use private frequency bands to facilitate intraspecific communication and identification of conspecifics (acoustic communication hypothesis, ACH). If so, animals should show increasing rates of misclassification with increasing overlap in frequency between their own calls and those used by sympatric heterospecifics. We tested this on the echolocation of the horseshoe bat, Rhinolophus capensis, using a classical habituation–dishabituation experiment in which we exposed R. capensis from two phonetic populations to echolocation calls of sympatric and allopatric horseshoe bat species (Rhinolophus clivosus and Rhinolophus damarensis) and different phonetic populations of R. capensis. As predicted by the ACH, R. capensis from both test populations were able to discriminate between their own calls and calls of the respective sympatric horseshoe bat species. However, only bats from one test population were able to discriminate between calls of allopatric heterospecifics and their own population when both were using the same frequency. The local acoustic signalling assemblages (ensemble of signals from sympatric conspecifics and heterospecifics) of the two populations differed in complexity as a result of contact with other phonetic populations and sympatric heterospecifics. We therefore propose that a hierarchy of discrimination ability has evolved within the same species. Frequency alone may be sufficient to assess species membership in relatively simple acoustic assemblages but the ability to use additional acoustic cues may have evolved in more complex acoustic assemblages to circumvent misidentifications as a result of the use of overlapping signals. When the acoustic signal design is under strong constraints as a result of dual functions and the available acoustic space is limited because of co-occurring species, species discrimination is mediated through improved sensory acuity in the receiver.

Karyotype Evolution in the Horseshoe Bat Rhinolophus sedulus by Whole-Arm Reciprocal Translocation (WART)

Robertsonian (centric) fusion or fission is one of the predominant modes of chromosomal rearrangement in karyotype evolution among mammals. However, in karyotypes composed of only bi-armed chromosomes, creation of new chromosomal arm combinations in one step is possible only via whole-arm reciprocal translocation (WART). Although this type of rearrangement has often been proposed to play an important role in chromosomal evolution, direct observations of WARTs remained rare, and, in most cases, were found in hybrids of chromosomal races in the genera Mus and Sorex. For the first time, we present the karyotype of the horseshoe bat species Rhinolophus sedulus (2n = 28, FNa = 52), where a WART between 2 metacentric autosomes was detected by G-banding and confirmed by FISH with painting probes of the vespertilionid bat Myotis myotis. Among the 6 specimens analyzed, 2 showed the heterozygous condition of the WART, 1 showed the presumed ancestral, and 3 specimens showed the derived homozygous state. As the existence of a hybrid zone at the sampling locality is thought to be rather improbable, the WART may indicate ongoing karyotype evolution in this taxon.

Bat cave and Hobbit hole, microbats of Liang Bua (Flores, Indonesia)

Liang Bua is a limestone cave on the island of Flores, Indonesia. Palaeontological and archaeological excavations at this site have unearthed one of the best insular records of fossil bats to date. The assemblage is numerically dominated by the horseshoe bat species Rhinolophus euryotis, followed by the leafnosed bat Hipposideros diadema. In addition to these species, remains of Rhinolophus simplex, Hipposideros sp. cf. H. sumbae, Murina sp. aff. M. florium, Taphozous sp., Kerivoula sp. and Miniopterus sp. have also been recovered. Two taxa found in Liang Bua, Rhinolophus euryotis and Miniopterus sp., are not apparent in the modern bat fauna of Flores. Some specimens, found during the more recent excavations, are known from specific depths. Two age-groups are represented. The stratigraphically oldest is 74–61 ka and includes Murina sp. aff. M. florium and Rhinolophus euryotis. Conversely, the younger, Holocene, assemblage is much more diverse. Despite the relatively small amount of microbat material collected thus far, it displays a higher diversity than the rodents from the site. This is presumably related to the better dispersal ability of bats relative to other, non-volant mammals.

Behavioral responses to echolocation calls from sympatric heterospecific bats: implications for interspecific competition

Mutual recognition is the product of species coexistence, and has direct effects on survival and reproduction of animals. Bats are able to discriminate between sympatric different heterospecifics based on their echolocation calls, which has been shown both in free-flying and captive bats. To date, however, the factors that may determine the behavioral responses of bats to echolocation calls from sympatric heterospecifics have rarely been tested, especially under well-controlled conditions in captive bats. Hence, we aimed at tackling this question by performing playback experiments (habituation–dishabituation) with three horseshoe bat species within the constant-frequency bat guild, which included big-eared horseshoe bats (Rhinolophus macrotis), Blyth’s horseshoe bats (Rhinolophus lepidus), and Chinese horseshoe bats (Rhinolophus sinicus). We studied the behavioral responses of these three species to echolocation calls of conspecifics, to other two species, and to another heterospecifics bat, Stoliczka’s trident bat (Asellisus stoliczkanus), which also belongs to this guild. We found that the three rhinolophid species displayed a series of distinct behaviors to heterospecific echolocation but few to conspecific calls after habituation, suggesting that they may have been able to discriminate sympatric heterospecific echolocation calls from those of conspecifics. Interestingly, the behavioral responses to heterospecific calls were positively correlated with the interspecific overlap index in trophic niche, whereas call design had only a minor effect. This implies that the behavioral responses of these bats to heterospecific echolocation calls may be related to the degree of interspecific food competition.

The diet of four species of horseshoe bat (Chiroptera: Rhinolophidae) in a mountainous region of Algeria: evidence for gleaning

We analyzed the diet of four rhinolophids ( Rhinolophus ferrumequinum, R. hipposideros, R. euryale and R. blasii ) in the region of Kabylia Babors, in northern Algeria. Between March 2007 and January 2008 we sampled droppings at ten sites and carried out a morphological identification of prey remains. Three main groups of Arthropoda (Insecta, Chilopoda and Araneida) were preyed upon but frequencies varied across species. The insect prey most frequently preyed upon by R. ferrumequinum are Diptera (34.56%), mainly including Culicidae (10.40 %), Chironomidae / Ceratopogonidae (10.94 %) and Tipulidae (4.28 %), and also Lepidoptera (24.13 %). In R. hipposideros droppings we found Diptera (41.58 %) , Chironomidae / Ceratopogonidae ( 9.68 %) and Tipulidae (6.45 %). Also common were Lepidoptera (moths; 21.14 %) and Hemiptera (11.68 %). R.euryale ate mainly Diptera (29.00 %), Chironomidae / Ceratopogonidae (7.14 %) and Tipulidae (5.71 %). The order Lepidoptera (moths) was also well represented (19.08 %). R. blasii preyed on two groups of Arthropoda: Insecta (96.87 %) and Chilopoda (4.34 %). The most consumed insect prey was Diptera (37.50 %), mainly Chironomidae / Ceratopogonidae ( 9.38 %), Culicidae, Anisopodidae and Sphaeroceridae (6.25 %). The order Trichoptera was also well represented in its diet (15.63 %) and Lepidoptera accounted for 12.50 % . The most interesting aspect of this study was that Chilopoda appeared in the diet of all species and that, albeit rarely, R. ferrumequinum also ate spiders. This result suggests that all species could glean prey from substrate, most probably as a strategy to better exploit the open habitats typical of the study area. Download the complete issue.

Systematics and biogeography of the arcuate horseshoe bat species complex (Chiroptera, Rhinolophidae)

The present study sheds light on species delimitation in what has been previously described as Rhinolophus arcuatus, a morphologically conservative bat species complex nominally distributed throughout archipelagic South‐East Asia from New Guinea to Sumatra. Given that rhinolophids tend to be relatively weak fliers, hence have low vagility, we hypothesized that some specimens attributed to R. arcuatus, but originating from geographically disjunct populations, may in fact represent distinct species. To test this hypothesis, we examined specimens attributed to R. arcuatus as well as to other species in the Rhinolophus euryotis species group using both morphological techniques and mitochondrial cytochrome b and control region sequences. Careful morphological analysis reveals heretofore cryptic but nevertheless distinct, species‐level morphological differences among specimens derived from geographically isolated locations. Furthermore, molecular data illuminate the existence of several species‐level sequence divergences among specimens heretofore attributed to R. arcuatus. These analyses similarly suggest the existence of additional species in other South‐East Asian Rhinolophus taxa previously considered monotypic. We suggest at least one description to be undertaken of a previously unrecognized species as well as the elevation of several others from sub‐specific to specific status.

New look on the geographical variation in Rhinolophus clivosus with description of a new horseshoe bat species from Cyrenaica, Libya

New look on the geographical variation in Rhinolophus clivosus with description of a new horseshoe bat species from Cyrenaica, Libya

Cave bats of the central west coast and southern section of the Northwest Panay Peninsula, Panay Island, the Philippines

Bats (order Chiroptera) form a large proportion of the species-rich mammalian fauna of the Philippines, and while the threats posed to these animals are well documented, for many species there is currently insufficient data to enable even a basic assessment of their conservation status. This is true for Panay Island, located in the Western Visayas region of the archipelago, where the need for surveying remaining suitable bat habitat has been identified as a priority. Between 5 April and 9 May 2011 a survey of 21 caves was undertaken on Panay, along the central section of the west coast of the island and within the southern section of the Northwest Panay Peninsula. Survey methods included visual observations, emergence counts and the recording of echolocation calls. Of the caves surveyed, 19 were found to support bats or show signs of their use, and at least 12 different species were observed. Three large maternity colonies of the Common Rousette Rousettus amplexicaudatus and two of the Dusky Roundleaf Bat Hipposideros ater were noted as having particular significance in terms of their conservation value for local populations. Potential maternity colonies of Asian Lesser False Vampire Megaderma spasma, Black-bearded Tomb Bat Taphozous melanopogon and Diadem Roundleaf Bat Hipposideros diadema were also observed but not confirmed. M. spasma was the most frequently encountered species, occurring in small numbers at five different caves. Other species/genera encountered in small numbers were the Arcuate Horseshoe Bat Rhinolophus arcuatus, Common Short-nosed Fruit Bat Cynopterus brachyotis, Philippine Sheath-tailed Bat Emballonura alecto, Yellow-faced Horseshoe Bat Rhinolophus virgo, Bent-wing (Miniopterus) and Myotis bat species, and at least one other Horseshoe (Rhinolophus) bat species. Ten of the caves were confirmed to support multiple bat species. An indication of current threats and recommendations for further survey and management priorities are provided.

Horseshoe Bats Recognise the Sex of Conspecifics from Their Echolocation Calls

Echolocation calls produced by bats contain information on the emitter that could be used in social interactions. Nevertheless, little is known about the whether such information actually is being used by bats. Here we tested the ability of two horseshoe bat species (Rhinolophus mehelyi and R. euryale; n = 20 each) to discriminate conspecific male from conspecific female echolocation calls and vice versa. Behavioural data from a habituation-discrimination paradigm revealed that both species were able to recognize the sex of conspecifics from their calls, providing evidence that cues on the sex of the emitter are encoded in echolocation signals. While our data support that the sex-specific acoustic cues are not (just) the horseshoe bats' constant-frequency call part, their nature remains enigmatic. This is the second study to show that bats can recognize the caller's gender from echolocation calls and the first to do so for ‘high-duty-cycle bats’; those that use calls with a prominent constant-frequency (CF) component.

Variability in echolocation call intensity in a community of horseshoe bats: a role for resource partitioning or communication?

BackgroundOnly recently data on bat echolocation call intensities is starting to accumulate. Yet, intensity is an ecologically crucial parameter, as it determines the extent of the bats' perceptual space and, specifically, prey detection distance. Interspecifically, we thus asked whether sympatric, congeneric bat species differ in call intensities and whether differences play a role for niche differentiation. Specifically, we investigated whether R. mehelyi that calls at a frequency clearly above what is predicted by allometry, compensates for frequency-dependent loss in detection distance by using elevated call intensity. Maximum echolocation call intensities might depend on body size or condition and thus be used as an honest signal of quality for intraspecific communication. We for the first time investigated whether a size-intensity relation is present in echolocating bats.Methodology/Principal FindingsWe measured maximum call intensities and frequencies for all five European horseshoe bat species. Maximum intensity differed among species largely due to R. euryale. Furthermore, we found no compensation for frequency-dependent loss in detection distance in R. mehelyi. Intraspecifically, there is a negative correlation between forearm lengths and intensity in R. euryale and a trend for a negative correlation between body condition index and intensity in R. ferrumequinum. In R. hipposideros, females had 8 dB higher intensities than males. There were no correlations with body size or sex differences and intensity for the other species.Conclusions/SignificanceBased on call intensity and frequency measurements, we estimated echolocation ranges for our study community. These suggest that intensity differences result in different prey detection distances and thus likely play some role for resource access.It is interesting and at first glance counter-intuitive that, where a correlation was found, smaller bats called louder than large individuals. Such negative relationship between size or condition and vocal amplitude may indicate an as yet unknown physiological or sexual selection pressure.

Behavioral Evidence for Community‐Wide Species Discrimination from Echolocation Calls in Bats

Recognizing species identity is crucial for many aspects of animal life and is often mediated by acoustic signals. Although most animals are able to distinguish acoustic signals of their own species from other sympatrically occurring species, it is yet unknown whether animals can distinguish among acoustic signals of different closely related sympatric species. In this context, echolocating bats are a particularly interesting model system: their echolocation system evolved primarily for spatial orientation and foraging, but recent studies indicate that echolocation also has an important communicative function. Yet, the role of echolocation calls for species discrimination and thus potentially for interspecific communication has not been investigated. Using a behavioral discrimination assay, we found that two species of wild horseshoe bats could discriminate calls of their own species from those of three sympatric congeneric species. We further show that the bats were able to discriminate between echolocation calls of different congeneric species from the local community. In both cases, discrimination ability was high despite strong overlap of species' call frequency bands. This study provides the first experimental evidence for species discrimination based on echolocation calls. On a more general level, it shows for the first time that animals can distinguish among acoustic signals of different closely related and ecologically similar species from their local community.

Historical male‐mediated introgression in horseshoe bats revealed by multilocus DNA sequence data

Instances of hybridization between mammalian taxa in the wild are rarely documented. To test for introgression between sibling species of horseshoe bat (Rhinolophus yunanensis and R. pearsoni) and two subspecies of the latter (R. p. pearsoni and R. p. chinensis), we sequenced two mtDNA and two ncDNA markers in individuals sampled from multiple localities within their overlapping ranges. The interspecific mtDNA gene tree corresponded to the expected taxonomic divisions, and coalescent-based analyses suggested divergence occurred around 4 MYA. However, these relationships strongly conflicted with those recovered from two independent nuclear gene trees, in which R. yunanensis clustered with R. p. pearsoni to the exclusion of R. p. chinensis. This geographically widespread discordance is best explained by large-scale historical introgression of ncDNA from R. yunanensis to R. pearsoni by male-mediated exchange in mixed species colonies during Pleistocene glacial periods, when ranges may have contracted and overlapped more than at present. Further species tree-gene tree conflicts were detected between R. p. pearsoni and R. p. chinensis, also indicating past and/or current introgression in their overlapping regions. However, here the patterns point to asymmetric mtDNA introgression without ncDNA introgression. Analyses of coalescence times indicate this exchange has occurred subsequent to the divergence of these subspecies from their common ancestor. Our work highlights the importance of using multiple data sets for reconstructing phylogeographic histories and resolving taxonomic relationships.