X Chromosome Evolution in Cetartiodactyla

The phenomenon of a remarkable conservation of the X chromosome in eutherian mammals has been first described by Susumu Ohno in 1964. A notable exception is the cetartiodactyl X chromosome, which varies widely in morphology and G-banding pattern between species. It is hypothesized that this sex chromosome has undergone multiple rearrangements that changed the centromere position and the order of syntenic segments over the last 80 million years of Cetartiodactyla speciation. To investigate its evolution we have selected 26 evolutionarily conserved bacterial artificial chromosome (BAC) clones from the cattle CHORI-240 library evenly distributed along the cattle X chromosome. High-resolution BAC maps of the X chromosome on a representative range of cetartiodactyl species from different branches: pig (Suidae), alpaca (Camelidae), gray whale (Cetacea), hippopotamus (Hippopotamidae), Java mouse-deer (Tragulidae), pronghorn (Antilocapridae), Siberian musk deer (Moschidae), and giraffe (Giraffidae) were obtained by fluorescent in situ hybridization. To trace the X chromosome evolution during fast radiation in specious families, we performed mapping in several cervids (moose, Siberian roe deer, fallow deer, and Pere David’s deer) and bovid (muskox, goat, sheep, sable antelope, and cattle) species. We have identified three major conserved synteny blocks and rearrangements in different cetartiodactyl lineages and found that the recently described phenomenon of the evolutionary new centromere emergence has taken place in the X chromosome evolution of Cetartiodactyla at least five times. We propose the structure of the putative ancestral cetartiodactyl X chromosome by reconstructing the order of syntenic segments and centromere position for key groups.


Species
The list of studied species with scientific and common names, diploid chromosome number, and source of cell lines is presented in the Table 1. All cell lines belong to the cell cultures collection of general biological purpose (No. 0310-2016-0002) of Institute of Molecular and Cellular Biology Siberian Branch of the Russian Academy of Sciences.

BAC Clones
Using the cattle genome assembly version from October 2011 (Baylor Btau_4.6.1/bosTau7) in UCSC Genome Browser [28], X chromosome-located BAC clones were manually chosen from the CHORI-240 BAC library from the "BACPAC Resource Center" (BPRC, the Children's Hospital Oakland Research Institute in Oakland, CA, USA). To download information in the Genome Browser about the localization of BACs of appropriate size (length of insertion varied from 50-300 kb), a custom track in Browser Extensible Data (BED) format was created [29]. BAC clones with appropriate insert sizes (50-300 kbp) and genetic content (unique genes, less repetitive elements) were selected. BAC sequence conservation was estimated from phyloP data [30] in the human genome ("Conservation" track in GRCh37/hg19 assembly). Genome coordinates were converted from cow to human using the Batch Coordinate Conversion (liftOver tool) in UCSC Genome Browser. Thus, 73 BAC clones evenly distributed on cattle X chromosome (2-5 Mbp gaps) were selected. For each of the manually selected 73 BACs, we defined various genomic features selected to increase the probability of a clone to hybridize with metaphase spreads of distant cetartiodactyl species. To do so, we calculated protein coding genes, cattle genes orthologous to human, GC content, and repetitive sequences in each of the selected BAC clones. By using multiple alignments, including all available cetartiodactyl genomes, we calculated the nucleotide conservation scores and conserved elements using phastCons [31]. Then, we compared the characteristics of four BACs that had previously worked on distant species with all the 73 BACs by using the classification tree from the CART algorithm [32]. A total of 51 BACs were selected to have a high probability of hybridization to distant species. These BACs contained less than 48% of repetitive sequence and more than 20% of conserved elements. A subset of 26 of these BAC clones that were evenly distributed along the cattle X chromosome with a median distance of 5 Mb were hybridized on all cetartiodactyl species studied here. Table 2 lists the CHORI-240 cattle X chromosome BAC clones used in this study. A single BAC clone (CH240-316D2) is the same as used by Fröhlich et al. [25].
BAC DNA was isolated using the Plasmid DNA Isolation Kit (BiosSilica, Novosibirsk, Russia) and amplified with GenomePlex Whole Genome Amplification kit (Sigma-Aldrich Co., St. Louis, MO, USA). Labeling of BAC DNA was performed using GenomePlex WGA Reamplification Kit (Sigma-Aldrich Co., St. Louis, MO, USA) by incorporating biotin-16-dUTP or digoxigenin-dUTP (Roche, Basel, Switzerland). The quality of produced BAC probes was controlled by FISH localization on cattle chromosomes.

Bioinformatics Analysis
An analysis in UCSC Genome Browser was performed to establish the order of CHORI-240 BAC clones on X chromosomes of one cetartiodactyl species (sheep) and four species from out-group mammalian orders (Perissodactyla, Primates, Rodentia). BAC positions in these genomes were obtained using Batch Coordinate Conversion (liftOver) in the UCSC Genome Browser that converts genome coordinates between assemblies. The cattle genome assembly (Bos_taurus_UMD3.1.1/bosTau8) was used as a reference. Sequences coordinates of all BAC clones were calculated in human (GR ch38/hg 38), mouse (GRC m38/mm10), rat (RGSC 6.0/rn6, except 386M8, which is disrupted in this genome), horse (Broad/equCab2), and sheep (ISGC Oar_v3.1/oviAri3) genomes.

Ancestral Chromosome Deduction
The morphology and conservative block orientation of the ancestral X chromosome were deduced using maximum parsimony by comparing X chromosomes across the top branches of Cetartiodactyla and assuming the most common variant to be ancestral for the order. Once the provisional ancestral chromosome was identified, we detected whether the extant X chromosome and the suggested ancestral form differ by inversions (change of BAC order) or/and by centromere repositioning (change of centromere position without change in BAC order).

BACs Localization
We investigated the X chromosome structure across major branches of Cetartiodactyla represented by 18 species from four non-ruminant (Suidae, Camelidae, Eschrichtiidae (Cetacea), Hippopotamidae) and six ruminant (Tragulidae, Antilocapridae, Giraffidae, Moschidae, Cervidae, and Bovidae) families ( Table 1). The order of 26 labeled cattle BAC clones was established on the X chromosomes of each of 18 species by a series of pairwise FISH experiments ( Table 2). In total, comparative analyses of BAC orders across 18 species revealed three major chromosomal conservative segments, which were numbered and designated with colors used throughout the paper: X Syntenic Block 1 (13 BACs, XSB1, pink); X Syntenic Block 2 (seven BACs, XSB2, yellow), and; X Syntenic Block 3 (six BACs, XSB3, blue).

Intrachromosome Rearrangements
Comparative analysis of the order of BAC on the X chromosomes of 18 species identified three key scenarios that likely took place in the course of the cetartiodactyl X chromosomes' evolution.

1.
Conservation: no change in the BAC order and no change of the centromere position.
We identified a group of four basal cetartiodactyl species (gray whale (ERO), common hippopotamus (HAM), alpaca (LPA), and pig (SSC)) that have an identical order of the BACs and the same relative position of the centromere (located in XSB1).

2.
Centromere repositioning: conserved BAC order, changes in the centromere position. Centromere repositions have been shown in roe deer, and mouse-deer, resulting in metacentric (Siberian roe deer (CPY)) and acrocentric (Java mouse-deer (TJA)) X chromosomes, respectively. This event took place prior to a formation of some lineage specific ancestral chromosomes (RAX (Ruminant Ancestral X), AAX (Antilopinae Ancestral X), and CEAX (Cervinae Ancestral X)), indicating that centromere repositioning is one of the key rearrangements of the ruminant X: while maintaining a conserved order of the segments there was a displacement of the centromere ( Figure 1).

BACs Localization
We investigated the X chromosome structure across major branches of Cetartiodactyla represented by 18 species from four non-ruminant (Suidae, Camelidae, Eschrichtiidae (Cetacea), Hippopotamidae) and six ruminant (Tragulidae, Antilocapridae, Giraffidae, Moschidae, Cervidae, and Bovidae) families ( Table 1). The order of 26 labeled cattle BAC clones was established on the X chromosomes of each of 18 species by a series of pairwise FISH experiments ( Table 2). In total, comparative analyses of BAC orders across 18 species revealed three major chromosomal conservative segments, which were numbered and designated with colors used throughout the paper: X Syntenic Block 1 (13 BACs, XSB1, pink); X Syntenic Block 2 (seven BACs, XSB2, yellow), and; X Syntenic Block 3 (six BACs, XSB3, blue).

Intrachromosome Rearrangements
Comparative analysis of the order of BAC on the X chromosomes of 18 species identified three key scenarios that likely took place in the course of the cetartiodactyl X chromosomes' evolution.
1. Conservation: no change in the BAC order and no change of the centromere position. We identified a group of four basal cetartiodactyl species (gray whale (ERO), common hippopotamus (HAM), alpaca (LPA), and pig (SSC)) that have an identical order of the BACs and the same relative position of the centromere (located in XSB1). 2. Centromere repositioning: conserved BAC order, changes in the centromere position.
Centromere repositions have been shown in roe deer, and mouse-deer, resulting in metacentric (Siberian roe deer (CPY)) and acrocentric (Java mouse-deer (TJA)) X chromosomes, respectively. This event took place prior to a formation of some lineage specific ancestral chromosomes (RAX (Ruminant Ancestral X), AAX (Antilopinae Ancestral X), and CEAX (Cervinae Ancestral X)), indicating that centromere repositioning is one of the key rearrangements of the ruminant X: while maintaining a conserved order of the segments there was a displacement of the centromere (Figure 1)   Table 1.
Taken together, we found that inversions (paracentric and pericentric) and centromere shifts were key rearrangements in the course of X chromosome evolution in Cetartiodactyla. In addition to the described rearrangements, the nucleolar organizing region (containing clusters of 18S and 28S rDNA genes) were localized on the short arm of both X and Y sex chromosomes of the Java mouse-deer (TJA) [33].  Table 1.
Taken together, we found that inversions (paracentric and pericentric) and centromere shifts were key rearrangements in the course of X chromosome evolution in Cetartiodactyla. In addition to the described rearrangements, the nucleolar organizing region (containing clusters of 18S and 28S rDNA genes) were localized on the short arm of both X and Y sex chromosomes of the Java mouse-deer (TJA) [33].  Chromosome rearrangements were identified by changes in BAC order. Three major conservative segments are designated by different colors: pink-X syntenic block 1; yellow-X syntenic block 2, and; blue-X syntenic block 3. Individual BAC clones are shown with a different color in small colored circles on corresponding conservative segment. Centromere position is indicated by a black circle. The orientation of the conservative segments is indicated by the white arrowhead. Ancestral associations are shown in black rectangle (Cetartiodactyla ancestral X (CAX), Ruminantia ancestral X (RAX), Pecora ancestral X (PAX), Antilopinae ancestral X (AAX), Cervinae ancestral X (CEAX)). CR: centromere reposition. Inv: inversion.

Bioinformatic Analysis of Mammalian X chromosomes
To evaluate the unique conservation of mammalian X chromosomes [3] we calculated the coordinates of 26 BAC clone sequences in four Boreoeutherian non-cetartiodactyl genomes represented by Euarchontoglires: human (Primates); mouse, and rat (Rodentia), and; Laurasiatheria: horse (Perissodactyla). We have observed that three X chromosome syntenic blocks (XSB) found in Cetartiodactyla are conserved in Laurasiatheria and also in Euarchontoglires, indicating common Boreoeutherian structure of the X chromosome. It was previously reported that human, horse, and pig X chromosomes have similar gene order [3]. In general, this observation was confirmed by liftOver analyses (Table 3). We have identified several small inversions in XSB1 (human, horse) and in XSB2 (horse) in comparison to CAX. Interestingly, XSB1 is the most derived segment outside of Cetartiodactyla, no rearrangements in BACs order in the cetartiodactyl species were detected within this block. According to our data, XSB2 is highly conserved in non-cetartiodactyl species, while in ruminants there are inversions inside of this syntenic block (CHI, OMO, sheep (OAR)) and exchange inversions between XSB2 and XSB3 (TJA and DDA).
We also aligned the BAC clone sequences to another cetartiodactyl genome, the domestic sheep. We observed the same BAC order as in all analyzed Caprini species except for a small inversion in XSB3. The FISH with relevant BAC clones confirmed the presence of this inversion in the sheep genome.

Ancestral X chromosome
The phenomenon of X chromosome conservation in eutherian mammals was first proposed by Susumu Ohno and was based solely on its size similarity across a wide range of species [1]. High similarity in G-banding pattern led to the hypothesis that not only size and gene content [34] but also gene order is conserved on the X chromosomes of most eutherian mammals, and this was later confirmed by fine gene mapping [3,[35][36][37][38]. Remarkably, the submetacentric X chromosome morphology defined by the location of the centromere is also largely conserved across mammals. Some slight changes of otherwise conserved X chromosomes were observed in several orders, such as the difference in the distance between homologous genes between human and alpaca [39], or a shift in centromere position without a change of the gene order in Afrotheria [37]. Still, the lack or low level of rearrangements of the X chromosome in comparison to the active exchanges on autosomes during over 150 million years of eutherian evolution represents an interesting phenomenon. Comparative G-banding analysis had identified the classical chromosome X morphology and banding pattern common to most eutherian species [2]. Similar submetacentric morphology and gene order were also found in non-ruminant cetartiodactyls. A high level of X chromosome conservation was shown in Suinae [3,40], Tylopoda [41][42][43], and Cetacea [44]. Nevertheless, using G-banding analysis [4] and high-resolution mapping with BACs [25] or region specific probes, [12] intrachromosomal rearrangements were uncovered in Ruminantia species. Compared with the previous study, we have expanded the number of BACs to 26 and the species list to 18 in order to define conservative blocks and their orientation, to identify rearrangements across species, and to reconstruct the ancestral cetartiodactyl X chromosome. The analyses of BAC order across major families of Cetartiodactyla revealed three syntenic blocks on the X chromosome that in general correspond to the conserved segments reported by Fröhlich and coauthors [25].
Using available FISH and bioinformatic data on the order of cattle BACs in the genomes of different species, we were able to investigate the phenomenon of the conservation of the X chromosome in eutherian mammals represented by four superorders: Laurasiatheria; Euarchontoglires; Afrotheria, and; Xenarthra [45]. Three conserved syntenic blocks identified here can be traced in Boreoeutherians (Laurasiatheria and Euarchontoglires), and possibly in all eutherians, considering reports on Afrotheria X chromosome conserved gene order [37] ( Table 2). The eutherian X chromosome ancestral condition (EUX) is represented by a submetacentric chromosome with the centromere located in XSB1. Bioinformatic analysis in outgroup species shows a common change of BAC order in XSB1 on human and horse X chromosomes. Supposedly, an inversion on EUX had occurred in the ancestor of Cetartiodactyla prior the radiation of this order. This ancestral condition was revealed in all non-ruminant cetartiodactyls and named here Cetartiodactyla Ancestral X (CAX). We confirmed the conservation of the X chromosome in basal branches of Cetartiodactyla. It occurs in Suidae (pig), Camelidae (alpaca), and Cetacea (gray whale) ( Table 2 and Figure 3). Cetacea is a sister taxon to Hippopatamidae and is characterized by extremely conserved karyotypes across the whole infraorder and by uniform X chromosome morphology and banding pattern [11,46]. The Hippopotamidae X chromosome also displays the same morphology and the gene order [8,25]. However, it should be emphasized that there are some unresolved cases of the X chromosome changes in these basal groups that would require additional investigation, for example, the X chromosome of Tayassu pecari (Suinae, Taysuidae) has been changed due to a centromere reposition [40].
chromosome with the centromere located in XSB1. Bioinformatic analysis in outgroup species shows a common change of BAC order in XSB1 on human and horse X chromosomes. Supposedly, an inversion on EUX had occurred in the ancestor of Cetartiodactyla prior the radiation of this order. This ancestral condition was revealed in all non-ruminant cetartiodactyls and named here Cetartiodactyla Ancestral X (CAX). We confirmed the conservation of the X chromosome in basal branches of Cetartiodactyla. It occurs in Suidae (pig), Camelidae (alpaca), and Cetacea (gray whale) ( Table 2 and Figure 3). Cetacea is a sister taxon to Hippopatamidae and is characterized by extremely conserved karyotypes across the whole infraorder and by uniform X chromosome morphology and banding pattern [11,46]. The Hippopotamidae X chromosome also displays the same morphology and the gene order [8,25]. However, it should be emphasized that there are some unresolved cases of the X chromosome changes in these basal groups that would require additional investigation, for example, the X chromosome of Tayassu pecari (Suinae, Taysuidae) has been changed due to a centromere reposition [40].

Ancestral Form of Ruminantia-Pecora X-Chromosome
Contrary to the conservation of the X chromosome in Suidae-Camelidae-Whippomorpha, we found that multiple rearrangements occurred during the radiation of other cetartiodactyl branches. We suggest that in the Ruminantia an ancestral centromere reposition led to changes of the X chromosome morphology from submetacentric to metacentric forming the Ruminantia Ancestral X-chromosome (RAX) (Figures 2 and 3). Both ancestral forms (CAX and RAX) have same intrachromosomal structure and differ only by centromere position. The RAX form of the X chromosome is also preserved in many basal Pecora branches: Giraffidae (GCA); Moschidae (MMO); and in the Capreolini (AAL) subfamily of Cervidae. Only in the basal Pecoran family Antilocapridae, an inversion turned the ancestral metacentric X chromosome into an acrocentric element ( Figure 2). Thus we expect that the Ancestral Ruminant and the Ancestral Pecoran X chromosomes have the same structure: RAX=PAX.

Ancestral Form of Ruminantia-Pecora X-Chromosome
Contrary to the conservation of the X chromosome in Suidae-Camelidae-Whippomorpha, we found that multiple rearrangements occurred during the radiation of other cetartiodactyl branches. We suggest that in the Ruminantia an ancestral centromere reposition led to changes of the X chromosome morphology from submetacentric to metacentric forming the Ruminantia Ancestral X-chromosome (RAX) (Figures 2 and 3). Both ancestral forms (CAX and RAX) have same intrachromosomal structure and differ only by centromere position. The RAX form of the X chromosome is also preserved in many basal Pecora branches: Giraffidae (GCA); Moschidae (MMO); and in the Capreolini (AAL) subfamily of Cervidae. Only in the basal Pecoran family Antilocapridae, an inversion turned the ancestral metacentric X chromosome into an acrocentric element (Figure 2). Thus we expect that the Ancestral Ruminant and the Ancestral Pecoran X chromosomes have the same structure: RAX=PAX.
In the Tragulidae, the basal and the only non-Pecora ruminant group, we found a major centromere reposition resulted in the formation of an acrocentric X. Also, two kinds of inversions (SB-flip and synteny block exchange) affect syntenic block structure in the Tragulidae. These rearrangements create unique arrangement of the three syntenic blocks in the Java mouse-deer. This arrangement may occur across all tragulids, but requires confirmation in other Tragulus species.

Cervidae
There is a great variation in X chromosome morphology among cervids. Two cervid subfamilies, Capreolinae and Cervinae, exhibit a notably differing extent of sex chromosome conservation. The only detected rearrangement was a centromere shift in CPY. G-banding pattern comparison of Capreolinae X chromosomes otherwise indicates a uniform metacentric morphology [48] and suggests a similar disposition of conservative syntenic blocks.
In contrast, Cervinae is characterized by a variety of rearrangements on the X chromosome: centromere repositioning, SB flips, and many inversions disrupting the XSB2. The Cervinae Ancestral X-chromosome (CEAX) was formed by a centromere reposition and a SB flip of XSB2. Inversions change this ancestral form in EDA by SB flip of XSB3 and in DDA by the splitting of XSB2 (Figure 2). Also in the same subfamily, a translocation of an autosome to the X chromosome was reported in several Muntiacini species [7,[49][50][51][52]. In total, this indicates that the level of X chromosome variation is increased in Cervinae and is caused not only by inversions and centromere repositioning but also by autosome to sex chromosome translocations.

Bovidae
The family Bovidae includes two major branches: Bovinae and Antilopinae [53]. Earlier cytogenetic studies identified two types of morphological diversity of X chromosome in Bovidae: a caprine type (acrocentric, suni type) and a bovine type (submetacentric) [12,54]. The bovine type of X chromosome was likely formed from the ancestral pecoran X (PAX) by two inversions. This form is retained in cattle (BTA) and American bison (BBI). Cytogenetic data for other studied Bovinae species demonstrated same submetacentric X chromosome morphology [48]. There are independent autosome translocations in two branches (Tragelaphini and Bosephalini) altering the bovine type X chromosome [12,23,48,55,56]. The notable exceptions are the Bubalina lineage, oryx and kudu (Tragelaphilini), whose X chromosomes have acrocentric morphology (designated as eland-type acrocentric based on eland, kudu, and nyala X chromosomes [12]).
Centromere reposition and inversion events resulted in the formation of an acrocentric Antilopinae Ancestral X-chromosome (AAX) (Figure 2) from PAX. Therefore the X of the sable antelope (HNI) could likely represent an ancestral form for all Antilopinae. Moreover, comparative analyses based on published karyotypes supports the theory that the X chromosome in antelopes is largely conserved, retaining the same morphology and banding pattern [48,57]. The exceptions are autosome to X chromosome translocations found in several Antilopini species [48,58]. In the Caprini lineage there is an additional inversion within the XSB3 (OMO, CHI, OAR). The bioinformatic and FISH analyses of X chromosome of OAR indicated that the inversion between 128C9 and 229I15 is an apomorphic phylogenetic marker for Caprini.

X chromosome Rearrangements
All X chromosome rearrangements discovered here are in agreement with the current phylogenetic tree (Figure 3), and some of them could be used as cytogenetic markers for different Cetartiodactyla groups. Therefore, we suggest that our BAC clone set can serve as a precise instrument for a further search for cytogenetic X chromosome markers in Bovidae. The independent autosome to sex chromosome translocations that occurred in several Bovidae and Cervidae branches require special attention because they increase the previously identified rapid rate of evolution of the structure of the cetartiodactyl X chromosome [7,12,[49][50][51][52]55,56].
The BAC clones that mark the borders of three conserved segments delineate regions of frequent chromosome rearrangements in cetartiodactyl X, indicating a breakpoint reuse phenomenon [59]. Several BAC clones were involved at least twice in the intrachromosomal rearrangements found here, suggesting breakpoint reuse: 108D16 and 214A3; 514O22 and 316D2; 229I15 and 103E10. We found that the regions surroundings these BACs in the cattle genome are often gene sparse. It was previously shown that chromosomal regions with evolutionary breakpoint in amniotes are enriched for structural variations (segmental duplications, copy number variants, and indels), retrotransposons, zinc finger genes, and single nucleotide polymorphisms [60]. Further investigation is required to find precise points of evolutionary chromosome breakage on the Cetatiodactyla X and to define common genomic features underlying chromosome rearrangements.
Another mammalian order characterized by the increased rate of X chromosome evolution is Rodentia. Heterochromatin expansion, amplification of tandem repeats, inversions [61], centromere reposition [62], and autosome to sex chromosome translocations [63] were shown to be involved in rearrangements of X chromosome in rodents. Comparative chromosomal analysis of X chromosomes was performed by microdissection in the Microtus genus. Rubtsov with coauthors postulated that intrachromosomal rearrangements are associated with large clusters of intrachromosomal duplications and/or repeated DNA sequences which were present in ancestral species but have subsequently disappeared during evolution [61]. We hypothesize that similar processes were involved in evolution of X chromosome in Ruminantia. Some genomic events possibly took place in the ruminant ancestor that launched multiple chromosomal rearrangements of the conservative eutherian X chromosome. Insertions of mobile repetitive elements such as long and short interspersed nuclear elements (LINE and SINE were probably involved in synteny breaks on this sex chromosome [64]. It is possible that this transforming genomic event had happend in or around the XSB2 area which demonstrates highest rate of inversions in Ruminantia. In total, nine paracentric, two pericentric inversions, and five centromere reposition events have been revealed in Cetartyodactyla X chromosome evolution based on the analysis of 18 species. The eutherian and cetartiodactyl ancestral X differ only by one small inversion; one additional rearrangement is proposed to derive the Ruminantia ancestral X (RAX). Most other identified rearrangements happend during the remaining 55 million years of ruminant's radiation. The cow X chromosome was formed by at least two rearrangements that distinguish it from PAX, corresponding to a rate of rearrangements of approximately 1 per 15 million years. This is comparable to 1 rearrangement per 10 million years postulated for autosomal evolution among most mammalian orders found by chromosome painting [65]. These findings are consistent with the rate of X chromosome evolution in Ruminantia being at least twice as high as in X chromosomes of average eutherian mammalian group.

Conclusions
High-resolution X chromosome maps of cetartiodactyl species provide unique information about evolution of intrachromosomal rearrangements. Three conserved syntenic blocks have been identified. We postulate that inversions and centromere repositioning were two key types of rearrangements in course of cetartiodactyl X chromosome evolution. The detailed analysis of the BAC order across multiple species by FISH mapping and bioinformatic analysis allowed the reconstruction of a putative cetartiodactyl ancestral X chromosome. The basal cetartiodactyl group of non-ruminants (pigs, camels, whales, and hippos) share this metacentric ancestral type of X chromosome. The submetacentric ancestral Ruminantia X chromosome was likely formed by simple centromere shift but it retained the ancestral intrachromosomal structure. Currently observed X chromosome morphological variation was formed by inversions and centromere repositioning during 55 million years of ruminant evolution. Chromosome rearrangements supporting the taxonomic status of ruminant families and subfamilies were found by mapping 26 BAC clones specific to the X chromosome. The rate of X-specific rearrangements in Ruminantia significantly exceeds that among eutherian mammals.