previously been defined as ‘‘polymorphic’’ for the northern white- cheeked gibbon [17,18] without any concrete population analysis done [24]. Our investigation is the first study with a substantial number of Nomascus individuals, taking into consideration that these are very rare apes. All tested NLE individuals were homozygous for this inversion. We additionally found that southern white-cheeked gibbons (NLS) do not share this inversion with NLE, in disagreement with Couturier and Lernould [17]. NLS has a different geographic distribution than NLE, as they inhabit Southern Laos, central Vietnam and possibly northeast Cambodia [25] whereas NLE is found in Northern Laos, Northwestern Vietnam and Southeastern China [3]. Following our results, it seems likely that NLS does not carry the NLE7 inversion. To explain this difference with the finding of Couturier and Lernould we hypothesize a sampling error. There are many reasons why we were intrigued by this chromosomal rearrange- ment and pursuit its characterization. First, this inversion is not shared by other members of the same genus and it is more likely that the most recent chromosomal rearrangement occurred in NLE. Taking into consideration the chromosomal theory of speciation, we wondered if this inversion could have been responsible for causing reproductive isolation in this gibbon population and then drive a speciation event [26]. Further studies on sequence divergence within the population will be necessary to exploit this possibility. Second, an important outcome of our study is that, given its taxon-specificity, this chromosomal breakpoint may be use as one of the markers to distinguish the northern white-cheeked gibbon from other taxa or hybrids, serving as a tool for conservation purposes. Several cooperative breeding programs are currently in place for endangered gibbon species. Breeding gibbons in captivity can have the ultimate goal of releasing gibbons in a protected native habitat after creating a viable gene pool [3]. Many breeding and conservation centers have difficulties in identifying which gibbon species or subspecies that they house, and in determining which individual gibbon would benefit in their breeding program. This is a very important issue for species as rare as the northern white-cheeked gibbon. In a recent publication, Mootnick [3] commented on all the complications that can arise when conservation or rescue centers need to identify some of the gibbon taxa. One of the confusing factors existing in gibbon taxonomy was ambiguous or incomplete descriptions found in some publications. Karyotyping represents a powerful method to distinguish between different species where other morphological traits are unclear. Unfortunately, most breeding centers do not have the training or equipment for genetic analysis. Another limitation is chromosomal preparation and the availability of fresh blood from each individual. In the case of endangered species, this becomes a recurring limiting factor to take into account. Recently a new approach called ‘‘DNA barcoding’’ [27,28] emerged in the conservation and forensic community, demonstrating the need to complement classic taxonomy with other tools. This method proposes the use of a single locus that can be amplified through specific primers and sequences; sequences are then introduced in a database in order to classify species or identify new species. Since it was introduced, this approach generated controversial opinions in the scientific community and many experts argued that DNA barcoding could produce misleading results if not accompanied by a full taxonomic revision [26]. The sequence that is used to ‘‘barcode’’ animals is the mitochondrial cytochrome oxidase I (coxI) gene which seems to have the features required for such analysis: it contains sufficient variability between species, it is short enough to be analyzed in one experiment and it contains conserved regions that can be used to design universal primers [27]. The clear advantage of this method is the need for minimal technical Gibbon Chromosomal Inversion support, as only one PCR reaction is necessary. However, by analyzing only a maternally inherited gene, possible hybrids remain undetected. Our approach can be seen as a combination between classic cytogenetics and the DNA barcoding method. In our case, only few PCR experiments are required to discern similar species but the target of such amplification is a synteny breakpoint and not a gene. One of our long term goals is to identify at least one species- specific marker for each gibbon species in order to make them available to the conservation community. It is noteworthy that most of our PCR experiments were done on ‘‘low quality’’ DNA samples but were still successful. This feature is quite important when only materials collected in the wild are available, which are mostly incompatible with cytogenetic analysis. The use of a chromosomal rearrangement as a marker has the advantage of being uncoupled from population sequence variations but based on a simple presence/absence analysis. Additionally, as we are planning to characterize the sequences spanning the complete set of chromosomal rearrangements for all the gibbon species, these data will help to clarify the phylogenetic relationships between gibbon species. We are aware, however, that chromosomal rearrangements cannot be isolated criteria to identify different gibbon taxa and the complete assessment has to be based also on morphological and other molecular data. Nevertheless, our PCR approach represents a first screening step to retrieve information that otherwise would require fresh blood samples and cytogenetic expertise, which in many cases are not available. Materials and Methods Fluorescent in situ hybridization Chromosome preparations for all the gibbon species in this study were obtained from peripheral blood following standard procedures. Briefly, blood was incubated with cell culture media and phytohemagglutinin (GIBCO) for 72 hours (37uC, 5 % CO2). Colcemid was then added (final concentration 0.05 ug/ml) and cells were harvested after a 1 hour incubation. Cells were spun down by centrifugation, the media was discarded and the pellet was resuspended in 8ml of hypotonic solution. After incubating for 20 minutes, the standard fixative solution (1 part Acetic Acid, 3 parts Methanol) was added and cells were centrifuged at 2500rpm for 5 minutes. The pellet was washed with fixative solution and cells were kept at 4uC overnight. DNA from BACs was extracted using PureLink Miniprep kit (Invitrogen) as previously described [14]. Images were acquired using Nikon 80i microscope, equipped with CCD camera Cool Snap HQ2 (Photometrics) and software Nis Elements Br (NIKON). Elaboration of the images was done using Photoshop. Samples For the present study, DNA from each individual of S. syndactylus, H. leuconedys, H. moloch, H. agilis, H. lar, H. muelleri, H. albibarbis and H. pileatus as well as from 2 individuals of N. nasutus (eastern black gibbon), 6 individuals of N. concolor (western black gibbon), 24 individuals of N. l. leucogenys, 5 individuals of N. l. siki and 14 individuals of N. gabriellae were examined (Table 2). All study specimens were identified by pelage coloration, additional external characteristics, and geographical distribution for gibbons living in their native habitat. Moreover, mainly wild-born gibbons or samples collected during field surveys in local houses were tested. Captive-born specimens were only studied if the species identification of their parents was possible. Total genomic DNA from blood and tissue was extracted using the DNeasy Blood & Tissue Kit from Qiagen. Hair follicle cells were directly PLoS ONE | www.plosone.org 6 March 2009 | Volume 4 | Issue 3 | e4999