The mitochondrial D-loop is a robust maternal-species identifier in gibbons (Hylobatidae)

The mitochondrial D-loop is a robust maternal-species identifier in gibbons (Hylobatidae) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The mitochondrial D-loop is a robust maternal-species identifier in gibbons (Hylobatidae) Lauren Lansdowne, Kazunari Matsudaira, Takafumi Ishida, Sîan McHugh, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5039233/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 04 Jul, 2025 Read the published version in Conservation Genetics Resources → Version 1 posted 9 You are reading this latest preprint version Abstract Some gibbon species are difficult to distinguish phenotypically. This issue is compounded by recent changes in the gibbon taxonomy, which have sometimes occurred long after individuals were taken from the wild for captive breeding programmes. Furthermore, gibbon species within the Hylobates and Nomascus genera have been documented to hybridize to produce viable, fertile offspring, both in captivity and in the wild. This raises the possibility of cryptic hybrids within captive populations, threatening the genetic integrity of these genera. Phylogenetic methods using the mitochondrial D-loop and cox1 gene were compared to genetically identify gibbon species. Both loci identified lineages with cryptic hybrid ancestry. However, the D-loop outperformed the cox1 gene, providing higher resolution, particularly for Nomascus spp ., which for some species were not monophyletic when using the cox1 gene. The D-loop also revealed a significantly higher number of unique sequences, making it more suitable for investigations into relatedness, such as female dispersal patterns. Gibbons Genetics Species Hybrids Phylogenetics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Taxonomy and Evolution Gibbons (Hylobatidae) are a family in the order Primates, known as the small or lesser apes, that with the great apes (Hominidae) form the superfamily of apes (Hominoidea) (Groves, 2001 ; Roos, 2016 ). It has been estimated that the gibbons last shared a common ancestor with the great apes 16–20 Million Years Ago (MYA) (Roos, 2016 ). The gibbon family is divided into four genera ( Hoolock , Hylobates , Nomascus , Symphalangus ), on the basis of chromosome complement and karyotype (ranging from 2n = 38 to 2n = 52) (Roos, 2016 ).Within these genera 20 species are currently recognised (Mittermeier, Rylands and Wilson, 2013 ; IUCN, 2020 ). It has been estimated that the current species diversity within the gibbons arose during the last ~ 15MYA (Matsudaira and Ishida, 2010 ; Thinh, Mootnick, Geissmann, et al. , 2010). In addition, there is genetic evidence that some radiations occurred as recently as 2MYA (Thinh, Mootnick, Geissmann, et al. , 2010; Carbone et al., 2014 ). All gibbon species are currently listed as Vulnerable, Endangered, or Critically Endangered by the International Union for Conservation of Nature (IUCN) (IUCN, 2020 ). Resolving the evolutionary history of these taxa has proven complex and controversial. Numerous phylogenetic studies of the Hylobatidae have been conducted using mitochondrial DNA (mtDNA) genes and regions, such as Cytochrome b (Chatterjee, 2006 ; Thinh, Mootnick, Geissmann, et al. , 2010; Thinh, Rawson, et al. , 2010), ND3-ND4 (Takacs et al., 2005 ), the D-loop/Control Region (Roos and Geissmann, 2001 ; Monda et al., 2007 ; Whittaker, Morales and Melnick, 2007 ), and the whole mitochondrial genome (Chan et al., 2010 ; Matsudaira and Ishida, 2010 , 2021 ; Fan et al., 2017 ). However, these studies have failed to resolve the gibbon phylogeny, with different studies producing conflicting results. Whilst the four genera are consistently monophyletic, their branching order varies, and relationships between species remain unclear. For example, using mitochondrial D-loop sequence data, Whittaker, Morales and Melnick ( 2007 ) found Hoolock to be basal, followed by Hylobates , with Symphalangus and Nomascus being the youngest sister genera. However, Roos and Geissmann ( 2001 ) found Nomascus to be basal, with Hoolock and Hylobates being the youngest and sister genera. Takacs et al. ( 2005 ), also found Hoolock to be basal, based on mitochondrial ND3-ND4 sequence data, but Hylobates and Symphalangus were sister genera, and the youngest. Analyses of nuclear DNA have, to date, likewise failed to produce a single phylogeny (Wall et al., 2013 ; Carbone et al., 2014 ; Veeramah et al., 2015 ; Shi and Yang, 2018 ). Hybrids Fertile intra-genera gibbon hybrids have been recorded both in captivity and in the wild (Brockelman and Schilling, 1984 ; Brokelman and Gittins, 1984 ; Tenaza, 1985 ; Marshall and Brockelman, 1986 ; Marshall and Sugardjito, 1986 ; Couturier and Lersould, 1991 ; Van Tuinen et al., 1999 ; Matsudaira et al., 2013 ; Nie et al., 2018 ). In addition, viable inter-genera hybrids have been documented in captivity (Myers and Shafer, 1979 ; Hirai et al., 2007 ). Such successful hybridisation events within genera demonstrate a low level of biological reproductive isolation. These observations indicate how closely related the different species are, particularly within the same genus. This level of recent relatedness is likely contributing to the difficulty in resolving the gibbon phylogeny, despite consistent effort. Furthermore, some gibbon species appear phenotypically so similar to one another, that they can be difficult to distinguish, even for specialists (Geissmann, 1995 ; Mootnick, 2006 ; Thinh, Rawson, et al. , 2010; Nie et al., 2018 ). This represents a particular problem for conservation centers like zoos, who may unknowingly pair individuals of different species, creating hybrids, which then continue to breed, as the institutions believe them to be of the same species (Geissmann, 1995 ). Changes in the recognised gibbon taxonomy since the founding of captive populations may exacerbate this issue. Furthermore, as hybrids occur in the wild, it is also possible for zoos to import wild-caught individuals without realising they are hybrids, particularly if the provenance of the individual is unknown (Mootnick, 2006 ). Utility of mtDNA Whilst the evolutionary history and relationships of the gibbon species may not yet have been determined, mtDNA phylogenetic studies do demonstrate that the genera and species frequently group in a monophyletic manner (though precise relationships vary depending on the marker analysed), making such analyses potentially useful for species identification. Mitochondrial DNA is especially suited to such studies because of its high mutation rate relative to nuclear DNA, providing a higher level of phylogenetic resolution at or below the species level (Wilson et al., 1985 ). Other practical advantages of mtDNA are that it can be recovered from degraded genetic samples such as hair, faecal samples or stomach contents, allowing use in field studies and forensics applications (Johnson, 2010 ; Spencer, Schmidt and Hummel, 2010 ). Mitochondrial cox1 gene The mitochondrial cox1 gene has been proposed as a universal genetic marker (‘DNA barcode’) to identify taxonomic groups (Hebert et al., 2003 ; DeSalle, Egan and Siddall, 2005 ), including primates (Lorenz et al., 2005 ; Hajibabaei, Singer and Hickey, 2006 ; Nijman and Aliabadian, 2010 ). However, to date, it has not yet been tested as a DNA barcode to specifically identify gibbon species. Concerns have been raised as to the suitability of cox1 as a truly universal species identifier, as it is an essential gene, imposing limitations upon how many functional variations can exist (DeSalle, Egan and Siddall, 2005 ). The reliability of DNA barcoding, utilising cox1 , for species across the primate order has been assessed by Hajibabaei, Singer and Hickey ( 2006 ). These authors concluded that while cox1 may provide adequate data to assign samples to known species, it was insufficient to assign novel species or to resolve deeper phylogenetic relationships (Hajibabaei, Singer and Hickey, 2006 ). The authors determined that cox1 is most suitable in cases without taxonomic ambiguity but maybe inadequate in cases of historical hybridisation, a point of view shared by Krishnamurthy and Francis ( 2012 ). As noted by Moritz and Cicero ( 2004 ) closely related taxa have not yet been sufficiently sampled to determine whether cox1 can differentiate between species that have recently diverged. These concerns may render cox1 an unreliable marker for gibbon species identification. One of the aims of the research reported here was to address this question. Mitochondrial D-loop The non-coding mitochondrial D-loop may be more suited to species identification for gibbons as it is a more rapidly evolving part of the mitochondrial genome (Garza and Woodruff, 1992 ; Avise, 2000 ; Roos and Geissmann, 2001 ; Whittaker, Morales and Melnick, 2007 ). Importantly, as discussed above, gibbon species are apparently very recently diverged, and therefore may require a more variable locus to provide sufficient phylogenetic resolution for species identification, which the mitochondrial D-loop could offer (Whittaker, Morales and Melnick, 2007 ). The aim of this work was to use the mitochondrial genome as a species identifier by conducting phylogenetic analyses of sequence data. Additionally, we wish to test whether the ‘standard’ barcode, the cox1 gene, provides sufficient resolution for gibbon species identification, or whether the more variable D-loop is required in some cases. Materials and Methods Samples A total of 140 blood, tissue, hair, and FTA blood-spot samples were acquired from 20 institutions within the European Association of Zoos and Aquaria (EAZA) (see Table 1 ). All invasive samples were residual material collected during medical procedures, or from autopsies. 73 samples from cultured gibbon cell lines, held at the Department of Biological Sciences, Graduate School of Science, at the University of Tokyo, were also analysed for the following species: Symphalangus syndactylus (n = 11), Nomascus leucogenys (n = 2), Nomascus siki (n = 1), Hylobates muelleri (n = 3), Hylobates abbotti (n = 2) Hylobates lar (n = 35), Hylobates pileatus (n = 9), Hylobates moloch (n = 1), Hylobates albibarbis (n = 4), Hylobates agilis (n = 5). Table 1 Number of samples for each species by sample type from EAZA institutions Species No. of samples by type Extracted DNA Blood Tissue Hair FTA blood-spots TOTAL Symphalangus syndactylus 1 23 1 5 0 30 Nomascus leucogenys 1 9 0 9 0 19 Nomascus gabriellae 0 5 0 3 0 8 N. leucogenys x N. gabriellae 0 1 1 0 0 2 Nomascus siki 0 0 0 4 0 4 Hylobates agilis 0 8 0 0 0 8 Hylobates lar 0 9 1 3 0 13 Hylobates pileatus 0 11 1 4 0 16 Hylobates moloch 0 0 0 0 40 40 TOTALS 2 66 4 28 40 140 DNA Extraction For blood and tissue samples, DNA was extracted using the Maxwell® 16 Automated Nucleic Acid and Protein Extraction System (Promega, Madison WI, USA), with the Maxwell® 16 Blood DNA Purification Kit, following the manufacturer’s instructions. Samples were loaded into the extraction cartridges in a dedicated pre-PCR Class II laminar flow hood (Walker Safety Cabinets, Glossop, UK). For hair samples DNA extractions were performed in a dedicated pre-PCR clean room, in a laminar flow cabinet (Starlab, Milton Keynes, UK), which was UV irradiated prior to each use. All pipettes and consumables were kept exclusively in the pre-PCR laminar flow cabinets, and so were also regularly UV irradiated. Nitrile gloves were UV irradiated prior to use. DNA was extracted using the QIAamp (Qiagen, Manchester, UK) DNA Investigator Kit, following the manufacturer’s instructions, with the following modifications: lysis at 56 o C took place overnight (approximately 18 hours), and the elution step was performed twice to increase yield. Samples were then concentrated using Amicon Ultra-0.5 mL Centrifugal Filters (Merck Millipore. Watford, UK), washing with 150µl TMT (10mM Tris-HCl, pH8.0). For the FTA blood-spot cards DNA was extracted using the Qiagen DNA Investigator kit, following the manufacturer’s instructions, in a dedicated pre-PCR Class II laminar flow hood. PCR Amplification The mitochondrial D-loop (nucleotides 1,319-1,898 of the NomLeu3 reference, Genbank assembly accession GCA_000146795.3) was amplified using primers GIBDLF3 (5’-CTTCACCCTCAGCACCCAAAGC-3’) & GIBDLR4 (5’-GGGTGATAGGCCTGTGATC-3’) (Andayani et al., 2001 ). The mitochondrial cox1 gene (nucleotides 2,001–2,832 of the NomLeu3 reference) was amplified using primers LLGIBCOX1_F (5’-CTGGTTATTCTCCACAAACC-3’) & LLGIBCOX1_R (5’-GAAGCCAATTGATATTATGGC-3’). DNA sequencing All PCR amplicons were directly sequenced via Sanger sequencing (Source BioScience, Nottingham, UK), using the amplifying primers. Alignment and phylogenetics MAFFT (v.7.490) alignments (Katoh et al., 2002 ; Katoh and Standley, 2013 ) were generated in Geneious Prime (Kearse et al., 2018 ) (algorithm G-INS-I, scoring matrix 200PAM/k = 2, gap open penalty = 2.8, offset value = 0.5) together with reference sequences (Genbank Accession numbers provided in figures; reference sequence for N. annamensis provided by C. Roos at the German Primate Center), and manually refined where appropriate. For D-loop, sequencing amplicons ranged from 190–596bp (mean 567bp). The final alignment length was 605bp. For the cox1 gene sequencing amplicons ranged from 791–832bp, and the final alignment length was 832bp. The alignments were used to generate Maximum-Likelihood phylogenetic trees using PhyML (Guindon, Dufayard and Lefort, 2010 ), in Geneious Prime (Kearse et al., 2018 ) with 100 bootstrap replicates of the data. Results All unique sequences have been submitted to Genbank, accession numbers: PQ186079-186184. D-loop Samples For this locus, 121 samples were sequenced from the EAZA collection, and 73 from the Department of Biological Sciences, Graduate School of Science, University of Tokyo. Matrilines All but one of the 48 documented matrilines (as recorded in the EAZA studbooks) had a unique D-loop sequence. The SIA18 and SIA23 matrilines had identical sequences. These matrilines are not recorded as being related. However, in both cases the founder parentage is unknown, so it cannot be determined whether these individuals have identical amplified D-loop haplotypes by chance or are identical by descent (IBD). Two samples from the Tokyo collection also had identical sequences: LAR_G712 and LAR_DZ10. Additionally, two samples from the EAZA collection were found to have identical D-loop sequences as two samples from the Tokyo collection: LAR05 (EAZA) and LAR_G110 (Tokyo), and PIL12 (EAZA) and PIL_KZ15 (Tokyo). As no pedigree information is available for the Tokyo samples, again, it cannot be determined whether they are IBD. One of each unique sequence was included in the alignment for the phylogenetic tree. Phylogenetic analysis As shown in Fig. 1 . Hylobates form one clade, with Symphalangus , Hoolock , and Nomascus in the other, with Symphalangus and Nomascus clustering as sister taxa. Within the Hylobates clade H. agilis and H. albibarbis are sister to all other species. In the second major clade H. klossii is separate, H. pileatus and H. lar are sister taxa, and H. moloch forms a sub-clade with sister taxa H. muelleri and H. abbotti . Within the Nomascus clade N. concolor separates first, with N. siki , N. leucogenys , N. annamensis , N. gabriellae , and N. hainanus forming another clade. Within this clade. N. leucogenys and N. siki are sister taxa, as are N. annamensis and N. gabriellae , which form a sub-clade with H. hainanus . Species identification The average bootstrap support at the species nodes is 86.3%, ranging from 61% − 100% (N.B. Symphalangus syndactylus is not included in this calculation as it is the only species in the genus, nor are Hoolock spp . as the tree contains only six reference sequences from Genbank for the entire genus). For the D-loop, 111/116 sequences grouped with their documented species. However, three purported H. agilis sequences grouped as H. lar (see Fig. 2 ). Bootstrap support for the H. lar clade is 89%. Additionally, two documented N. gabriellae sequences did not group as expected. The GAB02 sequence grouped as N. siki rather than N. gabriellae (see Fig. 3 ). Bootstrap support for this clade is 79%. The GAB09 sequence grouped with N. annamensis , with 87% support (see Fig. 3 ). cox1 Samples For this locus, 83 samples were successfully amplified sequenced from the EAZA collection. Matrilines Compared to the D-loop, far fewer cox1 matrilines had unique sequences. Of the 41 documented matrilines tested, only 26 had unique sequences. One of each unique sequence was included in the phylogenetic tree. Phylogenetic analysis As shown in Fig. 4 , the branching order for cox1 was different to that obtained with the D-loop: Nomascus splits in to one clade, with the second clade separating Hoolock , and Symphalangus and Hylobates being sister taxa. Species identification Within Hylobates bootstrap support values are not available for H. klossii , H. abbotti , or H. albibarbis as only a single reference sequence is available for each (N.B. see Discussion below regarding possible misidentification of H. abbotti / H. muelleri sequences). For the remaining species, the average support at the species nodes is 97.6% (range 91%-100%). Within Nomascus , only a single sequence is available for N. concolor , no sequences are available for N. hainanus , and N. leucogenys and N. siki do not group monophyletically (see Fig. 4 ). Support for N. gabriellae is 52%, and 71% for N. annamensis . All cox1 sequences grouped as expected, with only three exceptions: consistent with the D-loop data, AG02 grouped as H. lar (see Fig. 5 ), GAB02 grouped within the clade containing N. siki , and GAB09 grouped with the N. annamensis reference sequence (see Fig. 6 ). Discussion Identification of species and hybrids Overall, 96% (111/116) of individuals were unambiguously assigned to their provenance asserted species, via their mitochondrial D-loop sequences. This demonstrates that the D-loop performs well as a maternal species identifier for gibbons, in the tested species and samples. Specific exceptions to species identification were limited to two species. Three H. agilis individuals clustered with H. lar ; one N. gabriellae sequence clustered with N. siki , and a second N. gabriellae matriline grouped with N. annamensis . As the remaining 96% of sequences grouped as expected we are confident that these results indicate genuine mixed-species ancestry in these matrilines. Furthermore, bootstrap support at the species nodes is generally high (mean 86.3%, median 88%). The main exception to this robust species discrimination is the clade containing H. agilis and H. albibarbis . Historically H. albibarbis was considered a sub-species of H. agilis (Lyon, 1911 ), but was raised to full species level by Groves ( 2001 ), and this was supported by cytogenetic and molecular genetic analyses (Hirai et al., 2005 , 2009 ). H. albibarbis and H. agilis are currently recognised as distinct species by the IUCN (IUCN, 2020 ). However, the sequences reported here do not group as monophyletic taxa. The sequences are grouped together in a single clade, with 74% bootstrap support, which suggests they may be the same species, as previously recognised. The purported H. albibarbis sequences cluster together in a group within this clade, with moderate bootstrap support (61%), suggesting the existence of a sub-species. However, the chromosomal translocation between the two taxa (Hirai et al., 2005 ), and their geographic isolation ( H. agilis is found on the Malaysian peninsular and the island of Sumatra, while H. albibarbis is found only on the island of Borneo), support separate species status. Furthermore, recent work by Matsudaira and Ishida ( 2021 ) found nuclear data also support distinction of H. agilis and H. albibarbis . It is possible that the two species have diverged so recently as to have accumulated limited phylogenetic signal. Excluding these two taxa, species-level bootstrap support across Hylobates and Nomascus is strong (mean 89.4%, median 90%). H. agilis and H. lar have been documented to produce fertile hybrid offspring, both in the wild and in captivity (Brokelman and Gittins, 1984 ; Van Tuinen et al., 1999 ). Mixed ancestry is therefore a plausible explanation for the anomalous H. agilis results. No pedigree information is available for the two cell line samples from Tokyo (AG_G80 and AG_JMC1). However, nuclear data confirm hybrid ancestry in the case of AG_G80 (Matsudaira and Ishida, 2021 ). These data showed the majority of the nuclear SNVs were of H. agilis (the documented species), with some H. lar . The levels of H. lar introgression were low, suggesting the hybridisation event was not recent (i.e. in the wild, rather than recently in captivity). The data presented here confirm the H. lar introgression in this individual, and demonstrate that the introgression comes from the maternal lineage. In the case of the EAZA individual AG02, the identity of the dam is unknown. It is possible a hybrid was accidentally created in captivity, if the dam was misidentified, although as these two species are readily distinguishable phenotypically, we consider this to be unlikely. Alternatively, the founding female taken from the wild could have been an H. lar x H. agilis hybrid, with the physical appearance of H. agilis , similar to the case of AG_G80, leading to misidentification in the studbook. Similarly, N. siki x N. gabriellae hybrids have been documented in captivity (Nie et al., 2018 ), and suspected in the wild (Geissmann et al., 2000 ; Mootnick, 2006 ). Male N. siki and N. gabriellae individuals may be readily distinguished by their white and buff cheek colouration respectively. Females of these species however are very similar in appearance and can be difficult to distinguish reliably (Thinh, Rawson, et al. , 2010; Harding, 2012 ). If we assume our sequencing data to be reliable, the anomalous grouping of GAB02 with N. siki may therefore be either the result of a hybrid individual being taken from the wild, or the founding female (the dam of GAB02) being a misidentified N. siki , particularly if their geographic origin was unknown, or incorrectly documented. Historically N. annamensis was considered a sub-species of N. gabriellae , but was raised to full species status in 2010 (Thinh, Mootnick, Thanh, et al. , 2010), 13 years after the founding female of the GAB09 matriline was taken into captivity. With only N. gabriellae being recognised at that time, this female could only have been identified as such. Furthermore, females of these species are indistinguishable phenotypically (Thinh, Mootnick, Thanh, et al. , 2010), so it is unlikely the misidentification would have been diagnosed later, leading to unavoidable, if accidental, hybridization. Implications for breeding programmes As H. agilis are no longer bred for conservation by EAZA, these results do not have significant consequences for the remaining H. agilis population in the European collection (12 individuals remaining as of September 2023). However, the data reported here show H. lar introgression in three different lineages of H. agilis (one in EAZA, and two of the samples from the University of Tokyo), indicating the EAZA case is not isolated. It would therefore be prudent for other global breeding programmes to investigate their H. agilis groups, as well as H. lar and H. pileatus , as these species are also known to hybridize in the wild (discussed above). However, the captive N. gabriellae population are part of active conservation breeding programmes, and so these results could have implications for these groups. The data reported here indicate N. siki and N. annamensis ancestry in two N. gabriellae lineages, and it is the stated policy of EAZA to avoid the breeding of hybrids (EAZA, 2013 ). It is important to note that these data are not conclusive evidence of introgression, for several reasons. Firstly, the resolution of N. leucogenys , N. siki , N. gabriellae , and N. annamensis remains uncertain. While these taxa are recognised as four distinct species by the IUCN (IUCN, 2020 ), it is noted that N. annamensis has been considered a sub-species of N. gabriellae , and that N. gabriellae and N. siki have historically been considered sub-species of N. leucogenys . The IUCN further notes that N. siki may not be a true species at all, but rather may be a naturally occurring hybrid of N. leucogenys and N. gabriellae . Significant overlap between these purported species was found in phylogenetic analysis of the mitochondrial Cytochrome b gene by Thinh, Rawson, et al. , (2010). Our analysis of the D-loop, however, demonstrates clear distinctions between these four taxa (Fig. 3 ). This finding suggests that the individuals of the GAB02 and GAB09 lineages are genuinely of mixed-species ancestry. When considering possible implications for the N. gabriellae breeding programme, it should also be noted that these data do not reveal the proportion of the current generation’s genome which might be affected. The youngest generation is three generations removed from the founding females. Theoretically, if all other breeding has been with true N. gabriellae individuals, then approximately 12.5% of the current genome would be expected to be N. siki or N. annamensis depending on the inheritance of alleles after random segregation at meiosis. Furthermore, if the founding female was a hybrid this proportion would be lower still. In the absence of sequence data from variable nuclear loci, this proportion cannot be estimated. Cryptic relatedness 97% of documented matrilines for all species were found to have a unique sequence for the D-loop amplicon analysed here. Within the EAZA collection tested, unexpected identical sequences were limited to three cases, one for S. syndactylus , one for H. lar , and one for H. pileatus . Incomplete pedigree information prevents a definitive conclusion as to whether these matrilines are related. However, given the high number of unique haplotypes identified in this study (n = 113), we consider this possibility most likely. D-loop vs cox1 Within Hylobates , where multiple cox1 sequences are available, bootstrap support for species clusters is high (91%-100%). However, H. abbotti does not group separately from H. muelleri , unlike in the D-loop phylogenetic tree. This may an artefact of only having a single sequence available for H. abbotti , which is therefore grouped with H. muelleri as the most closely related sequences. Alternatively, the reference sequences used may be erroneously labelled. In Chan et al. ( 2010 ), where the sequences were published, they were labelled as H. muelleri , however, the study does not document H. funereus or H. abbotti samples. It is possible therefore that some or all of the reference sequences are in fact H. abbotti . As with the D-loop phylogenetic tree, the single H. albibarbis sequence groups within H. agilis , although it does have a longer branch length (Fig. 5 ) suggesting greater divergence. The Nomascus clade is not clearly delineated by cox1 compared to the D-loop, in particular N. leucogenys , and N. siki , which do not group monophyletically. This makes cox1 unsuitable for species identification for these species. The results for AG02 and GAB09 are concordant with the results for the D-loop, being identified as H. lar and N. annamensis respectively. The sequence for GAB02 also groups within the clade containing N. siki . In contrast to the D-loop, matrilines are not readily distinguished using cox1 sequences. It is therefore not recommended for studies in which ascertaining relatedness is important, such as in captive populations, or studying female dispersal in the wild. Instead, the D-loop should be used in preference. Additionally, the D-loop had a higher successful amplification rate for the lower-quality hair samples compared to cox1 (68% and 59% respectively). This may be due to the smaller amplicon size of the D-loop compared to cox1 (~ 560bp and ~ 790bp respectively). This makes the D-loop a more attractive target locus for situations where only lower quality samples such as hair are available, as is often the case in wildlife rescue centers. Conclusions and future work Based on the data reported here we recommend the D-loop should be used for gibbon maternal species identification, due to its higher resolution compared to cox1 . In particular, we do not recommend the use of cox1 for species identification for Nomascus spp . as N. leucogenys and N. siki are not monophyletic when using this locus. By contrast, the D-loop sequences cluster clearly along recognised species boundaries, with strong support. The possible exception to this recommendation, is in the case of H. albibarbis , as discussed above. Two recognised species are currently absent from this work: Hylobates funereus , and Nomascus nasutus. (N.B. at the time of writing, two sequences for N. nasutus were published, but were significantly shorter than the other sequences used in these analyses, and so were excluded). Sequencing of individuals of known provenance for these species would provide the opportunity to construct a complete phylogenetic tree and most likely enable reliable maternal species identification for any individual. A significant limitation of mitochondrial sequences is that they are maternally inherited. As a result, any introgression which has occurred in the paternal line will remain undetected. Therefore, markers from the nuclear genome which can identify species and possible hybrids from both the maternal and paternal lineages are highly desirable. Whole genome sequencing would be ideal as not only could hybrids be detected, but also the proportion of introgression quantified. However, such methods are currently impractical in terms of cost, time, data analysis, and sample requirements. One approach to this problem are nuclear markers, utilising species-specific retrotransposon insertions, which can be detected with simple PCR and gel electrophoresis technology (Lansdowne, 2022 ). While phylogenetics using the mitochondrial D-loop is limited to maternal lineages, it is nevertheless a robust method, which can assign the maternal species origin of an individual with a high degree of confidence. Where introgression has occurred on the maternal side, it can also highlight cases of cryptic hybrids, as demonstrated here. We recommend use of the D-loop for gibbon species identification, rather than cox1 , due to the greater resolution provided, the higher amplification rates for low quality samples, and the higher number of observed haplotypes which enable lineage determination. Declarations Funding Funding was provided by the BBSRC, the University of Leicester, and Twycross Zoo. Competing Interests The authors have no relevant financial or non-financial interests to disclose. Compliance with Ethical Standards All invasive samples were residual material collected either during medical procedures or autopsies, in compliance with each institutions’ standards, and with support from the EAZA Gibbon Taxon Advisory Group. Author Contribution The study was designed by L.L. and R.B. Data collection was performed by L.L., K.M., and S.M. Data analysis was performed by L.L. Samples at the University of Tokyo were provided by T.I. The first draft of the manuscript was written by L.L. and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Acknowledgement We would like to thank Twycross Zoo (UK) for their support and collaboration, and also the other institutions who generously provided samples: Apenheul Primate Park (Netherlands), Howletts and Port Lympne (UK), Borås Zoo (Sweden), Bristol Zoo (UK), Cotswold Wildlife Park (UK), German Primate Center (Germany), Marwell Zoo (UK), Mulhouse Zoological and Botanical Park (France), Paignton Zoo (UK), Parc Animalier d'Auvergne (France), Reserve Africaine de Sigean (France), Selwo Aventura (Spain), Serengeti Park (Germany), Zoo Boissiere (France), Zoo de Cerza (France), Zoo de la Besancon Citadelle (France), Zoopark Erfurt (Germany), ZSL (UK), Zurich Zoo (Switzerland). Funding was provided by the BBSRC, the University of Leicester, and Twycross Zoo. 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Vietnamese J Primatol 1(4):1–12 Thinh VN, Mootnick AR, Geissmann T et al (2010) ‘Mitochondrial evidence for multiple radiations in the evolutionary history of small apes’, BMC Evolutionary Biology , 10(1). https://doi.org/10.1186/1471-2148-10-74 Thinh VN, Rawson B et al (2010) ‘Phylogeny and distribution of crested gibbons (genus Nomascus) based on mitochondrial cytochrome b gene sequence data’, American Journal of Primatology , 72(12), pp. 1047–1054. https://doi.org/10.1002/ajp.20861 Van Tuinen P et al (1999) ‘Complex, compound inversion/translocation polymorphism in an ape: Presumptive intermediate stage in the karyotypic evolution of the agile gibbon Hylobates agilis’, American Journal of Physical Anthropology , 110(2), pp. 129–142. https://doi.org/10.1002/(SICI)1096-8644(199910)110:23.0.CO;2-M Veeramah KR et al (2015) ‘Examining phylogenetic relationships among gibbon genera using whole genome sequence data using an approximate bayesian computation approach’, Genetics , 200(1), pp. 295–308. https://doi.org/10.1534/genetics.115.174425 Wall JD et al (2013) ‘Incomplete Lineage Sorting Is Common in Extant Gibbon Genera’, PLoS ONE , 8(1), pp. 1–5. https://doi.org/10.1371/journal.pone.0053682 Whittaker DJ, Morales JC, Melnick DJ (2007) ‘Resolution of the Hylobates phylogeny: Congruence of mitochondrial D-loop sequences with molecular, behavioral, and morphological data sets’, Molecular Phylogenetics and Evolution , 45(2), pp. 620–628. https://doi.org/10.1016/j.ympev.2007.08.009 Wilson AC et al (1985) Mitochondrial DNA and two perspectives on evolutionary genetics. 26:375–400 Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5039233","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":353962138,"identity":"dbe9484d-2237-469b-a117-a93e7167bf5d","order_by":0,"name":"Lauren Lansdowne","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABA0lEQVRIiWNgGAWjYBACPigtAyIkQAQ/AwMbXi0wWR64FskGkrUYHCCkhf2M2YePOxh4+Ge3P7zxse2OnPGN5GcPPlQwyPOLHcCuhSfHeObMMww8EnfOGFvObHtmbHYjzdxwxhkGw5mzE3A4LMeYmbcN6LAbOWzSvNsOJ267kWAmDRRJMLiNQwv/G2Pmv0At8jfSn4G01G+ekf4NvxYJoC2MQC0GYMO3HU4wkMghYIvEs2LG3jYJHsMbOUC//DsM9MabMskZZyRw+oWfP3kzw882Gzm5G+kPb3w4c1ievz19m8SHCht5fmnsWqBAAoktkIAuQhDwHyBF9SgYBaNgFIwAAAB1BFZnF2n24AAAAABJRU5ErkJggg==","orcid":"","institution":"University of Leicester","correspondingAuthor":true,"prefix":"","firstName":"Lauren","middleName":"","lastName":"Lansdowne","suffix":""},{"id":353962140,"identity":"0b274698-e47e-4d56-9be6-5db4307aa74f","order_by":1,"name":"Kazunari Matsudaira","email":"","orcid":"","institution":"University of Tokyo","correspondingAuthor":false,"prefix":"","firstName":"Kazunari","middleName":"","lastName":"Matsudaira","suffix":""},{"id":353962142,"identity":"20825ef8-214f-4953-a4b8-bd2f9fdada2e","order_by":2,"name":"Takafumi Ishida","email":"","orcid":"","institution":"University of Tokyo","correspondingAuthor":false,"prefix":"","firstName":"Takafumi","middleName":"","lastName":"Ishida","suffix":""},{"id":353962145,"identity":"f55a9c8f-85db-47e8-9600-93316f64882d","order_by":3,"name":"Sîan McHugh","email":"","orcid":"","institution":"University of Leicester","correspondingAuthor":false,"prefix":"","firstName":"Sîan","middleName":"","lastName":"McHugh","suffix":""},{"id":353962146,"identity":"e1873dfe-9f49-4d3a-a2f5-623edeaf4156","order_by":4,"name":"Richard M. Badge","email":"","orcid":"","institution":"University of Leicester","correspondingAuthor":false,"prefix":"","firstName":"Richard","middleName":"M.","lastName":"Badge","suffix":""}],"badges":[],"createdAt":"2024-09-05 15:16:36","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5039233/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5039233/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s12686-025-01392-8","type":"published","date":"2025-07-04T15:57:58+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":66386251,"identity":"2370bf0c-9739-4127-af36-8c478322f1ad","added_by":"auto","created_at":"2024-10-11 08:10:49","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":68041,"visible":true,"origin":"","legend":"\u003cp\u003eMaximum Likelihood Phylogenetic tree of gibbons, based on mitochondrial D-loop amplicon sequence data (n=135). Branch labels indicate bootstrap support values (100 replicates) for the genus and species clades. Scale bar is 0.1 nucleotide substitutions per site. Terminal branches with three or more sequences have been collapsed for clarity\u003c/p\u003e","description":"","filename":"Fig1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5039233/v1/42dbc7196e62df87948b8633.jpeg"},{"id":66384881,"identity":"ac455d22-1c2a-41a6-a1ba-282c1076f562","added_by":"auto","created_at":"2024-10-11 07:54:49","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":51404,"visible":true,"origin":"","legend":"\u003cp\u003eSection of the ML Phylogenetic tree showing the Hylobates clade for mitochondrial D-loop sequence data (n=79). Branch labels indicate bootstrap support values (100 replicates) for species clades. Stars indicate anomalous results for documented H. agilis samples, which cluster with H. lar. Scale bar is 0.04 nucleotide substitutions per site\u003c/p\u003e","description":"","filename":"Fig2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5039233/v1/e813e2ec3cb3fe065c0e8a2c.jpeg"},{"id":66385271,"identity":"018d4e71-d6a3-4957-b69a-f691eaac416a","added_by":"auto","created_at":"2024-10-11 08:02:49","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":47838,"visible":true,"origin":"","legend":"\u003cp\u003eSection of the ML Phylogenetic tree showing the Nomascus clade for mitochondrial D-loop sequence data (n=26). Branch labels indicate bootstrap support values (100 replicates) for species clades. Stars indicate anomalous results for documented N. gabriellae samples, described in text. Scale bar is 0.05 nucleotide substitutions per site\u003c/p\u003e","description":"","filename":"Fig3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5039233/v1/240580874acaecc0a624a83b.jpeg"},{"id":66385269,"identity":"0d4a5d63-9dc8-435d-9458-4aebbbd5d8bc","added_by":"auto","created_at":"2024-10-11 08:02:49","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":69548,"visible":true,"origin":"","legend":"\u003cp\u003eMaximum Likelihood Phylogenetic tree of gibbons, based on mitochondrial cox1 gene sequence data (n=56). Branch labels indicate bootstrap support values (100 replicates) for the genus and species clades. Scale bar is 0.03 nucleotide substitutions per site. Terminal branches with three or more sequences have been collapsed for clarity\u003c/p\u003e","description":"","filename":"Fig4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5039233/v1/8b5a486161c91803aec264d7.jpeg"},{"id":66384884,"identity":"8a677f10-4843-482e-8207-f275d37f9790","added_by":"auto","created_at":"2024-10-11 07:54:49","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":45286,"visible":true,"origin":"","legend":"\u003cp\u003eSection of the ML Phylogenetic tree showing the Hylobates clade for mitochondrial cox1 sequence data (n=29). Branch labels indicate bootstrap support values (100 replicates) for species clades. Star indicates anomalous result for documented H. agilis sample, which clusters with H. lar. Scale bar is 0.02 nucleotide substitutions per site\u003c/p\u003e","description":"","filename":"Fig5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5039233/v1/d514425dd93a39525f951113.jpeg"},{"id":66384886,"identity":"4fc6b948-5d35-44cc-ae66-2a4f43af2c49","added_by":"auto","created_at":"2024-10-11 07:54:49","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":36374,"visible":true,"origin":"","legend":"\u003cp\u003eSection of the ML Phylogenetic tree showing the Nomascus clade for mitochondrial cox1 sequence data (n=15). Branch labels indicate bootstrap support values (100 replicates) for species clades. Stars indicate anomalous results for documented N. gabriellae samples, described in text. Scale bar is 0.008 nucleotide substitutions per site\u003c/p\u003e","description":"","filename":"Fig6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5039233/v1/a7ca49483a20f80a7b112780.jpeg"},{"id":86179102,"identity":"d6c99cb0-0885-4177-9fbb-828812b229ef","added_by":"auto","created_at":"2025-07-07 16:15:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1200592,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5039233/v1/236edc73-c0d9-47f5-9447-e4dc6e65197a.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The mitochondrial D-loop is a robust maternal-species identifier in gibbons (Hylobatidae)","fulltext":[{"header":"Introduction","content":"\u003cdiv id=\"Sec2\" class=\"Section2\"\u003e \u003ch2\u003eTaxonomy and Evolution\u003c/h2\u003e \u003cp\u003eGibbons (Hylobatidae) are a family in the order Primates, known as the small or lesser apes, that with the great apes (Hominidae) form the superfamily of apes (Hominoidea) (Groves, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Roos, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). It has been estimated that the gibbons last shared a common ancestor with the great apes 16\u0026ndash;20\u0026nbsp;Million Years Ago (MYA) (Roos, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The gibbon family is divided into four genera (\u003cem\u003eHoolock\u003c/em\u003e, \u003cem\u003eHylobates\u003c/em\u003e, \u003cem\u003eNomascus\u003c/em\u003e, \u003cem\u003eSymphalangus\u003c/em\u003e), on the basis of chromosome complement and karyotype (ranging from 2n\u0026thinsp;=\u0026thinsp;38 to 2n\u0026thinsp;=\u0026thinsp;52) (Roos, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).Within these genera 20 species are currently recognised (Mittermeier, Rylands and Wilson, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; IUCN, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). It has been estimated that the current species diversity within the gibbons arose during the last\u0026thinsp;~\u0026thinsp;15MYA (Matsudaira and Ishida, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Thinh, Mootnick, Geissmann, \u003cem\u003eet al.\u003c/em\u003e, 2010). In addition, there is genetic evidence that some radiations occurred as recently as 2MYA (Thinh, Mootnick, Geissmann, \u003cem\u003eet al.\u003c/em\u003e, 2010; Carbone et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). All gibbon species are currently listed as Vulnerable, Endangered, or Critically Endangered by the International Union for Conservation of Nature (IUCN) (IUCN, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eResolving the evolutionary history of these taxa has proven complex and controversial. Numerous phylogenetic studies of the Hylobatidae have been conducted using mitochondrial DNA (mtDNA) genes and regions, such as \u003cem\u003eCytochrome b\u003c/em\u003e (Chatterjee, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Thinh, Mootnick, Geissmann, \u003cem\u003eet al.\u003c/em\u003e, 2010; Thinh, Rawson, \u003cem\u003eet al.\u003c/em\u003e, 2010), ND3-ND4 (Takacs et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), the D-loop/Control Region (Roos and Geissmann, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Monda et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Whittaker, Morales and Melnick, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), and the whole mitochondrial genome (Chan et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Matsudaira and Ishida, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2010\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Fan et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). However, these studies have failed to resolve the gibbon phylogeny, with different studies producing conflicting results. Whilst the four genera are consistently monophyletic, their branching order varies, and relationships between species remain unclear. For example, using mitochondrial D-loop sequence data, Whittaker, Morales and Melnick (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) found \u003cem\u003eHoolock\u003c/em\u003e to be basal, followed by \u003cem\u003eHylobates\u003c/em\u003e, with \u003cem\u003eSymphalangus\u003c/em\u003e and \u003cem\u003eNomascus\u003c/em\u003e being the youngest sister genera. However, Roos and Geissmann (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2001\u003c/span\u003e) found \u003cem\u003eNomascus\u003c/em\u003e to be basal, with \u003cem\u003eHoolock\u003c/em\u003e and \u003cem\u003eHylobates\u003c/em\u003e being the youngest and sister genera. Takacs et al. (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), also found \u003cem\u003eHoolock\u003c/em\u003e to be basal, based on mitochondrial ND3-ND4 sequence data, but \u003cem\u003eHylobates\u003c/em\u003e and \u003cem\u003eSymphalangus\u003c/em\u003e were sister genera, and the youngest. Analyses of nuclear DNA have, to date, likewise failed to produce a single phylogeny (Wall et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Carbone et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Veeramah et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Shi and Yang, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eHybrids\u003c/h2\u003e \u003cp\u003eFertile intra-genera gibbon hybrids have been recorded both in captivity and in the wild (Brockelman and Schilling, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1984\u003c/span\u003e; Brokelman and Gittins, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1984\u003c/span\u003e; Tenaza, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e1985\u003c/span\u003e; Marshall and Brockelman, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1986\u003c/span\u003e; Marshall and Sugardjito, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1986\u003c/span\u003e; Couturier and Lersould, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1991\u003c/span\u003e; Van Tuinen et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Matsudaira et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Nie et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In addition, viable inter-genera hybrids have been documented in captivity (Myers and Shafer, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1979\u003c/span\u003e; Hirai et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Such successful hybridisation events within genera demonstrate a low level of biological reproductive isolation. These observations indicate how closely related the different species are, particularly within the same genus. This level of recent relatedness is likely contributing to the difficulty in resolving the gibbon phylogeny, despite consistent effort.\u003c/p\u003e \u003cp\u003eFurthermore, some gibbon species appear phenotypically so similar to one another, that they can be difficult to distinguish, even for specialists (Geissmann, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Mootnick, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Thinh, Rawson, \u003cem\u003eet al.\u003c/em\u003e, 2010; Nie et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). This represents a particular problem for conservation centers like zoos, who may unknowingly pair individuals of different species, creating hybrids, which then continue to breed, as the institutions believe them to be of the same species (Geissmann, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). Changes in the recognised gibbon taxonomy since the founding of captive populations may exacerbate this issue. Furthermore, as hybrids occur in the wild, it is also possible for zoos to import wild-caught individuals without realising they are hybrids, particularly if the provenance of the individual is unknown (Mootnick, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eUtility of mtDNA\u003c/h2\u003e \u003cp\u003eWhilst the evolutionary history and relationships of the gibbon species may not yet have been determined, mtDNA phylogenetic studies do demonstrate that the genera and species frequently group in a monophyletic manner (though precise relationships vary depending on the marker analysed), making such analyses potentially useful for species identification. Mitochondrial DNA is especially suited to such studies because of its high mutation rate relative to nuclear DNA, providing a higher level of phylogenetic resolution at or below the species level (Wilson et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e1985\u003c/span\u003e). Other practical advantages of mtDNA are that it can be recovered from degraded genetic samples such as hair, faecal samples or stomach contents, allowing use in field studies and forensics applications (Johnson, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Spencer, Schmidt and Hummel, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eMitochondrial\u003c/em\u003e cox1 \u003cem\u003egene\u003c/em\u003e\u003c/p\u003e \u003cp\u003eThe mitochondrial \u003cem\u003ecox1\u003c/em\u003e gene has been proposed as a universal genetic marker (\u0026lsquo;DNA barcode\u0026rsquo;) to identify taxonomic groups (Hebert et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; DeSalle, Egan and Siddall, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), including primates (Lorenz et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Hajibabaei, Singer and Hickey, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Nijman and Aliabadian, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). However, to date, it has not yet been tested as a DNA barcode to specifically identify gibbon species.\u003c/p\u003e \u003cp\u003eConcerns have been raised as to the suitability of \u003cem\u003ecox1\u003c/em\u003e as a truly universal species identifier, as it is an essential gene, imposing limitations upon how many functional variations can exist (DeSalle, Egan and Siddall, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). The reliability of DNA barcoding, utilising \u003cem\u003ecox1\u003c/em\u003e, for species across the primate order has been assessed by Hajibabaei, Singer and Hickey (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). These authors concluded that while \u003cem\u003ecox1\u003c/em\u003e may provide adequate data to assign samples to known species, it was insufficient to assign novel species or to resolve deeper phylogenetic relationships (Hajibabaei, Singer and Hickey, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). The authors determined that \u003cem\u003ecox1\u003c/em\u003e is most suitable in cases without taxonomic ambiguity but maybe inadequate in cases of historical hybridisation, a point of view shared by Krishnamurthy and Francis (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). As noted by Moritz and Cicero (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2004\u003c/span\u003e) closely related taxa have not yet been sufficiently sampled to determine whether \u003cem\u003ecox1\u003c/em\u003e can differentiate between species that have recently diverged. These concerns may render \u003cem\u003ecox1\u003c/em\u003e an unreliable marker for gibbon species identification. One of the aims of the research reported here was to address this question.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eMitochondrial D-loop\u003c/h2\u003e \u003cp\u003eThe non-coding mitochondrial D-loop may be more suited to species identification for gibbons as it is a more rapidly evolving part of the mitochondrial genome (Garza and Woodruff, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1992\u003c/span\u003e; Avise, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Roos and Geissmann, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Whittaker, Morales and Melnick, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Importantly, as discussed above, gibbon species are apparently very recently diverged, and therefore may require a more variable locus to provide sufficient phylogenetic resolution for species identification, which the mitochondrial D-loop could offer (Whittaker, Morales and Melnick, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe aim of this work was to use the mitochondrial genome as a species identifier by conducting phylogenetic analyses of sequence data. Additionally, we wish to test whether the \u0026lsquo;standard\u0026rsquo; barcode, the \u003cem\u003ecox1\u003c/em\u003e gene, provides sufficient resolution for gibbon species identification, or whether the more variable D-loop is required in some cases.\u003c/p\u003e \u003c/div\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eSamples\u003c/h2\u003e \u003cp\u003eA total of 140 blood, tissue, hair, and FTA blood-spot samples were acquired from 20 institutions within the European Association of Zoos and Aquaria (EAZA) (see Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). All invasive samples were residual material collected during medical procedures, or from autopsies. 73 samples from cultured gibbon cell lines, held at the Department of Biological Sciences, Graduate School of Science, at the University of Tokyo, were also analysed for the following species: \u003cem\u003eSymphalangus syndactylus\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;11), \u003cem\u003eNomascus leucogenys\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;2), \u003cem\u003eNomascus siki\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;1), \u003cem\u003eHylobates muelleri\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;3), \u003cem\u003eHylobates abbotti\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;2) \u003cem\u003eHylobates lar\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;35), \u003cem\u003eHylobates pileatus\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;9), \u003cem\u003eHylobates moloch\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;1), \u003cem\u003eHylobates albibarbis\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;4), \u003cem\u003eHylobates agilis\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;5).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eNumber of samples for each species by sample type from EAZA institutions\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSpecies\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"6\" nameend=\"c7\" namest=\"c2\"\u003e \u003cp\u003eNo. of samples by type\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eExtracted DNA\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBlood\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTissue\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHair\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eFTA blood-spots\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eTOTAL\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSymphalangus syndactylus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e30\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eNomascus leucogenys\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e19\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eNomascus gabriellae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e8\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eN. leucogenys x N. gabriellae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eNomascus siki\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eHylobates agilis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e8\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eHylobates lar\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e13\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eHylobates pileatus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e16\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eHylobates moloch\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e40\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTOTALS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e66\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e28\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e40\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e140\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eDNA Extraction\u003c/h2\u003e \u003cp\u003eFor blood and tissue samples, DNA was extracted using the Maxwell\u0026reg; 16 Automated Nucleic Acid and Protein Extraction System (Promega, Madison WI, USA), with the Maxwell\u0026reg; 16 Blood DNA Purification Kit, following the manufacturer\u0026rsquo;s instructions. Samples were loaded into the extraction cartridges in a dedicated pre-PCR Class II laminar flow hood (Walker Safety Cabinets, Glossop, UK). For hair samples DNA extractions were performed in a dedicated pre-PCR clean room, in a laminar flow cabinet (Starlab, Milton Keynes, UK), which was UV irradiated prior to each use. All pipettes and consumables were kept exclusively in the pre-PCR laminar flow cabinets, and so were also regularly UV irradiated. Nitrile gloves were UV irradiated prior to use. DNA was extracted using the QIAamp (Qiagen, Manchester, UK) DNA Investigator Kit, following the manufacturer\u0026rsquo;s instructions, with the following modifications: lysis at 56\u003csup\u003eo\u003c/sup\u003eC took place overnight (approximately 18 hours), and the elution step was performed twice to increase yield. Samples were then concentrated using Amicon Ultra-0.5 mL Centrifugal Filters (Merck Millipore. Watford, UK), washing with 150\u0026micro;l TMT (10mM Tris-HCl, pH8.0). For the FTA blood-spot cards DNA was extracted using the Qiagen DNA Investigator kit, following the manufacturer\u0026rsquo;s instructions, in a dedicated pre-PCR Class II laminar flow hood.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003ePCR Amplification\u003c/h2\u003e \u003cp\u003eThe mitochondrial D-loop (nucleotides 1,319-1,898 of the NomLeu3 reference, Genbank assembly accession GCA_000146795.3) was amplified using primers GIBDLF3 (5\u0026rsquo;-CTTCACCCTCAGCACCCAAAGC-3\u0026rsquo;) \u0026amp; GIBDLR4 (5\u0026rsquo;-GGGTGATAGGCCTGTGATC-3\u0026rsquo;) (Andayani et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). The mitochondrial \u003cem\u003ecox1\u003c/em\u003e gene (nucleotides 2,001\u0026ndash;2,832 of the NomLeu3 reference) was amplified using primers LLGIBCOX1_F (5\u0026rsquo;-CTGGTTATTCTCCACAAACC-3\u0026rsquo;) \u0026amp; LLGIBCOX1_R (5\u0026rsquo;-GAAGCCAATTGATATTATGGC-3\u0026rsquo;).\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003eDNA sequencing\u003c/h2\u003e \u003cp\u003eAll PCR amplicons were directly sequenced via Sanger sequencing (Source BioScience, Nottingham, UK), using the amplifying primers.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eAlignment and phylogenetics\u003c/h2\u003e \u003cp\u003eMAFFT (v.7.490) alignments (Katoh et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Katoh and Standley, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) were generated in Geneious Prime (Kearse et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) (algorithm G-INS-I, scoring matrix 200PAM/k\u0026thinsp;=\u0026thinsp;2, gap open penalty\u0026thinsp;=\u0026thinsp;2.8, offset value\u0026thinsp;=\u0026thinsp;0.5) together with reference sequences (Genbank Accession numbers provided in figures; reference sequence for \u003cem\u003eN. annamensis\u003c/em\u003e provided by C. Roos at the German Primate Center), and manually refined where appropriate. For D-loop, sequencing amplicons ranged from 190\u0026ndash;596bp (mean 567bp). The final alignment length was 605bp. For the \u003cem\u003ecox1\u003c/em\u003e gene sequencing amplicons ranged from 791\u0026ndash;832bp, and the final alignment length was 832bp. The alignments were used to generate Maximum-Likelihood phylogenetic trees using PhyML (Guindon, Dufayard and Lefort, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), in Geneious Prime (Kearse et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) with 100 bootstrap replicates of the data.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eAll unique sequences have been submitted to Genbank, accession numbers: PQ186079-186184.\u003c/p\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eD-loop\u003c/h2\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003eSamples\u003c/h2\u003e \u003cp\u003eFor this locus, 121 samples were sequenced from the EAZA collection, and 73 from the Department of Biological Sciences, Graduate School of Science, University of Tokyo.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eMatrilines\u003c/h2\u003e \u003cp\u003eAll but one of the 48 documented matrilines (as recorded in the EAZA studbooks) had a unique D-loop sequence. The SIA18 and SIA23 matrilines had identical sequences. These matrilines are not recorded as being related. However, in both cases the founder parentage is unknown, so it cannot be determined whether these individuals have identical amplified D-loop haplotypes by chance or are identical by descent (IBD). Two samples from the Tokyo collection also had identical sequences: LAR_G712 and LAR_DZ10. Additionally, two samples from the EAZA collection were found to have identical D-loop sequences as two samples from the Tokyo collection: LAR05 (EAZA) and LAR_G110 (Tokyo), and PIL12 (EAZA) and PIL_KZ15 (Tokyo). As no pedigree information is available for the Tokyo samples, again, it cannot be determined whether they are IBD. One of each unique sequence was included in the alignment for the phylogenetic tree.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic analysis\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. \u003cem\u003eHylobates\u003c/em\u003e form one clade, with \u003cem\u003eSymphalangus\u003c/em\u003e, \u003cem\u003eHoolock\u003c/em\u003e, and \u003cem\u003eNomascus\u003c/em\u003e in the other, with \u003cem\u003eSymphalangus\u003c/em\u003e and \u003cem\u003eNomascus\u003c/em\u003e clustering as sister taxa. Within the \u003cem\u003eHylobates\u003c/em\u003e clade \u003cem\u003eH. agilis\u003c/em\u003e and \u003cem\u003eH. albibarbis\u003c/em\u003e are sister to all other species. In the second major clade \u003cem\u003eH. klossii\u003c/em\u003e is separate, \u003cem\u003eH. pileatus\u003c/em\u003e and \u003cem\u003eH. lar\u003c/em\u003e are sister taxa, and \u003cem\u003eH. moloch\u003c/em\u003e forms a sub-clade with sister taxa \u003cem\u003eH. muelleri\u003c/em\u003e and \u003cem\u003eH. abbotti\u003c/em\u003e. Within the \u003cem\u003eNomascus\u003c/em\u003e clade \u003cem\u003eN. concolor\u003c/em\u003e separates first, with \u003cem\u003eN. siki\u003c/em\u003e, \u003cem\u003eN. leucogenys\u003c/em\u003e, \u003cem\u003eN. annamensis\u003c/em\u003e, \u003cem\u003eN. gabriellae\u003c/em\u003e, and \u003cem\u003eN. hainanus\u003c/em\u003e forming another clade. Within this clade. \u003cem\u003eN. leucogenys\u003c/em\u003e and \u003cem\u003eN. siki\u003c/em\u003e are sister taxa, as are \u003cem\u003eN. annamensis\u003c/em\u003e and \u003cem\u003eN. gabriellae\u003c/em\u003e, which form a sub-clade with \u003cem\u003eH. hainanus\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eSpecies identification\u003c/h2\u003e \u003cp\u003eThe average bootstrap support at the species nodes is 86.3%, ranging from 61% \u0026minus;\u0026thinsp;100% (N.B. \u003cem\u003eSymphalangus syndactylus\u003c/em\u003e is not included in this calculation as it is the only species in the genus, nor are \u003cem\u003eHoolock spp\u003c/em\u003e. as the tree contains only six reference sequences from Genbank for the entire genus).\u003c/p\u003e \u003cp\u003eFor the D-loop, 111/116 sequences grouped with their documented species. However, three purported \u003cem\u003eH. agilis\u003c/em\u003e sequences grouped as \u003cem\u003eH. lar\u003c/em\u003e (see Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Bootstrap support for the \u003cem\u003eH. lar\u003c/em\u003e clade is 89%. Additionally, two documented \u003cem\u003eN. gabriellae\u003c/em\u003e sequences did not group as expected. The GAB02 sequence grouped as \u003cem\u003eN. siki\u003c/em\u003e rather than \u003cem\u003eN. gabriellae\u003c/em\u003e (see Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Bootstrap support for this clade is 79%. The GAB09 sequence grouped with \u003cem\u003eN. annamensis\u003c/em\u003e, with 87% support (see Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003ecox1\u003c/h2\u003e \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e \u003ch2\u003eSamples\u003c/h2\u003e \u003cp\u003eFor this locus, 83 samples were successfully amplified sequenced from the EAZA collection.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eMatrilines\u003c/h2\u003e \u003cp\u003eCompared to the D-loop, far fewer \u003cem\u003ecox1\u003c/em\u003e matrilines had unique sequences. Of the 41 documented matrilines tested, only 26 had unique sequences. One of each unique sequence was included in the phylogenetic tree.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic analysis\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, the branching order for \u003cem\u003ecox1\u003c/em\u003e was different to that obtained with the D-loop: \u003cem\u003eNomascus\u003c/em\u003e splits in to one clade, with the second clade separating \u003cem\u003eHoolock\u003c/em\u003e, and \u003cem\u003eSymphalangus\u003c/em\u003e and \u003cem\u003eHylobates\u003c/em\u003e being sister taxa.\u003c/p\u003e\u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eSpecies identification\u003c/h2\u003e \u003cp\u003eWithin \u003cem\u003eHylobates\u003c/em\u003e bootstrap support values are not available for \u003cem\u003eH. klossii\u003c/em\u003e, \u003cem\u003eH. abbotti\u003c/em\u003e, or \u003cem\u003eH. albibarbis\u003c/em\u003e as only a single reference sequence is available for each (N.B. see Discussion below regarding possible misidentification of \u003cem\u003eH. abbotti\u003c/em\u003e/\u003cem\u003eH. muelleri\u003c/em\u003e sequences). For the remaining species, the average support at the species nodes is 97.6% (range 91%-100%). Within \u003cem\u003eNomascus\u003c/em\u003e, only a single sequence is available for \u003cem\u003eN. concolor\u003c/em\u003e, no sequences are available for \u003cem\u003eN. hainanus\u003c/em\u003e, and \u003cem\u003eN. leucogenys\u003c/em\u003e and \u003cem\u003eN. siki\u003c/em\u003e do not group monophyletically (see Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Support for \u003cem\u003eN. gabriellae\u003c/em\u003e is 52%, and 71% for \u003cem\u003eN. annamensis\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eAll \u003cem\u003ecox1\u003c/em\u003e sequences grouped as expected, with only three exceptions: consistent with the D-loop data, AG02 grouped as \u003cem\u003eH. lar\u003c/em\u003e (see Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), GAB02 grouped within the clade containing \u003cem\u003eN. siki\u003c/em\u003e, and GAB09 grouped with the \u003cem\u003eN. annamensis\u003c/em\u003e reference sequence (see Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eIdentification of species and hybrids\u003c/h2\u003e \u003cp\u003eOverall, 96% (111/116) of individuals were unambiguously assigned to their provenance asserted species, via their mitochondrial D-loop sequences. This demonstrates that the D-loop performs well as a maternal species identifier for gibbons, in the tested species and samples. Specific exceptions to species identification were limited to two species. Three \u003cem\u003eH. agilis\u003c/em\u003e individuals clustered with \u003cem\u003eH. lar\u003c/em\u003e; one \u003cem\u003eN. gabriellae\u003c/em\u003e sequence clustered with \u003cem\u003eN. siki\u003c/em\u003e, and a second \u003cem\u003eN. gabriellae\u003c/em\u003e matriline grouped with \u003cem\u003eN. annamensis\u003c/em\u003e. As the remaining 96% of sequences grouped as expected we are confident that these results indicate genuine mixed-species ancestry in these matrilines. Furthermore, bootstrap support at the species nodes is generally high (mean 86.3%, median 88%).\u003c/p\u003e \u003cp\u003eThe main exception to this robust species discrimination is the clade containing \u003cem\u003eH. agilis\u003c/em\u003e and \u003cem\u003eH. albibarbis\u003c/em\u003e. Historically \u003cem\u003eH. albibarbis\u003c/em\u003e was considered a sub-species of \u003cem\u003eH. agilis\u003c/em\u003e (Lyon, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1911\u003c/span\u003e), but was raised to full species level by Groves (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), and this was supported by cytogenetic and molecular genetic analyses (Hirai et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2005\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). \u003cem\u003eH. albibarbis\u003c/em\u003e and \u003cem\u003eH. agilis\u003c/em\u003e are currently recognised as distinct species by the IUCN (IUCN, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, the sequences reported here do not group as monophyletic taxa. The sequences are grouped together in a single clade, with 74% bootstrap support, which suggests they may be the same species, as previously recognised. The purported \u003cem\u003eH. albibarbis\u003c/em\u003e sequences cluster together in a group within this clade, with moderate bootstrap support (61%), suggesting the existence of a sub-species. However, the chromosomal translocation between the two taxa (Hirai et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), and their geographic isolation (\u003cem\u003eH. agilis\u003c/em\u003e is found on the Malaysian peninsular and the island of Sumatra, while \u003cem\u003eH. albibarbis\u003c/em\u003e is found only on the island of Borneo), support separate species status. Furthermore, recent work by Matsudaira and Ishida (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) found nuclear data also support distinction of \u003cem\u003eH. agilis\u003c/em\u003e and \u003cem\u003eH. albibarbis\u003c/em\u003e. It is possible that the two species have diverged so recently as to have accumulated limited phylogenetic signal. Excluding these two taxa, species-level bootstrap support across \u003cem\u003eHylobates\u003c/em\u003e and \u003cem\u003eNomascus\u003c/em\u003e is strong (mean 89.4%, median 90%).\u003c/p\u003e \u003cp\u003e \u003cem\u003eH. agilis\u003c/em\u003e and \u003cem\u003eH. lar\u003c/em\u003e have been documented to produce fertile hybrid offspring, both in the wild and in captivity (Brokelman and Gittins, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1984\u003c/span\u003e; Van Tuinen et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). Mixed ancestry is therefore a plausible explanation for the anomalous \u003cem\u003eH. agilis\u003c/em\u003e results. No pedigree information is available for the two cell line samples from Tokyo (AG_G80 and AG_JMC1). However, nuclear data confirm hybrid ancestry in the case of AG_G80 (Matsudaira and Ishida, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). These data showed the majority of the nuclear SNVs were of \u003cem\u003eH. agilis\u003c/em\u003e (the documented species), with some \u003cem\u003eH. lar\u003c/em\u003e. The levels of \u003cem\u003eH. lar\u003c/em\u003e introgression were low, suggesting the hybridisation event was not recent (i.e. in the wild, rather than recently in captivity). The data presented here confirm the \u003cem\u003eH. lar\u003c/em\u003e introgression in this individual, and demonstrate that the introgression comes from the maternal lineage. In the case of the EAZA individual AG02, the identity of the dam is unknown. It is possible a hybrid was accidentally created in captivity, if the dam was misidentified, although as these two species are readily distinguishable phenotypically, we consider this to be unlikely. Alternatively, the founding female taken from the wild could have been an \u003cem\u003eH. lar\u003c/em\u003e x \u003cem\u003eH. agilis\u003c/em\u003e hybrid, with the physical appearance of \u003cem\u003eH. agilis\u003c/em\u003e, similar to the case of AG_G80, leading to misidentification in the studbook.\u003c/p\u003e \u003cp\u003eSimilarly, \u003cem\u003eN. siki\u003c/em\u003e x \u003cem\u003eN. gabriellae\u003c/em\u003e hybrids have been documented in captivity (Nie et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), and suspected in the wild (Geissmann et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Mootnick, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Male \u003cem\u003eN. siki\u003c/em\u003e and \u003cem\u003eN. gabriellae\u003c/em\u003e individuals may be readily distinguished by their white and buff cheek colouration respectively. Females of these species however are very similar in appearance and can be difficult to distinguish reliably (Thinh, Rawson, \u003cem\u003eet al.\u003c/em\u003e, 2010; Harding, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). If we assume our sequencing data to be reliable, the anomalous grouping of GAB02 with \u003cem\u003eN. siki\u003c/em\u003e may therefore be either the result of a hybrid individual being taken from the wild, or the founding female (the dam of GAB02) being a misidentified \u003cem\u003eN. siki\u003c/em\u003e, particularly if their geographic origin was unknown, or incorrectly documented.\u003c/p\u003e \u003cp\u003eHistorically \u003cem\u003eN. annamensis\u003c/em\u003e was considered a sub-species of \u003cem\u003eN. gabriellae\u003c/em\u003e, but was raised to full species status in 2010 (Thinh, Mootnick, Thanh, \u003cem\u003eet al.\u003c/em\u003e, 2010), 13 years after the founding female of the GAB09 matriline was taken into captivity. With only \u003cem\u003eN. gabriellae\u003c/em\u003e being recognised at that time, this female could only have been identified as such. Furthermore, females of these species are indistinguishable phenotypically (Thinh, Mootnick, Thanh, \u003cem\u003eet al.\u003c/em\u003e, 2010), so it is unlikely the misidentification would have been diagnosed later, leading to unavoidable, if accidental, hybridization.\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eImplications for breeding programmes\u003c/h2\u003e \u003cp\u003eAs \u003cem\u003eH. agilis\u003c/em\u003e are no longer bred for conservation by EAZA, these results do not have significant consequences for the remaining \u003cem\u003eH. agilis\u003c/em\u003e population in the European collection (12 individuals remaining as of September 2023). However, the data reported here show \u003cem\u003eH. lar\u003c/em\u003e introgression in three different lineages of \u003cem\u003eH. agilis\u003c/em\u003e (one in EAZA, and two of the samples from the University of Tokyo), indicating the EAZA case is not isolated. It would therefore be prudent for other global breeding programmes to investigate their \u003cem\u003eH. agilis\u003c/em\u003e groups, as well as \u003cem\u003eH. lar\u003c/em\u003e and \u003cem\u003eH. pileatus\u003c/em\u003e, as these species are also known to hybridize in the wild (discussed above).\u003c/p\u003e \u003cp\u003eHowever, the captive \u003cem\u003eN. gabriellae\u003c/em\u003e population are part of active conservation breeding programmes, and so these results could have implications for these groups. The data reported here indicate \u003cem\u003eN. siki\u003c/em\u003e and \u003cem\u003eN. annamensis\u003c/em\u003e ancestry in two \u003cem\u003eN. gabriellae\u003c/em\u003e lineages, and it is the stated policy of EAZA to avoid the breeding of hybrids (EAZA, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). It is important to note that these data are not conclusive evidence of introgression, for several reasons. Firstly, the resolution of \u003cem\u003eN. leucogenys\u003c/em\u003e, \u003cem\u003eN. siki\u003c/em\u003e, \u003cem\u003eN. gabriellae\u003c/em\u003e, and \u003cem\u003eN. annamensis\u003c/em\u003e remains uncertain. While these taxa are recognised as four distinct species by the IUCN (IUCN, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), it is noted that \u003cem\u003eN. annamensis\u003c/em\u003e has been considered a sub-species of \u003cem\u003eN. gabriellae\u003c/em\u003e, and that \u003cem\u003eN. gabriellae\u003c/em\u003e and \u003cem\u003eN. siki\u003c/em\u003e have historically been considered sub-species of \u003cem\u003eN. leucogenys\u003c/em\u003e. The IUCN further notes that \u003cem\u003eN. siki\u003c/em\u003e may not be a true species at all, but rather may be a naturally occurring hybrid of \u003cem\u003eN. leucogenys\u003c/em\u003e and \u003cem\u003eN. gabriellae\u003c/em\u003e. Significant overlap between these purported species was found in phylogenetic analysis of the mitochondrial \u003cem\u003eCytochrome b\u003c/em\u003e gene by Thinh, Rawson, \u003cem\u003eet al.\u003c/em\u003e, (2010). Our analysis of the D-loop, however, demonstrates clear distinctions between these four taxa (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This finding suggests that the individuals of the GAB02 and GAB09 lineages are genuinely of mixed-species ancestry.\u003c/p\u003e \u003cp\u003eWhen considering possible implications for the \u003cem\u003eN. gabriellae\u003c/em\u003e breeding programme, it should also be noted that these data do not reveal the proportion of the current generation’s genome which might be affected. The youngest generation is three generations removed from the founding females. Theoretically, if all other breeding has been with true \u003cem\u003eN. gabriellae\u003c/em\u003e individuals, then approximately 12.5% of the current genome would be expected to be \u003cem\u003eN. siki\u003c/em\u003e or \u003cem\u003eN. annamensis\u003c/em\u003e depending on the inheritance of alleles after random segregation at meiosis. Furthermore, if the founding female was a hybrid this proportion would be lower still. In the absence of sequence data from variable nuclear loci, this proportion cannot be estimated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eCryptic relatedness\u003c/h2\u003e \u003cp\u003e97% of documented matrilines for all species were found to have a unique sequence for the D-loop amplicon analysed here. Within the EAZA collection tested, unexpected identical sequences were limited to three cases, one for \u003cem\u003eS. syndactylus\u003c/em\u003e, one for \u003cem\u003eH. lar\u003c/em\u003e, and one for \u003cem\u003eH. pileatus\u003c/em\u003e. Incomplete pedigree information prevents a definitive conclusion as to whether these matrilines are related. However, given the high number of unique haplotypes identified in this study (n = 113), we consider this possibility most likely.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eD-loop vs cox1\u003c/h2\u003e \u003cp\u003eWithin \u003cem\u003eHylobates\u003c/em\u003e, where multiple \u003cem\u003ecox1\u003c/em\u003e sequences are available, bootstrap support for species clusters is high (91%-100%). However, \u003cem\u003eH. abbotti\u003c/em\u003e does not group separately from \u003cem\u003eH. muelleri\u003c/em\u003e, unlike in the D-loop phylogenetic tree. This may an artefact of only having a single sequence available for \u003cem\u003eH. abbotti\u003c/em\u003e, which is therefore grouped with \u003cem\u003eH. muelleri\u003c/em\u003e as the most closely related sequences. Alternatively, the reference sequences used may be erroneously labelled. In Chan et al. (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), where the sequences were published, they were labelled as \u003cem\u003eH. muelleri\u003c/em\u003e, however, the study does not document \u003cem\u003eH. funereus\u003c/em\u003e or \u003cem\u003eH. abbotti\u003c/em\u003e samples. It is possible therefore that some or all of the reference sequences are in fact \u003cem\u003eH. abbotti\u003c/em\u003e. As with the D-loop phylogenetic tree, the single \u003cem\u003eH. albibarbis\u003c/em\u003e sequence groups within \u003cem\u003eH. agilis\u003c/em\u003e, although it does have a longer branch length (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) suggesting greater divergence. The \u003cem\u003eNomascus\u003c/em\u003e clade is not clearly delineated by \u003cem\u003ecox1\u003c/em\u003e compared to the D-loop, in particular \u003cem\u003eN. leucogenys\u003c/em\u003e, and \u003cem\u003eN. siki\u003c/em\u003e, which do not group monophyletically. This makes \u003cem\u003ecox1\u003c/em\u003e unsuitable for species identification for these species. The results for AG02 and GAB09 are concordant with the results for the D-loop, being identified as \u003cem\u003eH. lar\u003c/em\u003e and \u003cem\u003eN. annamensis\u003c/em\u003e respectively. The sequence for GAB02 also groups within the clade containing \u003cem\u003eN. siki\u003c/em\u003e. In contrast to the D-loop, matrilines are not readily distinguished using \u003cem\u003ecox1\u003c/em\u003e sequences. It is therefore not recommended for studies in which ascertaining relatedness is important, such as in captive populations, or studying female dispersal in the wild. Instead, the D-loop should be used in preference. Additionally, the D-loop had a higher successful amplification rate for the lower-quality hair samples compared to \u003cem\u003ecox1\u003c/em\u003e (68% and 59% respectively). This may be due to the smaller amplicon size of the D-loop compared to \u003cem\u003ecox1\u003c/em\u003e (~ 560bp and ~ 790bp respectively). This makes the D-loop a more attractive target locus for situations where only lower quality samples such as hair are available, as is often the case in wildlife rescue centers.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e "},{"header":"Conclusions and future work","content":"\u003cp\u003eBased on the data reported here we recommend the D-loop should be used for gibbon maternal species identification, due to its higher resolution compared to \u003cem\u003ecox1\u003c/em\u003e. In particular, we do not recommend the use of \u003cem\u003ecox1\u003c/em\u003e for species identification for \u003cem\u003eNomascus spp\u003c/em\u003e. as \u003cem\u003eN. leucogenys\u003c/em\u003e and \u003cem\u003eN. siki\u003c/em\u003e are not monophyletic when using this locus. By contrast, the D-loop sequences cluster clearly along recognised species boundaries, with strong support. The possible exception to this recommendation, is in the case of \u003cem\u003eH. albibarbis\u003c/em\u003e, as discussed above. Two recognised species are currently absent from this work: \u003cem\u003eHylobates funereus\u003c/em\u003e, and \u003cem\u003eNomascus nasutus.\u003c/em\u003e (N.B. at the time of writing, two sequences for \u003cem\u003eN. nasutus\u003c/em\u003e were published, but were significantly shorter than the other sequences used in these analyses, and so were excluded). Sequencing of individuals of known provenance for these species would provide the opportunity to construct a complete phylogenetic tree and most likely enable reliable maternal species identification for any individual.\u003c/p\u003e\u003cp\u003eA significant limitation of mitochondrial sequences is that they are maternally inherited. As a result, any introgression which has occurred in the paternal line will remain undetected. Therefore, markers from the nuclear genome which can identify species and possible hybrids from both the maternal and paternal lineages are highly desirable. Whole genome sequencing would be ideal as not only could hybrids be detected, but also the proportion of introgression quantified. However, such methods are currently impractical in terms of cost, time, data analysis, and sample requirements. One approach to this problem are nuclear markers, utilising species-specific retrotransposon insertions, which can be detected with simple PCR and gel electrophoresis technology (Lansdowne, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eWhile phylogenetics using the mitochondrial D-loop is limited to maternal lineages, it is nevertheless a robust method, which can assign the maternal species origin of an individual with a high degree of confidence. Where introgression has occurred on the maternal side, it can also highlight cases of cryptic hybrids, as demonstrated here. We recommend use of the D-loop for gibbon species identification, rather than \u003cem\u003ecox1\u003c/em\u003e, due to the greater resolution provided, the higher amplification rates for low quality samples, and the higher number of observed haplotypes which enable lineage determination.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eFunding was provided by the BBSRC, the University of Leicester, and Twycross Zoo.\u003c/p\u003e \u003ch2\u003eCompeting Interests\u003c/h2\u003e \u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e \u003ch2\u003eCompliance with Ethical Standards\u003c/h2\u003e \u003cp\u003eAll invasive samples were residual material collected either during medical procedures or autopsies, in compliance with each institutions\u0026rsquo; standards, and with support from the EAZA Gibbon Taxon Advisory Group.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eThe study was designed by L.L. and R.B. Data collection was performed by L.L., K.M., and S.M. Data analysis was performed by L.L. Samples at the University of Tokyo were provided by T.I. The first draft of the manuscript was written by L.L. and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe would like to thank Twycross Zoo (UK) for their support and collaboration, and also the other institutions who generously provided samples: Apenheul Primate Park (Netherlands), Howletts and Port Lympne (UK), Bor\u0026aring;s Zoo (Sweden), Bristol Zoo (UK), Cotswold Wildlife Park (UK), German Primate Center (Germany), Marwell Zoo (UK), Mulhouse Zoological and Botanical Park (France), Paignton Zoo (UK), Parc Animalier d'Auvergne (France), Reserve Africaine de Sigean (France), Selwo Aventura (Spain), Serengeti Park (Germany), Zoo Boissiere (France), Zoo de Cerza (France), Zoo de la Besancon Citadelle (France), Zoopark Erfurt (Germany), ZSL (UK), Zurich Zoo (Switzerland). Funding was provided by the BBSRC, the University of Leicester, and Twycross Zoo. We would also like to thank Tony King and Jane Hopper at The Aspinall Foundation for the provision of additional DNA sequences.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eSequences generated during the current study may be found on Genbank (Accession numbers: PQ186079-186184).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAndayani N et al (2001) \u0026lsquo;Genetic variability in mtDNA of the silvery gibbon: Implications for the conservation of a critically endangered species\u0026rsquo;, \u003cem\u003eConservation Biology\u003c/em\u003e, 15(3), pp. 770\u0026ndash;775. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1046/j.1523-1739.2001.015003770.x\u003c/span\u003e\u003cspan address=\"10.1046/j.1523-1739.2001.015003770.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAvise JC (2000) Phylogeography: The History and Formation of Species. 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26:375\u0026ndash;400\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"conservation-genetics-resources","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cogr","sideBox":"Learn more about [Conservation Genetics Resources](https://www.springer.com/journal/12686)","snPcode":"12686","submissionUrl":"https://submission.nature.com/new-submission/12686/3","title":"Conservation Genetics Resources","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Gibbons, Genetics, Species, Hybrids, Phylogenetics","lastPublishedDoi":"10.21203/rs.3.rs-5039233/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5039233/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSome gibbon species are difficult to distinguish phenotypically. This issue is compounded by recent changes in the gibbon taxonomy, which have sometimes occurred long after individuals were taken from the wild for captive breeding programmes. Furthermore, gibbon species within the \u003cem\u003eHylobates\u003c/em\u003e and \u003cem\u003eNomascus\u003c/em\u003e genera have been documented to hybridize to produce viable, fertile offspring, both in captivity and in the wild. This raises the possibility of cryptic hybrids within captive populations, threatening the genetic integrity of these genera. Phylogenetic methods using the mitochondrial D-loop and \u003cem\u003ecox1\u003c/em\u003e gene were compared to genetically identify gibbon species. Both loci identified lineages with cryptic hybrid ancestry. However, the D-loop outperformed the \u003cem\u003ecox1\u003c/em\u003e gene, providing higher resolution, particularly for \u003cem\u003eNomascus spp\u003c/em\u003e., which for some species were not monophyletic when using the \u003cem\u003ecox1\u003c/em\u003e gene. The D-loop also revealed a significantly higher number of unique sequences, making it more suitable for investigations into relatedness, such as female dispersal patterns.\u003c/p\u003e","manuscriptTitle":"The mitochondrial D-loop is a robust maternal-species identifier in gibbons (Hylobatidae)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-11 07:54:45","doi":"10.21203/rs.3.rs-5039233/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-04-17T03:40:48+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-28T14:24:06+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-27T12:34:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"284232383291293966351037412373742539278","date":"2025-02-24T15:02:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"24794601344931888684465564270034749791","date":"2025-02-24T12:45:22+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-10-28T10:29:24+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-09-06T08:50:15+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-09-06T08:49:20+00:00","index":"","fulltext":""},{"type":"submitted","content":"Conservation Genetics Resources","date":"2024-09-05T15:15:16+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"conservation-genetics-resources","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cogr","sideBox":"Learn more about [Conservation Genetics Resources](https://www.springer.com/journal/12686)","snPcode":"12686","submissionUrl":"https://submission.nature.com/new-submission/12686/3","title":"Conservation Genetics Resources","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"89084afb-2886-408a-88b2-24336d2cf0ca","owner":[],"postedDate":"October 11th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-07-07T16:05:10+00:00","versionOfRecord":{"articleIdentity":"rs-5039233","link":"https://doi.org/10.1007/s12686-025-01392-8","journal":{"identity":"conservation-genetics-resources","isVorOnly":false,"title":"Conservation Genetics Resources"},"publishedOn":"2025-07-04 15:57:58","publishedOnDateReadable":"July 4th, 2025"},"versionCreatedAt":"2024-10-11 07:54:45","video":"","vorDoi":"10.1007/s12686-025-01392-8","vorDoiUrl":"https://doi.org/10.1007/s12686-025-01392-8","workflowStages":[]},"version":"v1","identity":"rs-5039233","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5039233","identity":"rs-5039233","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}