Trachypithecus phayrei (TPH), also known as Phayre’s leaf monkey or Phayre’s lutung (Behie and Groves, 2016), belongs to old-world monkeys (OWMs), family Cercopithecidae, subfamily Colobinae—the latter including an African and an Asian clade. The genus Trachypithecus comprises 17 species with one Asian colobine—TPH (Pinthong et al., 2018). TPH is widely distributed in continental Southeast Asia including India, Bangladesh, Western Myanmar, Thailand, Laos, Vietnam, and Southern China (Muul, 2002). It is important to notice that genus TPH was initially denominated with different Latin names, such as Semnopithecus phayrei and Presbytis phayrei, before the current name came into use (Gupta and Kumar, 1994).

The pedigree and chromosomal evolution of Hominidae has been principally and roughly resolved in previous cytogenetic and molecular cytogenetic studies; however, some gaps remain, including the karyotype of TPH (Stanyon et al., 2008). The latter was first described in 1970 as 2n = 44 (Hsu and Benirschke, 1970). In 1981, G banding revealed for a male TPH the karyotype composition is as follows: 22 (M) + 18 (SM) + 2 (A), XX (SM) (Chen et al., 1981). In 1998, chromosomal homologies between human and TPH chromosomes were established by FISH applying human whole chromosome paintings. This revealed unique reciprocal translocations corresponding to chromosomes of (Homo sapiens) HSA 1 and 19, and HSA 6 and 16 as well as fusions of HSA 14 and 15 and HSA 21 and 22 (Nie et al., 1998). In 2018, the subspecies TPH crepuscula was studied by GTG-banding and NOR staining (Pinthong et al., 2018).

Accordingly, up to now, there have been few or neither really comprehensive nor high-resolution FISH-banding–based (Mrasek et al., 2001; Liehr and Claussen, 2002; Weise et al., 2008) comparative molecular cytogenetic reports on homologies between HSA and TPH chromosomes. Thus, here, the first detailed comparative chromosomal map of TPH compared to HSA is presented, established by MCB and complementary heterochromtin- and one locus-specific probe(s). Furthermore, the results obtained in TPH were compared to karyotypes of Macaques (such as Macaca fascicularis = MMU) (Fan et al., 2014) and Silvery lutung (Trachypithecus cristatus = TCR) (Xiaobo et al., 2013), which were studied by identical high-resolution molecular cytogenetic approaches. Additionally, the relationship of ECBs with human fragile sites was analyzed.

Results obtained in molecular cytogenetic studies are summarized in Figure 1 and Table 1.

Overall, the majority of TPH chromosomes are completely homologous to one of the human chromosomes; exceptions are chromosomal exchanges that took place as follows: TPH 4 and 5 (homologous to HSA 1 and 19), TPH 12 (homologous to HSA 14 and 15), TPH 15 and 18 (homologous to HSA 6 and 16), and TPH 21 (homologous to HSA 21 and 22). The centromeric positions could be identified at the sub-band level for all 23 TPH chromosomes. In the following chromosomes, the TPH centromeric positions were the same as in HSA: TPH 2 (= HSA 5), TPH 4 (= HSA 1), TPH 5 (= HSA 19), TPH 6 (= HSA 10), TPH 7 (= HSA 17), TPH 8 (= HSA 11), TPH 11 (= HSA 7), TPH 16 (= HSA 12), TPH X (= HSA X), and TPH Y (= HSA Y). Centromere positions shifted compared to HSA as follows: TPH 1 (HSA 3q26), TPH 3 (HSA 4q21.2), TPH 9 (HSA 13q11.1), TPH 10 (HSA 18q21.32), TPH 12 (HSA 15q26.3/14q11.1), TPH 13 (HSA 8p12), TPH 14 (HSA 2q24.3), TPH 15 (HSA 16p11.2), TPH 17 (HSA 9q33.2), TPH 18 (HSA 6q24.3), TPH 19 (HSA 2p14), TPH 20 (HSA 20q13.3), and TPH 21 (HSA 21q22.3/22q11.1).

Furthermore, repetitive DNA was identified by human heterochromatin-specific probes as follows: the repetitive sequence D1Z5 located in HSA 1q11-q12 was not present in TPH 4 or TPH 5, while the region being present in human as the band 19p12/19q12 could be found in TPH 5. The human hemi-heterochromatic region 9p12/9q13 was located on the long arm of TPH 17, while D9Z3 (HSA 9q12) and D16Z3 (HSA 16q11.2) were not detectable in TPH. NOR signals can be found in the centromere region of TPH 21. Repetitive DNA in the human male in Yq12 also was observed in TPH Y. Overall, only HSA chromosomes 4 (TPH 3), 8 (TPH 13), 12 (TPH 16), X (TPH X), and Y (TPH Y) were completely unaltered during evolution between these two relatively distantly related species among OWMs.

Table 2 summarizes 50 ECBs observed in TPH in this study, which were identified according to the homologous regions in HSA. In addition, the characterized TPH breakpoints were compared with previously reported ones in TCR and in other macaque species using the MCB approach (Table 2).

The co-localization of ECBs among TPH, TCR, and in macaque species are listed with respect to HSA in Table 2. Out of 50 ECBs mapped in TPH, 29 (58%) and 18 (36%) coincided with ECBs in TCR and macaques, respectively (Figure 2; Table 3). Moreover, 41 (82%) reported ECBs in TPH co-localized with human fragile sites (Figure 2; Table 4).

MCB combined with heterochromatin- and a locus-specific probe is suited best to characterize basic karyotypic features in primates, as shown in our previous studies (Mrasek et al., 2001; Fan et al., 2014; Fan et al., 2015; Xiaobo et al., 2013; Sangpakdee et al., 2018). In this study, the first comprehensive characterization of the karyotype of TPH was done; and a comparison with that in TCR and macaques was performed accordingly (Fan et al., 2014; Xiaobo et al., 2013). Our results confirmed and refined previous cytogenetic studies of TPH chromosomes, which were at a much lower resolution (Nie et al., 1998; Pinthong et al., 2018). These results extended to a detailed characterization of all TPH chromosomes aligned to HSA by MCB, that were not available before (Dutrillaux et al., 1979; Rhesus Macaque Genome Sequencing and Analysis Consortium et al., 2007). NOR was mapped to ECBs/fusion points of HSA 14 and HSA 15 (corresponding to TPH 12) confirming previous results (Pinthong et al., 2018). Compared to the basic Hominidea karyotype, five chromosomes remained unchanged in TPH, namely chromosomes 3, 13, 16, X, and Y, similar to those in TPH (Pinthong et al., 2018) and related species (Misceo et al., 2008). In addition, compared to HSA, complex chromosomal rearrangements (Table 1) first described here took place during the evolutionary process when the common ancestor of HSA and TPH diverged and may further continue.

ECBs must have undergone breaking and rejoining of double-strand breaks (Tsai and Lieber, 2010). These evolutionary conserved chromosomal changes could have been driven by several factors, such as the intrinsic instability of segmental duplications (SDs) enriched in the flanking regions of ECBs. SDs have been suggested to have a significant impact on genome plasticity during the evolution of primate chromosomes in previous studies (Kehrer-Sawatzki and Cooper, 2008). It is suggested that SDs within recombination hotspots might mediate non-allelic homologous recombination (NAHR). For example, two homologous SDs on the same chromosome, but in opposite orientation, could be the bases of an inversion. If SDs are in direct orientation, NAHR results in duplication and/or deletion as reported in human microdeletion-/microduplication syndromes and bases of copy-number variant regions (CNV’s) in human (Liehr, 2021). SDs located on different chromosomes can be the bases of NAHR-mediated chromosomal translocations (Tsai and Lieber, 2010; Gu et al., 2008).

While in previous reports, there were no detailed and characterized centromeric regions of TPH in corresponding reviews on OWMs (Ventura et al., 2004; Ventura et al., 2007; Stanyon et al., 2008), here, a first clue was possible about positions of centromeric regions in TPH (Table 1), that is, 56.5% TPH centromere positions shifted and 43.5% centromere positions were conserved compared to HSA. This is similar to the situation in TCR, that is, conserved centromeres in TPH kept their positions during evolution from common ancestors. However, these conserved centromeric regions normally do not have identical alphoid sequences as in HSA (Rocchi et al., 2012), and neo-centromeres are preferentially formed most often in gene deserts (Lomiento et al., 2008).

There are 29 identical ECBs in TPH and in TCR, and 17 ECBs are in concordance with those in macaque species. Moreover, 11 identical ECBs were identified in TPH, in TCR, and in macaque species (Tables 3 and 4). These findings are useful for the reconstruction of a common ancestral karyotype in further studies by applying, for example, locus-specific FISH-probes and/or sequencing of the TPH genome. In total, 41 (82%) of reported 50 ECBs in TPH corresponded to human fragile sites, which is in concordance to previous observations in TCR that ECB regions are highly connected to common FS locations (Francis, 2002, Mrasek et al., 2010; Fungtammasan et al., 2012). It has been suggested that FSs are low-stability regions, supporting their potential role in the formation of evolutionary chromosomal rearrangements (Mishmar et al., 1998). In this connection, others suggested the involvement of the cellular checkpoints proteins ATR and BRCA1, which are also critical for the expression of FSs (Casper et al., 2002; Arlt et al., 2006; Glover, 2006). Also, comparative analyses showed that the co-localization of ECBs in TPH/TCR with human FSs revealed no differences, indicating that Asian langurs are karyotypically closely related (Alekseyev and Pevzner, 2010).

In conclusion, the presented TPH karyotype and comparison to other langurs and macaques provided new insights into chromosomal evolution. It is another stepping stone in primate evolution research.