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536 Forum TRENDS in Genetics Vol.17 No.9 September 2001

Science Chronicle Oranges and : clues to the of from molecular markers Gloria A. Moore

Go into any grocery store and one is difficult to ascertain centers of origin and the types available were probably bitter confronted with an array of Citrus : diversity because of natural and perhaps and used mostly as condiments. Columbus oranges, , mandarins (), human-aided hybridization, wide and Ponce de Leon carried various Citrus lemons and limes. This is rich bounty for the dispersion, and the paucity of remaining to the New World in the late 1400s shopper,but taxonomists are perplexed as to wild Citrus stands. At the moment, the and early 1500s, as did others. The how to classify the various kinds of Citrus that center of origin and diversity of Citrus and Portuguese introduced one or more have existed since antiquity.Now, thanks to its related genera is generally considered superior types of sweet into new genetic and molecular biological to be Southeast Asia, especially east India, Europe, probably in the 16th century. techniques, the relationships between these north Burma and southwest , but Although mandarins had been cultivated fruit are being unraveled and show that there possibly ranging from northeastern India in China and Japan from ancient times, are probably only three true . eastward through the Malay archipelago, the first mandarin varieties were brought north into China and Japan, and south to to England in 1805 by Sir Abraham Hume, Until the mid 1970s, Citrus taxonomists Australia3–8. The wild relatives of Citrus and they only subsequently spread to the based their conclusions solely on are native to Southeast Asia, the Mediterranean region7,8. morphological and geographical data. East Indian Archipelago, New Guinea, All Citrus types except the Citrus relative This led to major disagreements on the Melanesia, New Caledonia and . the are highly sensitive to classification of species within the Citrus The exact routes of dispersion of freezing; this limits their growth range, and subgenus (Box 1). One definition of a species Citrus from its origin are also unknown. the could not survive unprotected in is that when populations of two kinds occur However, Citrus is mentioned in the most parts of Europe. Therefore, together without interbreeding, they are ancient writings of many cultures4,8. The and other devices for citriculture were different species. Thus, some ‘joiners’believe oldest known reference to Citrus appears developed. European sailors became aware that all Citrus types belong to one large in Sanskrit literature that dates to before of the benefits of Citrus consumption in species because they are all graft-compatible 800 BC; descriptions in Chinese, Greek and preventing scurvy during their voyages, and and, were it not for the reproductive barriers Roman literature followed8. It is believed they carried Citrus fruit for consumption described below, they are all interfertile. that some Citrus types, including , and and trees to along trade Swingle, in 1943, devised a system for Citrus sour oranges and lemons, spread slowly routes. Citrus culture proliferated in Florida where ten species in the subgenus Citrus (from 500 to 1300 AD) through wide areas, in the late 1700s, when the first commercial were recognized (Table 1)1,2. However, including into Europe, by successive shipments were made. At about the same Tanaka3 (the ultimate ‘splitter’) defined waves of invaders and travelers – Muslim time, Citrus was introduced into California, 147 different species in 1954. A major armies, Arab traders, Crusaders and although commercial production only difference in these two systems was in others moving along trade routes. began there much later4. how mandarins were treated; Swingle Although some Citrus trees were A second factor that makes taxonomic placed all mandarins except C. tachibana, grown in Europe during these centuries, classification difficult is the reproductive a wild species from Japan, and C. indica, a wild species from India, in C. reticulata, Glossary whereas Tanaka separated mandarins into 36 species. The vast difference in Accession: A distinct type or of plant, not necessarily a ; e.g. a breeding line or a related wild number of species recognized in these two species. : Reproduction of a plant without any form of sexual union. Obligate apomixis, where reproduction systems and some intermediate ones occurs by apomixes only; facultative apomixis, where reproduction can occur by apomixes or normal sexual reflected opposing theories on what degree reproduction. of morphological difference justified Cultivar: A term contracted from ‘cultivated variety’; a plant type that is clearly distinguished from any other type by any characters (morphological, physiological, cytological, chemical, or any others), and that, when species status and whether presumed reproduced (sexually or asexually), retains its distinguishing characters. A variant cultivar is one that differs hybrids among naturally occurring forms from others in the group of for a specific characteristic, such as an isozyme genotype. should be given species status. Discontinuity of traits: A distinct profile of characteristics maintained in a species by reproductive isolation. Several characteristics of the Citrus Intraspecific affinity: The degree of similarity, measured statistically by correlation analyses, of given characters within members of a species. subgenus made taxonomic classification Introgression: Hybridization between donor and recipient parents, followed by multiple crosses to the recipient on the basis of morphology and geography parent such that only a relatively small part of the genome of the donor parent is ultimately transferred. especially difficult. First, Citrus is an Jaccard coefficient: A statistical measurement of similarity of attributes between samples. Rootstock: The system, the bottom part of a plant propagated by . ancient crop, with records of human Trihybrid intergeneric cross: A cross involving three original parental types, with at least one of the parents from cultivation extending back to at least a different than the others. 2100 BC (Refs 4,5). Therefore, it has been

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Box. 1. Taxonomic classification

The hierarchical groupings used by taxonomists, using an ), Eremocitrus (Australian desert lime) and . example from Citrus, that of Citrus sinensis, the sweet orange. The genus Citrus is divided into two subgenera. The common Most taxonomists include six closely related genera in the cultivated types of this fruit are placed in the subgenus subtribe of the ‘true citrus fruit trees’ – Citrus, Pocirus (trifolate Citrus (Eucitrus). Species of the subgenus do not bear orange), Fortunella (), Microcitrus (Australian wild edible fruit.

Family Subfamily Tribe Subtribe Genus Subgenus Species Variety Aurantiodeae Citrinae Citrinae Citrus Eucitrus C. sinensis Valencia TRENDS in Genetics

biology of the genus. Many Citrus types mutations (‘sports’) in the genus. Beneficial development of various biochemical and reproduce asexually by through a ones can be vegetatively propagated, and molecular markers has provided some process called nucellar embryony7. The might, in fact, be ancient in origin. Thus, the answers. In particular, DNA markers, with nucellus, a temporary nutritive tissue in biological concept of speciation where there their phenotypic neutrality, abundance developing seeds, gives rise to ‘nucellar is exchange of genes between members of a and imperviousness to environmental ’that are genetically identical to species and barriers to such an exchange conditions, have been most useful (Box 2). the plant on which the fruit is developing. between species is difficult to apply to the Many nucellar embryos can be present, Citrus genus9. Because of these conflicts, C. aurantifolia Christm. (limes) although usually only two or three develop the term Citrus ‘biotype’will be used in The limes include both acid and sweet to maturity; Citrus types that display this this discussion when proper designation varieties1. In addition, the sour limes consist trait are referred to as polyembryonic. Very as a species is not clear10. A biotype is all of two kinds, the small-fruited Mexican frequently, the nucellar embryos out- individuals that have the same genotype, (West Indian, Key) type, and the large- compete the single zygotic (sexual) except for mutations. Because the offspring fruited Tahiti (Persian) lime, which is for space or nutrients in the . Thus, of an apomictally perpetuated biotype are triploid and therefore seedless. Limes are when a Citrus seed is planted, the resulting asexually produced, these individuals are one of the Citrus biotypes that are believed seedling often has the same genetic collectively a clone. to be apomictically perpetuated10. There composition as the from which it arose. Fortunately, modern techniques have are very few varieties, and these are fairly This aspect of Citrus reproduction been instrumental in deciphering the homogenous; seven CULTIVARS have invariant greatly complicates taxonomic analyses taxonomic situation in Citrus. In the mid isozyme genotypes at four loci11. However, of the genus. One the one hand, Citrus 1970s, Barrett and Rhodes performed a limes are also relatively heterozygous; they contains an enormous degree of variation, comprehensive phylogenetic study that have heterozygous genotypes at seven of with abundant natural hybridization evaluated 146 morphological and ten isozyme loci evaluated11,12. Barrett and giving rise to a wide range of phenotypes, biochemical tree, , and fruit Rhodes10 suggested that the lime probably suggesting one species with many characteristics10. This study, and a second arose from a TRIHYBRID INTERGENERIC CROSS . On the other hand, because this one8, suggested that only three Citrus involving C. medica (), C. grandis type of facultative APOMIXIS (see Glossary) types, citron (C. medica), mandarin (pummelo) and a Microcitrus species. is so widespread in the genus, exchange (C. reticulata) and pummelo (C. grandis; Nicolosi et al.13 found that all RAPD and of genes is often prevented, leading to now C. maxima) constituted valid species. SCAR markers present in Mexican lime are reproductive isolation, a requirement for How, then, did the other Citrus types arise also present in citron or in C. species designation. Furthermore, there and maintain their integrity? How closely (a small-flowered papeda, a Citrus relative is an extremely high rate of and limb related are the various Citrus types? The that produces inedible fruit), suggesting

Table 1. Citrus types designated as species by Swingle3 Swingle taxonomya Common name Examples of varieties

C. aurantifolia Christm. Lime Mexican, Key West Indian, Key, Bearss C. aurantium L. Sour orange Chinotto, Seville C. indica Tan. An Indian species that is not cultivated C. maxima Merril; formerly C. grandis Osbeck Pummelo, shaddock Acidless, Thong Dee C. limon (L.) Burm. f. Femminello, Lisbon C. medica L. Citron , Corsican C. paradisi Macf. Grapefruit Marsh, Redblush, Flame C. reticulata Blanco Mandarin, , Dancy, Satsuma C. sinensis Osbeck Sweet orange Valencia, Washington navel, Shamouti C. tachibana (Mak.) Tan. A Japanese species that is not cultivated aIn this binomial classification, Citrus is the genus; aurantifolia, aurantium, etc. is the species; and Chistm., L., etc. is the authority.

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to be maintained by facultative apomixis; Box 2. Types of DNA marker used in the analysis of thus, sour orange is also not a true species. Inter-simple sequence repeat (ISSR): PCR amplification of DNA using a single primer Barrett and Rhodes10 believed sour ′ ′ composed of a microsatellite sequence such as (CA)8 anchored at the 5 or 3 end by two oranges to be of predominantly to four arbitrary nucleotides. C. reticulata (mandarin) genotype

Microsatellite probes: Short repetitive nucleotide sequences (e.g. GTG5) used either as INTROGRESSED with genes from C. grandis radiolabelled probes for hybridization with restricted citrus DNA or as single primers in (pummelo). This is supported by SCAR PCR reactions. and RAPD analyses13. DNA Random amplified polymorphic DNA (RAPD): Generation of a typically dominant analyses (Southern blots probed with marker through the use of a single, small, random-sequenced primer in a PCR reaction. labeled tobacco chloroplast DNA or PCR Restriction fragment length polymorphism (RFLP): Variation in band size following amplification of chloroplast sequences cutting of DNA with a specific restriction enzyme, electrophoresis and hybridization using ‘universal’ primers) revealed that with a specific probe. C. aurantium, C. limon, C. paradisi, Sequence characterized amplified region (SCAR): A polymorphic band, e.g. from a RAPD C. sinensis and C. grandis had the same reaction, that is sequenced, so that specific PCR primers for the band can be designed. Southern hybridization pattern, whereas C. reticulata and C. medica each had a unique pattern13,15. Pummelo appeared that these two species were the parents. oranges); the bittersweet orange, whose to be the maternal parent, which is not That citron is a parent of limes is fruit is similar to the common type, but unexpected because it is monoembryonic supported by RFLP data14. less acidic; and the variant bitter oranges and produces zygotic seedlings13. that are grown primarily as ornamentals Barrett and Rhodes10 point out that C. aurantium L. (sour oranges) and for their , from which oil the kind and number of differences There are three types of fruit grouped is extracted1. Barrett and Rhodes10 found among clones of cultivated Citrus types within the highly apomictic sour oranges: INTRASPECIFIC AFFINITY (correlation might be partially explained by the needs the common type that is used principally coefficients of traits measured) to be of the people who domesticated and as a ROOTSTOCK for sweet oranges and other quite high within common sour oranges, cultivated them. Sour oranges, for Citrus biotypes, and for the preparation of although the biotype appeared highly example, were used in ancient times marmalade (the well-known Seville heterozygous. This condition was believed for flavoring, perfumery and medicine. Although these uses continue in a minor way today, the primary modern use of sour oranges is as a rootstock, where × uniformity of type is required. This would militate against selection of variants. Supporting this theory, of 15 ACCESSIONS Pummelo Sweet Orange examined, two had a variant isozyme genotype; the others were identical11. The bergamot is a grown for its distinctive rind oil used in Earl Grey tea and in perfume. According to RAPD and SCAR markers, bergamot is a hybrid of Duncan, Walters citron and sour orange13. (seedy, white) C. grandis (L.) Osbeck; C. maxima Merril (pummelos) Marsh Foster The pummelo is a dessert fruit in Southeast (seedless, white) (seedy, pink) Asia, but is grown elsewhere1 mostly as a curiosity. This largest fruit of the Citrus genus can be pigmented or unpigmented, and acid or acidless. Pummelos are one of Redblush 10 (seedless, red) the three Citrus types Barrett and Rhodes proposed as a true species. This was because pummelos are monoembryonic, rather than being facultative apomicts, Thompson Ruby, Ray Ruby, Flame (seedless, pink) (seedless, very red) yet have a well-developed DISCONTINUITY TRENDS in Genetics OF TRAITS from other Citrus types (i.e. reproductive isolation). Although pummelo Fig. 1. Grapefruit is unusual in the genus Citrus because its history is mostly documented. The original grapefruit is primarily cross-pollinating, some clones biotype originated in the Caribbean, most probably by a natural hybridization between pummelo and sweet produce relatively vigorous selfed progeny. 13,17 orange, perhaps followed by introgression back to pummelo . The fruit of this type were white-fleshed and very 10 seedy. All other grapefruit cultivars arose as mutations, selected either for being seedless or for having Barrett and Rhodes found intraspecific increasingly red fruit color. affinity to be quite high in this Citrus type,

http://tig.trends.com Forum TRENDS in Genetics Vol.17 No.9 September 2001 539 and this, along with the propensity for self- C. paradisi Macf. (grapefruit) fertilization, was believed to be because the Grapefruit is the one Citrus biotype in which species was relatively homozygous. Isozyme a hybrid origin and subsequent selection for data supports this; pummelo was mutants is well documented (Fig. 1). RAPD homozygous at all ten loci examined11,12. and SCAR marker data indicate that C. grandis could be separated from other grapefruit was derived from a backcross C. aurantium C. sinensis Citrus types by its unique ISSR banding between sweet orange and pummelo, as do Sour orange Sweet orange patterns16. Of 14 accessions, only three had historical and morphological data13,17. Not variant isozyme patterns11. unexpectedly, intraspecific affinity was very high in this biotype10. When isozymes were C. limon (L.) Burm. f. (lemons) used to examine 13 cultivars, no variation The lemons consist of the common, acid was detected11; when 1230 ISSR markers C. paradisi Grapefruit varieties and a few sweet or acidless were used to characterize seven cultivars, types1. Lemons were used medicinally in one was different from the norm, which 18 ancient times; presently, they are mostly was attributed to mutation . C. reticulata C. grandis used for juice and flavoring. Barrett and Surprisingly, grapefruit might be rather Mandarin Pummelo Rhodes10 found intraspecific affinity to be homozygous; eight of ten isozyme loci were quite high in this species. They speculate homozygous in this biotype11. that because present needs are met by a C. aurantifolia uniform product, selection in this biotype C. reticulata Blanco (mandarins) Lime has favored the unique, original apomictic Mandarins are the most phenotypically type, with only minor mutational heterogeneous group in Citrus; both variations. Isozyme data supports this; monoembryonic and polyembryonic clones Other C. medica of 16 accessions, only two had a variant exist, as do self-fertile and self- Citron isozyme genotype11. incompatible types1. This suggested to Barrett and Rhodes10 speculated that Barrett and Rhodes10 that a broader-based, lemons are a complex hybrid similar to more-complex heterozygosity was present C. limon limes but carrying a greater proportion than was found in the other facultatively Lemon of citron genes. Molecular marker data apomictic types. Therefore, this was the TRENDS in Genetics indicates that lemon originated from citron third Citrus type assigned species status. and sour orange, with sour orange being the Sweet mandarin types have been used for Fig. 2. The most probable relationships among the maternal parent13. When ISSR markers dessert fruit since ancient times, although different Citrus types. were examined, a total of 12 polymorphic sour types have been used as rootstocks, fragments generated by seven primers and for flavorings and medicine. Thus, it is sweet oranges are recognized: the were detected among six lemon cultivars, difficult to assess the relative importance of common, or blond, orange, which is the suggesting a possible polyphyletic origin genetic versus mutational variation in the most important and of which there are (development from more than one ancestral complex history of this species. many varieties; the acidless orange, of type) for lemon16. Six of ten isozyme loci SCAR and RAPD analyses did not minor importance; the , examined were heterozygous11,12. allow the identification of progenitor which has a red pigmentation in the flesh type13. Although the molecular marker due to the accumulation of anthocyanins; C. medica L. (citrons) data reveal great heterogeneity within and the navel orange, grown for fresh The citrons also fall into acid and sweet this group, it supports species consumption1. They can also be classes, with several varieties extant for status11,13,16,19,20. For example, when categorized on the basis of season of each class1. Citrons are grown mostly RAPD markers were used to evaluate maturity as early, mid-season, or late. for their peel, which is then candied. genetic similarity among 35 mandarin Barrett and Rhodes10 observed much Historically, the fruit had great religious accessions, the minimum JACCARD lower intraspecific affinity in this type significance for some cultures. Citron is COEFFICIENT was 0.77, which indicates a than in any of the other types they the second type Barrett and Rhodes10 high genetic similarity in this group. The considered facultative apomicts (sour advanced as a true species. It is researchers proposed that the , lemon and grapefruit); in other monoembryonic. It is also relatively group is a single species, composed of words, there was a relatively great homozygous; eight of ten isozyme loci were several genetically different individuals diversity in phenotype. Some studies homozygous, and of six cultivars, one and a great number of hybrids20. Finally, indicate that genetically, sweet oranges was variant11,12. Molecular marker data C. reticulata has a unique chloroplast are a biotype. Chromosome banding support species status13. Citron had a banding pattern, again suggesting that it patterns of ten clones were heterozygous unique chloroplast hybridization should have species status15. and invariant21. When three pattern13,15, also supporting species microsatellite probes were assayed, no status. Interestingly, the chloroplast C. sinensis Osbeck (sweet oranges) differences were detected among ten data13 indicate that citron always acted as Sweet orange, the most widely grown cultivars19. This points to a monophyletic the male parent – unexpected given the and consumed Citrus type, presents origin for sweet orange followed by monoembryonic nature of this species. something of a mystery. Four kinds of somatic mutation and selection of

http://tig.trends.com 540 Forum TRENDS in Genetics Vol.17 No.9 September 2001 desirable clones19. However, when four and abiotic stresses and for horticultural (Janick, J. and Moore, J.N., eds), pp. 257–323, isozyme loci were used to examine improvements such as increased fruit John Wiley & Sons 8 Scora, R.W. (1975) On the history and origin of 21 cultivars, there were seven variants quality. However, might then Citrus. Bull. Torrey Bot. Club 102, 369–375 11 at one locus . Further, of 31 cultivars, face many generations of crossing and 9 Vardi, A. and Spiegel-Roy, P. (1978) Citrus 14 differed from the basic ISSR profile of selection to return to an edible type or one breeding, taxonomy and the species problem. In about 1230 fragments by one to four ISSR that fits industry standards. This makes Proceedings of the International Society of bands18. Fang and Roose18 believed these modern genetic techniques, such as Citriculture (Cary, P.R., ed.) pp. 51–57, International Society of Citriculture differences originated by mutation. genetic transformation, attractive for 10 Barrett, H.C. and Rhodes, A.M. (1976) A However, it seems unusual that small the improvement of these economically numerical taxonomic study of affinity mutations could be revealed at such a important and aesthetically pleasing fruit. relationships in cultivated Citrus and its close frequency using molecular markers. relatives. System. Bot. 1, 105–136 Like sour oranges, sweet oranges are Acknowledgements 11 Torres, A.M. et al. (1978) Leaf isozymes as genetic markers in Citrus. Am. J. Bot. 65, 869–881 thought to be predominantly of C. reticulata This is Fla. Agr. Exp. Stn. Series 12 Torres, A.M. et al. (1982) Citrus isozymes: (mandarin) genotype introgressed with No. N-02086. Genetics and distinguishing nucellar from zygotic C. grandis10,13. The sweet orange and sour seedlings. J. Hered. 73, 335–339 orange biotypes are thought to have a Gloria A. Moore 13 Nicolosi, E. et al. (2000) Citrus phylogeny and genetic origin of important species as investigated parallel but separate origin, with their Horticultural Sciences Dept, by molecular markers. Theor. Appl. Genet. differences stemming from parentage Plant Molecular and Cellular Biology Program, 100, 1155–1166 from separate subspecies from within Institute of Food and Agricultural Sciences, 14 Federici, C.T. et al. (1998) Phylogenetic the polytypic C. reticulata. Similar PO Box 110690, University of Florida, relationships within the genus Citrus microsattelite patterns observed with sweet Gainesville, FL 32611, USA. (Rutaceae) and related genera as revealed by RFLP and RAPD analysis. Theor. Appl. Genet. oranges and mandarins agree with the close e-mail: [email protected] 96, 812–822 19 phylogenetic relationships of these species . References 15 Green, R.M. et al. (1986) The plastome of Citrus. 1 Swingle, W.T. and Reece, P.C. (1967) The of Physical map, variation among Citrus cultivars Conclusions Citrus and its wild relatives. In The Citrus and species and comparison with related genera. Historical records and genetic analyses Industry. (Vol I) (Reuther, W. et al., eds), Theor. Appl. Genet. 72, 170–177 16 Fang, D. et al. (1998) Phylogenetic relationships lead to the conclusion that there are three pp. 190–423, University of California Press 2 Swingle, W.T. (1943) The botany of Citrus and its among selected Citrus germplasm accessions true species in the genus Citrus (citron, wild relatives of the orange subfamily. In The revealed by inter-simple sequence repeat (ISSR) mandarin and pummelo) and additional Citrus Industry. (Vol I) ( Webber, H.J. and markers. J. Am. Soc. Hort. Sci. 123, 612–617 cultivated biotypes (Fig. 2). Oranges, Batchelor, L.D., eds), pp. 129–474, University of 17 Gmitter, F.G., Jr (1995) Origin, evolution and lemons, limes and grapefruit, although California Press breeding of the grapefruit. Plant . Rev. 13, 345–363 disseminated throughout the world by 3 Tanaka, T. (1954) Species Problem in Citrus. Japanese Society for the Promotion of Science 18 Fang, D.Q. and Roose, M.L. (1997) Identification men in both ancient and modern times, 4 Webber, H.J. (1967) History and development of of closely related Citrus cultivars with inter- each have a narrow genetic base. . In The Citrus Industry. simple sequence repeat markers. Theor. Appl. Cultivated types were selected by men (Vol. 1) (Reuther, W. et al., eds), pp. 1–39, Genet. 95, 408–417 and propagated by grafting or nucellar University of California Press 19 Luro, F. et al. (1995) DNA amplified 5 Scora, R.W. (1988) Biochemistry, taxonomy and fingerprinting, a useful tool for determination of seedlings. The variation within these evolution of modern cultivated Citrus. In genetic origin and diversity analysis in Citrus. biotypes is due primarily to somatic Proceedings of the International Society of Hort. Sci. 30, 1063–1067 mutations. This genetic homogeneity is Citriculture (Goren, R. and Mendel, K., eds), 20 Coletta Filho, H.D. et al. (1998) Analysis of the reinforced by consumer expectations and pp. 277–289, International Society of Citriculture genetic diversity among mandarins (Citrus spp.) industry regulations. Within the genus, 6 Gmitter, F.G. and Hu, X. (1990) The possible role using RAPD markers. Euphytica 102, 133–139 of , China, in the origin of contemporary 21 Pedrosa, A. et al. (2000) Cytological and in related genera, there is genetic Citrus species (Rutaceae). Econ. Bot. 44, 237–277 heterozygosity and the hybrid origin of sweet variability that a can exploit; for 7 Soost, R.K. and Roose, M.L. (1996) Citrus. In orange [Citrus sinensis (L.) Osbeck]. Theor. Appl. instance, to create resistance to both biotic Fruit Breeding, Volume I: Tree and Tropical Fruits Genet. 100, 361–367

Rosa Beddington

Rosa Beddington, who served on the editorial board of Trends in Genetics from 1993, died on May 18th 2001 following a prolonged battle against breast cancer. She was just 45. A distinguished embryologist, she made a great contribution to the understanding of anterior–posterior axis formation and of the function of mammalian extra-embryonic tissues in head formation.

Full obituaries can be found in (2001) 105, 709–710 and Nature (2001) 412, 138. Photograph courtesy of The Medical Research Council National Institute for Medical Research

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