For smaller mammals, careful examination of prepared specimens and comparison with reference material in museums is often required to confirm identifications using standard morphological approaches. This can be a particular challenge for field workers in Southeast Asia because critical reference material, such as type specimens, is scattered in the world's museums.
Since its initial proposal as a tool for rapid identification of species  , DNA barcoding has gained considerable validation. Among terrestrial vertebrates, this approach has been shown to be effective in the identification of amphibians  , North American birds  ,  , Neotropical birds  and Neotropical small mammals  , . For bats in Guyana, all 87 currently recognized species could be uniquely identified by DNA barcodes .
DNA barcodes are also proving to be a useful tool for identifying genetically distinct units worthy of more intense taxonomic study. In this paper, we examine the value of DNA barcodes for enhancing our knowledge of the distribution and taxonomy of Southeast Asian mammals and facilitating conservation planning using the bat fauna as a model system. Specifically, we examine three main questions: 1 the extent to which currently recognized taxa can be uniquely identified using DNA barcodes; 2 the extent to which currently recognized species show deep genetic divides which may be suggestive of previously unrecognized species; and 3 whether DNA barcodes show evidence of geographic differentiation within widespread species suggestive of lineages that should be considered as separate units in conservation planning.
We obtained DNA barcodes from specimens representing morphologically distinct species from Asia, predominantly from southern China, Laos, Vietnam, peninsular Malaysia and Borneo Figure 1. Of these, species were assigned names based on currently published taxonomy, while an additional 15 species were recognized as morphologically distinct, but were either undescribed or could not be assigned appropriate names based on available reference material.
Most species out of were represented by multiple specimens, with an average of 12 specimens per species; six species were represented by more than 50 specimens. The majority of specimens came from Vietnam , Laos , southern China and Malaysia with smaller numbers from other countries. Nearly all species could be uniquely identified based on DNA barcodes with the exception of four pairs of closely related congeneric species see Figures 2 , 3 , 4 , 5 , 6 and 7.
In a few additional cases, species were distinct, but were very similar genetically, such as Pteropus vampyrus and P. However, in most cases, interspecific genetic distances were large, with the minimum genetic distance to the nearest species averaging Blue indicates clusters of specimens that include more than one species that could not be resolved.
Red indicates taxa with deep intra-specific divides that potentially represent distinct species. Species identities are based on usually recognized morphological characters Francis , but do not correspond to genetic differences. These two forms were readily separated by size and echolocation calls. The specimens came from Laos, Vietnam, Myanmar and southern China. The outlying specimen at the bottom is from southern Vietnam and may prove to represent something different. The four species groups that could not be clearly distinguished based on barcodes each had slightly different patterns of haplotype divergence.
Specimens referred to Macroglossus minimus and M. Cynopterus horsfieldi had three specimens with a diagnostic genotype, and two with a genotype that was 3. Rhinolophus macrotis and R.
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Myotis annamiticus , although morphologically distinct  , differed by only 0. The amount of genetic differentiation among species varied among the 7 families or subfamilies for which we had more than 5 species represented Figure 8. Mean minimum inter-specific distances ranged from a low of 8. Among the Murininae and Kerivoulinae, which are each dominated by one genus Murina and Kerivoula respectively , interspecific nearest neighbour distances were almost invariably very high, even between species that are morphologically very difficult to distinguish.
Families with fewer than 5 species represented Miniopteridae, Megadermidae, Nycteridae, Emballonuridae are not shown. Some taxa had multiple clusters; one species R. The number of distinct haplogroups and their degree of genetic divergence within species differed among families Figure 9 , with families having the highest degree of interspecific variation also having high levels of intraspecific variation. Results are presented separately for each subfamily or family.
Most widespread species for which we had samples from multiple geographic areas showed substantial geographic variation in DNA barcodes Figure The comparisons include 21 species from both peninsular Malaysia and Borneo, and 13 species from Malaysia 11 from Peninsular Malaysia and two from Borneo and Indochina. Our analyses indicate that DNA barcodes are an effective tool for both differentiating and identifying species of bats in Southeast Asia.
Although this study has only examined bats, early results from some of our team suggest that barcodes are similarly effective at differentiating other mammals in the region, including rodents and insectivores C. M Francis, J. Eger, A. Borisenko, unpublished data. This suggests that DNA barcoding will enhance the effectiveness and efficiency of both conservation planning and research activities for all mammals in the region by assisting with species delineation and identification.
Our study has revealed that mammalian biodiversity in this region, at least measured in genetic terms, is much higher than previously recognized. Most widespread taxa showed substantial geographic variation in their barcode sequences, with populations from different regions such as peninsular Malaysia and Borneo often being genetically distinct Figure Although not all of the genetic divides that we detected necessarily represent new species, they do represent lineages with long histories of evolutionary independence, at least among maternal lineages.
Furthermore, we only sampled part of the fauna that is shared among regions. For example, most of the approximately bat species reported from peninsular Malaysia also occur in Borneo, but we only sampled 21 species from both areas. High levels of genetic differentiation can also be anticipated among the many islands within the Philippine and Indonesian archipelagos, most of which we did not sample.
Currently, many species are thought to be shared among numerous islands, but, assuming similar levels of biogeographic separation to the regions we did sample, we anticipate that many of those will prove to represent genetically distinct lineages. Similar levels of divergence may also occur among widely separated areas on the mainland, as noted by the results of our comparisons between peninsular Malaysia and Indochina.
Sequencing tissue samples from these regions, either from existing or new collections, should be a high priority for understanding speciation in south-east Asia. We also found several sympatric lineages showing deep genetic divergence and anticipate many more will be discovered with further sampling. Even allowing for the fact that some of these branches do not represent distinct species see below , we suspect that bat species diversity in south-east Asia is at least twice that currently recognized. This reassessment suggests a much higher level of endemism than currently recognized, a conclusion with significant implications for conservation planning.
Adequate conservation of biodiversity in Southeast Asia requires protection for the complete suite of species within each geographic subregion, through a combination of protected areas and effective conservation in anthropogenic landscapes. Apparently widespread species are unlikely to be adequately protected by the designation of just one or a few reserves. Distinct biogeographic regions such as peninsular Malaysia, Borneo, and Indochina must be viewed as separate units for conservation planning.
The same is likely to be true for many of the islands in Indonesia and the Philippines. Evidence of genetic differentiation in several species within Indochina suggests that it may be necessary to define areas of conservation importance at smaller scales, such as different ecoregions. The observed high levels of genetic divergence suggest that, in addition to increased taxonomic effort to define species, ecological studies and field surveys are needed in each region to determine the ecological and conservation requirements of each species and any genetically divergent lineages.
Genetically distinct populations, whether or not they are considered different species, are likely to have distinctive ecological requirements which need to be studied to ensure effective conservation planning. Our data support the utility of DNA barcodes as a tool for field researchers carrying out faunal surveys  as well as ecological or behavioural studies. Despite the recent availability of comprehensive regional field guides e. For some species, their taxonomic identification can only be validated by careful examination of internal characters such as skull or baculum shape  , .
By contrast, sufficient tissue for DNA analysis can be collected from a live mammal through a small biopsy e. When working in protected areas where collecting is not possible, or when carrying out behavioural studies of live animals, DNA barcodes recovered from biopsy samples will allow validation of identifications at a relatively low cost with a high degree of confidence.
The use of DNA barcodes as an identification tool by field workers requires the prior construction of a carefully validated reference database matching DNA barcodes to professionally curated specimens identified through traditional taxonomic work. Although our study has produced an initial dataset, most linked to museum vouchers, much additional work is needed to complete the database. We have not yet sampled all currently recognized species, and the results of our study suggest the likelihood of many undescribed taxa and further genetic variants to be discovered, especially in new geographic areas.
When carrying out distributional surveys, especially in new areas, we recommend retaining a representative set of voucher specimens for deposit in a properly curated and publicly accessible collection because of the likely discovery of new taxa or genetically distinct populations.
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Tissue samples for molecular analysis should also be obtained from these specimens and preserved using appropriate protocols. DNA barcodes should then be promptly sequenced and published on shared international databases such as BOLD  to ensure the rapid sharing of knowledge about this diversity to aid in conservation planning.
DNA barcodes can also help taxonomists by facilitating comparison with other taxonomic material, even at a distance e. One of the challenges for a mammal taxonomist working in Southeast Asia is that most of the larger and reliably identified reference collections are scattered among museums, primarily in North America and Europe. Travel to those collections, or shipping specimens for comparison, is becoming increasingly difficult and expensive. In contrast, with ongoing improvements in technology, high quality sequences can be obtained cheaply from very small tissue samples.
Moreover, the digital nature of genetic information makes DNA barcodes readily comparable through internationally accessible online data portals such as BOLD  or GenBank. Sequences in the BOLD database that are associated with specimen records linked to museum vouchers are the most valuable as reference material. By confirming the identification of specimens through DNA barcodes, local museums can establish reference collections that can serve as a basis for future research including the description of new species.
DNA barcodes can also facilitate international collaboration. For example, Bates et al. Finally, DNA barcodes are a valuable tool for highlighting areas in need of further taxonomic research. Baker and Bradley  noted that divergent mtDNA sequences are often an indicator of unrecognized genetic species. However, we agree with Baker and Bradley  that a simple threshold value, especially one based on a single gene, is not a sufficient basis for species recognition.
A variety of processes including incomplete lineage sorting or introgression through a past hybridization event could lead to high levels of genetic variation within species  , . Female philopatry to breeding locations could lead to differentiation of mtDNA lines, even if there is extensive interbreeding and nuclear gene flow due to male dispersal. Variation in mtDNA can be retained for long periods if there is no selective pressure against it .
Nevertheless, substantial divergence in DNA barcodes can help to identify priority groups for further taxonomic study using other characters including morphology, behaviour e. Regardless of which characters are used to identify new species, DNA analyses supplement, but do not replace traditional morphological studies. Morphological examination of type specimens is still needed in most cases to determine whether the taxon already has a name. For example, if animals formerly regarded as conspecific are shown to represent two or more species, the original type must be examined to determine which of the newly proposed forms represents the original species name.
In many cases, the types of closely related taxa must also be examined, including those currently considered as synonyms or subspecies. Early taxonomists working in Southeast Asia often coined names for different populations or even different colour morphs that were later synonymized; some of these may well prove to be valid taxa. Ideally, DNA barcodes should be obtained from all type material, and we urge researchers describing new taxa to ensure that properly preserved tissue samples and DNA barcodes are available for their type series, especially the holotype.
Because most extant types were collected before the introduction of molecular techniques, many were preserved as dried skins, sometimes with added preservatives such as arsenic, while others were fixed in formalin before storage in alcohol.
The Role of DNA Barcodes in Understanding and Conservation of Mammal Diversity in Southeast Asia
Furthermore, work with archival DNA requires a special laboratory setting and care to avoid contamination . Finally, many museum curators remain reluctant to allow destructive tissue sampling of types for DNA extraction until analytic protocols are improved. As a consequence, DNA barcodes are not available for most mammal type specimens, so morphological comparisons remain the only available approach. We conclude that DNA barcodes will greatly facilitate the challenge of properly describing and mapping biodiversity in Southeast Asia for the benefit of conservation.
Such work is urgently needed because, despite evidence of high levels of genetic diversity within many species, conservationists and politicians still focus their effort around named species, as do data compendia such as the IUCN Red List . Given the urgency for robust conservation actions within this region, where many habitats have already been lost, we hope that the use of DNA barcodes and public access of such information through Web portals will encourage the intensified taxonomic effort needed to describe and catalogue this diversity and ultimately to aid its protection.
All tissue samples came from specimens that had already been collected as part of other biodiversity studies which had been carried out with appropriate permissions from local authorities. Much of the survey work was carried out by teams involving one or more of the authors of this paper, although some material was provided by additional researchers listed in the acknowledgements.
Bats were trapped in the field using a variety of methods including mist nets, harp traps  , and flap traps  for free-flying bats, as well as capture by hand or with small nets from roosts in caves, trees or buildings. Bats were measured and weighed and given a preliminary identification in the field. Tissue samples were largely taken from bats that were euthanized and preserved as museum specimens. Vouchers were prepared either as a dry skin and skeleton, or preserved in alcohol, usually after initial fixation in formalin. A few tissue samples were obtained from wing punches  taken from live bats that were subsequently released.
However, few of these older preserved samples amplified successfully, and those that did often yielded only short sequences. DNA extraction and sequencing success varied with the source of the tissue, the mode of preservation, and with the species group, but we did not track success rates due to changes in analytical protocols over the four years of this study.
New primer cocktails were developed over the course of the study, improving sequencing success. In addition, accurate records were not always available on the tissue type or preservation methods used. As a result, for this paper we only consider samples that were successfully sequenced.
Bats were identified based on morphological criteria described in key taxonomic references including Corbet and Hill  , Payne et al. In most cases, taxonomy has been updated to match Simmons  except for species that have been described or recognized more recently or for which our own research indicates an alternative name is more appropriate. In most cases, specimens were initially identified in the field and then confirmed through subsequent examination of museum skulls and dental characters.
In several instances, conflicts between DNA barcode results and initial identification of a specimen prompted its re-examination and a correction in the identification. We also detected several cases where tissue samples had been mixed up or mislabelled. In most cases the solution was easily deduced and the error was corrected. For example, if two representatives of morphologically distinct species collected at the same time had sequences that matched each other's species, we assumed they had been transposed and corrected the records.
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In a few other cases where an error seemed highly probable, but the cause could not be unambiguously determined, the data record was omitted from analysis. Sequence analysis was carried out at the Canadian Centre for DNA Barcoding using standard high-throughput barcoding protocols . Learn more. Abstract: Depressed mammal densities characterize the interior of many Southeast Asian protected areas, and are the result of commercial and subsistence hunting.
Local people are part of this problem but can participate in solutions through improved partnerships that incorporate local knowledge into problem diagnosis. We illustrate the practical details of initiating such a partnership through our work in a Thai wildlife sanctuary. Many protected areas in Southeast Asia present similar opportunities. Commercial hunting contributed heavily to extensive population declines for most species, and subsistence hunting was locally significant for some small carnivores, leaf monkeys, and deer.
Workshops thus clarified which species were at highest risk of local extinction, where the most threatened populations were, and causes for these patterns. Most important, they advanced a shared problem definition, thereby unlocking opportunities for collaboration. As a result, local people and sanctuary managers have increased communication, initiated joint monitoring and patrolling, and established wildlife recovery zones. Using local knowledge has limitations, but the process of engaging local people promotes collaborative action that large mammals in Southeast Asia need.
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