Phylogenomics of the dog and fox family (Canidae, Carnivora) revealed by chromosome painting

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1 Chromosome Research (2008) 16:129Y143 # Springer 2007 DOI: /s Phylogenomics of the dog and fox family (Canidae, Carnivora) revealed by chromosome painting Alexander S. Graphodatsky 1 *, Polina L. Perelman 1,2, Natalya V. Sokolovskaya 1, Violetta R. Beklemisheva 1, Natalya A. Serdukova 1, Gauthier Dobigny 3, Stephen J. O_Brien 2, Malcolm A. Ferguson-Smith 4 & Fengtang Yang 5 1 Institute of Cytology and Genetics, SB RAS, Novosibirsk, Russia; graf@bionet.nsc.ru; 2 Laboratory of Genomic Diversity, National Cancer Institute, Frederick, MD 21702, USA; 3 Institut de Recherche pour le Développement, Centre de Biologie et Gestion des Populations, Campus International de Baillarguet, CS30016, Montferrier-sur-Lez, France; 4 Cambridge Resource Centre for Comparative Genomics, Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB3 0ES, UK; 5 The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK * Correspondence Key words: Canidae, Carnivora, chromosome maps, chromosome painting, evolution, genome, phylogeny Abstract Canid species (dogs and foxes) have highly rearranged karyotypes and thus represent a challenge for conventional comparative cytogenetic studies. Among them, the domestic dog is one of the best-mapped species in mammals, constituting an ideal reference genome for comparative genomic study. Here we report the results of genomewide comparative mapping of dog chromosome-specific probes onto chromosomes of the dhole, fennec fox, and gray fox, as well as the mapping of red fox chromosome-specific probes onto chromosomes of the corsac fox. We also present an integrated comparative chromosome map between the species studied here and all canids studied previously. The integrated map demonstrates an extensive conservation of whole chromosome arms across different canid species. In addition, we have generated a comprehensive genome phylogeny for the Canidae on the basis of the chromosome rearrangements revealed by comparative painting. This genome phylogeny has provided new insights into the karyotypic relationships among the canids. Our results, together with published data, allow the formulation of a likely Canidae ancestral karyotype (CAK, 2n = 82), and reveal that at least 6Y24 chromosomal fission/fusion events are needed to convert the CAK karyotype to that of the modern canids. Abbreviations ACK Ancestral Carnivore Karyotype ALA Alopex lagopus, Arctic fox CAK CAL CFA Canidae Ancestral Karyotype Cuon alpinus, dhole Canis familiaris, domestic dog Electronic supplementary material The online version of this article (doi: /s ) contains supplementary material, which is available to authorized users.

2 130 A. S. Graphodatsky et al. CTH FZE HSA HMA MYa NPRp NPRv TOR UCI VCO VVE VVU Introduction Cerdocyon thous, crab-eating fox Fennecus zerda, fennec fox Homo sapiens, human Helarctos malayanus, Malaysian sun bear million years ago Nycteruetes procyonoides procyonoides, Chinese raccoon dog Nycteruetes procyonoides viverrinus, Japanese raccoon dog Tremarctos ornatus, spectacled bear Urocyon cinereoargenteus, gray fox Vulpes corsac, corsac fox Vulpes velox, kit fox Vulpes vulpes, red fox The domestic dog, red fox and 34 other fox and dog species belong to the same family (Canidae) and diverged from a common ancestor some 10Y12 million years ago (Wayne et al. 1997). Lindblad- Toh et al. (2005) stated Fman_s best friend, Canis familiaris, occupies a special niche in genomics. The unique breeding history of the domestic dog provides an unparalleled opportunity to explore the genetic basis of disease susceptibility, morphological variation and behavioral traits._ The dog has well-developed meiotic and radiation hybrid linkage maps (Breen et al. 2004), and the canine genome sequence (Lindblad-Toh et al. 2005) anchor to these maps. Significant progress was made also in developing a meiotic linkage map of chromosomes for another closely related species, the red fox, Vulpes vulpes (Kukekova et al. 2007). All extant canids so far studied by genomic methods have shown unique peculiarity in relation to other carnivore species investigated hitherto. While the karyotypes of felids, mustelids, ursids, pinnipeds, etc, closely resemble the ancestral karyotype of the mammalian founder, the dog and fox karyotypes are among the most rearranged in mammals. Human chromosome probes detected 30Y35 homologous regions on the chromosomes of cats, weasels, lesser panda, pinnipeds, civets, and hyenas and 43Y45 regions in the karyotypes of bears and giant panda, while more than 70 regions were detected in the canine karyotypes (Yang et al. 1999, Yang & Graphodatsky 2004, Graphodatsky 2007). These data, along with comparative gene mapping data in mouse and human, indicate that so far only the murids (including the laboratory mouse) have more rearranged karyotypes than the canids (Yang et al. 1999, Graphodatsky 2007). Almost all conserved segments that are characteristic for the ancestor of mammals and, in particular, the carnivores have broken into several pieces in the canine genome (Yang et al. 1999). The domestic dog, with the highest diploid number and the most highly rearranged karyotype as well as the most advanced genome map in Carnivora, constitutes the ideal reference species for high-resolution comparative genomic analysis of carnivores. The use of paint probes derived from the dog chromosomes provides us with unprecedentedly high resolution for comparative cytogenetic analysis of carnivore genome organization. Although comparative chromosome painting studies have confirmed the majority of early findings based on comparative banding (Graphodatsky &Radjabli1981,Mäkinen & Gustavsson 1982, Wayne et al. 1987a,b, Graphodatsky et al. 1995), they did reveal many erroneous assignments in interspecies homology, in addition to providing a genome-wide view of chromosomal correspondence. There is thus a need to further verify those early comparative cytogenetic data by molecular cytogenetic approaches (Graphodatsky et al. 2001). Recently, painting probes derived from a dog, red fox and Japanese raccoon dog have been applied to comparative cytogenetics of some canids, including the domestic dog, red fox, Arctic fox, crabeating fox, and Chinese and Japanese raccoon dogs (Yang et al. 1999, Graphodatsky et al. 2000a, 2001; Nash et al. 2001, Trifonov et al. 2002, Nie et al. 2003). By the combination of multiple sets of molecular data, complete phylogenies for Canidae species have been proposed (Bininda-Emonds et al. 1999, Vila et al. 1999, Bardeleben et al. 2005, Lindblad-Toh et al. 2005). But these trees show only the phylogenetic relationships among different taxa with no information on changes in genome organization. Here we describe the mapping of dog chromosome-specific probes to the chromosomes of dhole, fennec fox, and gray fox, as well as the assignment of red fox chromosome-specific probes to the chromosomes of corsac fox, the construction of a comparative map of chromosomes of these species, and its integration with the comparative cytogenetic map of all previously studied canids. On the basis of these data, we performed a phylogenomic analysis as well as a mapping of the chromosomal changes onto a previously published molecular topology (Lindblad- Toh et al. 2005). We discuss the evolution of the

3 Phylogenomics of the dog and fox family 131 canid genome architecture in the different lineages and reconstruct the Canid Ancestral Karyotype (CAK). Materials and methods Metaphase preparations and G-banding The fennec fox and gray fox metaphase preparations were made from established fibroblast cell lines (Laboratory of Genomic Diversity, National Cancer Institute, Frederick, MD, USA). The metaphases from the dhole and corsac fox (both animals from the Novosibirsk Zoo) were prepared from peripheral blood cultures stimulated with a combination of mitogens: pokeweed, phytohemagglutinin and conconavalin A (Gibco). G-banding followed the method described in Graphodatsky et al. (1995). Previously prepared chromosomal suspensions of other species were used in other FISH experiments: red fox, Vulpes vulpes, Arctic fox, Alopex lagopus, Chinese raccoon dog, Nyctereutes procyonoides procyonoides, and Japanese raccoon dog, Nyctereutes procyonoides viverrinus (Graphodatsky et al. 2000a, 2001). Table 1 lists the studied species, their scientific and common names, and references on Zoo-FISH studies. Fluorescent in situ hybridization (FISH) The sets of dog and red fox painting probes used in this study have been described previously (Yang et al. 1999, Graphodatsky et al. 2000a). G-banding prior to FISH was performed using trypsin treatment (Seabright 1971). FISH was performed following previously published protocols (Yang et al. 1999, Graphodatsky et al. 2000a). Phylogenomic reconstruction Following Dobigny et al. (2004), the differences in genome architecture were used to define 88 binary characters that correspond to synteny association or disruption. Dog chromosomes were used as a reference to define all genome-wide homologies between 10 canid species as well as 2 ursid species, Tremarctos ornatus (TOR) and Helarctos malayanus (HMA), that were used as outgroups (Tian et al. 2004, Yang & Graphodatsky 2004). The resulting matrix is provided in the supplementary material as Table S1. Importantly, the 18a/38 and 18b/38 characters could not be unambiguously identified in the gray fox, and were then coded as F?_ for this species. Similarly, all four Vulpes karyotypes display a 18a/38/18b association which may result from an inversion or insertion involving a previously existing 18a/18b. We chose to code this particular character as F?_ for these four taxa. This matrix was then investigated through a maximum parsimony analysis (heuristic search, Branch Swapping Option in PAUP 4.0b; Swofford 1998). Chromosome changes were polarized a posteriori using the outgroup comparison criteria, and chromosomal changes were then mapped onto the inferred tree. Global statistics (Consistency Index (CI) and Retention Index (RI)) were calculated, and support was evaluated through a 1000-replicate bootstrap analysis. Table 1. List of canid species studied by the Zoo-FISH technique Species and abbreviation Common name 2n Reference Canis familiaris (CFA) Domestic dog 78 Yang et al. (1999); Graphodatsky et al. (2000a) Cuon alpinus (CAL) Dhole 78 This study Cerdocyon thous (CTH) Crab-eating fox 74 Nash et al. (2001) Alopex lagopus (ALA) Arctic fox 50 Graphodatsky et al. (2000a); Nash et al. (2001) Vulpes velox (VVE) Kit fox 50 Nash et al. (2001) Vulpes vulpes (VVU) Red fox 34 + B_s Yang et al. (1999); Graphodatsky et al. (2000a) Vulpes corsac (VCO) Cape fox 36 This study Fennecus zerda (FZE) Fennec fox 64 This study Nycteruetes procyonoides procyonoides (NPRp) Chinese raccoon dog 38 + B_s Nie et al. (2003) Nycteruetes procyonoides viverrinus (NPRv) Japanese raccoon dog 54 + B_s Graphodatsky et al. (2001); Nash et al. (2001) Urocyon cinereoargenteus (UCI) Gray fox 66 This study

4 132 A. S. Graphodatsky et al. Results Chromosome painting of dog and fox probes onto canids The complete set of dog chromosomal probes was successfully hybridized to the G-banded metaphase spreads of dhole, Cuon alpinus, fennec fox, Fennecus zerda, gray fox, Urocyon cinereoargenteus. Examples of fluorescence in situ hybridization are shown in Figure 1. In the dhole, Cuon alpinus (CAL, 2n = 78), the 39 probes, corresponding to the 38 autosomes plus the X chromosome of domestic dog, Canis familiaris (CFA, 2n = 78), each mapped to one chromosome only (Figures 1 and 2, Table 2) delineating 39 homologous segments in total. G-banding comparison shows that the dhole and dog have identical karyotypes. It is noteworthy that many dog paints produced signals in the heterochromatic centromeric regions of dhole chromosomes. In the fennec fox, Fennecus zerda (FZE, 2n = 64), as in the dhole genome, most dog probes each mapped to one segment of the fennec chromosomes. However, chromosome 1, 13, 18, and 19 probes each hybridized to two discrete regions (Figures 1 and 3a, Supplementary Figure S1 and Table 2). Figure 3a shows a comparative map of the G-banded chromosomes of the fennec fox and domestic dog defined by Zoo-FISH. In the gray fox, Urocyon cinereoargenteus (UCI, 2n = 66), most dog probes each mapped to one segment, although chromosome 1, 2, 13, 18, and 19 probes each hybridized to two segments (Figures 1 and 3b, Supplementary Figure S1 and Table 2). In total, all dog probes delineated 44 homologous segments in U. cinereoargenteus. Figure 3b shows a comparative map of the G-banded chromosomes of the gray fox and domestic dog defined by Zoo-FISH. In the corsac fox, Vulpes corsac (VCO, 2n = 36), the 18 probes, corresponding to the autosomes and the X chromosome of the red fox, Vulpes vulpes (VVU, 2n = Y8B_s), each hybridized to two segments (Figures 1 and 4a and Table 2). The red fox chromosome VVU 6 probe painted to the VCO 6q and VCO 11q, with very bright additional signals at the telomeric regions of VCO 6 and 11 and six other VCO autosomes. This pattern suggests that this red fox probe contains sequences amplified in the corsac fox genome in the form of appreciable telomeric heterochromatic blocks (Figure 1). In the other canids studied so far, this paint, however, did not produce any additional signals, except for autosomal areas, homologous to the p and q-arms of VVU6. Red fox and corsac fox belong to the same genus, Vulpes, but they do not share a single entire autosome. Our results of comparative analysis of banding patterns of homologous chromosomes defined by painting are presented in Figure 4b and confirm that the karyotypes of corsac and red fox could have evolved through centric and tandem fusions of ancestral chromosomes, but with different combinations in the V. vulpes and V. corsac branches. The results of reciprocal chromosome painting between the red fox and dog established a comparative chromosome map illustrating the one-to-one correspondence between the chromosomes of these two canid species (Yang et al. 1999). This map allows us to extrapolate the chromosomal correspondence with the dog from the red fox genome on those of the corsac fox genome (Table 2; Figure 4a). Data presented here have also revealed a few erroneous homology assignments inferred previously between chromosomes of the Arctic fox and red fox (Graphodatsky et al. 2000a). Previously it was suggested that chromosome 2 of the red fox (VVU) was homologous to three chromosomes of the Arctic fox based on the painting of dog chromosomespecific probes (Graphodatsky et al. 2000a). However, direct localization of VVU2 probe on Arctic fox chromosomes (Figure 1) has detected only two homologous segments (ALA12 and ALA8q). Our new painting results have further demonstrated the following chromosomal correspondence: (1) the Arctic fox chromosome ALA12 corresponds to the VVU2p of the red fox and CFA13a and CFA9 of the dog; (2) ALA9 corresponds to one half of the VVU13 and CFA13b and CFA34; (3) ALA11 corresponds to parts of the VVU3 and VVU5 or CFA36, CFA18a, CFA38, CFA18b, respectively (Figure 5; Table 2). Phylogenomic analysis One single tree (L = 89, CI = 0.989, RI = 0.982) was retrieved by our maximum-parsimony analysis (Supplementary Figure S2 and Table S1, Figure 6, blue frame). All characters but one were found synapomorphic (n = 27) or autapomorphic (n = 60). The wolflike (dog, dhole and crab-eating fox) and foxes +

5 Phylogenomics of the dog and fox family 133 Figure 1. Examples of chromosome painting onto G-banded metaphases. Images on the right of panels (ayf) represent the G-banded metaphases, while the images on the left show the same spreads after FISH. Paint specific for domestic dog chromosome 1 (CFA1) was hybridized onto dhole chromosome 1 (a), paints specific for dog chromosome CFA18(green) and CFA38 (red) were hybridized onto gray fox chromosomes 4 and 16 (b) and fennec fox chromosome 10 (c). Paint specific for red fox chromosome 16 (VVU16) was hybridized onto corsac fox chromosomes 10 and 14 (d). Paint specific for red fox chromosome 6 (VVU6) was hybridized onto corsac fox chromosomes 2, 4, 5, 6, 7, 8, 9, 10, 11 (e, see explanation in the text). Paint specific for red fox chromosome 2 (VVU2) was hybridized onto arctic fox chromosomes 8 and 12 (f). Chromosome numbers of painted chromosomes are indicated on the banded metaphases. Scale bar = 10 2m.

6 134 A. S. Graphodatsky et al. Figure 2. Summary of the hybridization pattern of domestic dog probes onto G-banded dhole chromosomes. Black lines show homologous segments of domestic dog chromosomes. Dhole chromosomes are numbered below and domestic dog chromosome homology is shown laterally.

7 Phylogenomics of the dog and fox family 135 Table 2. Chromosomal correspondence between domestic dog (CFA), dhole (CAL), crab-eating fox (CTH), red fox (VVU), corsac fox (VCO), Arctic fox (ALA), fennec fox (FZE), raccoon dog (NPR) and gray fox (UCI) CFA CAL CTH VVU VCO ALA FZE NPR UCI p 6p 6p 21 7p 18 19a 19a 21a 4p 6q 6p 21 7p 18 19b 19b 21b 5p 4q 24q 22 10q 2 1a 1a 1a 5p 4p 24q 22 10q 21 1b 1b 1b 1p 4qter 2q 6 3q q 16q 9q 2q 1q 19 13a 13a 18a 13p 16p 9p 2p 1q 19 13b 13b 18b 2p 1p 12p 9 2q p 1p 12q 9 13q q 13q 8q 7 14q q 14q 4q 8 2q q 5q 3q 3 4q q 1q 10q 5 5q c 12q 1q 10p 5 3p q 9q 3p 14 8q q 15q 1p 11 7q q 6q 6q 12 8q q 10q 5q 16 12q p 7q 7q 15 9q q 2q 1q 4 15q q 2q 1q 4 10p q 7p 13q 18 1p q 12p 23q 19 9p p 2p 4p 1q 18q q 17pq 5p 17 11q 14 18a 18a 24a 5 q qter 11q ter 11pa 16 18b 18b 24b 5q qprox 11q prox 11pb b 5q 3q 11q 10 13p q 8p 7p 23 4p p 11p 15q 24 10p p 11q 16q 25 8p q 10p 19q 26 2p p 5p 18q 27 12p q 8q 2p 13 6q q 8p 2p 13 6q p 15p 14q 1p 3p p 9p 21q 29 1q p 12q 8p 20 6p p 3p 17q 28 5p p 14p 20q 31 6q p 13p 22q 30 17q a 3p 3p 11p 3 17q 15 X X X X X X X X X Y Y Y Y Y Y Y Y Chromosomal signatures discussed in the text are marked by various colors. raccoon dogs (red, corsac, Arctic and fennec foxes, sister to the two raccoon dog species) clades were retrieved as sister lineages, whereas the gray fox was basal to all canids investigated here (Figure 6). Global support was very high (CI = 0.989, RI = 0.982), confirming the very low level of homoplasy. Bootstrap values were moderate to very high (BP = 61Y100; see Figure 6), something that is expected with characters of this morphological-like type (Dobigny et al. 2004). However, it should be emphasized here that the unidentified characters involving segments 18a, 18b and 38 (coded as F?_ in our matrix) are

8 136 A. S. Graphodatsky et al. Figure 3. G-banded karyotype of fennec fox and gray fox. Summary of hybridization pattern of domestic dog probes onto G-banded fennec fox and gray fox chromosomes. Genome-wide correspondence between G-banded chromosomes of the fennec fox and domestic dog (a) and the gray fox and domestic dog (b). Fennec and gray fox chromosomes are numbered below and domestic dog chromosome homology is shown laterally. Note the high degree of G-band conservation between homologous chromosomes or chromosome arms defined by painting homology. critical for the resolution of the grouping of raccoondogs with foxes, as well as for the basal branching of the gray fox. Indeed, if these latter characters are coded as simple presence/absence of segmental associations of 18a/18b, 18a/38 and 18b/38, thus considering that the breakpoints in the different combinations observed are truly homologous, the consensus topology retrieved consists in an unresolved polytomy of the wolf-like, the foxes, the raccoon-dogs and the gray fox lineages (data not shown). Discussion Chromosome evolution in carnivores is currently one of the best examples for species belonging to the same mammalian order but with contrasting genome organizations, i.e. highly conserved versus highly rearranged karyotypes. Karyotypic relationships between the major carnivore families (Felidae, Ursidae, Canidae, Mustelidae, Hyaenidae, Viverridae, Pinnipedia) and their relationships with humans have been extensively revisited using comparative painting (Rettenberger et al. 1995, Froenicke et al. 1997, Hameister et al. 1997, Wienberg et al. 1997, Nash et al. 1998, 2001, Breen et al. 1999, Yang et al. 1999, 2000, Cavagna et al. 2000, Graphodatsky et al. 2000a, 2001, 2002, Tian et al. 2004, Nie et al. 2002, 2003, Yang & Graphodatsky 2004, Perelman et al. 2005). The consensus ancestral carnivore karyotype (ACK) was proposed to have 2n = 42 (Murphy et al. 2001; see Table 3 for their correspondences with human and dog). The comparative map between canids demonstrates that the genomes of canid species each consist of 42 highly conserved autosomal segments, with 34 segments each equivalent to a single dog chromosome. Dog chromosomes 1, 13, 18, and 19 each consist of two conserved ancestral segments. Comparison of the distribution of the 42 conserved

9 Phylogenomics of the dog and fox family 137 Figure 4. G-banded karyotype of corsac fox. Summary of hybridization pattern of red fox probes onto G-banded corsac fox chromosomes. Black lines show homology with red fox (on the right) and domestic dog chromosomes (on the left) (a). Genome-scale correspondence of G-banded corsac and red fox chromosomes. Corsac fox chromosomes are numbered below and red fox chromosome homology is shown laterally (b). Note the high degree of G-band conservation between homologous chromosomes or chromosome arms defined by painting homology.

10 138 A. S. Graphodatsky et al. Figure 5. Chromosomal signatures marking some Canidae lineages. A comparative map of domestic dog (CFA) chromosomes 1a/19a (a), 13a/34 (b), 5/37 (c), 9/13b (d), 12/30 (e), 25/31 (f), 18a/38/18b (g), 19a/ (h), 35/36 (i) and their corresponding chromosomes in red fox (VVU), corsac fox (VCO), arctic fox (ALA), fennec fox (FZE), gray fox (UCI), Japanese raccoon dog (NPRv) and Chinese raccoon dog (NPRp) based on FISH results and G-banding comparison. Chromosome numbers are indicated below, above or beside chromosomes. Homologous segments are linked by lines. segments in canids and other mammals reveals some contiguous segment combinations, each consisting of two conserved segments defined by the dog paints, thus suggesting that they have a more ancient origin than their individual component segments (Table 2). The distribution patterns of these contiguous segment combinations provide further insight into the phylogeny of the Canidae family and indicate that the synteny and linkage groups, represented by each dog chromosome, have evolved most recently in relation to the Canidae ancestor. The CFA 13a/34 and CFA19a/32 have been found in the foxes and raccoon dogs, as well as in all non-canid carnivores (Graphodatsky et al. 2000a,b, 2001, Yang et al. 2000, Yang & Graphodatsky 2004, Tian et al. 2004, Perelman et al. 2005), humans (Yang et al. 1999), and the pig (Biltueva et al. 2004), suggesting that they could have formed in the Eutheria ancestor. Hence, we propose that the Canidae Ancestral Karyotype (CAK) consists of 40 autosomal elements plus XY chromosomes (Figure 6). However, we have not excluded that the number of CAK autosomal elements could be more than 40. CFA chromosome 2 is homologous to two fragments in the gray fox, and so we cannot exclude the presence of these CFA 2 homologous segments in the CAK. This uncertainty remains to be clarified by hybridizing the painting probes of the gray fox to outgroup species. For the sake of convenience, here we assume that the CAK of the modern canids had a chromosome number of

11 Phylogenomics of the dog and fox family 139 2n = 82. Appearance of this CAK chromosomal complement could have descended from ACK via a huge number of rearrangements, at least 42 fissions and 25 fusions (Figure 6). All these rearrangements took place during the past 45Y50 million years, before the appearance of the modern Canidae lineage at 10Y12 million years ago (MYa) (Bininda-Emonds et al. 1999, 2007). Several phylogenies for the extant Canidae have been proposed by combining multiple molecular and morphology datasets (Bininda-Emonds et al. 1999, Vila et al. 1999, Bardeleben et al. 2005, Lindblad-Toh et al. 2005). These studies suggest a few alternative groupings among some lineages in Canidae. We have chosen the latest Canidae phylogeny that is based on 15 kb of exon and intron sequence (Lindblad-Toh et al. 2005), and have superimposed our data on the evolutionary chromosomal rearrangements revealed by chromosome painting onto this phylogeny (Figure 6 and Table 2). According to this phylogeny, the gray fox lineage seems to be the most primitive among extant canids. Our chromosomal data and cladistic analysis provide further support to the basal position of the gray fox, which is in agreement with a North American origin of living canids about 10 MYa (Lindblad-Toh et al. 2005). The distribution pattern of conserved segments suggests that at least one fission (break) and nine fusions are needed to convert the CAK karyotype to that of the gray fox. Among these 11 rearrangements, only one fusion (CFA18a + 38) is shared in a number of other canids. Probably, this CFA18a + 18b fusion separates all other canids from the gray fox lineage (Figure 6). This fusion signature is present in the wolf-like, South American and fox-like canids. The CFA19aY32 and CFA13aY34 fissions and CFA19a + 19b, CFA13a + 13b and CFA1a + 1b fusions are the signature rearrangements that have occurred during the origin of the most recent common ancestor of dog-like (domestic dog and dhole) canids and South American (crab-eating fox) canids. The tandem fusion of CFA is characteristic for the crab-eating fox karyotype (Nash et al. 2001; Figure 6, Table 2). The CFA19b + 1a chromosome fusion took place prior to the divergence of raccoon dogs and fox-like canid branches (Figures 5 and 6). The raccoon dog branch is separated from other canids by 12 centric and tandem chromosomal fusions. Eight additional centric fusions distinguish karyotypes of Japanese and Chinese raccoon dogs (Nie et al. 2003; Figure 6). This branch of two raccoon dogs received 100-bootstrap support in cladistic analysis. The chromosomal rearrangements CFA18a b, CFA13b + 9, CFA5 + 37, CFA , and CFA are characteristic for the fox-like canids, as they are present in the chromosomal sets of all five fox-like species including red fox, corsac, kit fox, arctic fox and fennec (Table 2; Figures 3, 5, and 6). These five fusions were possibly present in the fox-like ancestor with 2n = 68. The fennec fox karyotype has evolved through two characteristic fusions (CFA and CFA36 + 4), while the red fox has evolved through 17 apomorphic fusions (Figures 3, 5, and 6; Table 2). The fox and raccoon dogs shared the fusion CFA that has been postulated to be present at the ancestor of extant canids previously (Graphodatsky et al. 2001). Nevertheless, this association is absent in all other studied species. Scrutiny of banding and painting data suggests that this fusion could have taken place independently several times in different lineages. In the red fox lineage we detect tandem fusion of the CFA36 centromeric region with the telomere end of CFA35, whereas in raccoon dogs the centromere end of CFA35 attaches to the telomere end of CFA36 (Figure 5). The kit fox and arctic fox have identical 2n = 50 karyotypes (Nash et al. 2001). This karyotype could have originated through nine fusions from the proposed fox-like ancestor. The arctic fox karyotype is characterized by having a centric fusion polymorphism (Gustavsson & Sundt 1967, Graphodatsky et al. 2000a) The chromosomal complement of the arctic fox with 2n = 48, 49 was formed by incorporating an additional fusion: CFA1a/19b with CFA 15. None of these fusions coincides with those at the red fox lineage (Figures 5 and 6; Table 2). The corsac fox karyotype was formed as a result of 16 fusions. None of these fusions is the same as those in the red fox lineage. Two fusions, CFA18a/38/ 18b + CFA36 and CFA19a/32 + CFA8, are identical to those in the arctic fox lineage (Figure 5). These data thus suggest a close phylogenetic relationship between the corsac and arctic fox lineages, and their shared isolation from the red fox branch. Our phylogenomic analysis by PAUP has also suggested a close relationship between the Arctic and corsac foxes (Figure 6, frame). Only one particular rearrangement seems to contradict our previous conclusion (Graphodatsky et al.

12 140 A. S. Graphodatsky et al. 2001, Nie et al. 2003) that argues for the presence of the CFA 18a/18b association, found in dog-like canids and in raccoon dogs, in the common ancestral karyotype of extant canids. The insertion of CFA38 between CFA18a and18b seen in the foxes (Yang et al. 1999, Graphodatsky et al. 2000a) was regarded as a derived rearrangement. The detection of 18a/38 association in the gray fox (this study) confounds this inference. Although it is possible that this rearrangement (CFA18a + 38) has been acquired in the gray fox and fox-like branches independently, we cannot exclude that the 18a/38 association could have appeared at the basal branch of canid phylogeny. In the latter case, we have to assume independent fissions in the 18a/38/18b chromosome in the branches of dog-like canids and raccoon dogs. However, it is noteworthy that the 18a/18b chromosomes in dog and raccoon dog karyotypes differ

13 Phylogenomics of the dog and fox family 141 R Figure 6. Karyotypic relationships of the Canidae. Chromosome structural changes defined by chromosome painting and banding (see explanation in the text) mapped to the canids phylogenetic tree proposed by Lindblad-Toh et al. (2005). Each chromosome (in brackets) of the Ancestral Carnivora Karyotype (ACK, 2n = 42; Murphy et al. 2001); at the bottom of the figure in a dark blue frame is shown as a set of domestic dog chromosomes. Above in a red frame are shown the chromosomes of the Canidae Ancestral Karyotype (CAK, 2n = 82), formed from ACK as a result of 42 fissions and 25 fusions. Chromosomal signatures CFA19a/32 and 13a/34 are shown through slashes. Above on branches of the tree, in a red frame, chromosomal fusions (+) and fissions (j) are indicated. At the branching points the most probable numbers of chromosomes are shown. In red frames above animal pictograms the chromosomal rearrangements are shown that took place during the formation of the chromosomal complements of the Japanese raccoon dog, the red fox, corsac fox, and also arctic fox plus kit fox. The dark blue arrow indicates the rearrangement of chromosomes 19b/1a + 15 that determines the polymorphism in the arctic fox, 2n = 48Y50. Chromosomal fusions that partly contradict this phylogeny are shown in light red, green, blue and yellow frames (see Discussion). The most probable phylogeny of arctic fox, kit fox, corsac fox and red fox is shown in the black frame. The R numbers indicate the number of chromosomal fusions/fissions separating each lineage from CAK. The divergence time, as millions of years ago (MYa), is indicated for some tree nodes as discussed in Lindblad-Toh et al. (2005). One of the most parsimonious trees retrieved from cladistic analysis of chromosomal characters identified in our study is shown in the blue frame (see Results, Supplementary Figure S2, and Supplementary Table S1). Bootstrap values are indicated in italic. slightly in banding patterns, thus indicating a different origin (Figure 5). In our phylogenomic analysis the coding of this particular arrangement (18a/38, 18b/38, 18a/18b and the associated 18a/38/ 18b) was ambiguous and could not be resolved. If we assume that the insertion of CFA38 into CFA18a/18b happened in corsac, arctic, red and fennec foxes then the topology of the tree from cladistic analysis is completely resolved and looks the same as the tree proposed by Lindblad-Toh et al. (2005) (Figure 6, frame in blue). Table 3. Chromosomal correspondence between the domestic dog (CFA), human (HSA) and Ancestral Carnivora Karyotype (ACK) ACK HSA CFA 1 19p, 3, 21 (20,27,30,35,23), 2 4, 8p (16,28,15,19,32,13,3), 3 5 (11,2,3,4), 4 6 (37,12,1), 5 14, 15 (3,25,8), 6 10, 12p-q, 22q (2,29,10,15), 7 20, 2p-q (24,10,17), 8 2q (19,36,33,28), 9 7p-q (18,14,16), 10 1p-q (5,2,15,5,6,17), 11 10q (4,31), (5,21,18), 13 12q, 22, 18 (26,7,1), 14 9 (1,11,9), (22,28), (5,9), 17 1q (7,38), 18 8q (34,13), 19 19q, 16q (1,2,5), 20 7q, 16p (6), X X X We propose that a fission of CFA 2 has occurred in the gray fox lineage. However, it is impossible to exclude that the ancestor had CFA2a and CFA2b chromosomes and that the fusion of CFA2a and 2b occurred after the divergence of the core Canidae from the gray fox lineage. All chromosome rearrangements described above are fusion and/or fission events. It is obvious that during karyotype evolution of Canidae these types of rearrangements have been abundant. However, pericentric inversions and/or centromere repositioning also seem to have played a significant role in the karyotype evolution of Canidae. For example, the chromosome UCI19 (CFA32/19a) of gray fox is acrocentric. In the raccoon dog, this chromosome participated in tandem and centric fusions with the formation of bi-armed chromosome NPR1. Although in red, arctic, corsac and fennec foxes this chromosome is also bi-armed, it was formed as a result of inversion or centromere shift. This rearrangement, along with a set of fusions, defines the fox-like clade. Some bi-armed chromosomes of the arctic fox, for example ALA10 and ALA12, are also formed as a result of pericentric inversions compared with their homologues in the genomes of other foxes (Figure 5). Our study confirms the radical genomic reshuffles that have occurred in the evolution of the ancient Canidae. During the 35Y45 million years of canid evolution before the occurrence of extant species 10 million years ago, no fewer than 67 rearrangements occurred. This is much more than in any other family within Carnivora. However, during 10 million years of evolution of the extant canid karyotype, rearrangements are very extensive too, especially in the

14 142 A. S. Graphodatsky et al. fox-like group and raccoon dogs. The karyotype of the Japanese raccoon dog differs from the karyotype of the common ancestor (CAK) by at least 22 rearrangements, the corsac fox by 23 rearrangements, and the red fox by 24 rearrangements. Our cytogenetic data confirm the natural grouping of dog-like canids and their affinity with the branch of South American foxes. This branch has 100- bootstrap support in cladistic analysis. The grouping of a fox-like branch, with a basal placement for the fennec fox, is also supported by cytogenetic signatures. Cladistic analysis also builds a fox-like branch with highest support, but relations within the group remain polytomic with three branches: corsac fox + arctic fox, red fox, and fennec fox. At the morphological level, all modern molecular phylogenies show some uncertainty as to the positions for raccoon dogs and gray fox branches. Authors in papers published in the same year give different positioning of raccoon dogs and gray fox in relation to dog-like and fox-like branches (Bardeleben et al. 2005, Lindblad-Toh et al. 2005). Chromosomal data also do not give an unambiguous view on the phylogenetic position of the genera Nyctereutes and Urocyon. Cladistic analysis was also unable to resolve this issue with high supportive indices. We hope that similar genome mapping efforts as seen in the dog (Lindblad-Toh et al. 2005) will be applied to other canids. Naturally, the increase of the number of studied Canidae species will lead to an improvement in our understanding of the chromosomal and genomic evolution of this family. 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