First detailed reconstruction of the karyotype of Trachypithecus cristatus (Mammalia: Cercopithecidae)

Background The chromosomal homologies of human (Homo sapiens = HSA) and silvered leaf monkey (Trachypithecus cristatus = TCR) have been previously studied by classical chromosome staining and by fluorescence in situ hybridization (FISH) applying chromosome-specific DNA probes of all human chromosomes in the 1980s and 1990s, respectively. Results However, as the resolution of these techniques is limited we used multicolor banding (MCB) at an ~250-band level, and other selected human DNA probes to establish a detailed chromosomal map of TCR. Therefore it was possible to precisely determine evolutionary conserved breakpoints, orientation of segments and distribution of specific regions in TCR compared to HSA. Overall, 69 evolutionary conserved breakpoints including chromosomal segments, which failed to be resolved in previous reports, were exactly identified and characterized. Conclusions This work also represents the first molecular cytogenetic one characterizing a multiple sex chromosome system with a male karyotype 44,XY1Y2. The obtained results are compared to other available data for old world monkeys and drawbacks in hominoid evolution are discussed.

The karyotype of TCR was described in 1970 as 2n = 44 [5]. In the 1980s, chromosome banding analysis were used in TCR [6][7][8], including comparative R-banding of three different species of Colobus genus [6]. In 1983, G and Q banding were applied to analyze the banding patterns of a female TCR [7]. One year later, male TCR was characterized as carrying an evolutionary conserved translocation involving the Y chromosome and two autosomes [9]. Furthermore, since 1997 chromosomal homologies between human chromosomes and TCR has been established by fluorescence in situ hybridization (FISH) [10]. However, as whole chromosome paints have only a limited resolution [11], we established a detailed comparative chromosome map of TCR primarily based on multicolor banding (MCB). The potential of this approach in order to clarify and to resolve evolutionary conserved chromosomal rearrangements was already shown by our group for other primates [12][13][14].

Results
MCB results are summarized in Figure 1 and in Tables 1  and 2. Representative results of probes specific for all acrocentric short arms in HSA, the NOR-region and SRY are also shown in Figure 1. As outlined in Table 1 the  majority of TCR chromosomes are completely homologous  to one of the human chromosomes, exceptions are  only TCR 5 (homologous to HSA 14 and 15), TCR 6 and 8 (homologous to HSA 1 and 19), TCR 9 and 16 (homologous to HSA 6 and 16), TCR 15 (homologous to HSA 21 and 22), and TCR Y1 and Y2 (homologous to HSA Y and 5).
The centromeric positions could be narrowed down to the subband level for all 24 TCR chromosomes (Table 1). In the following chromosomes the TCR centromeric None of the aforementioned TCR centromeric regions that kept their position during evolution compared to human showed positive FISH-signals with any of the used HSA centromere specific probes (data not shown). Also, none of the other human heterochromatin specific probes from HCM probe set gave any specific signals in TCR, with 2 exceptions: NOR-specific signals were observed in TCR 5 (at fusion of HSA 14q11.2 and 15q26.3) (Figure 1) and the probes midi54 ( Figure 1) and midi 36 (data not shown); the latter two located on the distal end of the long arm of TCR 14.
In the literature there were 81 TCR specific heterochromatic insertions and/ or additions to chromosomes reported ( Figure 2). In this study only 25 of them were confirmed and mapped ( Figure 2 and Table 1, last column). Table 2 summarizes the 69 evolutionary conserved breakpoints observed in TCR in this study; they are given according to the homologous regions in HSA. Only HSA chromosomes X (TCR X) and 8 (TCR 7) are completely unaltered during evolution from a common ancestor of HSA and TCR. All other homologous of TCR chromosomes have undergone one (HSA 12, 14, 16, 18, 21 and 22), two (HSA 4 and 15), three (HSA Y, 11, 13, 17, 19, and 20), four (HSA 1 and 6), five (HSA 2), six (HSA 5, and 10), or nine (HSA 3) evolutionary conserved break events during speciation in respect to the human karyotype.
Besides, the characterized breakpoints of TCR are compared with such previously mapped in Hylobates lar (HLA) and Gorilla gorilla (GGO) using MCB approach (Table 2). Again, an alignment of the breakpoint and their positioning in GTG-light bands and their spatial proximity to human fragile sites was done.

Discussion
The present study represents the first one that comprehensively characterizes the karyotype of TCR. In general, previous homologies of HSA and TCR chromosomes could be confirmed [10]. However, in this study, homologous regions for TCR chromosomes 4, 10, 11, 14, 17, 18 and 21 (that were not studied before) [10] were specifically aligned to their HSA-homologous. In contrast to [10] NOR was mapped here to the fusion points of HSA 14 and HSA 15, i.e. TCR 5 and not TCR 15. In our two studied individuals derived from Thailand, no differences in TCR 1 banding pattern were seen, which is in accordance with the literature [10]. For the first time, the exact breakpoints could be determined for the extremely rearranged karyotype of TCR, in comparison to HSA. In fact, 69 evolutionary conserved breakpoints were determined in a male TCR and confirmed in a female individual, excluding Y 1 and Y 2 chromosomes, obviously.
In this study no special attention was given to the centromeric regions of TCR, i.e. they were not detailed characterized as in other studies e.g. by [15] or [16]. However, a first impression is provided in which centromeres kept their positions during evolution from common ancestors to HSA and TCR and were neo-centromeres (Table 1), i.e.~50% of them stayed at the same positions and~50% moved either in one of the two species or in both. As expected from the literature [17], even the centromeric regions that kept their positions did not have the identical alphoid sequences in HSA and TCR.
Concerning evolution it is interesting to report that in TCR and in HLA 11 evolutionary conserved breakpoints are identical and 15 more are most likely in concordance to each other. Even more interesting, 6 identical and 2 most likely identical evolutionary conserved breakpoints were identified in TCR and in GGO ( Table 2). These findings need to be confirmed in further studies by locusspecific probes, and if confirmed, they will be very useful for the reconstruction of a common ancestral karyotype. Compared to the postulated Hominidea ancestral karyotype proposed by [25], only four chromosomes remained unchanged in TCR, i.e. chromosomes 4, 7, 11 and X, eleven chromosomes underwent only intrachromosomal changes like inversions (TCR 2, 3, 10,12,13,14,17,18,19,20,21) and two TCR chromosomes resulted from a fusion of ancestral chromosomes (TCR 5 and 15).
Interestingly, the regions between TCR 1 and TCR Y 1 and TCR Y 2 being homologous to HSA 5 were shown to be subject to different evolutionary conserved rearrangements. Broadly speaking, TCR Y1 is homologous to TCR 1p and TCR Y2 to TCR 1q. However, each arm of chromosome TCR 1 underwent a further paracentric inversion, most likely being important to separate the sex chromosomes from the chromosome 1 during meiosis. Thus, a XY 1 Y 2 sex chromosome system is present in TCR, and not an X 1 X 2 Y 1 Y 2 system as initially suggested [10]. However, as in TCR from Indonesia, two other forms of TCR 1 chromosome could be found [10]. Therefore, the existence of an X 1 X 2 Y 1 Y 2 system cannot be completely excluded by this study. The sex determination system in mammals is usually highly conserved as XY-system. However, multiple sex chromosome systems, like the one present in TCR and few other apes [27,28] are exceptionally found in some species of e.g. the orders Insectivora, Chiroptera, Artiodactyla, Rodentia [29], and in marsupials [30]. In general, constitutional Y-chromosome / autosome translocations in human appear de novo and have a deleterious effect and, although the infertility is the only common feature, other clinical symptoms can also be observed depending on the involved breakpoints [31]. In such cases, the infertility is thought to be a result of disruption of the sex vesicle during meiosis [32]. From this point of view it is hard to imagine conditions which are in favor of developing a multiple sex-from an XY-chromosome system. On the other hand, in population genetic models of Y-autosome and X-autosome rearrangements the population can gain a selective advantage under a wide range of conditions. If they can invade the population, Y-autosome rearrangements always spread to fixation, whereas X-autosome rearrangements may be maintained as stable polymorphisms" [33]. The XY 1 Y 2 sex chromosome system observed in TCR fits well into the suggestions of [34] that (i) female meiotic drive is the major contribution to the evolution of neo-sex chromosomes and (ii) that "in mammals, the XY 1 Y 2 sex chromosome system is more prevalent in species with karyotypes of more biarmed chromosomes" rather than in species with acrocentric chromosomes. Research on meiotic behavior of such sex systems is scarce; however, one study on Bolivian owl monkey (Aotus spec.) showed that no XY pairing was observable but the Y-chromosomes formed trivalents with an autosome during gametogenesis [27].

Conclusions
In conclusion, the presented comparative map of TCR karyotype gives new insights into primate evolution and can be used as a starting point for further detailed analyses. Evolutionary conserved breakpoints, TCR-specific heterochromatic regions, centromeric sequences as well as the sex chromosome system can be fruitful fields of research in near future.

Cell culture and chromosomal preparation
Immortalized lymphoblast cell lines derived from a male and female TCR, were provided by the Khon Kaen University, Thailand. Culture techniques and chromosome preparation followed standard protocols.

Microscopic evaluation
Images were captured using an Axioplan II microscope (Carl Zeiss Jena GmbH, Germany) equipped with filter sets for DAPI, FITC, TR, SO, Cy5 and DEAC. Image analysis was done using pseudocolor banding and fluorochrome profiles of the ISIS digital FISH imaging system (Meta Systems Hard & Software GmbH, Altlussheim, Germany). At least 10 up to 20 metaphases were recorded, derived from a male and a female TCR for each applied probe and probe set.