Comparative genome maps of the pangolin, hedgehog, sloth, anteater ...

Chromosome Research (2006) 14:283–296 DOI : 10.1007/s10577-006-1045-6

# Springer 2006

Comparative genome maps of the pangolin, hedgehog, sloth, anteater and human revealed by cross-species chromosome painting: further insight into the ancestral karyotype and genome evolution of eutherian mammals Fengtang Yang1,6*, Alexander S. Graphodatsky2, Tangliang Li1, Beiyuan Fu3, Gauthier Dobigny4, Jinghuan Wang1, Polina L. Perelman2, Natalya A. Serdukova2, Weiting Su1, Patricia CM O’Brien3, Yingxiang Wang1, Malcolm A. Ferguson-Smith3, Vitaly Volobouev5 & Wenhui Nie1* 1 Key Laboratory of Cellular and Molecular Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650223, PR China; E-mail: [email protected], [email protected]; 2Institute of Cytology and Genetics, SB RAS, Novosibirsk 630090, Russia; 3Centre for Veterinary Sciences, University of Cambridge, Madingley Road, Cambridge CB3 0ES, UK; 4Institut de Recherche pour le D eveloppement, Centre de Biologie et Gestion des Populations, Campus International de Baillarguet, CS30016, 34988 Montferrier-surLez, France; 5Mus eum National d’Histoire Naturelle, Origine, Structure et Evolution de la Biodiversit e, 55, rue Buffon, 75005 Paris, France; 6Current address: Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK * Correspondence Received 19 January 2006. Received in revised form and accepted for publication by Herbert Macgregor 16 February 2006

Key words: ancestral karyotype, Carnivora, chromosome painting, Eulipotyphla, Pholidota, Xenarthra

Abstract To better understand the evolution of genome organization of eutherian mammals, comparative maps based on chromosome painting have been constructed between human and representative species of three eutherian orders: Xenarthra, Pholidota, and Eulipotyphla, as well as between representative species of the Carnivora and Pholidota. These maps demonstrate the conservation of such syntenic segment associations as HSA3/21, 4/8, 7/16, 12/22, 14/15 and 16/19 in Eulipotyphla, Pholidota and Xenarthra and thus further consolidate the notion that they form part of the ancestral karyotype of the eutherian mammals. Our study has revealed many potential ancestral syntenic associations of human chromosomal segments that serve to link the families as well as orders within the major superordinial eutherian clades defined by molecular markers. The HSA2/8 and 7/10 associations could be the cytogenetic signatures that unite the Xenarthrans, while the HSA1/19p could be a putative signature that links the Afrotheria and Xenarthra. But caution is required in the interpretation of apparently shared syntenic associations as detailed analyses also show examples of apparent convergent evolution that differ in breakpoints and extent of the involved segments.

Introduction Pangolins, hedgehogs, armadillos and anteaters are among the more peculiar mammalian species having unique specialized anatomical features adapted to insect-eating. Partly due to the resulting morphology,

their phylogenetic affinities have long puzzled biologists. The hedgehogs (Order Eulipotyphla) and pangolins (Order Pholidota) are of northern hemisphere origin, while the armadillos and anteaters, together with the sloths (Order Xenarthra) are of southern hemisphere origin (Patterson & Pascual

284 1972, Delsuc et al. 2002). Morphology-based phylogenies generally identify the Xenartha as the basal eutherian clade (McKenna 1975, Shoshani & McKenna 1998), but Novacek (1992) placed pangolins as basal sisters to the Xenarthra. Recent molecular phylogenetic studies, however, have reshaped the eutherian tree by resolving the 18 extant mammalian orders into four superordinal clades: (1) Afrotheria (elephants, manatees, hyraxes, tenrecs, golden moles, aardvark and elephant-shrews); (2) Xenarthra (sloths, anteaters and armadillos); (3) Euarchontoglires (rabbits, rodents, flying lemurs, tree shrews and primates); and (4) Laurasiatheria (core insectivores, bats, cetartiodactyls, perissodactyls, carnivores and pangolins), with the latter two clades being referred to as the Boreoeutheria (Murphy et al. 2001a, see reviews in Murphy et al. 2004, Springer et al. 2004). Within the Laurasiatheria, the Eulipotyphla (i.e. the core insectivores including the hedgehogs, shrews and moles, Douady et al. 2002) form the basal clade, while the Pholidota are placed as the sister group to the Carnivora (Murphy et al. 2001a, 2004, Springer et al. 2004). The molecular ordinal relationships are now supported by overwhelming sequence evidence and RGC (i.e. rare genomic changes sensu Rokas & Holland 2000, see review by Murphy et al. 2004), but the root of the placental tree remains ambiguous. The mtDNA analysis of Arnason et al. (2002) suggests that hedgehogs are the earliest offshoot of the placental tree, although Lin et al. (2002) found that mtDNA trees could recover the same four clades as nuclear genes when the outgroup taxa are removed. Furthermore, several nodes in the Laurasiatheria remain to be defined unambiguously (Murphy et al. 2004, Springer et al. 2004) In addition to the molecular approach, crossspecies chromosome painting has been shown to be a highly prolific approach that can help to further verify the current placental phylogenetic relationships (Scherthan et al. 1994, Yang et al. 1995). Cross-species chromosome painting has enabled the rapid establishment of genome-wide comparative chromosome maps among species without any gene mapping data, in particular representative species of

F. Yang et al. less-studied mammalian orders (Yang et al. 1999, 2000, 2003, reviewed in Murphy et al. 2001b, Wienberg 2004). The comparative chromosome maps provide not only information on comparative genome organizations and evolutionary chromosome rearrangements that have shaped the genome organization of eutherian mammals, but also insights into the ancestral karyotype and cytogenetic signatures that characterize each phylogenetic lineage (O’Brien et al. 1999, Yang et al. 2003, Wienberg 2004). The systematic application of human chromosome-specific painting probes to representative species of diverse mammalian orders holds the promise that the genomes of representative species can be linked with the human genome as the reference. This will allow homology assessment throughout placental mammals, hence providing an integrative view of eutherian genome evolution. Such comprehensive comparison has hardly been possible using conventional cytogenetic approaches. Up to now, 13 of the 18 eutherian orders are represented by genome-wide comparative maps with human (reviewed in Fro¨nicke 2005), but one order (Xenarthra) is only partially covered with HSA 1, 7, 16 and 19 probes (Murphy et al. 2003, Richard et al. 2003) and four orders (Dermoptera, Pholidota, Hyracoidea and Sirenia) have no published map available. In such a framework, genome rearrangements, as revealed through chromosome painting, can serve as valuable phylogenetic markers (reviewed in Murphy et al. 2004). Recent comparative data between human and four Afrotheria species (elephants, aardvark, elephant-shrews and golden moles) have provided cytogenetic signatures (i.e. such shared syntenic associations as HSA3/21/5 and 1/19p) that unite Afrotheria (Fro¨nicke et al. 2003, Yang et al. 2003, Robinson et al. 2004), whereas this grouping has hardly any support from the morphological evidence (Madsen et al. 2001, Murphy et al. 2001a). Here we have extended cross-species chromosome painting with human chromosome-specific paints to the Javan pangolin (Manis javanica, 2n = 38), longeared hedgehog (Hemiechinus auritus, 2n = 48), twotoed sloth (Choloepus didactylus, 2n = 65), and tree anteater (Tamandua tetradactyla, 2n = 54), repreb

Figure 1. Examples of cross-species chromosome painting with human (Homo sapiens, HSA) and stone marten (Martes foina, MFO) chromosome-specific painting probes. (a) Simultaneous hybridization of HSA 1 (green) and 19 (red) to Manis javanica metaphase; (b) hybridization of MFO 9 probe (red) to M. javanica metaphase; (c) hybridization of HSA16 (green) and 19 (red) probes to Hemiechinus auritus metaphase; (d) hybridization of HSA 17 probes (red) to Hemiechinus auritus metaphase; (e) hybridization of HSA 1 (green) and 19 (red) to Choloepus didactylus metaphase; (f) hybridization of HSA 1 (green) and 19 (red) to Tamandua tetradactyla metaphase.

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286 senting three distinct mammalian orders (Pholidota, Eulipotyphla and Xenarthra) that have not been, or only partly, investigated up to now. Furthermore, paints from the stone marten (Martes foina, 2n = 38), a species from the order Carnivora with highly conserved genome organization (Nie et al. 2002) were also extended to the Javan pangolin (M. javanica). In doing so we intended to: (1) establish comparative cytogenetic maps that would facilitate future cloning of important candidate genes underlying the special adaptive evolution of these species, as well as advance comparative and evolutionary genomics studies; (2) search for the cytogenetic signatures that could be used to consolidate the groupings of and within the Afrotheria, Xenarthra and Laurasiatheria; (3) provide further insight into the hypothetical ancestral eutherian karyotype as previously proposed (Chowdhary et al. 1998, Fro¨nicke et al. 2003, Richard et al. 2003, Yang et al. 2003, Svartman et al. 2004, Wienberg 2004).

Materials and methods Cell culture, metaphase preparation and G-banding Fibroblast cell lines derived from one male Javan pangolin (M. javanica, MJA, from Kunming, Yunnan, P.R. China, KCB200311S), long-eared hedgehog (H. auritus, HAU, from Novosibirsk, Russia and Kunming, Yunnan, P. R. China, KCB88023), two-toed sloth (C. didactylus, CDI, born in the Budapest Zoo from parents from an unknown area in Brazil), and a female tree anteater (T. tetradactyla, TTE, from French Guyana) were established from skin biopsies. Cell culture, metaphase preparations and G-banding were carried out following conventional methods as previously described (Yang et al. 2003). Karyotypes of the Javan pangolin and long-eared hedgehog were arranged according to relative chromosomal size, from the largest to the smallest, while the chromosomes of the two-toed sloth and tree anteater were numbered based on recently published G-banded karyotypes (Dobigny et al. 2005). Fluorescence in-situ hybridization (FISH) Chromosome-specific painting probes derived from degenerate oligonucleotide PCR (DOP-PCR,

Telenius et al. 1992) amplification of flow-sorted chromosomes of the human (Yang et al. 1997) and stone marten (Nie et al. 2002) were used to delimit homologous chromosomes or chromosomal segments in the genomes of the Javan pangolin, long-eared hedgehog, two-toed sloth and tree anteater, following the methods described previously (Yang et al. 2003).

Results Painting the Javan pangolin (MJA) genome with human (HSA) and stone marten (MFO) probes To delimit the evolutionary conserved regions among the pangolin, human and stone marten, whole sets of human and stone marten painting probes (without the Y probes) were hybridized onto Javan pangolin metaphases. The FISH examples and summary of hybridization patterns of all probes are shown in Figures 1a, b, and 2, respectively. Six human chro– mosome probes (HSA9, 14, 17, 18, 20 and X) each delimited one homologous segment. Paint probes derived from 17 human chromosomes (HSA1-8, 10Y13, 15, 16, 19, 21 and 22) each detected two to four homologous segments in the pangolin genome. Human probes revealed three putative inversions involving two pangolin chromosomes (MJA3 and 10) homologous to HSA 3/21 and 7/16. Human segmental associations such as HSA 3/21, 4/8, 7/16, 12/22, 14/15 and 16/19 that were proposed as ancestral to all eutherian mammals (Chowdhary et al. 1998, Haig 1999, Yang et al. 1999, 2003, Murphy et al. 2001b, Richard et al. 2003, Svartman et al. 2004, Wienberg 2004) were also present in the pangolin genome. In addition to these ancestral mammalian conserved syntenic associations, we found a further 16 human adjacent segment associations (HSA 1/5, 1/11, 1/17, 2/5, 2/8, 2/10, 3/12, 3/4, 4/20, 5/13, 6/19, 7/11, 8/10, 9/10, 12/16 and 13/18) in the pangolin genome, with all but four of these associations (HSA2/5, HSA3/12 on MJA15q, HSA8/10 and HSA6/19) involving centromeres. In total, 22 human autosomal painting probes identified 47 conserved segments in the pangolin genome. Painting probes derived from seven stone marten chromosomes (MFO 11, 12, 14, 15, 17, 18 and the X) each defined one pangolin homologous segment or chromosome; eight stone marten painting probes

Genome organizations of the pangolin, sloth, anteater and hedgehog (MFO 4, 5, 6, 7, 8, 10, 13 and 16) each detected two pangolin homologous segments; three stone marten painting probes (MFO 1, 2 and 9) each painted three homologous segments; stone marten chromosome 3 probe delineated four homologous segments in the pangolin genome. In total, all 18 stone marten autosomal probes revealed 35 conserved segments (Figure 2). Further integration with previous reciprocal painting results between stone marten and cat (Nie et al. 2002), and cat and human (Wienberg et al. 1997, Yang et al. 2000) allows for the deduction of

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sub-chromosomal homologies between the human and Javan pangolin (Figure 2). Painting the long-eared hedgehog (HAU) genome with human (HSA) probes To delimit the conserved segments between human and long-eared hedgehog, painting probes derived from 22 human autosomes and the X chromosome were hybridized onto the HAU metaphases. The FISH examples and summary of hybridization pat-

Figure 2. The genome-wide correspondence between Javan pangolin (Manis javanica, MJA), human and stone marten (Martes foina, MFO), with the G-banded karyotype of M. javanica as the reference. The numbers below each homologous pair identify the MJA chromosomes; homology with human (HSA) and M. foina is indicated to the right of each MJA chromosomal pair. Note that the subchromosomal correspondence was inferred from the published comparative maps among human, cat, dog, and stone marten (Wienberg et al. 1997, Yang et al. 2000, Nie et al. 2002).

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Figure 3. The genome-wide correspondence between the long-eared hedgehog (Hemiechinus auritus, HAU) and human (HSA), with the Gbanded karyotype of H. auritus as the reference. The numbers below each homologous pair identify the HAU chromosomes; homology with human (HSA) is indicated to the right of each HAU chromosomal pair.

terns of all probes are shown in Figures 1c, d and 3, respectively. In total, the human probes defined 60 homologous segments in the long-eared hedgehog genome. Ten human painting probes (HSA 1Y5, 10, 12, 14, 15 and 19) each painted three to six homologous segments in the long-eared hedgehog. Eight human paints (HSA 6Y8, 11, 13, 16, 17 and 22) each defined two homologous long-eared hedgehog segments. Only five human painting probes (HSA 9, 18, 20, 21 and X) each delimited one homologous longeared hedgehog segment. The patterns of human probes revealed putative inversions on the long-eared hedgehog chromosomes 2, 6, 7, 9, 11 and 16. All the ancient human segmental combinations such as HSA 3/21, 4/8, 7/16, 12/22, 14/15, and 16/19 were also present in hedgehogs. Beside those conserved ancestral syntenic associations, a further 20 associations (HSA 1/3, 1/4, 1/5, 1/6, 2/4, 2/22, 3/5, 3/10, 3/17, 4/5, 4/16, 5/8, 5/13, 5/19, 7/18, 8/10, 10/12, 10/20, 11/15, and 12/13) were detected in the long-eared hedgehog genome, with about half of these associations involving centromeres.

Painting the two-toed sloth (CDI) genome with human (HSA) probes The 22 human autosomal probes and the X probe were hybridized onto the chromosomes of two-toed sloth. The FISH example of HSA1 and 19 probes is shown in Figure 1e; the hybridization patterns of all probes were summarized on the G-banded karyotype of a male two-toed sloth (Figure 4). Paints from eight human chromosomes (HSA 9, 13, 15, 17, 18, 20, 21 and X) each defined one single conserved chromosome or one chromosomal segment in the two-toed sloth. Human chromosome 2 probe gave four signals on three two-toed sloth chromosomes, while the remaining human painting probes delimited two or three conserved segments. In total, 22 human autosomal paints revealed 42 conserved syntenies in the two-toed sloth haploid genome. All the ancestral human segmental combinations mentioned above, except for HSA 16/19, were also found in the twotoed sloth genome. Only two derived associations (HSA 2/8 and HSA7/10) were detected. The patterns

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Figure 4. The genome-wide correspondence between the two-toed sloth (Choloepus didactylus, CDI) and human (HSA), with the G-banded karyotype of C. didactylus as the reference. The numbers below each homologous pair identify the CDI chromosomes; homology with human (HSA) is indicated to the right of each CDI chromosomal pair.

of human probes revealed one putative inversion on CDI chromosome 18. Surprisingly, in addition to the entire CDI X, the HSA X also gave signals on the long arm of CDI Y chromosome but with variable signal intensity as compared with that on the X chromosome, indicating the existence of some homology between the CDI Y and X. Such a hybridization pattern of human X probe painting the Y chromosome from species of the other orders has never been reported in mammals studied so far, with the exception of cross-hybridization to the pseudoautosomal region between closely related species.

Painting the tree anteater (TTE) genome with the human (HSA) probes The 22 human autosomal probes and the X probe were hybridized onto the chromosomes of tree

anteater. The FISH example of HSA1 and 19 probes is shown in Figure 1f; the hybridization patterns of all probes were summarized on the G-banded haploid karyotype of tree anteater (Figure 5). Painting probes derived from eight human chromosomes (HSA 9, 11, 13, 17, 18, 20, 21 and X) each delimited one tree anteater segment or chromosome. Human chromosome 4 painting probe hybridized onto five different tree anteater chromosomes (TTE 21, 22, 23, 25 and 26). The other human painting probes each painted two or three tree anteater chromosomes or chromosomal segments. In total, 22 human autosomal paints revealed 44 segments of conserved synteny in the tree anteater genome. All the ancient human segment combinations were also present in the tree anteater. In addition to these ancient combinations, we found nine derived associations (HSA 1/9, 1/13, 1/19, 2/8, 3/6, 3/12, 7/10, 7/20 and 8/17) with four of them (HSA1/9, 1/13, 3/6, and 8/17) involving centromeres.

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Figure 5. The genome-wide correspondence among the tree anteater (Tamandua tetradactyla, TTE) and human (HSA), with the haploid Gbanded karyotype of T. tetradactyla as the reference. The numbers below each chromosome identify the TTE chromosomes; homology with human (HSA) is indicated to the right of each TTE chromosome.

The patterns of human probes revealed one putative inversion on TTE 3.

Discussion We have established, for the first time, genome-wide comparative chromosome maps between human and Javan pangolin, long-eared hedgehog, two-toed sloth and tree anteater, representing four eutherian orders. We also present the first comparative map between representatives from the orders Carnivora and Pholidota. Together with the previously published comparative maps between human and 12 other placental orders, this leaves only three eutherian orders (i.e. Dermoptera, Hyracoidea and Sirenia) uncharted.

Furthermore, the establishment of a comparative map between human and Xenarthra, which represents one of the four superordinal clades, ensures that all of the four major superordinal clades are now represented in the phylogenetic reconstruction of eutherian genome evolution. The integration of these maps with the human genome as the reference has provided a near-complete pictorial illustration of the evolutionary history of genome organization of extant eutherian mammals and, in particular, new insights into the ancestral mammalian karyotype and the major characteristic genome reshuffles in each phylogenetic lineage. Despite the lack of painting data from the immediate outgroup (i.e. marsupials) of eutherian mammals, the findings of the putative ancestral syntenic associations of HSA3/21, 4/8,

Genome organizations of the pangolin, sloth, anteater and hedgehog 7/16, 12/22, 14/15 and 16/19 in the representative species of three more eutherian orders, in particular, from the Xenarthra, further consolidate the notion that they form part of the ancestral karyotype of the eutherian mammals. Karyotypic relationships within Xenarthra and cytogenetic signatures The Xenarthra, which is considered as one of the four superordinal groups, comprises four families: Dasypodidae (armadillos), Myrmecophagidae (anteaters), Bradypodidae (three-toed sloths) and Megalonychidae (two-toed sloths) (Nowak 1999). Both morphology and sequence data sets supported the monophyly of all four lineages (Delsuc et al. 2002). A recent comparative chromosome painting study on the Xenarthra with two-toed sloth probes provided insights into karyotypic evolution, although the data were not complete due to three chromosomes missing in the flow-sorts. The results showed that the two- and three-toed sloth species display similar karyotypes that are rather different from that of the anteater (Dobigny et al. 2005). The establishment of homology maps between human, the two-toed sloth and the tree anteater allows us to further clarify the karyotypic relationships in the Xenarthra and, more importantly, between the Xenarthra and other eutherian orders. Our data from human paints showed that these two Xenarthran species display conserved genome organizations. In the two-toed sloth, 24 of the 32 autosomes are each homologous to one human chromosome or chromosomal segment, the remaining autosomes being each homologous to one ancestral human syntenic segmental association. Therefore, it seems that the main type of chromosomal rearrangement in the two-toed sloth lineage is the fissions of the ancestral eutherian syntenies, leading to the high diploid number seen in this species. Notably the HSA 16/19 association that has been retained in many mammals has been disrupted into two chromosomes in the two-toed sloth genome. In contrast, in the tree anteater, which has a lower diploid number than the two-toed sloth, nine derived human chromosomal segment associations were identified. The HSA 2/8 and 7/10 associations represent the two derived segmental associations shared by the two-toed sloth and tree anteater. Thus, they could constitute the

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first cytogenetic signatures for the Xenarthra (i.e. the last supra-ordinal placental clade unmapped with human probes). The conservation of CDI 6 + 22 association in three-toed sloth (Bradypus tridactylus) and six-banded armadillo (Euphractus sexcinctus) (Dobigny et al. 2005) provides further evidence for the HSA7/10 syntenic association as a cytogenetic signature for the Xenarthran. But the existence of HSA 2/8 association in B. tridactylus and E. sexcinctus remains inconclusive due to the incomplete coverage of B. tridactylus and E. sexcinctus genome by the CDI paints. The HSA 1/19p found in the tree anteater is especially noteworthy. This association has been found previously in four Afrotheria species (aardvark, elephants, golden mole and elephant-shrew, Fro¨nicke et al. 2003, Yang et al. 2003, Robinson et al. 2004, Svartman et al. 2004). While Fro¨nicke et al. (2003) and Robinson et al. (2004) suggested that the HSA1/ 19p association was one of two unique associations to support the Afrotheria, Yang et al. (2003) proposed that the HSA1/19p association was one chromosome of the ancestral eutherian karyotype. The homologous segment of human 19 combined to the homologue of human chromosome 1 in the tree anteater probably originated from human 19p, since the ancient association HSA16q/19q is retained on tree anteater chromosome 16. The finding of HSA 1/19p associations in both Afrotheria and Xenarthra, i.e. two basal clades of the Eutheria, suggests that HSA 1/19p could be present in the common ancestor of Afrotheria and Xenarthra, and so probably ancestral to all eutherians. The existence of HSA19p as one individual chromosome in the sloth and armadillo genomes would thus rather represent a derived condition. Searching for cytogenetic signatures in the Laurasiatheria The Pholidota and Eulipotyphla are the two critical orders for understanding the evolutionary relationships within the superordinal clade Laurasiatheria that also shelters the Chiroptera, Perissodactyla, and the Cetartiodactyla. Up to now, molecular phylogenetic study has so far failed to resolve unambiguously the three major nodes concerning the relationships between Cetartiodactyla, Perissodactyla, Carnivora and Chiroptera (see Murphy et al. 2004, Springer et al.

292 2004). The establishment of genome-wide comparative maps for the Pholidota and Eulipotyphla representatives, together with the previously published comparative maps for the Chiroptera, Carnivora, Perissodactyla and Cetartiodactyla representatives, makes the Laurasiatheria the first of the four placental superordinal clades that has complete taxonomic sampling coverage. The integrated analysis of comparative chromosome maps, to some extent, has allowed the identification of major chromosomal rearrangements specific for each lineage in the Laurasiathera. Below are listed the major segmental associations found in each lineage with the putative ancestral syntenic associations for eutherian mammals excluded: Cetacea (dolphin): 3/6, 5/19p, 7/20, 18/22 (Bielec et al. 1998) Artiodactyla: Cattle and muntjac: HSA1/2, 1q/10q, 2p/9, 4/12, 5/19p, 8/9, 10p/20 (Yang et al. 1997, Itoh et al. 2005) Pig: HSA1q/7, 1q/8q, 1p/18, 1p/19q, 2q/4qter, 2q/8p, 4/12, 5q/19p, 8p/20, 9/10p, 9/15, 10q/22, 11/19p, 14/18 (Biltueva et al. 2004, Lahbib-Mansais et al. 2005) Perissodactyla: HSA 1q/10q, 5/19p-distal, 11/19pproximal (Yang et al. 2003, 2004, BrinkmeyerLangford et al. 2005) Carnivora: HSA 2p-q/20, 3/19p, 18/22q-proximal (Rettenberger et al. 1995, Wienberg et al. 1997, Fro¨nicke et al. 1997, Yang et al. 1999, 2000, Cavagna et al. 2000, Perelman et al. 2005) Pholidota: HSA1q/5, 1q/11, 2p/5, 4p+q/20, 5/13, 6/19p, 7/11, 8q/10p (this study) Chiroptera: HSA1/6; 4/11, 4/19p, 8/13, 11/12, 18/20 (Volleth et al. 1999, 2002) Eulipotyphla: HSA1/5, 1/11, 3/19p, 4/20, 5/19p, 8/10, 8/13 (Dixkens et al. 1998, Ye et al. 2006, this study) At first glimpse the HSA5/19p association could be the cytogenetic signature that unites the Laurasiatheria because of the wide occurrence of HSA5/19p in the ingroup orders including the Perissodactyla, Artiodactyla, Cetaceans, and Eulipotyphla (the longeared hedgehog only). We, however, believe that this notion should be treated with caution since the HSA5/19p association is absent in the Chiroptera

F. Yang et al. and Carnivora studied to date, in addition to its absence in the other three superordinal clades that served as the outgroup of the Laurasiatheria clade. The HSA19p has disassociated with HSA5 in the Carnivora and Chiroptera, forming the HSA3/19p and 4/19p association, respectively. At the moment we cannot exclude the possibility that the HSA5/19 association found in the long-eared hedgehog, which has a highly rearranged genome, resulted from a convergent evolution since the other Eulipotyphlans (i.e. common shrew, shrew hedgehog and the Asiatic short-tailed shrew) do not retain the HSA5/19 association (Ye et al. 2006). As further evidence the HSA5 chromosome is fragmented into five fragments in the hedgehog genome (thus enhancing the probability of becoming fused to other fragments, including the HSA19) whereas it has only been found to break into one or a few fragments in most placental species investigated so far. Therefore, the HSA5/19 association most likely represents a signature that unites the Perissodactyla, Artiodactyla and Cetaceans. So far we have found no cytogenetic signature for Cetartiodactyla (Cetacea + Artiodactyla). In the Equidae, the HSA19p broke into two segments (19p-distal and 19p-proximal), forming two associations HSA5/19p-distal and HSA11/19p-proximal (Yang et al. 2003, 2004, Brinkmeyer-Langford et al. 2005). The comparative painting results between Burchell’s zebra and rhinoceros (Trifonov et al. 2003) suggest the existence of HSA5/19-distal and 11/19p-proximal in Rhinocerotidae also. Therefore, the disassociation of HSA19p to two segments and subsequent formation of HSA5/19-distal and 11/19p-proximal seem to have occurred during the divergence of the most recent common ancestor of perissodactyls. Nevertheless, the HSA11 and 19p homologous segments are linked together on pig chromosome 2, but dissociated in the Bovidae and Cervidae representatives. Alternatively the finding of HSA11/19p association in Suidae, Equidae and Rhinocerotidae may suggest that the HSA11/19p could be present in the most recent common ancestor of Perissodactyla and Artiodactyla. However, as in the case of HSA5/19p, we cannot exclude the possibility of the HSA11/19p association found in the Suidae being a convergent rearrangement. In summary, we believe that HSA5/19p and, to a lesser extent, the HSA11/19p, represent the likely synapo-

Genome organizations of the pangolin, sloth, anteater and hedgehog morphies for the grouping of Perissodactyla + Cetartiodactyla (i.e. the Euungulata or true ungulates sensu Waddell et al. 2001). Recently the HSA1q/10q association, identified previously by cross-species chromosome painting in the canids (chromosomes 4 in both the dog and red fox, Yang et al. 1999) and equids (horse chromosome 1 and its corresponding chromosomes in other equids), has been found in cattle (chromosome 28) by comparative RH mapping (Itoh et al. 2005). The HSA1q/10q thus seems to be the signature supportive of the grouping of Perissodactyls + Cetartiodactyls + Carnivora. Nevertheless it is noteworthy that the HSA1q segment involved is very small, about 10.7 Mb in length according to comparative gene mapping (Haig 2005). Therefore we cannot rule out that the absence of HSA1q/10q in the dolphin, pangolin and core insectivores and bats was due to the technical limitation of cross-species chromosome painting in resolving small segments. Further high-resolution comparative mapping is needed to determine the presence or absence of HSA1q/10q in these species. Additionally, the HSA12/22/18 association (equivalent to cat D3) that has been conserved in all feliformia carnivores studied so far (Perelman et al. 2005) has been retained in the dolphin also (Bielec et al. 1998), but has not been detected in the Artiodactyla and Perissodactyla. The HSA12/18 link thus could be present in common ancestors of Carnivora and Cetacea or alternatively they could have evolved independently. The Chiroptera is the only lineage that apparently does not share a single derived association with any representative species from the other Laurasiatheria orders so far studied. The position of Pholidota As for the phylogenetic position of the order Pholidota, molecular analyses based on nuclear and mitochondrial genes sequence data placed Pholidota as the sister group to Carnivora although species in these two orders share little morphological similarities (Arnason et al. 2002, Springer et al. 2004). Comparative maps between human and nine carnivore species (i.e. domestic cat, domestic dog, domestic ferret, harbor seal, American mink, giant panda, spotted hyena, masked palm civet and stone marten) have been established by multidirectional cross-

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species chromosome painting (Rettenberger et al. 1995, Fro¨nicke et al. 1997, Wienberg et al. 1997, Nash et al. 1998, Cavagna et al. 2000, Graphodatsky et al. 2000, Yang et al. 2000, Perelman et al. 2005, Nie et al. unpublished data). It has been demonstrated that the stone marten has retained a highly conserved genome that is still close to the proposed ancestral Carnivora karyotype (Nie et al. 2002). Surprisingly, the availability of comparative data from the core insectivores and pangolin, however, has brought more complexity rather than helping to clarify the phylogenetic relationships between the orders Pholidota and Carnivora. The comparison shows the absence of the Carnivora signature association of HSA3/19p in the pangolin, besides the existence of two apparently shared segmental associations between the Javan pangolin and a few carnivore species (i.e. HSA 1/11 shared by the pangolin and giant panda, and 5/13 shared by the pangolin and domestic cat). Considering that the HSA 1/11 association was also found in shrew-hedgehog (Neotetracus sinensis, Ye et al. 2006), and that the HSA5/13 association is also present in the longeared hedgehog, the HSA1/11 and HSA5/13 associations are thus unlikely to be a shared derived character that unites the Carnivora and Pholidota. Furthermore comparative chromosome painting shows that Pholidota and Eulipotyphla (i.e. the core insectivores) share four chromosome associations (i.e. HSA1/5, HSA4/8/2, HSA8/10 and in particular, the HSA4/20 association that has been suggested as a potential cytogenetic signature for the Eulipotyphla; Ye et al. 2006) and that the common shrew seems to have retained the HSA3/19p association that is present in the Carnivora. Considering that most of these shared associations could be explained by centric fusions of ancestral segments, we thus favor the hypothesis that they have evolved independently since (1) it is well known that centric fusions are prone to convergent evolution, and (2) a sister relationship between the Eulipotyphla and the Pholidota would contradict the Pholidota + Carnivora grouping that is strongly supported by some molecular evidence, including a common 363-bp deletion in the APOB gene (Amrine-Madsen et al. 2003). Alternatively, some of the shared associations such as the HSA3/19p and 4/20 could represent the ancestral chromosome rearrangements that originated

294 during the divergence of the most recent common ancestor of Eulipotyphla, Carnivora and Pholidota, thus supporting an alternative polytomy of Carnivora + Pholidota + Eulipotyphla. Furthermore, the same HSA3/19p association is also present in the squirrels which are near the basal branch of Rodentia in the Euarchontoglires clade (Stanyon et al. 2003, Li et al. 2004), suggesting an even more ancient origin for the HSA3/19p association (perhaps representing the synapomorphic rearrangement that occurred before the divergence of Laurasiatherian and Euarchontoglires lineages). Cytogenetic signatures and convergence in eutherian mammals There is a wide occurrence of coincidental shared segmental associations (markers are syntenic in two species for reasons other than uninterrupted ancestral linkage, Haig 1999) in eutherian mammals, sometimes involving Laurasiatherian species; for instance, the HSA 2/8/4 association observed on pangolin MJA13. The HSA2p-q/8p/4 association has been demonstrated to be a shared derived character for Afroinsectiphilia (aardvark, golden mole and elephant-shrew, Robinson et al. 2004). However, the results of multidirectional chromosome painting (between pangolin and stone marten, this study; stone marten and cat, Nie et al. 2002; cat and dog, Yang et al. 2000; dog and aardvark, Yang, unpublished data), shows that the segment of human 2 combined to the counterpart of HSA 8/4 in the pangolin differed from that combined to HSA 8/4 in the Afrotheria. The former originated from the segment homologous to cat C1q (HSA2q), while the latter came from the segment homologous to cat A3q (HSA2p-q-proximal). As a consequence, the HSA 2/8/4 association appearing in both pangolin and Afroinsectiphilia has different evolutionary origins. The other example is the HSA 10p/12/22 that is found in both Afrotheria and Carnivora as compared with HSA10/12p-q /22 in the long-eared hedgehog (HAU20). Close scrutiny shows that the HSA10/12/ 22 found on HAU20 is not the same as that found in the Afrotheria and Carnivora. The HSA12/22 in the HSA10/12/22 combination of the latter is HSA12pter-q/22q-distal, while it most likely involves 12qter/ 22q-proximal in the long-eared hedgehog based on banding comparison. Further examples for such convergent evolution include the HSA4/19p found

F. Yang et al. in the bats (Volleth et al. 2002) and the 4/19q in the Eulemur macaco macaco (Mu¨ller et al. 1997) and the HSA16q/19q found in most non-primate mammals and the HSA16q/19p found in the E. m. macaco and E. fulvus mayottensis (Mu¨ller et al. 1997). The most salient example of convergent evolution due to nonRobertsonian translocation is the HSA1/19p found in the Afrotherians so far studied and the tree anteater, versus the HSA1/19q found in the prosimians (Nie et al. 2006). Of course, none of these associations can serve as a cytogenetic signature (i.e. shared derived chromosomal character) that indicates a close phylogenetic relationship as they are in fact non-homologous characters. It should be pointed out that conservation of syntenic segment associations emphasizes the common origin of the fusion points (inheritance by descent) and the retention of fusion points in most, if not all, descendant species. As evidenced in the HSA3/21, 4/8, 12/22, 14/15, and 7b/16p, although the synteny of each composite segment could be disrupted by inter and intrachromosomal rearrangements during karyotypic evolution, the fusion points have been conserved throughout evolution (Robinson & Seiffert 2004). For instance, the HSA3 segment in the HSA3/21 association may have broken into several segments on different lineages of the Eutheria. It is still possible to detect the existence of HSA3/21 link (however small it might be) in all representative species of extant eutherian orders. Great caution should, however, be excised when dealing with apparently Fconserved_ syntenic associations when reverse painting data are absent, in particular, those associations derived from centric fusion of two conserved segments. It is noteworthy that none of the highly conserved ancestral syntenic associations of eutherian mammals is derived from centric fusions apart from the HSA16q/19q association. The frequent occurrence of centric fusions in mammalian karyotypic evolution underscores the importance of reciprocal painting, in particular the comparative sequencing of the breakpoints of the shared syntenic segmental combinations, since only such studies will elucidate the origin of the syntenic segment combinations. In summary, the findings of many shared syntenic associations among different ingroup orders of the Laurasiatheria clade seem to support the existence of three distinct phylogenetic lineages in the Laurasiatheria clade (i.e. Carnivora + Pholidota + Eulipoty-

Genome organizations of the pangolin, sloth, anteater and hedgehog phla/Cetartiodactyla + Perissodactyla/Chiroptera), but the wide occurrence of apparently convergent syntenic associations has so far prevented an unambiguous resolution to the puzzling phylogenetic nodes previously unresolved by molecular markers.

Acknowledgements This work was partly supported by the China National Natural Science Foundation grants to F.Y. and W.N. (Nos. 30025026 and 30270719), a Wellcome Trust grant to M.A.F-S, as well as MCB, RFBR and BOE grants to A.S.G.

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