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.