Comparative Chromosome Painting in Six Species of Oligoryzomys (Rodentia, Sigmodontinae) and the Karyotype Evolution of the Genus

Oligoryzomys belongs to the tribe Oryzomyini, and contains about 22 species. Diploid numbers range from 2n = 44 in Oligoryzomys sp. 2 to 2n = 72 in O. utiaritensis and phylogenetic relationships are not well defined. The high morphological convergence leads to misidentification of taxonomic entities and the species are often identified by chromosomal characters. Until now, the genus has been studied only by classical cytogenetic approaches. To understand the chromosomal evolution of Oligoryzomys, we developed chromosome probes from a female of Oligoryzomys moojeni (OMO) with 2n = 70 and hybridized to other five Oligoryzomys species. The probes painted 31 segments on O. fornesi (OFO) with 2n = 62; 32 segments on O. microtis (OMI), 2n = 64; 33 segments on O. nigripes (ONI), 2n = 62 and on O. rupestris (ORU), 2n = 46; and 34 on Oligoryzomys sp. 2 (OSP), 2n = 44. OMO probes 4 and 5 showed a syntenic association in O. fornesi, O. microtis and O. nigripes and were also presented in the same pair, although disrupted, in O. rupestris and Oligoryzomys sp. 2. Concerning O. rupestris and Oligoryzomys sp. 2, species with the lowest diploid numbers of the genus, a total of 8 probes hybridized to 11 segments on the largest pair of ORU 1 and 9 probes hybridized to 12 segments on OSP 1. Also, OMO 6 painted three segments in ORU, corresponding to the proximal segment of ORU 2q, and the whole of ORU 19 and 20. In OSP, the segment corresponding to ORU 20 was homologous to OSP 1p. OMO X showed signals of hybridization in both X and Y chromosomes. Extensive chromosomal rearrangements, that could not be detected by classical cytogenetic techniques, such as pericentric inversions or repositioning of centromeres, Robertsonian rearrangements and tandem fusions/fissions, as well as gain/activation or loss/inactivation of centromeres and telomeric sequences have driven the huge genome reshuffling in these closely related species.


Introduction
Rodents of the subfamily Sigmodontinae represent one of the most complex groups of New World mammals [1]. Traditionally, Sigmodontinae have been divided into tribes [2,3], and nowadays nine tribes are recognized, in addition to several incertae sedis genera that could not be grouped in any tribe [1,3].
Oryzomyini is the most diverse Sigmodontinae tribe and this is reflected in morphological, ecological and chromosomal variations which lead to taxonomic controversies [4,5]. One of the most complex genus in terms of taxonomic issues and number of species is Oligoryzomys.
Oligoryzomys is a speciose genus widely distributed from Mexico to Argentina, [6]. Phylogenetic analyses using morphology, allozymes and DNA sequences agree on the monophyly of the genus although the hierarchical relationships among species are not well established [7][8][9][10][11]. Recently, Agrellos et al. [12] based on nuclear (intron 7 of the beta-fibrinogen) and mitochondrial (cytocrome b) gene sequences, found O. microtis to be the sister group of all other Oligoryzomys species while O. moojeni and O. utiaritensis clustered together in the more derived clade.
The high morphological similarity generates difficulties in establishing the real number of species [12,13]. Heretofore, 22 species have been recognized but taxonomic problems still persist. Because of this, most species of the genus have been diagnosed by diploid (2n) and fundamental number (FN), instead of morphology [11,14].
Silva and Yonenaga-Yassuda [15] described the karyotypes of O. rupestris (2n = 46, FN = 52) and Oligoryzomys sp. 2 (2n = 44, FN = 52) and suggested that centric fusion was responsible for differences in diploid number between these species. In addition, when compared with other Oligoryzomys species, O. rupestris and Oligoryzomys sp. 2 present very distinctive karyotypes, with macro-and micro-chromosomes [15,16]. In contrast, the majority Oligoryzomys species possess chromosomes with a gradual variation in size and mostly with acrocentric morphology.
Heretofore, cytogenetic studies in Oligoryzomys used only banding patterns and fluorescent in situ hybridization (FISH) with telomeric probes [15,16]. However, cytogenetic comparison using banding patterns may not be informative enough when it comes to species with highly divergent genomes, such as Oligoryzomys [19,20].
This work brings to light a more refined perspective on karyotype evolution of Oligoryzomys as it is the first study using chromosome painting with species-specific probes in such a large number of species. The aim of this work is to investigate chromosome homologies among Oligoryzomys species and infer the rearrangements that have occurred during karyotype evolution, based on a published molecular phylogeny of the genus [12].

Material and Methods Sample
The sample comprises seven species of Oligoryzomys from different localities of Brazil (Table 1) [15]. Animals were euthanized according to the protocol of the "Animal experimentation ethics" [21]. The experiments were conducted according to the Committee on the Ethics of Animal Experiments of the Instituto Butantan (Comissão de ética no uso de animais do Instituto Butantan-permit number: 242/05). Skins and skulls were deposited at the Museu de Zoologia da Universidade de São Paulo (MZUSP). Spread metaphases of OFO, OMI, OMO, ONI, ORU and OSP were obtained from cell cultures, and in the case of OFL, from bone marrow.

Generation of Oligoryzomys moojeni (OMO) chromosome paints
Specific painting probes were generated from fibroblast cultures of a female of O. moojeni, with 2n = 70 and heteromorphic X chromosomes, in the Cambridge Resource Centre for Comparative Genomics, Department of Veterinary Medicine, University of Cambridge, UK. The whole chromosome probes were made by degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) as previously described [22,23]. Briefly, the chromosomes were prepared as described and stained with Hoechst 33258 (2 μg/ml) and Chromomycin A3 (40 μg/ml) in the presence of magnesium sulfate (2.5 mmol/l) for 2 h. Sodium sulfite (25 mmol/l) and sodium citrate (10 mmol/l) were added 15 min prior to flow sorting. Chromosome sorting was performed using a dual-laser cell sorter (MoFlo; Beckman Coulter). Approximately 400 chromosomes were sorted from each peak in the flow karyotypes directly into PCR tubes containing 30 μl of distilled water. Each sample was amplified by DOP-PCR using the primer 6MW [22]. Primary PCR products were labeled with biotin-16-dUTP (Boehringer Mannheim) or fluorescein isothiocyanate (FITC)-12-dUTP (Amersham) by taking 1 μl of product to a second round of DOP-PCR using the same primer. The biotin probes were detected with avidin-Cy3 or avidin-FITC.

Results
Flow sorting of O. moojeni (2n = 70, XX) The karyotype of the female used for sorting had 32 acrocentric pairs (pair 1 is clearly the largest of the complement), two small biarmed pairs (33 and 34) and heteromorphic sex chromosomes: submetacentric (Xa) and subtelocentric (Xb) (Fig. 1a). The flow karyotype yielded 30 peaks in which only 23 hybridized to single pairs. Four peaks painted two pairs (one of them could be identified after double color hybridization) and three peaks painted three or four pairs. From that, 24 out of the 30 peaks could be identified (Fig. 1b) by fluorescent in situ hybridization to DAPI-banded metaphases (OMO Xa, Xb, 1-8, 9/10, 11-13, 16, 17, 25-30, 33, and 34). The double peak identified corresponded to pairs OMO 9/10. X chromosomes were separated into different peaks due to differences in size (detected after measurements) and in the amount of constitutive heterochromatin located in the short arm of both Xa and Xb, although Xa has a larger C+ block (data not shown). The remaining peaks could not be identified since they are composed of more than one pair and probably they overlap with other identified peaks; therefore they are not shown on Fig. 1b Fig. 2, including the pattern of hybridization found for each one. Also, a compilation of hybridization patterns is shown in Fig. 3. O. flavescens has 2n = 64-66 and FN = 66, with 29 acrocentric pairs decreasing in size  and two small biarmed pairs (30 and 31). Pair 1 is the largest of the complement (Fig. 2b). Sex chromosome polymorphisms have been described as well as 1 to 4 supernumeraries (Bs) [14,17].
The hybridization of OMO Xa and OMO Xb probes onto metaphases of O. flavescens, male, with two Bs, showed signals in OFL X entirely and in the short arm of the OFL Y. The karyotype of O. fornesi comprises 28 acrocentric pairs (pair 1 being the largest of the complement), one medium submetacentric (pair 29) and one small metacentric (pair 30) (Fig. 2c) [24].
The hybridization of OMO probes onto OMI metaphases revealed 32 homologous segments ( O. nigripes presents 2n = 62 with 11 biarmed pairs and 19 acrocentric pairs decreasing in size (Fig. 2e). The fundamental number of this species varies from 78 to 82 due to pericentric inversions in pairs 2, 3, 4 and 8, previously revealed by comparison of banding patterns [18].

Discussion
Zoo-FISH has been widely used for studies of chromosomal evolution, nevertheless this type of data is still scarce for Neotropical rodents of the subfamily Sigmodontinae. The pioneering work with Zoo-FISH in Sigmodontinae rodents was performed by Fagundes et al. [25] with microdissected chromosome 1 of Akodon cursor (2n = 16). This probe was hybridized onto A. cursor (2n = 14 and 2n = 15) and A. montensis (2n = 24) metaphases and revealed several homologies.
Hass et al. [26] used Mus musculus probes to establish chromosome homology among Akodon cursor, A. montensis, A. paranaensis and A. serrensis and reconstructed the phylogenetic relationship among these species using Oligoryzomys flavescens as the outgroup.
Rodents of the tribe Akodontini were also studied by Ventura et al. [27] using reciprocal chromosome painting with Akodon paranaensis, A. cursor and Akodon sp. n. probes. This work revealed extensive chromosome rearrangements among Akodon species.
Up to now, the only study with species-specific paints in Oryzomyini tribe was performed by Nagamachi et al. [28] using Hylaemys megacephalus (2n = 54) probes in metaphases of Cerradomys langguthi (2n = 46). Results showed a large number of chromosome rearrangements. However, the authors did not consider the position of both species on the Oryzomyini phylogeny [5], so the rearrangements were merely descriptive with no correlation with the evolutionary context.
In the present paper, comparative chromosome painting, using O. moojeni probes in metaphases of five other Oligoryzomys species revealed extensive genomic reshuffling between very closely related species. Of the 24 O. moojeni probes, 14 (OMO 2,3,7,8,11,12,16,17,25,26,27,29,30 and 34) Fig. 3). The remainder of the probes produced more than one hybridization signal in at least one species, showing that they are rearranged in the genomes of the species studied.
Independently of the use of a phylogeny to interpret the direction of the rearrangements, is it possible to infer by our hybridization results that extensive chromosomal rearrangements such as pericentric inversions or repositioning of centromeres, Robertsonian rearrangements and tandem fusion/ fission, gain/ activation or loss/inactivation of centromeres and telomeric sequences have occurred in these closely related species of Oligoryzomys.
Additionally, in order to infer the chromosome rearrangements directions that have occurred during the karyotype evolution of the genus, we compared our results of chromosome painting to the phylogeny of Oligoryzomys based on molecular data, published by Agrellos et al. [12], in which we highlighted the rearrangements obtained herein (Fig. 4, Table 2). This molecular phylogeny encompasses the highest number of species, including all the species studied herein, except for Oligoryzomys sp. 2 that represent an undescribed species. Chromosomal rearrangements in Oligoryzomys revealed by chromosome painting Comparative G-banding patterns have indicated total homology among the largest chromosome (pair 1) of five Oligoryzomys species [14]. However, in O. nigripes, cross-species chromosome painting with OMO 1 showed that this chromosome is homologous to two chromosomes pairs, ONI 1 and 28 (Table 2). Considering the current phylogeny, we can infer the occurrence of fission involving ONI 1 and ONI 28, followed by pericentric inversion or centromere repositioning events yielding the subtelocentric ONI 1.  Additionally, as showed in Table 2 (Fig. 6) (Fig. 7). In addition, ORU 2/ OSP 2 were hybridized by four probes and ORU 3/ OSP 3 by three probes (Fig. 8), showing that tandem fusion events occurred during karyotype differentiation of both species, providing the lowest known diploid number of the genus.
We also should bring here again the information regarding OMO 4 and OMO 5 probes, which are disrupted in all species studied and this situation cannot be showed clearly in Fig. 4 (see table 2).
Results indicate loss, inactivation or centromeric repositioning, since tandem fusions formed the three largest pairs of O. rupestris and Oligoryzomys sp. 2. In addition, as we were not able to use all probes, more chromosomes than observed here can be involved in tandem fusions in these pairs.
Chromosome painting corroborates previous data that centric fusion was the mechanism responsible for the difference in diploid number of both species [15] (Fig. 9).

Sex chromosomes homologies
Both OMO Xa and OMO Xb hybridized to the whole X chromosome of all species, corroborating that the mammalian X chromosome is conserved. In O. flavescens, OMO Xa and Xb did not show positive signals in supernumerary chromosomes, indicating that in the case of this species, no homology were found between Bs and X chromosome. In contrast, Silva and Yonenaga-Yassuda [29] showed homology of Nectomys squamipes B chromosome and the constitutive heterochromatin the X chromosome of this species.
In O. flavescens, O. fornesi, O. microtis, O. moojeni, O. nigripes and Oligoryzomys sp. 2, OMO Xa and OMO Xb showed homology with the euchromatic region of Y chromosomes (Fig. 10). This could be related to the pseudoautosomal region as described in other rodents of the subfamily Arvicolinae and Sigmodontinae as well [30][31][32].

Chromosome evolution in Oligoryzomys
The present study shows that complex rearrangements are involved in the karyotype evolution of Oligoryzomys species. Nevertheless, FISH data using telomeric probes demonstrate signals  [14]. One possible explanation is that telomeric repeats may have been eliminated by chromosome breakages [33]. On the other hand, the presence of interstitial telomeric sequences (ITS) could not mean that these sequences are involved in rearrangements, since ITS were also observed in highly conserved karyotypes [34] and blocks of constitutive heterochromatin [35,36].  There are relatively few studies that compare chromosome painting with a phylogeny based on molecular data. According to the phylogeny, we could infer the occurrence of many tandem fusion events, since single OMO probes (the most derived species of the genus) painted two or more regions of different chromosomes of Oligoryzomys microtis (that have diverged earlier in the phylogeny) ( Table 2).
However, the phylogenetic relationships of Oligoryzomys species remain unclear and this is the first phylogeny in which O. moojeni belongs to the most derived clade [12]. Still, we consider it important to integrate cytogenetic and molecular data in order to understand the karyotype evolution of such a complex genus as Oligoryzomys.

Conclusions
Chromosomal evolution data in Oligoryzomys is being presented for the first time. Cross species chromosome painting revealed an extensive chromosomal reorganization despite the absence of interstitial telomeric sequence. Oligoryzomys rupestris and Oligoryzomys sp. 2 are the species with the most rearranged karyotypes and at least 17 tandem fusions originate the three largerst pairs of both species. Besides, a centric fusion originates the difference in diploid number of both species (2n = 46 and 2n = 44, respectively). Chromosome painting in Oligoryzomys species shows that pericentric inversions, fissions, tandem and Robertsonian fusions, centromeric loss/ inactivation, gain/activation or repositioning have occurred during karyotype evolution of the genus.
Also, although it is not easy to determine the direction of chromosome change, it is possible to infer that in Oligoryzomys, chromosomal evolution has been associated with both, decrease and increase in diploid numbers, in contrast with other groups in which diploid number tends to decrease during their evolution. These data indicate that all those closely related species have experienced recent autosomal rearrangement.