A New Species of the Miniopterus Schreibersii Species Complex (chiroptera: Miniopteridae) from the Maghreb Region, North Africa

(English) We used an integrative approach combining cranio-dental characters, mitochondrial and nuclear data and acoustic data to show the presence in the genus Miniopterus of a cryptic species from the Maghreb region. This species was previously recognised as Miniopterus schreibersii (Kuhl, 1817). Miniopterus maghrebensis sp. nov. can be differentiated from M. schreibersii sensu stricto on the basis of cranial characters and from mitochondrial DNA and microsatellite evidence. Although slight external morphological and acoustic differences were noted between the two species, these criteria alone did not allow reliable species identification from live animals. Based on the specimens identified morphologically and/or genetically , the distribution range of M. maghrebensis sp. nov. extends from northern Morocco to south of the High Atlas Mountains and northern Tunisia. The new cryptic species is found in sympatry with M. schreibersii s.str. near coastal regions of North Africa. Abstract (French) Nous avons utilisé une approche intégrative combinant des analyses cranio-dentaires, des marqueurs moléculaires mito-chondriaux et nucléaires ainsi que des données acoustiques pour montrer la présence dans le genre Miniopterus d'une es-pèce cryptique en provenance du Maghreb. Cette espèce était auparavant reconnue en tant que Miniopterus schreibersii crâniaux ainsi que des marqueurs moléculaires mitochondriaux et des microsatellites. Bien que de petites différences mor-phologiques externes et acoustiques aient été notées entre les deux espèces, ces critères à eux seuls ne permettent pas d'identifier de manière fiable les animaux sur le terrain. Sur la base d'identifications morphologiques et/ou génétiques de spécimens, M. maghrebensis sp. nov. s'étends du nord du Maroc jusqu'au sud des montagnes du Haut Atlas et au nord de la Tunisie. Cette nouvelle espèce cryptique est trouvée en sympatrie avec M. schreibersii s.str. près des régions côtières d'Afrique du Nord.


Introduction
Species discoveries and descriptions have gone through different phases and since the 1960's the number of mammal species described per year has continually increased, and the 'species accumulation' curve shows no indication of reaching an asymptote (Reeder et al., 2007). The order Chiroptera follows this trend with, for example, 78 species formally described between July 1992 and June 2006 (Reeder et al., 2007). Within the order Chiroptera, the number of species per family varies greatly, from a single recognised species in the Craseonycteridae family  to over 400 species in the Vespertilionidae family (Simmons, 2005). The latter have recently been split into three families: Miniopteridae (Mein & Tupinier, 1977;Miller-Butterworth et al., 2007), Cistugidae (Lack et al., 2010) and Vespertilionidae s.str. Miniopterus is the only genus within the family Miniopteridae, which has been cited for decades as containing the mammal species with the widest distribution on earth Miniopterus schreibersii (Kuhl, 1817). Nevertheless, current taxonomic revisions suggest that M. schreibersii sensu lato represents a large complex from which several species have been recognized in the last decade. The latest change being the elevation of M. s. pallidus Thomas, 1907, as a full species (M. pallidus) following morphometric, mitochondrial and nuclear data (Furman et al., 2010;Bilgin et al., 2012). The type locality of Vespertilio schreibersii (=M. schreibersii) is Colmbäzer Höhle (=Kolumbács Cave), on the left bank of the Danube river, near the village of Coronini, Romania (Ansell & Topál, 1976). The latest taxonomic revision restricts the range of M. schreibersii to Europe, coastal Anatolia, the Levant, Cyprus, western Transcaucasia and North Africa (Šrámek et al., 2013). In Europe and North Africa (north of the Sahara), there is no other extant Miniopterus species known but M. schreibersii (Šrámek et al., 2013). It is however important to note that the morphological features in M. schreibersii and M. pallidus as well as across most members of this genus are strikingly similar; hence, it can be challenging to reliably identify cryptic species based on morphological characteristics only (e.g. Monadjem et al., 2010). As in other bat genera (e.g. Hipposideros; Thong et al., 2012a;, the implementation of molecular techniques in combination with morphological analyses has greatly helped the recognition and description of many cryptic species within Miniopterus (e.g. Goodman, 2009;Furman et al., 2010;Goodman et al., 2010;Bilgin et al., 2012;Monadjem et al., 2013;Šrámek et al., 2013).
Our study aims at describing a new species of Miniopterus from the Maghreb Region of northern Africa, providing further evidence that M. schreibersii s.str. has a narrower distribution range than previously thought.

Material and methods
Morphology. Data and analyses presented in the current study are based on the work and specimens published by Šrámek et al. (2013) and further investigations on the same specimens. All the measurements presented here followed the definition reported by Šrámek et al. (2013). The 282 examined specimens were collected from throughout the species range, including near the type locality, Kolumbács Cave ( Fig. 1). From now on (unless otherwise stated), we refer to M. schreibersii s.str. as defined by Šrámek et al. (2013), but with the exclusion of Miniopterus sp. nov. described herein. Museum abbreviations used are: NMP = National Museum in Prague, Czech Republic; EBD = Estación Biológica de Doñana, Sevilla, Spain. Specimen collection for the species description complied with the national laws of Morocco, from where the specimens originated.
Genetic data. Data presented in the current study are based on the work by Bilgin et al. (submitted) and further analyses of those data. Mitochondrial DNA sequences used in the present study originated from 12 countries: Russia (n=8), Lebanon (n=7), Cyprus (n=6), Albania (n=6), Romania (n=20; 16 of which from the type locality of M. schreibersii), Slovakia (n=9), Slovenia (n=3), Croatia (n=4), France (n=10), Spain (n=5), Morocco (n=22) and Tunisia (n=2) (Fig. 1). A total of 101 sequences of the tRNA Threonine, tRNA Proline and hypervariable region I of the Control Region (called HV1 hereafter) and 101 sequences of partial Cytochrome b (positions 201-734) were obtained as detailed in Bilgin et al. (submitted). The HV1 region was amplified and sequenced using primers C and E as described in Wilkinson & Chapman (1991). The partial Cytochrome b was amplified using primers Molcit-F (Ibáñez et al. 2006) and MVZ-16 (Smith & Patton 1993) as described in Ibáñez et al. (2006). For each individual, sequences of the partial Cytochrome b fragment and the HVI were concatenated (total length of 787 bp) and only unique haplotypes were used for further analyses (GenBank accession numbers KJ535784-KJ535823 [Cytochrome b] and KJ535824-KJ535861 [HVI]). A phylogenetic reconstruction was undertaken in BEAST v1.8.0 (Drummond & Rambaut, 2007) with strict molecular clock, Yule process as tree prior, and UPGMA tree as starting tree. The HKY+I model of sequence evolution was used as selected by the Akaike Information Criterion in jModeltest2 (Darriba et al., 2012). The program was run for 10 million generations and sampled every 500, of which 10% were discarded as burn-in. Effective sample sizes values were all > 4000 for all parameters, suggesting the MCMC run was sufficient to obtain valid estimates of the parameters. Four replicate analyses were carried with a different random seed number to ensure convergence on a similar tree topology and the results were then pooled. Sequence divergence was estimated between groups of interest using the K2P distance as calculated in MEGA 5.2 (Tamura et al., 2011).
Map showing the origin of specimens and samples investigated in this study with coloured shading delimiting the approximate distribution of M. schreibersii s.str. as recognised prior to our work (cf. Šrámek et al., 2013). Coloured dots represent localities from which we analysed morphological (green dot) or genetic data (blue dot), or both data types combined (dots half green and half blue A set of six microsatellite loci was genotyped for 58 individuals from Iberia and North Africa (26 belonging to the putative new species). Hardy-Weinberg and linkage equilibrium were checked in Genepop 4.0.10 (Rousset, 2008) and a sequential Bonferroni correction was applied to take into consideration multiple testing (Rice, 1989). Differentiation (Fst) between sampled colonies (M. schreibersii: Spain, Win-Timdouine; Miniopterus sp. nov.: Win-Timdouine [+Talmat], Kef Azigza Cave) was calculated and its significance tested in Genepop. As mitochondrial DNA results and Fst calculations suggested the presence of two taxa (cf. Results), we further investigated and characterised the extent of genetic differentiation between these two taxa by performing multivariate and Bayesian analyses. Principal component analysis (PCA) is a powerful tool to summarize multivariate (multiple loci and multiple alleles) genetic information into a few synthetic variables also called principal components, with the first components explaining the highest proportion of variance. These methods do not require assumptions about an underlying genetic model, such as the Hardy-Weinberg or linkage equilibriums (Jombart et al., 2009). Only individuals with data for at least five loci (n=56) were used to run a principal component analysis using the adegenet package v1.3-9.2 (Jombart, 2008) in R v3.0.2 (R Development Core Team, 2013). For the PCA, our data set was combined with a data set of eight individuals of M. schreibersii s.str. from Turkey (Bilgin et al., 2012). To investigate whether hybridisation is occurring between the two taxa, we used a model-based Bayesian statistical technique implemented in STRUCTURE v 2.3.4 (Pritchard et al., 2000). With K set to two, individuals were probabilistically assigned to taxa, or in the case of admixed ancestry, to both parental taxa. We ran the model with the length of the burn-in and MCMC (Markov chain Monte Carlo) chains of 50000 each. We used the admixture model and the correlated allele frequency between populations (Falush et al., 2003). Results presented are based on the average of ten runs.  (Šrámek et al., 2013; this study). Multiple calls per individual were processed through a Fast Fourier Transformation (1024 points, Hamming window, fast Fourier transformations calculated with 98.43% time overlap; software Avisoft-SASLab Pro version 5.2.07, Avisoft Bioacoustics, Germany). The detection of calls in the recordings, the time at the start and the end of the call were determined via the 'three thresholds' algorithm with thresholds set to -24 dB, -20 dB and -10 dB respectively. Peak frequency was characterized as the frequency at the location of the pulse with maximum amplitude. Start frequency was characterised as the frequency where the amplitude goes first below -5 dB from the peak frequency at the start of the call (e.g. when moving in reverse from the maximum up to higher frequencies). Similarly, end frequency was characterised as the frequency where the amplitude goes first below -5 dB from the peak frequency at the end of the call (e.g. when moving from the maximum down to lower frequencies). The interval between pulses was measured as the time elapsed from the start of the preceding to the start of the current pulse. All calculations were processed in R 3.0.2 (R Development Core Team, 2013). Genetic data. At the mitochondrial DNA level, all samples (n=80 sequences) from Russia, Lebanon, Cyprus, Albania, Romania, Slovakia, Slovenia, Croatia, France, Spain plus a subset of samples from Morocco and Tunisia (n=11 sequences) formed a well supported monophyletic clade (n=30 haplotypes) (Fig. 6). These include 10 sequences from the type locality of M. schreibersii in southeast Romania and are therefore referred to as M. schreibersii s.str. The Moroccan and Tunisian samples grouping in the M. schreibersii s.str. clade originated from sites within ca. 40 km from the coastal line. A second monophyletic and strongly supported clade (Bayesian posterior probability=1) was composed of 10 sequences representing four haplotypes. These 10 sequences all originated from individuals from Moroccan and Tunisian sites situated further than ca. 40 km from the costal line. This second clade corresponds to Miniopterus sp. nov. The average K2P distance between M. schreibersii s.str. and Miniopterus sp. nov. sequences was 1.2%.

Morphology
Nuclear DNA. All colonies were in Hardy-Weinberg equilibrium and no linkage was detected between any locus at any colony (Fisher's method; P>0.05). The Fst values between the four colonies investigated were all significantly different from zero (Fst range: 0.  Fig. 7) group with M. schreibersii s.str. individuals from Iberia (red triangles in Fig. 7) and were very clearly differentiated from syntopic Miniopterus sp. nov. (light blue dots in Fig. 7), further demonstrating the presence of two species. The absence of intermediate individuals in Fig. 7 and complete agreement between mitochondrial and nuclear DNA suggest that there were no hybrids between the two species in our data set.
The results of the model-based Bayesian analysis were in full agreement with the results of the PCA and showed consistently across the five runs (data not shown) that the two species are clearly differentiated, with individuals of both species M. schreibersii s.str. and Miniopterus sp. nov. having a high median posterior probability (0.9947 and 0.995 respectively) to belong to their respective species (Fig. 8). Three individuals of M. schreibersii s.str. (two from Win-Timdouine and one from Spain) had a posterior probability to belong to M. schreibersii s.str., which was slightly lower than others (range: 0.89-0.96); these could be signs of past hybridisation or more likely of a recent common ancestry. Hence all the genetic analyses are congruent with each other and provide strong evidence of two isolated and differentiated gene pools which are represented by Miniopterus schreibersii s.str. and Miniopterus sp. nov.
Acoustic data. The calls (n=43) of the hand released Miniopterus sp. nov. individuals (n=2; Win-Timdouine & Oued Emi Oggoug) were typical frequency modulated Miniopterus calls with low duty cycle (Fig. 9). The peak frequency globally varied from 51 to 55 kHz with call duration from 3.2 to 5.6 ms while start and end frequency averaged 74.69 and 50.36 kHz respectively (Table 1). Although the data set of Miniopterus sp. nov. from Morocco contained recordings from only two individuals, the temporal and frequency parameters of the calls largely overlapped with call parameters from 13 individuals of M. schreibersii s.str. recorded in southern France. Although the number of calls and recordings analysed here was limited, both in terms of numbers and geographic coverage, we did not detect any obvious differences between the calls of Miniopterus sp. nov. and those of M. schreibersii s.str.  Aulagnier and Destre (1985).
The dorsal pelage in M. maghrebensis sp. nov. is chestnut brown to dark greyish-brown, the ventral pelage is pale brown (Fig. 2). Ventral hairs are bicoloured, dark brown on the proximal parts, pale brown to buffy in the distal parts. The dorsal skin of ears and the naked parts of face are pale greyish-brown, the ventral skin of ears is almost without pigments, fleshy pale to pinkish (Fig. 2). Wing membranes are dark brown to dark greyish-brown. Based on the skull size (cf. Fig. 3), M. maghrebensis sp. nov. conforms to the Central European representatives of M. schreibersii s.str., however, is slightly larger than most of the south-European and Levantine specimens,         Compared to M. schreibersii s.str. from Europe and the Levant (see Šrámek et al., 2013) unicuspidal teeth are more robust in M. maghrebensis sp. nov. The cingulum on the palatal side of the first upper molar has a very distinctive concave fold between the protoconus and the hypoconus in M. maghrebensis sp. nov.
Etymology. The name maghrebensis refers to the region (the Maghreb; the region of northern Africa located between the Atlantic Ocean, the Mediterranean Sea, and the Sahara) where the new species was discovered. The proposed vernacular names are Maghrebian bent-wing bat (English), Minioptère du Maghreb (French), Murciélago Distribution. Genetic analyses confirmed its presence from northern Morocco to south of the High Atlas Mountains and northern Tunisia (Fig. 10) (Šrámek et al., 2013;Puechmaille et al., 2012b; this study); however, these individuals were originally referred to as M. schreibersii. Based on these data and the current extent of Miniopterus distribution in the region (Aulagnier & Destre, 1986;Kowalski & Rzebik-Kowalska, 1991;Dieuleveut et al., 2010;Puechmaille et al., 2012b), we present a hypothetical distribution map of both species. Nevertheless, all previous records of M. schreibersii from the Maghreb need to be re-evaluated as they could potentially belong to M. maghrebensis sp. nov. since both species are found in the region (Bilgin et al., submitted).

Discussion
The type locality of M. s. schreibersii is Kolumbács Cave, left bank of the River Danube, near Coronini, Romania; sensu Ansell & Topál (1976). All the Miniopterus specimens genetically characterised from the type locality and the surrounding region (South-eastern Europe) fall within the same clade. Additionally, all the specimens morphologically investigated from near the type locality in Romania/Bulgaria and from South-eastern Europe are morphologically similar, hence we consider these specimens as representing M. schreibersii s.str. The species is distributed in Europe, coastal Anatolia, the Levant, Cyprus, western Transcaucasia, and coastal North Africa. We reviewed the literature to identify earlier possible descriptions for a new Miniopterus species from the Maghreb. However, as all previous authors had agreed that the Maghrebian populations belong to the nominotypical form, a previously proposed species name does not appear to be available.
Although M. maghrebensis sp. nov. can be diagnosed based on cranio-dental characters as well as genetic evidence, it may prove difficult to diagnose the species in the field from live animals. Such a situation is rather common in bats as many cryptic species are only diagnosable in the field with subtle qualitative differences in external characters as it is the case in Europe with Pipistrellus pipistrellus and Pipistrellus pygmaeus or Plecotus species . Furthermore, the Miniopteridae family is notorious for its morphological stasis and having species with very similar morphology (Yoshiyuki, 1989), hence more research is necessary to study the external morphology of M. schreibersii and M. maghrebensis sp. nov., especially in areas of sympatry or parapatry to identify reliable diagnostic field characters. It is not unusual to have closely related bat species showing divergence in echolocation calls (Douangboubpha et al., 2010;Ith et al., 2011;Puechmaille et al., 2011;Thong et al., 2012b) and those differences can be used to discriminate species (Walters et al., 2012). Nevertheless, our analyses of echolocation calls from a limited number of individuals tend to suggest that M. maghrebensis sp. nov. and M. schreibersii s.str. have very similar echolocation calls, which would render their acoustic identification to species level difficult. It is however important to mention that the recordings were only from hand released individuals (which was necessary to confirm species identification), hence we only obtained a small portion of both species' vocal repertoire. Upon hand release, bats generally tend to emit short steep frequency modulated (FM) calls that are more typical of animals flying in cluttered habitat. These calls are generally the most difficult to identify to species as they show greater overlap between species than calls emitted when animals are flying in the open and producing shallow FM or quasi-constant frequency calls (Barataud, 2012). More research is needed to characterize the echolocation call repertoire of M. maghrebensis sp. nov. in greater detail and to search for diagnostic features that would differentiate it from M. schreibersii s.str.
Although the percentage of mitochondrial DNA divergence between the two species (K2P distance: 1.2% for Cytochrome b) is low, it is within the range of values previously reported for different species of mammals (Hebert et al., 2003), including bats (Clare et al., 2007;Francis et al., 2010). Some bat species distinct at the morphological level show low inter-specific divergence at mitochondrial DNA genes (<1% for COI; e.g. Myotis annamiticus and M. laniger; Francis et al., 2010), others share the same mitochondrial DNA despite being sympatric and having clear differences in their nuclear genome (e.g. Myotis myotis and M. blythii; Berthier et al., 2006), while others are paraphyletic based on mitochondrial DNA (e.g. Hipposideros armiger; Thong et al., 2012b). Intra-specific sequence divergence values below 2 to 5% have been commonly reported in the literature for mitochondrial DNA genes, but it should be emphasized that these values are only indicative guidelines, as it is clear that there is no biologically meaningful cut-off value separating intra-and inter-specific variability for any taxonomic group (e.g. Baker & Bradley, 2006;Luo et al., 2011). Importantly, and in addition to the mitochondrial DNA divergence between M. schreibersii s.str. and M. maghrebensis sp. nov., the set of six microsatellite loci provide evidence that the two species have separated and isolated gene pools, even when found in sympatry. North African M. schreibersii s.str. are genetically (at mitochondrial and nuclear levels) more closely related to Turkish conspecifics (situated >4000 km away) than they are to syntopic M. maghrebensis sp. nov. The absence of intermediate individuals between the M. schreibersii s.str. from Turkey and conspecifics from Iberia and North Africa in the PCA plot is very likely due to the absence of samples from across Europe in our study, which would be expected to fill this gap. The non-significant Fst between M. maghrebensis sp. nov. colonies found ca. 560 km apart (Win-Timdouine [+Talmat] versus Kef Azigza Cave) strongly suggests that the colonies we studied are not isolated from each other but represent a rather homogeneous gene pool. Hence although our sampling might not cover the entire range of M. maghrebensis sp. nov., it is likely that the species presents a rather homogeneous gene pool across its range. The absence of detected gene flow between the two species, hence the integrity of their respective gene pools, combined with homogeneous gene pools within species over large distances clearly demonstrate the presence of two separate species according to the biological species definition proposed by Mayr (1996): "Species are groups of interbreeding natural populations that are reproductively isolated from other such groups". In our case, the isolating mechanism by which reproductive isolation is effected is unknown, but it must be properties of individuals rather than simply geographic isolation as individuals from both species can be found in sympatry (e.g. at Win-Timdouine).
Although bats in North Africa have not received as much attention as those in Europe, a significant number of new taxa have recently been elevated to species rank or described from this region (Castella et al., 2000;Benda et al., 2004a;Benda et al., 2004b;Ibañez et al., 2006;Garcia-Mudarra et al., 2009;Juste et al., 2009;Benda & Vallo, 2012). Some taxa deserving full species rank identified through analyses of genetic data are still awaiting formal description (Garcia-Mudarra et al., 2009;Salicini et al., 2011;Puechmaille et al., 2012a), while the status of populations of several species remains unclear but strongly suggests the presence of further cryptic species (Hulva et al., 2010;Puechmaille et al., 2012b). This, in combination with patterns of species diversity recently uncovered in other taxonomic groups (Brito et al., 2014), lead us to believe that the current bat diversity in the Maghreb and more generally in North Africa might be underappreciated. As recently highlighted by Puechmaille et al. (2012b), the taxonomic status of bats in North Africa needs to be investigated and reviewed in detail. The combination of morphological, genetic (including nuclear DNA) and acoustic studies in conjunction with re-inspections of type specimens (when relevant) will greatly contribute to the clarification of the taxonomic status of bats species in the region and reveal the region's real diversity. Such studies will also largely improve our understanding of the phylogeography of many European species that so far have not included populations from North Africa (for some exceptions, see Juste et al., 2004;Flanders et al., 2009;Dool et al., 2013), populations which have potentially survived during ice ages and hence were refugial populations (Dool et al., 2013). A better understanding of biodiversity patterns in North Africa and across the Sahara-Sahel will also be of particular importance as the magnitude and velocity of climate change in deserts and xeric shrublands are predicted to be strong and fast (Loarie et al., 2009). Large protected areas may mitigate the problem in desert biomes, but to increase their efficiency in preserving biodiversity, these protected areas need to be designed based on patterns of biodiversity, hence the importance of resolving the taxonomy and phylogeography of organisms in the region.
Studying species ecology can also reveal some important differences between cryptic species, whether this relates to foraging ecology (Arlettaz, 1999;Davidson-Watts et al., 2006;2006b;Nicholls & Racey, 2006a) or roosting ecology (Ibañez et al., 2006;Puechmaille et al., 2012a). Studies of reproductive phenology and especially the timing of parturition commonly reveal asynchrony between closely related cryptic species as illustrated for Myotis myotis and Myotis blythii (Arlettaz et al., 2001). Kowalski & Rzebik-Kowalska (1991) report that in Algeria, parturition in Miniopterus lasts from mid-April until almost to the end of June, which suggests that the two species might have different breeding periods (with M. maghrebensis sp. nov. most likely being earlier than M. schreibersii s.str., for which birth takes place in Europe between mid-June and early July; Dietz et al., 2009). More research is needed to investigate these ecological and phenological differences between these two cryptic species, differences which might have been involved in the speciation process and that could now represent prezygotic isolating mechanisms.