Judith L. Eger

The role of DNA barcodes in understanding and conservation of mammal diversity in southeast Asia.

Background Southeast Asia is recognized as a region of very high biodiversity, much of which is currently at risk due to habitat loss and other threats. However, many aspects of this diversity, even for relatively well-known groups such as mammals, are poorly known, limiting ability to develop conservation plans. This study examines the value of DNA barcodes, sequences of the mitochondrial COI gene, to enhance understanding of mammalian diversity in the region and hence to aid conservation planning. Methodology and Principal Findings DNA barcodes were obtained from nearly 1900 specimens representing 165 recognized species of bats. All morphologically or acoustically distinct species, based on classical taxonomy, could be discriminated with DNA barcodes except four closely allied species pairs. Many currently recognized species contained multiple barcode lineages, often with deep divergence suggesting unrecognized species. In addition, most widespread species showed substantial genetic differentiation across their distributions. Our results suggest that mammal species richness within the region may be underestimated by at least 50%, and there are higher levels of endemism and greater intra-specific population structure than previously recognized. Conclusions DNA barcodes can aid conservation and research by assisting field workers in identifying species, by helping taxonomists determine species groups needing more detailed analysis, and by facilitating the recognition of the appropriate units and scales for conservation planning.

A bony connection signals laryngeal echolocation in bats

Echolocation is an active form of orientation in which animals emit sounds and then listen to reflected echoes of those sounds to form images of their surroundings in their brains. Although echolocation is usually associated with bats, it is not characteristic of all bats. Most echolocating bats produce signals in the larynx, but within one family of mainly non-echolocating species (Pteropodidae), a few species use echolocation sounds produced by tongue clicks. Here we demonstrate, using data obtained from micro-computed tomography scans of 26 species (n = 35 fluid-preserved bats), that proximal articulation of the stylohyal bone (part of the mammalian hyoid apparatus) with the tympanic bone always distinguishes laryngeally echolocating bats from all other bats (that is, non-echolocating pteropodids and those that echolocate with tongue clicks). In laryngeally echolocating bats, the proximal end of the stylohyal bone directly articulates with the tympanic bone and is often fused with it. Previous research on the morphology of the stylohyal bone in the oldest known fossil bat (Onychonycteris finneyi) suggested that it did not echolocate, but our findings suggest that O. finneyi may have used laryngeal echolocation because its stylohyal bones may have articulated with its tympanic bones. The present findings reopen basic questions about the timing and the origin of flight and echolocation in the early evolution of bats. Our data also provide an independent anatomical character by which to distinguish laryngeally echolocating bats from other bats.

Diversification and reproductive isolation: cryptic species in the only New World high-duty cycle bat, Pteronotus parnellii.

BackgroundMolecular techniques are increasingly employed to recognize the presence of cryptic species, even among commonly observed taxa. Previous studies have demonstrated that bats using high-duty cycle echolocation may be more likely to speciate quickly. Pteronotus parnellii is a widespread Neotropical bat and the only New World species to use high-duty cycle echolocation, a trait otherwise restricted to Old World taxa. Here we analyze morphological and acoustic variation and genetic divergence at the mitochondrial COI gene, the 7th intron region of the y-linked Dby gene and the nuclear recombination-activating gene 2, and provide extensive evidence that P. parnellii is actually a cryptic species complex.ResultsCentral American populations form a single species while three additional species exist in northern South America: one in Venezuela, Trinidad and western Guyana and two occupying sympatric ranges in Guyana and Suriname. Reproductive isolation appears nearly complete (only one potential hybrid individual found). The complex likely arose within the last ~6 million years with all taxa diverging quickly within the last ~1-2 million years, following a pattern consistent with the geological history of Central and northern South America. Significant variation in cranial measures and forearm length exists between three of the four groups, although no individual morphological character can discriminate these in the field. Acoustic analysis reveals small differences (5–10 kHz) in echolocation calls between allopatric cryptic taxa that are unlikely to provide access to different prey resources but are consistent with divergence by drift in allopatric species or through selection for social recognition.ConclusionsThis unique approach, considering morphological, acoustic and multi-locus genetic information inherited maternally, paternally and bi-parentally, provides strong support to conclusions about the cessation of gene flow and degree of reproductive isolation of these cryptic species.

Genetic Diversity of Northeastern Palaearctic Bats as Revealed by DNA Barcodes

Sequences of the DNA barcode region of the cytochrome oxidase subunit I gene were obtained from 3 8 species of northeastern Palaearctic bats to assess patterns of genetic diversity. These results confirmed earlier findings of deep phylogeographic splits in four pairs of vicariant species (Myotis daubentoniilpetax, M. nattererilbombinus, Plecotus aurituslognevi and Miniopterus schreibersiil fuliginosus) and suggested previously unreported splits within Eptesicus nilssoni and Myotis aurascens. DNA barcodes support all taxa raised to species rank in the past 25 years and suggest that an additional species — Myotis sibiricus — should be separated from Myotis brandtii. Major phylogeographic splits occur between European and Asian populations of Myotis aurascens, Rhinolophus ferrumequinum and Myotis frater; smaller scale splits are observed between insular and mainland populations in the Far East (M. frater, Myotis ikonnikovi and Murina ussuriensis) and also between southeastern Europe and Ciscaucasia (Myotis daubentonii, Plecotus auritus, and Pipistrellus pipistrellus). One confirmed case of sequence sharing was observed in our dataset — Eptesicus nilssoni/serotinus. This study corroborates the utility of DNA barcodes as a taxonomic assessment tool for bats.

A New Species from Southwestern China in the Afro-Palearctic Lineage of the Horseshoe Bats (Rhinolophus)

Abstract A new species of horseshoe bat (Chiroptera: Rhinolophidae) is described from southwestern China. The presence of a wedge-shaped sella and pointed connecting process of the nose leaf aligns the new species to the landeri group in the Afro-Palearctic lineage of Rhinolophus. However, the new species is distinctly separable from these allopatrically distributed species by its noticeably larger body size. Other sympatric large-sized species of Rhinolophus have rounded connecting processes. Molecular systematic analyses based on mitochondrial cytochrome-b sequences confirmed the affinity of the new species to the Afro-Palearctic lineage, but in a clade most closely related to the ferrumequinum, fumigatus, and maclaudi groups. Of these species, only R. ferrumequinum ranges into Asia and overlaps in distribution with the new species. R. ferrumequinum is similar in general body size and external appearance; however, the new species is distinct in the characteristics of the nose leaf, skull, and baculum. The presence of a new species from southwestern China in the Afro-Palearctic lineage indicates a more complex historical biogeographic scenario within Rhinolophus than previously known. The difficulties found in allocating the new species to one of the phenetically described traditional species groups stress the convenience of using a phylogenetically based systematic organization of the genus Rhinolophus.

A new species of tube-nosed bat Murina (Vespertilionidae, Chiroptera) from Vietnam

Abstract A new species of Murina is described from Lam Dong province, Vietnam. The new form is a very small tube-nosed bat with a forearm length less than 30 mm. Externally it looks similar to Harpiola isodon from which it is well differentiated by teeth shape. From other small Murina species the new species can be defined by pelage coloration and texture, longer nasal tubes, dark skin on muzzle and smaller anterior upper premolar. Provisional analysis of mitochondrial DNA sequence also supports its species status. This species is known only from mountainous forests of the Da Lat plateau.

Three New Species of Murina from Southern China (Chiroptera: Vespertilionidae)

The biodiversity of tube-nosed bats (Murininae) from Asia has been underestimated by over 50%. Since 2005, eight taxa have been documented as distinct from the 19 previously known species. We describe three new species of Marina collected in southern China between 2004 and 2007. These species differ morphologically, morphometrically, and genetically from previously described species of Marina. Morphological differences include pelage color, size, skull shape and tooth morphology. Analysis of mitochondrial DNA barcodes of the cytochrome c oxidase subunit I (COI) gene supports species status based on divergent phylogenetic lineages.

Canines make the difference: a new species of Hypsugo (Chiroptera: Vespertilionidae) from Laos and Vietnam

Hypsugo was regarded as a subgenus of Pipistrellus by many authors, but its generic distinctiveness is now widely accepted. According to recent taxonomic arrangements, nine species are known to occur in Southeast Asia. During the investigation of material recently collected from Lao PDR and Vietnam we identified an additional species and hence describe it here as Hypsugo dolichodon n. sp. It resembles H. pulveratus, but is larger with conspicuously long canines and differs considerably in the DNA barcode gene sequence.

Speciation processes in putative island endemic sister bat species: false impressions from mitochondrial DNA and microsatellite data

Cases of geographically restricted co‐occurring sister taxa are rare and may point to potential divergence with gene flow. The two bat species Murina gracilis and Murina recondita are both endemic to Taiwan and are putative sister species. To test for nonallopatric divergence and gene flow in these taxa, we generated sequences using Sanger and next‐generation sequencing, and combined these with microsatellite data for coalescent‐based analyses. MtDNA phylogenies supported the reciprocally monophyletic sister relationship between M. gracilis and M. recondita; however, clustering of microsatellite genotypes revealed several cases of species admixture suggesting possible introgression. Sequencing of microsatellite flanking regions revealed that admixture signatures stemmed from microsatellite allele homoplasy rather than recent introgressive hybridization, and also uncovered an unexpected sister relationship between M. recondita and the continental species Murina eleryi, to the exclusion of M. gracilis. To dissect the basis of these conflicts between ncDNA and mtDNA, we analysed sequences from 10 anonymous ncDNA loci with *beast and isolation‐with‐migration and found two distinct clades of M. eleryi, one of which was sister to M. recondita. We conclude that Taiwan was colonized by the ancestor of M. gracilis first, followed by the ancestor of M. recondita after a period of allopatric divergence. After colonization, the mitochondrial genome of M. recondita was replaced by that of the resident M. gracilis. This study illustrates how apparent signatures of sympatric divergence can arise from complex histories of allopatric divergence, colonization and hybridization, thus highlighting the need for rigorous analyses to distinguish between such scenarios.

Genetic distinctiveness of the greater long-tailed hamster, Tscherskia triton nestor (Rodentia: Mammalia), from Jeju Island, Korea: cytochrome oxidase I and cytochrome b sequence analyses

Abstract To examine the taxonomic status of Tscherskia triton nestor, which inhabits in Korea, we first obtained partial cytochrome oxidase I (COI) (657 bp) and cytochrome b (426 bp) sequences of T. t. nestor from the Korean Jeju Island and Tscherskia triton fuscipes from northeastern China, and these sequences were compared with the corresponding sequences of Tscherskia triton, available from GenBank. In the COI analysis, T. t. nestor from Jeju was distinct from T. t. fuscipe from northeastern China, with the average Jukes–Cantor distance of 3.45%, and in the cytochrome b analysis, T. t. nestor from Jeju was divergent from T. t. fuscipes from northeastern China and T. t. triton from the North China Plain, with the average distance of 3.88%. We conclude that these sequencing results on the distinctiveness of T. t. nestor from Jeju support the current classification of T. t. nestor from Korea, although further analyses with mitochondrial sequences of T. t. nestor from the mainland Korea and nuclear sequences of T. triton from Korea and China are necessary to confirm our findings. In addition, we considered genetically distinct T. t. triton from Jeju as an allopatric phylogroup, which needs special protection for its conservation. Additionally, we found the lack of cytochrome b sequences between T. t. fuscipes from northeastern China and T. t. triton in the North China Plain, although further systematic analyses to clarify the taxonomic status of T. t. fuscipes are necessary.