The use of molecular phylogenetic and morphological tools to identify cryptic and paraphyletic species : examples from the diminutive long-fingered bats (Chiroptera : Miniopteridae : Miniopterus) on Madagascar

Based on nearly complete (1125 bp) cytochrome-b sequence data and morphological characters, two new endemic species of Miniopterus are described from Madagascar that were previously identified as M. manavi. Using phylogenetic analysis, the basal nodes of major lineages in the Malagasy members of this genus are weakly supported, while, in most cases, the branches leading to each of the clades are well resolved. Miniopterus mahafaliensis, new species, occurs in the southwestern semidesert areas and M. brachytragos, new species, has a broad distribution across the northern half of the island, ranging across several different biomes. Phylogenetic inference indicates that these two new taxa are not closely related to M. manavi sensu stricto, with average genetic distances of 9.2% and 5.7% from this taxon, respectively. On the basis of this and previous revisions, the former M. manavi complex is now recognized to represent at least five taxa, which do not form a monophyletic group with respect to one another, and represent extraordinary examples of convergent evolution. Miniopterus brachytragos is closely related to the recently named M. aelleni, while M. mahafaliensis is not closely associated with any of these species. Molecular phylogenetic analysis was imperative to resolve the species limits of these taxa and morphology then provided the means to corroborate the recovered clades. There are localities on the island, specifically limestone karstic zones, where four species of the former M. manavi sensu lato complex occur in strict sympatry. These species often use the same day-roost caves and have similar external and craniodental measurements. This raises intriguing questions as to how these animals divide their worlds with regard to dietary regimes and foraging strategies, as well as their speciation history.

Specific status of populations on Madagascar referred to Miniopterus fraterculus (Chiroptera: Vespertilionidae), with description of a new species

A new species of bat of the genus Miniopterus is described from Madagascar based on a series of specimens taken in the Central Highlands of the island. This new species previously was identified as M. fraterculus, which is widespread in portions of eastern and southern Africa. Comparisons between these 2 taxa were further complicated because M. fraterculus occurs in portions of its range in sympatry with a morphologically similar species, M. natalensis. Based on specimen material and associated tissue samples from near the type localities of M. natalensis and M. fraterculus, as well as access to some of the critical type specimens, morphological and genetic molecular analyses were used to determine that Malagasy specimens previously assigned to M. fraterculus represent a previously unrecognized species of Miniopterus endemic to the island. Given that the habitat used by Miniopterus sp. nov. is not necessarily associated with native forest, that it has a broad distribution across the Central Highlands, and that it has been found in synanthropic situations, this species is not considered a conservation concern.

Patterns of morphological and genetic variation in the endemic Malagasy bat Miniopterus gleni (Chiroptera: Miniopteridae), with the description of a new species, M. griffithsi

Over the past decade, major advances have been made concerning the systematics and species diversity of Malagasy bats, largely based on specimens collected during inventories and associated morphological and molecular genetic studies. Herein we describe a new species of endemic bat from southern Madagascar, Miniopterus griffithsi sp. n., which is the sister taxa to Miniopterus gleni, a taxon described in 1995 (holotype from Sarodrano, just north of the Onilahy River in the southwest). Based on current information, M. griffithsi is found in the sub-arid bioclimatic zone, south of the Onilahy River, and M. gleni occurs in a variety of different bioclimatic zones, north of the Onilahy River to the northern portion of the island and on the near shore island of Ile Sainte Marie. The realization that M. griffithsi was a separate entity was first based on phylogeographic studies of the M. gleni complex. Comparisons using 397 bp of mitochondrial cytochrome b found a divergence of 1.2% within animals occurring across much of Madagascar north of the Onilahy River, 0.07% in those south of the Onilahy River, and 7.4% in populations separated by this river. Subsequently, morphological characters were identified that supported the specific separation of populations occurring south (M. griffithsi) and north of the Onilahy River (M. gleni), which include tragus shape, pelage coloration, and skull proportions.

Development and characterisation of 20 microsatellite loci isolated from the large bent-wing bat, Miniopterus schreibersii (Chiroptera: Miniopteridae) and their cross-taxa utility in the family Miniopteridae

The large bent-wing bat, Miniopterus schreibersii (Kuhl 1819), has a long history of taxonomic uncertainty and many populations are known to be in a state of decline. Microsatellite loci were developed for the taxonomic and population genetic assessment of the Australian complex of this species. Of the 33 primer sets designed for this research, seven (21%) were deemed suitably polymorphic for population-level analyses of the Australian taxa, with five (71%) of these loci revealing moderate to high levels of polymorphism (PIC = 0.56 to 0.91). The cross-taxa utility of the M. schreibersii microsatellite markers was assessed in the microbat (Chiroptera) family Miniopteridae. Sub-species and species covering the Miniopteridae’s global distribution (with the exception of the Middle East) were selected, numbering 25 taxa in total. Amplification was successful for 26 loci, of which 20 (77%) were polymorphic. High cross-taxa utility of markers was observed with amplification achieved for all taxa for between four (20%) and 20 (100%) loci, and polymorphism was considered moderate to high (PIC = 0.47–0.91) for 12 (60%) of these loci. The high cross-taxa utility of the microsatellites reported herein reveal versatile and cost-effective molecular markers, contributing an important genetic resource for the research and conservation of Miniopteridae species worldwide.

The use of molecular and morphological characters to resolve the taxonomic identity of cryptic species : the case of Miniopterus manavi (Chiroptera, Miniopteridae)

Based on recent molecular phylogenetic studies, the Old World bat family Miniopteridae, composed of species in the genus Miniopterus, has been shown to contain complex paraphyletic species, many of which are cryptic based on convergent morphological characters. Herein we resolve the phylogenetic relationships and taxonomy of the species complex M. manavi on Madagascar and in the Comoro Archipelago, where these animals occur in different bioclimatic zones. First using mitochondrial cytochrome-b sequence data to define clades and then morphology to corroborate the molecular data, including comparisons to type specimens, we demonstrate that animals identified as this taxon are a minimum of three species: M. manavi sensu stricto occurs in at least the central portion of the Central Highlands; M. griveaudi has a broad distribution in lowland northern and central western Madagascar and the Comoros (Anjouan and Grande Comore), and M. aelleni sp. n. has been found in northern and western Madagascar and the Comoros (Anjouan). In each case, these three clades were genetically divergent and monophyletic and the taxa are diagnosable based on different external and craniodental characters. One aspect that helped to define the systematics of this group was isolation of DNA from one of the paratypes of M. manavi collected in 1896 and new topotypic material. Miniopterus manavi is most closely allied to a recently described species, M. petersoni. At several localities, M. griveaudi and M. aelleni have been found in strict sympatry, and together with M. manavi sensu stricto show considerable convergence in morphological characters, but are not immediate sister taxa. In defining and resolving the systematics of cryptic species, such as miniopterid bats, the process of defining clades with molecular tools, segregating the specimens accordingly, and identifying corroborative morphological characters has been notably efficient.

Insights into the evolution of a cryptic radiation of bats: dispersal and ecological radiation of Malagasy Miniopterus (Chiroptera: Miniopteridae)

The past decade has seen a proliferation of new species of Miniopterus bats (family Miniopteridae) recognized from Madagascar and the neighboring Comoros archipelago. The interspecific relationships of these taxa, their colonization history, and the evolution of this presumed adaptive radiation have not been sufficiently explored. Using the mitochondrial cytochrome-b gene, we present a phylogeny of the Malagasy members of this widespread Old World genus, based on 218 sequences, of which 82 are new and 136 derived from previous studies. Phylogenetic analyses recovered 18 clades, which divide into five primary lineages: (1) M. griveaudi; (2) M. mahafaliensis, M. sororculus and X3; (3) M. majori, M. gleni and M. griffithsi; (4) M. brachytragos; M. aelleniA, and M. aelleniB; and (5) M. manavi and M. petersoni recovered as sister species, which were in turn linked to a group comprising M. egeri and five genetically distinct populations referred to herein as P3, P4, P5, P6 and P7. Beast analysis indicated that the initial divergence within the Malagasy Miniopterus radiation took place 4.5 Myr; most species diverged between 4 and 2.5 Myr, and a secondary period was between 1.25 and 1 Myr. DNA K2P-distances between recognized taxa ranged from 12.9% to 2.5% and intraspecific variation was less than 1.8%. Of the 18 identified clades, Latin binomials are only associated with 11, which indicates much greater differentiation than currently recognized for Malagasy Miniopterus. These data are placed in a context of the dispersal history of this genus on the island and patterns of ecological diversity.

A molecular and morphological investigation of species boundaries and phylogenetic relationships in Australian free-tailed bats Mormopterus (Chiroptera:Molossidae)

The taxonomic uncertainty surrounding several prominent genera of Australian microbat has been a long-standing impediment to research and conservation efforts on these groups. The free-tail bat genus Mormopterus is perhaps the most significant example, with a long history of acknowledged species-level confusion. This study uses a combined molecular and morphological approach to conduct a comprehensive assessment of species and subgeneric boundaries, between-species phylogenetic affinities and within-species phylogeographic structure in Australian members of Mormopterus. Phylogenetic analyses based on 759 base pairs of the NADH Dehydrogenase subunit 2 mitochondrial gene were concordant with species boundaries delineated using an expanded allozyme dataset and by phallic morphology, and also revealed strong phylogeographic structure within two species. The levels of divergence evident in the molecular and morphological analyses led us to recognise three subgenera within Australia: Micronomus, Setirostris subgen. nov. and Ozimops subgen. nov. Within Ozimops we recognise seven Australian species, three of which are new, and none are conspecific with Indo-Papuan species. The family Molossidae now comprises eleven species across three subgenera in Australia, making it the continent's second most speciose family of bats. © CSIRO 2014.

Description of a new species of the miniopterus aelleni group (chiroptera: miniopteridae) from upland areas of central and northern Madagascar

Recent molecular genetic work, combined with morphological comparisons, on Malagasy members of the bat genus Miniopterus (Family Miniopteridae), has uncovered a number of cryptic species. Based on recently collected specimens and associated tissues, we examine patterns of variation in M. aelleni, the holotype of which comes from Ankarana in northern Madagascar. Using molecular genetic (mitochondrial cytochrome b) and morphological characters we describe a new species, M. ambohitrensis sp. nov. In northern Madagascar, M. ambohitrensis and M. aelleni are allopatric, but occur in relatively close geographical contact (approximately 40 km direct line distance) with M. ambohitrensis found at Montagne d'Ambre in montane humid forest and M. aelleni sensu stricto at Ankarana in dry deciduous forest. Morphologically, this new taxon is differentiated from M. aelleni based on pelage coloration, external measurements, craniodental differences, and tragus shape. Comparisons using 725 bp of cytochrome b found a divergence of 1.1% within M. aelleni sensu stricto, 0.8% within M. ambohitrensis, and 3.3% between these two clades. The two sister species do not demonstrate acoustical differences based on recordings made in a flight cage. Miniopterus ambohitrensis is known from four localities in the northern and central portions of Madagascar, all from montane regions and across an elevational range from about 800 to 1600 m; its calculated "Extent of occurrence" is 15,143 km². It is possible that this species is at least partially migratory.

An integrative approach to characterize Malagasy bats of the subfamily Vespertilioninae Gray, 1821, with the description of a new species of Hypsugo

© 2015 The Linnean Society of London. Although important advances have been made in recent years in the taxonomy of different families and subfamilies of Malagasy bats, those belonging to the Vespertilioninae remain partially unresolved. Herein using a mitochondrial marker (cytochrome b) as the point of departure for 76 specimens of Malagasy vespers and appropriate African taxa, we diagnose the six taxa of this subfamily on the island by overlaying different morphological and bioacoustic characters on the clade structure of sequenced animals. The species include: endemic Neoromicia matroka, which is sister to African N. capensis; endemics N. malagasyensis and N. robertsi, which form sister species; a member of the genus Hypsugo, which is sister to African H. anchietae and described herein as new to science; Pipistrellus hesperidus for which Madagascar animals are genetically close but distinct from African populations of the same species; and endemic P. raceyi, which shows segregation of eastern (mesic) and western (dry) populations and its sister species relationships are unresolved. While the external and craniodental measurements, as well as bioacoustic variables, allow only partial differentiation of these six species of Vespertilioninae, molecular characters provide definitive separation of the taxa, as do male bacular morphology.