Reconstruction of the ancestral karyotype of eutherian mammals

Reconstruction of the ancestral karyotype of eutherian mammals

F. Richard1,2, M. Lombard1 & B. Dutrillaux1,3

1UMR 147 CNRS, Institut Curie, Section Recherche, 26 rue d’Ulm, 75248 Paris Cedex 05, France;Tel: 33 1 42 34 66 72; Fax: 33 1 42 34 66 74; E-mail: £[email protected];2Universite¤ de Versailles-Saint-Quentin, 45 avenue des Etats Unis, 78030 Versailles, France;3De¤ partement de Radiobiologie, CEA-DSV, 92265 Fontenay-aux-Roses (France)

Received 5 March 2003. Received in revised form and accepted for publication by Paul Popescu 20 April 2003

Key words: ancestral chromosomes, chromosome painting, comparative cytogenetics, Eutheria,£uorescence, in-situ hybridization, mammals

Abstract

Applying the parsimony principle, i.e. that chromosomes identical in species belonging to different taxawere likely to be present in their common ancestor, the ancestral karyotype of eutherian mammals (about100 million years old) was tentatively reconstructed. Comparing chromosome banding with all ZOO-FISHdata from literature or studied by us, this reconstruction can be proposed with only limited uncertainties.This karyotype comprised 50 chromosomes of which 40^42 were acrocentrics. Ten ancestral pairs ofchromosomes were homologous to a single human chromosome: 5, 6, 9, 11, 13, 17, 18, 20, X and Y (humannomenclature). Nine others were homologous to a part of a human chromosome: 1pþ q (proximal), 1q,2pþ q (proximal), 2q, part of 7, 8q, 10p, 10q and 19p (human nomenclature). Finally, seven pairs of chrom-osomes, homologs to human chromosomes 3 þ 21, 4 þ 8p, part of 7 þ 16p, part of 12 þ part of 22(twice), 14þ 15, 16qþ 19q, formed syntenies disrupted in man.

Introduction

In 1964, Ohno and coworkers reported that the Xchromosome banding pattern was highly con-served among mammals. During the followingdecades, development of chromosome bandingand comparative cytogenetics revealed that strongconservation of chromosome banding patternswas not limited to the X chromosome and thatmammals belonging to di¡erent families or ordersshared many conserved autosomes (Buckland &Evans 1978, Dutrillaux 1979b, Dutrillaux et al.1982). Identi¢cation of ancestral chromosomessigni¢cantly contributed to phylogenetic studies

and to the reconstruction of hypothetical ancestralkaryotypes for Catarrhini, Simii, all Primates andother mammalian orders (Dutrillaux et al. 1982,Dutrillaux & Couturier 1983, Petit et al. 1984) butuncertainties did remain. In particular, compari-son of small chromosomes from distantly relatedspecies was impossible. In some taxa, it has beenpossible to con¢rm and re¢ne chromosomebanding interpretations by comparative genemapping (O’Brien & Graves 1991). However, untilrecently, large-scale comparative gene mappingwas limited because it represents a long-lastingtask and because of the lack of probes covering thewhole genome of many species.

Chromosome Research 11: 605^618, 2003. 605# 2003 Kluwer Academic Publishers. Printed in the Netherlands

With the development of ZOO-FISH, inter-speci¢c comparisons have become easier and moreaccurate for identifying small chromosomes andinterchromosomal rearrangements involving seg-ments as small as 5^10 megabases. ZOO-FISHcon¢rms the strong conservation of synteny blocksduring chromosome evolution but cannot revealthe large number of intrachromosomal changes

which exist in many species. For instance, 25 of the29 structural rearrangements identi¢ed by chro-mosome banding between great apes and man areperi- or paracentric inversions (Dutrillaux 1979a),undetected by ZOO-FISH using whole chromo-some probes. Thus, an approach involving bothZOO-FISH and banding techniques is essential foran accurate study of chromosome evolution.

Table 1. Information about eutherian species considered.

Orders/suborders Latin names Codes English names R-banding and ZOO-FISH references

Artiodactyla *Bos taurus BTA Cattle (Gustavsson et al. 1976)/(Hayes 1995)Bubalus bubalis BBU River buffalo (Iannuzzi et al. 1990)/(Iannuzzi et al. 1998)Muntiacus muntjakvaginalis

MMV Indian muntjac ND/(Yang et al. 1997)

*Ovis aries OAR Sheep (Di Berardino et al. 1990)/(Burkin et al. 1997)*Sus scrofa SSC Pig (Ronne et al. 1995)/(Rettenberger et al. 1995)

Carnivora Felis cattus FCA Cat (Roubin et al. 1973)/(Wienberg et al. 1997)*Felis serval FSE Serval cat (Couturier & Dutrillaux 1986)/ND*Mustela vison MVI American mink (Couturier & Dutrillaux 1986)/

(Hameister et al. 1997)*Panthera leo PLE Lion (Roubin et al. 1973)/this study*Paradoxurushermaphroditus

PHE Palm civet (Dutrillaux & Couturier 1983)/this study

*Phoca vitulina PVI Harbor seal Unpublished/(Fronicke et al. 1997)

Cetacea Tursiops truncatus TTR Atlantic bottlenose dolphin ND/(Bielec et al. 1998)

Chiroptera Glossophaga soricina GSO Long-tongued Bat ND/(Volleth et al. 1999)

Edentata *Dasypus novemcinctus DNO Nine-banded armadillo This study

Insectivora *Sorex araneus SAR Common Shrew (Volobouev 1989)/(Dixkens et al. 1998)

Lagomorpha *Oryctolagus cuniculus OCU Old world rabbit (Dutrillaux et al. 1980/(Korstanje et al. 1999)*Lepus europaeus LEU European hare This study/ND

Perissodactyla *Equus caballus ECA Horse (Romagnano & Richer 1985)/(Raudsepp et al. 1996)

*Equus zebrahartmannae

EZH Mountain zebra (Richard et al. 2001)/(Richard et al. 2001)

Primates *Homo sapiens HSA ManCatarhini *Pongo pygmaeus PPY Orangutan (Dutrillaux et al. 1975)/(Jauch et al. 1992)

**Macaca fuscata MFU Japanese macaque ND/(Wienberg et al. 1992)*Papio hamadryas PHA Hamadryas baboon (Dutrillaux 1979a)/(Best et al. 1998)

Platyrrhini *Cebus capucinus CCA White-throated capuchin (Dutrillaux 1979b)/(Richard et al. 1996)Prosimii *Eulemur fulvus EFU Brown lemur (Rumpler & Dutrillaux 1976)/

(Muller et al. 1997)*Microcebus murinus MMU Lesser mouse lemur (Rumpler & Dutrillaux 1990)/ND

Rodentia *Menetes berdmorei MBE Indochinese ground squirrel (Petit et al. 1984)/(Richard et al. submitted)

Scandentia Tupaia belangeri TBE Northern tree-shrew (Toder et al. 1992)/(Muller et al. 1999)*Tupaia chinensis TCH Chinese tree-shrew (Toder et al. 1992)/this study

Personal ZOO-FISH data are indicated in bold. *Karyotypes of species studied by R-banding in our laboratory. **Karyotype similar tothat of Macaca sylvana (Dutrillaux et al. 1982). ND: not done.

606 F. Richard et al.

Another important step to reconstruct ancestralkaryotypes is the identi¢cation, among livingspecies, of those which have the most conservedchromosomes. This implies the comparison ofmany species within the same order and fromdi¡erent orders. For instance, among Rodentia,although the mouse has a highly derived kar-yotype, most investigators refer to this species.Petit and collaborators (1984) have shown that thechromosomes of other rodents i.e. Sciuridae(squirrels and associates) are much easier tocompare with those of other orders. Thus, studieson the chromosomal evolution of Rodentia wouldtake this taxon as reference.

Previous attempts to reconstruct the ancestralkaryotype of placental mammals have used eitherbanding data (Dutrillaux et al. 1981, 1982) orZOO-FISH and molecular data (Chowdhary et al.1998, Haig 1999, Murphy et al. 2001a, Yang et al.2003). Our objective is to extend these studies bycomparing published and personal ZOO-FISHdata on species belonging to eleven from thesixteen orders of living placental mammals andchromosome band patterns of more than 200eutherian species using our own published andunpublished data. Although a few uncertaintiesremain in the ancestral karyotype of eutherianmammals proposed here, it should provide a usefultool for further phylogenetic studies.

Material and methods

Species studied

Although chromosome-banding data exist formore than 200 eutherian species in our laboratory,we have selected 29 species from eleven mam-malian orders (Table 1) according to the avail-ability of ZOO-FISH data with humanchromosome paints, which exist only for a limitednumber of species. We have used published dataand also new data reported here for Panthera leo,Paradoxurus hermaphroditus (Carnivora), Men-etes berdmorei (Rodentia), Dasypus novemcinctus(Edentata, Figure 1a) and Tupaia chinensis(Scandentia). The ZOO-FISH and R-banding references used are described in Table 1. We also considered Lepus europaeus (Lagomorpha, Figure 1b)and Microcebus murinus (Primates) for R-bands

only, because they are close to two species forwhich ZOO-FISH data exist, O. cuniculus and E.fulvus, respectively, and their karyotypes are lessderived.

Chromosome banding and ZOO-FISH techniques

Chromosome banding techniques (RHG, RBG,GTG and CBG) were applied as described inDutrillaux & Couturier (1981).In-situ hybridization using whole human chro-

mosome probes (painting) was performed onmetaphase chromosomes. Human chromosomeprobes were either purchased from Cambio-Biosys(Compie' gne, France) or kindly provided byM. N. Guilly (CEA, DRR, Fontenay aux Roses,France). Brie£y, chromosome preparations werehybridized for at least 72 h at 37�C and thenwashed at 40�C for 10 min once in 50% formamide,2� SSC, pH 7 and once in 2� SSC, pH 7.Hybridized probes were revealed in green byindirect detection with antibiotin antibodies(10 mg/ml, Vector laboratories, Burlingame) fol-lowed by FITC-conjugated antigoat antibodies(5 mg/ml, P.A.R.I.S., France). Chromosomes werecounterstained in blue with DAPI (0.5 mg/ml) andobserved with an epi£uorescence microscope(Microphot-FXA, Nikon, Japan) equipped with aCCD camera (Photometrics, Tucson, Arizona,USA). They were identi¢ed as described inRichard et al. (1996) or using a capture software(Quips-Smart, Vysis, Downers Grove Il, USA) byreverse DAPI-banding (Figure 2).

Results

ZOO-FISH

Weadded to published data new results onP. leo,P.hermaphroditus (Carnivora), T. chinensis (Scan-dentia), and on two species belonging to orderswhich have never been described before by this app-roach, D. novemcinctus (Edentata) and M. berd-morei (Rodentia), presented in Table 2a and b andFigure 2.

Since human chromosome paints are the mostfrequently used in ZOO-FISH experiments, wetook human chromosomes as reference toappreciate the conservation of synteny blocks

Ancestral karyotype of eutherian mammals 607

among species and orders. We established, inTable 3, the smallest number of chromosomalsegments corresponding to each human chromo-some in the eleven orders examined. This tableallowed us to de¢ne two categories of chromo-somes:

(1) Those conserved as a single block of synteny inspecies belonging to more than three di¡erentorders i.e. the homoeologs to human chromo-somes (HSA) 3, 4, 5, 6, 9, 11, 13, 14, 15,17, 18, 20, 21 and X. It is very likely that theseshared blocks of synteny correspond to ances-tral conditions.

(2) Those disrupted into two or more fragments inspecies belonging to more than three orders,i.e. the homoeologs to HSA1, 2, 7, 8, 10, 12, 16,19 and 22. Based on the principle of parsimony,

we have considered that the combination withthe smallest number of fragments of similarsize (i.e. two; see Table 3) is likely to representthe ancestral form if species of di¡erent ordersshare these chromosomes.

This analysis has also allowed us to detect sevenshared syntenies involving sequences conservedwith two di¡erent human chromosomes: HSA3and 21, HSA4 and 8, HSA7 and 16, HSA12 and 22(twice), HSA14 and 15, and HSA16 and 19. Table 4summarizes their distribution among the elevenorders studied here. They were found in speciesbelonging to the majority of orders studied exceptfor the synteny involving sequences homoeologousto HSA4 and HSA8, found in ¢ve of the ordersstudied only. These combinations are expected torepresent ancestral groups of synteny disrupted inthe human lineage.

Figure 1. (a) R-banded karyotype of Dasypus novemcinctus (nine-banded armadillo). (b) R-banded karyotype of Lepus europaeus(european hare). This karyotype differs from that of O. cuniculus by the presence of four acrocentric chromosomes (LEU16, 17,18 and 19) instead of two metacentric chromosomes (OCU1 and 2). Three of these acrocentric chromosomes are similar to the ancestralchromosomes EUT 11, 12 and 15 (see Results). Thus, the karyotype of O. cuniculus derives from that of L. europaeus, because of twoRobertsonian translocations, and the karyotype of L. europaeus has retained many ancestral features.


Overall, we propose that the ancestral karyo-type for eutherian mammals (EUT) is composed oftwice 25 blocks of synteny i.e. 25 chromosomepairs. Figure 3 shows the correspondences withhuman chromosomes, except for chromosome Ynot considered here. Centromere positions arepartially deduced from ZOO-FISH data and wehave considered that, if similar submetacentricchromosomes are observed in species belonging to

di¡erent orders, the ancestral form is probablysubmetacentric and, if not, it is acrocentric. Below,we describe, for each eutherian ancestral chro-mosome, the pattern of synteny conservation ascompared to human chromosomes in the di¡erentspecies and orders considered in this study:

. EUT1: syntenic sequences conserved with partsof HSA4 and HSA8p exist in species belonging

Figure 2. Chromosome painting data revealing homoeologies between human chromosomes 6 (a, b), 12 (c, d), 14 (e, f, i, j) and 15 (g, h,k, l) and lion (PLE7, a, b), armadillo (DNO12 and DNO1, c, d, e, f, g, h), and palm civet (PHE3, i, j, k, l) chromosomes. Chromosomesare identi¢ed using reverse DAPI-banding (b, d, f, h, j, l). Scale bars¼ 10 mm.


to ¢ve orders (B. taurus, F. cattus, O. cuniculus,E. zebra and M. berdmorei, see Table 4), whileassociation of chromosomal segments con-served with the whole of HSA4 and HSA8pis present in all the carnivores studied (F. cattus,M. vison, P. hermaphroditus, P. leo and P. vitu-lina). The morphology of this chromosomeremains uncertain (Richard et al. 2001). To con-¢rm this ancestral synteny, studies of othereutherian orders would have to be done.

. EUT2: syntenic sequences conserved with partsof HSA14 and 15 exist in species in all the ordersexamined, including non-human Primates(Table 4). In addition, syntenies consisting ofsegments conserved with the entire HSA14and 15 are present in species belonging to ¢veorders: Carnivora (F. cattus, M. vison, P. herm-aphroditus and P. vitulina), Cetacea (T. trunca-tus), Edentata (D. novemcinctus), Primates(M. fuscata and P. hamadryas) and Rodentia

Table 2a. Number of chromosome segments corresponding to each human chromosome (HSA) in the different species studied.

HSA 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X

PLE (Car) 2 1 2PHE (Car) 1 1 2DNO (Ede) 3 1? 1? 1 1 2 1 1 1?MBE (Rod) 2 2 1 2 1 1 2 2 1 2 1 2 1 1 1 2 1 1 2 1 1 2 1TCH (Sca) 1? 2 1 2 1 2 2 1 1 2

Table 2b. Species studied sharing syntenies involving sequences corresponding to two human chromosomes (HSA).

Species studied PLE PHE DNO MBE TCH

HSA3-21 þ þ þ

HSA4-8 ? þ

HSA7-16 þ þ þ þ

HSA12-22 þ þ (x2) þ (x2)HSA14-15 þ þ þ þ

HSA16-19 þ þ

The species are indicated by a three-letter code (see Table 1).Car: Carnivora;Ede: Edentata;Rod: Rodentia; Sca: Scandentia.

Table 3. Number of chromosome segments conserved with each human chromosome (HSA) in the different orders examined.

HSA 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X

BTA/SSC (Art) 3 2 1 1 2 2 2 2 1 2 2 2 1 2 2 2 1 1 2 1 1 2 1FCA/PVI/PLE (Car) 2 2 1 1 1 1 2 2 1 2 1 2 1 1 1 2 1 1 2 1 1 2 1TTR (Cet) 1 2 2 1 1 2 2 1 1 1 1 2 1 1 1 2 1 1 2 1 1 2 1GSO (Chi) 3 2 2 2 2 2 2 2 1 2 2 2 1 1 2 2 1 1 2 1 1 2 1DNO (Ede) ^ ^ 3 ^ ^ ^ 1? ^ ^ ^ ^ 1? ^ 1 1 2 ^ 1 ^ ^ 1 1? 1SAR (Ins) 2 1 3 2 2 1 1 2 1 2 2 2 1 1 1 2 1 1 1 1 1 1 1OCU (Lag) 2 2 4 2 2 1 4 2 1 2 1 3 1 2 2 2 1 1 2 1 1 2 1EZE/ECA (Per) 4 3 2 2 2 2 2 2 2 2 2 3 1 2 1 2 1 1 2 1 1 2 1CCA/EFU (Pri) 3 2 1 1 1 1 2 2 1 2 1 1 1 1 2 2 1 1 1 1 1 1 1MBE (Rod) 2 2 1 2 1 1 2 2 1 2 1 2 1 1 1 2 1 1 2 1 1 2 1TBE/TCH (Sca) 2 2 4 4 2 2 2 1 1 2 2 2 1 1 2 2 1 1 2 1 1 2 1

For each human chromosome and within each order, we selected the species with the smallest number of conserved segments. These speciesare indicated by a three-letter code (see Table 1). For Primates, only Platyrrhini and Prosimii were considered. Art: Artiodactyla; Car:Carnivora; Cet: Cetacea; Chi: Chiroptera; Ede:Edentata; Ins: Insectivora; Lag: Lagomorpha; Per: Perissodactyla; Pri: Primates; Rod:Rodentia; Sca: Scandentia; ^: undetermined.


(M. berdmorei). The centromere position is fre-quently sub-distal, suggesting that EUT2 is sub-metacentric, with sequences corresponding toHSA15 in the pericentromeric region.

. EUT3: this chromosome is composed of synten-ic sequences conserved with the entire HSA6either as an acrocentric chromosome as inM. vison (Carnivora) or slightly di¡erent sub-metacentric chromosomes as in O. cuniculus(Lagomorpha), P. leo (Carnivora) and M. berd-morei (Rodentia). Thus, its morphologyremains uncertain.

. EUT4 and EUT5: these chromosomes corre-spond to associations of segments conservedwith HSA16q and 19q, and HSA16p and part

of HSA7, respectively. The most frequentlyobserved chromosome morphology is submeta-centric and thus we propose that EUT4 andEUT5 are submetacentric chromosomes, asdescribed in Richard et al. (2000).

. EUT6: association of syntenic sequences con-served with HSA3 and HSA21 and the originof this acrocentric ancestral chromosome havebeen discussed elsewhere (Maccarone et al.1992, Richard & Dutrillaux 1998, Muller et al.2000).It is likelythattheacrocentricmorphology,observed inS. scrofa, is the ancestral form.

. EUT7 to EUT12, EUT14 to EUT21, EUT23and EUT24: each of these chromosomes con-sists of segments conserved with parts of a single

Table 4. Orders sharing syntenies involving sequences conserved with two human chromosomes (HSA).

Orders Art Car Cet Chi Ede Ins Lag Per Pri Rod Sca

HSA3-21 þ þ þ þ þ þ þ þ þ þ þ

HSA4-8 þ þ ? þ þ þ

HSA7-16 þ þ þ þ þ þ þ þ



HSA16-19 þ þ þ þ þ þ þ þ þ þ

In Artiodactyla, Carnivora, Perissodactyla and Primates, these syntenies, when they exist, were observed in at least two species. Forabbreviations, see Table 2.

Figure 3. Karyotype of the eutherian ancestor reconstructed using ZOO-FISH data. EUT chromosome numbers are indicated undereach diagram and homoeologies with human chromosomes are indicated on the right of each pair. Morphology of EUT1, EUT3and EUT22 remains uncertain. 7a, 7b, 12a, 12b, 22a and 22b correspond to parts of HSA7, 12 and 22, respectively.


human chromosome or with a whole humanchromosome as indicated in Figure 3. In thesecases, we never observed two similar submeta-centric or metacentric forms in di¡erent orders;thus we assume that these ancestral chromo-somes are acrocentric.

. EUT13 and EUT22: association of syntenicsequences conserved with parts of HSA12 andHSA22 is present in species belonging to tenorders (Artiodactyla, Carnivora, Cetacea, Chir-

optera, Edentata, Lagomorpha, Perissodac-tyla, Primates, Rodentia and Scandentia; seeTable 4). This association is split into two chro-mosomes in B. taurus and S. scrofa (Artiodac-tyla), F. cattus and M. vison (Carnivora),T. truncatus (Cetacea), G. soricina (Chiroptera),E. fulvus (Primates), M. berdmorei (Rodentia)and T. belangeri (Scandentia). We consider thatEUT13 and EUT22 are acrocentric chromo-somes.

Figure 4. Comparisons, based on ZOO-FISH data (except for LEU, FSE and MMU), of R-banded chromosomes from various speciesexhibiting high similarities of band patterns. Three-letter codes indicate the species names (see Table 1); PPY: Pongo pygmaeus. Therelative position of conserved segments with HSA12 and 22 in TCH28 and MMU23 is unknown.


. EUTX: submetacentric X chromosomes exist inspecies belonging to all the orders examined;therefore it is likely that the morphology ofEUTX resembles that of HSAX.

Chromosome banding

In our laboratory, we have analyzed R-bandedkaryotypes of about 200 species belonging to allthe orders examined by ZOO-FISH exceptCetacea. The R-banded karyotypes of D.novemcinctus and L. europaeus are shown for the¢rst time in Figure 1. We have found that most ofkaryotypes fall into two main categories: one withkaryotypes containing many chromosomes withband patterns easily compared with those ofspecies of other orders and the other with kar-yotypes in which the chromosome band patternsare di⁄cult or impossible to compare with those ofspecies from other eutherian orders. Some Car-nivora, Lagomorpha (for instance, L. europaeus),Primate species and Rodentia species of theSciuridae family belong to the ¢rst category whileArtiodactyla, Perissodactyla, Insectivora, Chir-optera and other Rodentia species belong to thesecond category. Scandentia (for instanceT. chinensis) and Edentata (for instance D.novemcinctus) karyotypes constitute an inter-mediate category with only a few chromosomeseasily compared with those of the ¢rst category.

In the second category, the fact that ZOO-FISHreveals strong interspecies conservation of chro-mosomal segments in spite of di¡ering bandpatterns, indicates that several intrachromosomalrearrangements have occurred during evolution.On the contrary, when both ZOO-FISH andcomparative banding data con¢rm conservation ofentire chromosomes or large parts of chromo-somes, these represent ancestral forms. Forinstance, comparisons of some R-banded chro-mosomes, based on ZOO-FISH data, are pre-sented in Figure 4. It shows band patterns verysimilar for chromosomes of distant species.According to this high degree of band patternconservation, ancestral form could be proposed:the equivalent to LEU17 or MMU10 for EUT12,to CCA16, MMU5 or LEU16 for EUT11, toTCH9 or MMU7 for EUT13 and to DNO1 orPHE3 for EUT2. The morphology of EUT3

remains uncertain (see below). Table 5 lists thechromosomes and chromosome arms of speciesbelonging to the ¢rst (Carnivora, Lagomorpha,Rodentia, Primates) and the intermediate category(Edentata and Scandentia) which share similarband patterns for ancestral syntenies. In eachorder, species were selected for their karyotypehaving the highest number of conserved syntenies,according to ZOO-FISH and banding data or onlyR-banding data. These comparisons were used toreconstruct the R-banded karyotype of theeutherian ancestor, shown in Figure 5.

The corresponding chromosomes to 16 of the 25EUT chromosome pairs (i.e. EUT3^5, 8^14, 19^23and X) were observed in the karyotypes of speciesbelonging to at least three of the orders describedin Table 5 and Figure 4. For EUT3, in species inwhich the corresponding chromosome consists of aunique block of synteny, it is either distributedalong a single chromosome arm or along sub-metacentric chromosomes with slightly di¡erent Rband patterns (Figure 4). Although a submeta-centric EUT3 is proposed in Table 5, its cen-tromere position remains uncertain.

Band patterns for EUT2, 6, 7, 15, 16, 17 and 24are deduced from band similarities observed inspecies belonging to only two di¡erent orders. ForEUT2 and 7, homoeologous chromosomes wereobserved in di¡erent Carnivora species and inD. novemcinctus (see Table 5 and Figure 4). ForEUT6, MMU1 (Table 5) and chromosome 13 ofpig (Artiodactyla) exhibit a similar band pattern.For EUT15 homoeologous chromosomes wereobserved in di¡erent Carnivora and Lagomorphaspecies, and for EUT16 and 24, in some Lago-morpha and Primates species (see Table 5). ForEUT17, some Lagomorpha and some Cebidae(Primates; Dutrillaux et al. 1986) species show asimilar band pattern.

The band pattern proposed for EUT1 corre-sponds to a chromosomeobserved in oneorder only(Carnivora) and does not correspond to the mostparsimonious representation proposed in Figure 3.Thus, it is not certain. In the case of EUT18,probably an acrocentric chromosome, no similarband pattern was observed in species belonging todi¡erent orders. Thus, it is not possible to choosebetween the band pattern observed in some Car-nivora (for instance P. hermaphroditus, Figure 5,left), Lagomorpha (for instance L. europaeus,


Figure 5, right), Rodentia (M. berdmorei) andScandentia (T. belangeri or chinensis). Finally, itmustbenoticedthatacompletesyntenyofsequencesconserved with HSA1 is observed in T. truncatus.This chromosome is either the result of a convergentevolution or homoeologous to the ancestral form.Thus, it is possible that the ancestral karyotype ofeutherian mammals was composed of 48 chromo-somes only, with one pair of chromosomes homo-eologous to HSA1 instead of two, EUT8 and 18.Therefore, EUT chromosome morphologies and

band patterns proposed in Figure 5 are probablycorrect, and uncertain for EUT1, 3, 8 and 18.

Table 5 also shows that M. murinus with 15conserved chromosomes as compared to theeutherian ancestral karyotype is the species withthe least derived karyotype. When consideringchromosome arms, we observe that species fromCarnivora, Lagomorpha and Rodentia possessseveral conserved but translocated ancestralchromosomes. This indicates that Robertsoniantranslocations involving chromosomes of the

Table 5. Whole chromosomes or chromosome arms of species from six orders with similar banding patterns and corresponding presumedancestral chromosome (EUT).

Car Lag Rod Pri Ede Sca

EUT FCA PHE OCU LEU MBE EFU MMU DNO TBE/TCH

1? B1 12 B3 3 13a B2 2 12 6 84 E2 20 5 14 14 5 55 E3 10 6 15 15 86b 1q 17 A1q 12 118 1q 3 109 A2q 4q 6q 12 11 12

10 C1q 14 4q 8 811 1q 16 2q 11 512 1p 17 2p 1p 10 1q13 B4q 5q 5q 10 7 914 F2 17 3q 2q 1p 1715 A3q 6q 2q 1816 18 20 14 1517b 8q 4q1819 19 21 4p 15 16 24 18?20 D3q 9q 5q 16q 16 19 2521 A3p 6p 4p 3p 3p 17? 18 2q22 D3p 21 23 19 23 2823 B4p 5p 16p 11p 12p 26 2924 11p 13p 26X X X X X X X X XTotal number of

conserved chromosomes 7 9 7 10 4 10 15 9 6Total number of

conserved arms 9 5 9 6 11 2 0 0 2Total 16 14 16 16 15 12 15 9 8

Species are indicated by a three-letter code (see Table 1). Chromosome nomenclatures used for FCA, OCU, EFU and TBE are described inTable 1 (ZOO-FISH references) and for PHE, MBE, MMU and TCH in Table 1 (R-banding references). Chromosomes and arms withsimilar band patterns, according to ZOO-FISH and banding data, are in bold characters. aBand patterns of the pericentromeric regionsof these chromosomes seem to be different. bEUT6 morphology is also observed in S. scrofa (SSC13) and EUT7 in some Cebidae.Constitutive heterochromatin variation in PHE10 chromosome was not taken into consideration.


ancestral karyotype of eutherian mammals haveoccurred during evolution in these orders.

Discussion

We present the reconstruction of the ancestraleutherian karyotype based on ZOO-FISH andchromosome-banding data. It is composed of2n¼ 50 chromosomes, which is close to thenumber proposed by Matthey (1973) based on thefact that many placental mammals have a chro-mosome number around 48. If all sequenceshomoeologous to HSA1 were syntenic, theancestral karyotype would be of 48 chromosomes.Thus, it appears that conservation of chromosomenumber and syntenies has occurred with fewinterchromosomal rearrangements during the 100million years separating ancestral and livingspecies. Indeed, leaving aside intrachromosomalrearrangements, about 10 interchromosomalrearrangements and ¢ssions are su⁄cient toreconstruct the human karyotype from theancestral karyotype proposed here. In the early1980 s, di¡erent versions of the ancestral kar-yotype of eutherian mammals based oncomparisons of R-banded chromosomes were

reported (Dutrillaux et al. 1981, 1982). Inter-pretations varied slightly with regard to chro-mosome number (54 and 58) and the morphologyof a few chromosomes. However, the sameinterpretations were given for most of the largechromosomes and these agree with the presentones. Moreover, the correspondences with humanchromosomes, which were determined by bandingcomparisons for chromosomes EUT3, 7, 8, 10 to12, 14 to 17, 19, 20, 24 and X, have proven correct.Concerning EUT2, EUT6 and EUT13, theinterpretation for the band patterns was correctbut not for the correspondences with humanchromosomes. In addition, banding comparisonscould not detect rearrangements involving smallchromosomal fragments, which are now identi¢edby ZOO-FISH techniques. This is the case forEUT1 and 14 conserved with HSA4 and 8, EUT5conserved with HSA7b and 16p and EUT22conserved with HSA12 and 22. Finally, nobanding interpretation had been proposed forEUT4, 9, 18, 21 and 23.

Other studies based on ZOO-FISH data(Chowdhary et al. 1998, Yang et al. 2003) orcompiling gene mapping and ZOO-FISH data(Murphy et al. 2001b) led to more or less similareutherian ancestral karyotypes. Chowdhary et al.

Figure 5. Karyotype of the eutherian ancestor reconstructed with R-banded chromosomes from different species. ChromosomesEUT1, 2, 7, 18 (left) and X (left) come from P. hermaphroditus (PHE 1, 3, 12, 18 and X, respectively), chromosomes EUT3 (left),4, 5, 11, 12, 14, 15, 16, 17, 18 (right), 19, 20, 21, 22, 23 and X (right) from L. europaeus (LEU6, 14, 15, 16, 17, 2q, 18, 20, 4q, 11q,21, 5q, 3p, 23, 11p and X, respectively), chromosomes EUT6, 8, 9, 10, 13 and 24 from M. murinus (MMU1, 3, 8, 6, 7 and 25,respectively) and chromosome EUT3 (right) from T. chinensis (TCH8). The band pattern of EUT1, 3, 18 remains uncertain.


(1998) proposed 25 undisrupted segments in com-parison to human chromosomes, instead of the 32in the present study, and several contiguous syn-tenic sequences conserved with HSA3 and 21,HSA12 and 22, HSA14 and 15, and HSA16q and19q. The discrepancies between the ancestral kar-yotype reported here and that proposed by Murphyet al. (2001b) are limited to EUT1-3, 6, 7 and 12.These authors hypothesize, for reconstructingEUT1, 3, 6 and 12, thatCarnivora have retained themost ancestral chromosomes, which introduces abias. ForEUT2, the position of the centromere theypropose is erroneous, since ZOO-FISH reveals thatpericentromeric sequences are conserved withHSA15andnotHSA14, and forEUT7, they give noargument in favor of the submetacentric formpresented. Yang et al. (2003) proposed anotherancestral karyotype with 2n ¼ 44 chromosomesafter comparison between human and Afrotheriaspecies. The conservation of sequences homo-eologous to HSA1 in a unique block is clearlyproposed in this study. Furthermore, they pro-posed two additional syntenies involving HSA1and 19p, and HSA10p, 12 and 22.

The conservation of the ancestral genomestructure is also revealed by other features ratheruniform in a conserved segment, like the GþCcontent, SINE and LINE densities, and varying inthe neighboring segments. For instance, it is thecase of the synteny HSA3-21 observed in mousechromosome16showingmarkeddiscontinuities forthese features at the ends of the segment, con¢rm-ing the ancestral synteny (Mural et al. 2002).

Intrachomosomal rearrangements, like inver-sions and translocations, are not taken intoconsideration in this study but can enhance therate of rearrangements that have occurred betweenthe eutherian ancestor and living species. Forinstance, Murphy et al. (2000) show that rear-rangements in cat were three-fold higher whenintra- and interchromosomal rearrangements areconsidered. The importance of this kind of rear-rangement must be slightly superior for speciesbelonging to the second category de¢ned in thisstudy. But, molecular data con¢rm that order andorientation of the orthologous genes or sequencestested are highly conserved during evolution(Mural et al. 2002).

Other modi¢cations, not observed with ZOO-FISH and banding techniques, are involved in

genome evolution, like insertion of retroviralsequences or gene duplication. Gene duplicationsmay be more frequent than expected (Bailey et al.2001) and the large interspeci¢c variability in thenumber of olfactive receptors is one example(Trask et al. 1998). Another example is the pre-sence of multiple duplications and translocationsinvolving a variant melanin-concentrating hor-mone gene located on both p and q (p14.3 andq13.3) arms of human chromosome 5 (Viale et al.1998) while Macaca chromosome 5, althoughcytogenetically identical, carries a single copy ofthis gene. These recent duplications in the evo-lution of the Hominoidae lineage, pinpoint thelimitations of chromosome banding and ZOO-FISH comparisons.

Acknowledgements

We wish to thank M. N. Guilly for giving us someprobes and Dr. He¤ le' ne Hayes for her criticalreading of the manuscript and corrections.

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Reconstruction of the ancestral karyotype of eutherian mammals

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