Genome Size Variation and Plant Systematics - Annals of Botany

Annals of Botany 82 (Supplement A): 75-83, 1998Article No. bo980765

Genome Size Variation and Plant Systematics

DEEPAK OHRI

National Botanical Research Institute, Lucknow-226001, U.P., India

Received: 14 November 1997 Returned for revision: 17 March 1998 Accepted: 2 July 1998

The importance of genome size, as a useful taxonomic marker, has been stressed in many studies based on differentgroups. A critical appraisal of intraspecific variability reported in various species so far shows that the plant genomeis fairly stable (taking a narrow species concept) and is one of the salient features defining a biological species. Largedifferences in DNA amounts may exist at the infrageneric level which may be correlated with various adaptive traitsat nuclear, cellular, tissue and organismic levels. Therefore, being an adaptive character, a comparison of nuclear C-values in conjunction with other characters may provide a natural way to elucidate evolutionary relationships. In anumber of cases it is possible to study whether gain or loss of DNA has occurred during speciation which maycoincide with the existing taxonomic treatment and presumed evolutionary relationships within a narrow taxonomicgroup. The present account deals with many recent examples which highlight the importance of genome size inmicrosystematics. © 1998 Annals of Botany Company

Key words: Angiosperm genome size, DNA C-values, plant adaptation, intraspecific variation, polyploidy,interspecific variation, species relationships, evolution and systematics.

INTRODUCTION

The study of nuclear DNA amounts is crucial for the overallunderstanding of the genome of an organism. The continuedstrong interest in this field is being sustained by the fact thatremarkable differences in genome size exist at the infra-generic level which can be used to demarcate various taxa.Nuclear DNA amounts are now known for nearly 3000species of angiosperms representing 1 % of the globalangiosperm flora, and show over 600-fold variation in C-values (Bennett and Smith, 1976, 1991; Bennett, Smith andHeslop-Harrison, 1982; Bennett and Leitch, 1995, 1997).Moreover, wide variation is known to occur within manyfamilies and genera. This variation in genome size ir-respective of organismic complexity has been called the C-value paradox by Thomas (1971), where C stands forconstancy of DNA amount of unreplicated haploid genomeof an individual (Swift, 1950). This extra DNA has beenvariously considered to be constituted by 'junk', 'selfish' or'parasitic' sequences (Ohno, 1972; Orgel and Crick, 1980;Doolittle and Sapienza, 1980). However, a functional andadaptive role for this DNA is envisaged in the nucleotypictheory which shows that this non-informative DNA canproduce various phenotypic effects at nuclear, cellular,tissue and organismic levels (Bennett, 1972, 1973, 1985;Cavalier-Smith, 1985; Price, 1988 a). Interspecific variationin nuclear DNA is directly correlated with such organismictraits as minimum generation time (Bennett, 1972), growthin different latitudes and ecological conditions (Bennett,1976; Grime and Mowforth, 1982; Grime, 1990; Ohri,Fritsch and Hanelt, 1998), water relations in conifers (Ohriand Khoshoo 1986a; Wakamiya et al., 1993, 1996), seedsize (Thompson, 1990; Ohri et al., 1998), physiology(Jasienski and Bazzaz, 1995) and development (Bharathan,

0305-7364/98/0A0075 + 09 $30.00/0

1996). Therefore as an adaptive character, a comparison ofC-values provides a natural way to explain phylogeneticrelationships and systematics of narrow taxonomic groups(Price, 1976; Cavalier-Smith, 1985; Ohri and Khoshoo1986b; Raina, 1990).

INTRASPECIFIC VARIATION

Intraspecific variability in genome size at a constant basicchromosome number and type is a current issue of greatinterest. Reports in many plant species show a strikingrange of variation which was previously thought to occuronly between species (Table 1). It is now being increasinglyconsidered that the changes in genome size may not only berestricted to species divergence but also associated withvarious environmental conditions and developmental stagesaffecting different populations or individual plants(Price, 1988a, b, 1991; Bassi, 1990; Cavallini and Natali,1991; Natali et al., 1993; Johnston et al., 1996; Price andJohnston, 1996) which makes possible their use as definingcharacteristics for specific clones or genotypes (Jarret et al.,1995). Indeed, in many cases nucleotypic effects have beenshown to be associated with such variability in genome size(Bennett, 1985; Price, 1988 a, b; Ceccarelli et al., 1993, 1995;Minelli et al., 1996). However, as the following accountshows, many reports on intraspecific variability have beenrefuted on reinvestigation.

The intraspecific changes in genome size, being thereforesubject to natural selection, are naturally related with thoseleading to the divergence and evolution of species. Therefore,a thorough appraisal (Table 1) of such changes is requiredfor the application of genome size data to demarcateinfrageneric taxa and in microsystematics.

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Ohri-Genome Size Variation and Plant Systematics

TABLE 1. Chromosome number (2n) ploidy level (x), C DNA values, and the percentage intraspecific variation in 1C DNAamount reported in angiosperm species

VariationTaxon 2n x IC Range (pg) (%) Reference

MonocotyledonsBulbine bulbosa (R. Br.) Haw 46-48 4 1370-2960 116 Watson, 1987Festuca arundinacea Schreb. 42 6 605-893 32 Ceccarelli et al., 1992Gibasis venustula (Kunth) D. R. Hunt 12 2 544-866 59 Kenton, 1984Milium effusum L. 28 4 376-51 36 Bennett and Bennett, 1992Paspalum nicorae Parodi - - 135-153 13 Jarret et al., 1995Poa annua L. 28 4 c. 133-240 80 Grime, 1983Zea mays L. ssp. mays 20 2 246-303 23 Rayburn et al., 1985Zea mays L. ssp. mays 20 2 277 337 22 Rayburn and Auger, 1990Zea mays L. ssp. mays 20 2 293-343 15 Tito et al., 1991Zea mays L. ssp. mays 20 2 246-337 37 Laurie and Bennett, 1985Zea mays L. ssp. mexicana 20 2 263-322 22 Laurie and Bennett, 1985Zea mays L. ssp. parviglumis 20 2 280-3.10 11 Laurie and Bennett, 1985

DicotyledonsArachis duranensis Krap. & Gureg. 20 2 266-295 11 Singh et al., 1996Arachis hypogaea L. 40 4 513-591 15 Singh et al., 1996Armeria maritima (Mill.) Willd 18 2 4-10-4-54 11 Vekemans et al., 1996Cajanus cajan (L.) Millsp. 22 2 155-199 24 Ohri et al., 1994Collinsia verna Nutt. 14 2 184-714 288 Greenlee et al., 1984Fraxinus americana L. 46 2 229-314 37 Black and Bachmann, 1983Fraxinus americana L. 92 4 504-588 17 Black and Bachmann, 1983Fraxinus americana L. 138 6 666-981 47 Black and Bachmann, 1983Glycine canescens F. J. Hermann 40 2 095-121 27 Hammatt et al., 1991Glycine max (L.) Merr. 40 2 092-131 42 Doerschug et al., 1978Glycine max (L.) Merr. 40 2 109 168 54 Yamamoto and Nagato, 1984Glycine max (L.) Merr. 40 2 1 25-144 15 Graham et al., 1994Glycine max (L.) Merr. 40 2 127 143 11 Rayburn et al., 1997Glycine soja (L.) Sieb. & Zucc. 40 2 c. 143-182 27 Yamamoto and Nagato, 1984Gossypium hirsutum L. 52 4 254-302 19 Gomez et al., 1993Helianthus annuus L. 34 2 337 533 58 Cavallini et al., 1986Helianthus annuus L. 34 2 300-397 52 Michaelson et al., 1991Microseris bigelovii (Gray) Sch. Bip. 18 2 113 141 25 Price et al., 1981aMicroseris douglasii (DC.) Sch. Bip. 18 2 202-256 27 Price et al., 1981bPisum sativum L. 14 2 393-507 29 Cavallini and Natali, 1990Viciafaba L. 12 2 1094-1473 35 Ceccarelli et al., 1995

The most detailed studies in this respect have been madein Microseris (Price, Chambers and Bachmann, 1981, a, b;Price et al., 1986). Variation of over 20 % was detected in M.bigelovii (Price et al., 1981 a) and M. douglasii (Price et al.,1981 b). DNA contents of 222 plants of M. douglasiirepresenting geographically, ecologically and morpho-logically diverse populations in California showed a 14%variation between population means, with those havinghigher and lower DNA amounts restricted to mesic andxeric sites, respectively (Price et al., 1981 b). Similarly, in M.begelovii, populations with small genome size were found togrow at the latitudinal extremes of the species, indicatingthat small genome size has been selected in stressful andtime-limited environments (Price et al., 1981 a). However, ina further study on 210 plants of M. douglasii no consistentcorrelations were detected, though in one populationtemporal variation in DNA amounts was noticed accordingto precipitation in different years (Price et al., 1986).

A much greater range of variation (2C = 29-52 pg,80 %), at a constant chromosome number, has been foundbetween 42 families of seed progeny derived from establishedplants of Poa annua from a single British population(Mowforth and Grime, 1989). This variation showed a

positive relationship with cell size but was negativelycorrelated with seedling growth rate and therefore drymatter accumulation.

Similarly, a 32-3 % difference covering 35 natural popu-lations of hexaploid Festuca arundinacea was positivelycorrelated with mean temperature during the year, with thecoldest month at stations and with generation time, while itwas negatively correlated with latitude and germinationpower of seeds and growth rate (Ceccarelli, Falistocco andCionini, 1992; Ceccarelli et al., 1993). In further experiments,DNA contents in different accessions were found to increasesignificantly with increase in temperature of seed ger-mination, again indicating the presence of adaptable fluiddomains in the nuclear DNA (Ceccarelli et al., 1997).Helianthus annuus is another example of the presence of ahighly flexible genome. A variation of 581 % (Cavallini etal., 1986) and 32% (Michaelson et al., 1991) has beenreported among various cultivated varieties or lines.Significant variation (1925%) also occurs within lines(Cavallini et al., 1986; Michaelson et al., 1991) whilevariability exceeding 27 and 43 % has been detected amongleaves from different nodes of plants of two varieties(Michaelson et al., 1991). Further investigations showed

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that significant differences in nuclear DNA amounts aregenerated during early embryo formation and that theseremain stable, resulting in differences even among singleprogeny from a homozygous line. The developing embryosin a capitulum have a characteristic gradient of genomesizes, from the centre to the periphery of the head dependingupon the genome size of the mother plant (Cavallini et al.,1989). These differences, which are due to variation in aparticular family of medium repeated sequences, showpositive correlation with surface area of leaf epidermal cellsand generation time (Natali et al., 1993). Likewise, thegenome size of 39 populations of Vicia faba differing by34-6 %, shows positive correlation with germination powerof seeds and epicotyl growth rate, and negative correlationwith height of main stem and fresh weight of plants atanthesis (Ceccarelli et al., 1995; Minelli et al., 1996). Thesefindings suggest that the nuclear genome is in a state of fluxdue to the fluid domains (comprised of repeated sequences),which undergo rapid changes in response to differentmacro- or microenvironmental conditions.

It must be mentioned that C-value variation due toheterochromatin polymorphism, B-chromosomes, aneu-ploidy, polyploidy or hybridity is well understood and is notexceptional in angiosperm species. Zea mays is a typicalexample in which C-band number and amount of hetero-chromatin show a positive correlation with DNA content(Laurie and Bennett, 1985; Rayburn et al., 1985). Theseresults have been corroborated by later studies (Rayburnet al., 1989; Tito, Poggio and Naranjo, 1991). A significantnegative correlation between genome size and latitude(maturity zones) was also seen and interpreted as an effectof selection by man for early maturation and optimum yieldwithin climatic constraints (Laurie and Bennett, 1985;Rayburn et al., 1985). The correlation with altitude issomewhat complex; in maize populations from Arizona andNew Mexico the genome size is low at both low and highaltitudes while it is high in intermediate elevations (Rayburnand Auger, 1990; Rayburn, 1990). Further studies haveshown an increase in genome size (increased hetero-chromatin) in populations of maize which are both cold andfrost tolerant, but a decrease in genome size (decreasedheterochromatin) in only cold tolerant populations(McMurphy and Rayburn, 1991, 1992). Similarly, a signi-ficant positive correlation has also been reported betweenduration of maturity and DNA amount of 20 cultivars ofGlycine max (Graham, Nickell and Rayburn, 1994) butDNA variation has been questioned (see below).

Sixteen cultivars of Cajanus cajan showed 23-6 % variationin 4C values, ranging from 619 to 797 pg. A polyphyleticorigin of this crop has been proposed (Ladizinsky andHamel, 1980; Pundir and Singh, 1985; Jha and Ohri, 1996)which means that present pigeonpea cultivars are segregantsof parental wild Cajanus species with different DNAamounts (Ohri, unpubl. res.).

Many of the reports on intraspecific variation have,however, been disproved by subsequent studies. The mostthoroughly studied among these pertain to conifers, inwhich intraspecific variation reported earlier (Miksche,1971; El-Lakany and Sziklai, 1971; Dhir and Miksche,1974) was shown to be due to methodological errors and the

presence of B-chromosomes (Teoh and Rees, 1976;Greilhuber, 1988a). Similar are the cases of Hedera helix(Konig, Ebert and Greilhuber, 1987) and Sambucusracemosa (Greilhuber, 1988b). Some recent reports havealso been disproved by subsequent studies. For example, the1-29-fold variation in DNA amount among ten Italiancultivars and experimental lines of Pisum sativum (Cavalliniand Natali, 1990) was shown to be positively correlated withroot and stem growth during early developmental stages,root and epicotyl cell lengths and leaf epidermal cell areas(Cavallini et al., 1993). Greilhuber and Ebert (1994)however, found only 1-054-fold variation in 25 wildaccessions, land races and cultivars of Pisum sativum ofwidely different geographical origin. Repeat measurementsshowed that even this difference is non-reproducible. DNAconstancy was substantiated by flow cytometry of 15cultivars, landraces and wild accessions of P. sativumsubspecies (Baranyi and Greilhuber, 1995) and by a furtherstudy on 38 accessions (Baranyi and Greilhuber, 1996),including those analysed by Cavallini and Natali (1990).Baranyi and Greilhuber (1996) therefore, strongly refutedthe existence of an intraspecific variation and its associatednucleotypic effects in P. sativum. Similarly, the presence of1-15-fold variation in C-values of 20 cultivars of Glycinemax and its positive correlation with duration of maturity(Graham et al., 1994) has been disproved on reinvestigationof the same cultivars which show non-significant differences(Greilhuber and Obermeyer, 1997).

The extent and prevalence of intraspecific variabilitytherefore needs proper assessment. Often variation due tomethodological errors has been reported as genuinedifferences. In view of this the reports on intraspecificvariability need to be verified by independent studies beforethe stability of the plant genome can be finally defined.

POLYPLOIDY AND GENOME SIZE

Polyploidy occurs in 70 % of all the angiosperms and is oneof the important cytogenetic mechanisms of plant evolution(Goldblatt, 1980; Lewis, 1980). It is logical to suppose thatthe DNA amount increases in proportion to ploidy levelsand that the genome size of amphidiploids is the sum valueof its parents. However, there is controversy over whetherthe addition to nuclear DNA amount is proportional toploidy level or whether it shows some discrepancy. This mayresult from actual loss of DNA after polyploidization orfrom the underestimation of nuclear DNA content bymicrodensitometry because of higher DNA condensation inpolyploids (Verma and Rees, 1974; Karp, Rees and Jewell,1982).

DNA diminution per basic genome with increase inploidy level has been reported in Betula (Grant, 1969),Festuca (Seal, 1983), Leucanthemum (Marchi et al., 1983),Bulnesia (Poggio and Hunziker, 1986), Piper (Samuel,Smith and Bennett, 1986), Larrea (Poggio, Burghardt andHunziker, 1989), Bulbine (Watson, 1987) and Pratia(Murray, Cameron and Standring, 1992). Recently, Rainaet al. (1994) studied the changes in DNA content in CO andsubsequent generations of induced polyploids of Tephrosia,Vicia and Phlox. While DNA content increased in C of

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Tephrosia oxygona L. and T. purpurea (L.) Pers. it decreasedby 16-7 % of the expected doubled value in the COgenerationof Phlox drummondii Hook. and in C2 an overall reductionof 25 % was noticed. This loss of DNA was achieved byequal reduction in all chromosomes in the complement.This finding is significant in as much as it shows the precisemanner in which DNA is lost in a polyploid complement.

However, a positive correlation between DNA contentand polyploidy has been shown in many cases includingHordeum species and varieties (Bennett and Smith, 1971),Celosia species and cultivars (Nath, Ohri and Pal, 1992),cultivars of Bougainvillea (Ohri and Khoshoo, 1982),Chrysanthemum (Ohri, Nazeer and Pal, 1981), Narcissus,Hyacinthus and Tulipa (Brandham and West, 1993), Prunuspersica (L.) Batsch (Vance Baird, Estager and Wells, 1994)and species of Ipomoea (Ozias-Atkins and Jarret, 1994),Vaccinium (Costich et al., 1993) and Andropogon gerardiiVitman (Keeler et al., 1987). Moreover, the DNA content ofthe amphidiploids in wheat (Pegington and Rees, 1970),Brassica (Verma and Rees, 1974), Bougainvillea (Ohri andKhoshoo, 1982), Nicotiana (Narayan, 1987), Arachis (Singh,Raina and Singh, 1996), Glycine (Hammatt, Blackall andDavey, 1991) and Allium (Ohri et al., 1998) correspond tothe sum value estimated for parental species. These examplesemphasize nuclear DNA constancy for a given set ofchromosomes within a species.

INTERSPECIFIC VARIATION

If a narrow species concept is adopted, the genome size ofa species appears fairly stable and may, therefore, be auseful additional character in taxonomic and evolutionarystudies (Price, 1976; Ohri and Khoshoo, 1986b; Greilhuberand Ehrendorfer, 1988; Raina, 1990; Bennett and Leitch,1995). However, there appear to be at least two issues toconsider here: how well does the genome size variation fitwith existing taxonomic treatments and what is therelationship between genome size and evolutionary relation-ships?

Nuclear DNA measurements have proved to be veryeffective in delimiting infrageneric divisions in a number oftaxa. In Helianthus, 18 diploid species show a four-folddifference between the highest (H. agrestis Pollard, 2C =25-91 pg) and the lowest (H. neglectus Heiser, 2C = 641 pg)value. These differences show a close association with thephenetic groupings of the annual species (Schilling andHeiser, 1981) and with the phylogenetic trees based onisozyme and cpDNA analysis (Rieseberg et al., 1991).Another example is the South American genus Bulnesiawhere a six-fold difference between diploid species alsoagrees with the previous numerical and taxonomic studies(Poggio and Hunziker, 1986). Species that are closelyrelated have similar DNA values: B. retama (Gill ex Hook.& Arn.) Grisen-B. chilesis Gay (highest DNA values); B.foliosa Griseb.-B. schickendantzii Heiron (intermediatevalues); B. arborea (Jacq.) Engl.-B. carrapo (low values).Another diploid species, B. sarmientoi Lorentz is an isolatedelement with no close affinities and has the lowest DNAamount of all the diploid species. The octoploid B.bonariensis Griseb., which is also isolated, has the lowest

amount of nuclear DNA per genome of any species in thegenus. Similar observations can be made in Cicer andCajanus. Seven annual species of Cicer show a 1-95-foldvariation (Ohri and Pal, 1991). The lowest 2C DNA valueis shown by C. judaicum Boiss, which also has the mostasymmetrical karyotype, while the cultivated C. arietinumL. has the highest 2C DNA value (330-357 pg) and arelatively symmetrical karyotype. This indicates that DNAdiminution has taken place along with karyotypic evolution.The results show that annual species of Cicer can bearranged into three groups (Group I: C. judaicum; GroupII: C. cuneatum Hochst. & Rich, C. bijugum Rech., C.pinnatifidum Jaub & Spach, C. reticulatum Lad. and C.echinospermum, Dav.; Group III: C. arietinum) and thecultivated C. arietinum has a larger genome size than thewild species, which are weedy in nature (Ohri and Pal,1991). However, it is interesting that these three groups donot correspond with those based on crossability whichmeans that the differences in karyotypes and DNA amountsdo not necessarily affect species crossability (Ohri and Pal,1991). This contrasts with the situation in Cajanus where the4C DNA amounts of ten wild species correspond with thesectional classification and more or less reflect speciescrossability (Ohri, Jha and Kumar, 1994; Ohri unpubl. res.).This is also the case in Bougainvillea where interspecificcrossability is reflected by differences in DNA amounts. Ofthe three elemental species which have given rise to gardenbougainvilleas, B. peruviana (Humb & Bonp. (BB) (7.00 pg)form completely sterile hybrids with B. glabra Choisy (AA)(8-25 pg) and B. spectabilis Willd. (AIA1) (8-91 pg) also hasa significantly lower 2C DNA value than the latter twospecies (Ohri and Khoshoo, 1982).

In certain groups, karyotypic studies along with genomesize measurements may reveal diversity which is nototherwise apparent from morphological studies. An exampleof this is the Australian genus Bulbine, which is con-ventionally represented by two species, B. bulbosa (R. Br.)Haw. and B. semibarbata (R. Br.) Haw. Karyotype studiesand DNA measurements (Watson, 1987) have shown thatthe perennial group designated by B. bulbosa s. lat. actuallycomprises three entities. The first of these, the bulbosacomplex (B. bulbosa s. str.) has three ploidy levels (4x = 24,8x = 48, 12x = 72). Its octoploid has two times more DNAcontent than a 'rock lily' group (2n = 46) which in turn isseparate from another 46-chromosome 'Kroombit' formwhich has 29 % less DNA than the 'rock lily' group. Thethree entities within B. bulbosa s. lat. are therefore clearlyseparable on the basis of their genome size. These differencesare also supported by C-banding studies (Watson, 1987). Inthe annual group represented by B. semibarbata s. lat. thereis differentiation between 4x 'alata' (2n = 28) which hasslightly more DNA than closely related 26-chromosome'semibarbata' (Watson, 1987). Similarly, DNA C-valuessupport the consideration of Tephrosia taxa as separatespecies. T. leptostachya DC., T. hamiltonii Drumm. exGamble and T. wallichii, Grahm. which are considered to besynonyms of the diploid cytotype of T. purpurea (L.) Pers.,show 2C DNA amounts of 203, 342 and 329 pg, re-spectively, which are very different from that of T. purpurea(288 pg) (Raina, Srivastava and Rama Rao, 1986). In

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FIG. 1. Frequency diagrams representing quantitative 4C DNA variation pattern in various subgenera of genus Allium.

addition, T. incana (Roxb.) Wight & Arnot (2-64 pg) and T.subtriflora Hochst. ex Baker (271 pg) have been consideredas synonyms of T. villosa (L.) Pers. (2-54 pg) and T.multiflora Blatter & Hallb. (2-81 pg) respectively, and theymay also need to be considered as separate taxa on the basisof their DNA amounts (Raina et al., 1986). Likewise, thestudy of DNA amounts has helped to distinguish varioustaxa in the Vicia narbonensis L. and V. sativa L. complexes(Raina, 1990), which are considered as distinct species bysome authors. V. narbonensis and V. sativa complexes arecomposed of five (14-14-18-21 pg) and six (3-95-540 pg)species, respectively (Raina, 1990).

Genome size measurements may also help in the taxo-nomic placement of particular species. In Vicia, the DNAamounts of 19 recently-discovered species agree with therecent taxonomic revision of the genus (Maxted, Callimassiaand Bennett, 1991). V. dionysiensis, Mout. a relict and rarespecies, has been placed in a newly erected sectionMicrocarinae of subgenus Vicia by Maxted (1993). It wasearlier shown to be distantly allied to section Trigonellopsisof subgenus Vicilla. However, DNA amounts and chromo-some numbers of V. lunata (Borss & Bal.) Boiss (2n -= 14,7-33 pg) of section Trigonellopsis and V. dionysiensis (2n =12, 27'61 pg) are markedly different (Maxted et al., 1991).Furthermore, its link with section Hypechusa of subgenusVicia is confirmed because of the similarity in chromosomenumber and DNA amount. V. cuspidata Boiss. has beenseparated from section Vicia sensu stricto into the distinct

section Wiggersia (Maxted, 1993), which is supported bydifferences with regard to both its chromosome number andDNA amount (Maxted et al., 1991).

The other question of whether genomes grow or shrinkduring evolution is more problematic. Both increases anddecreases of DNA amount with specialization and evolutionseem to have occurred. In the Bulnesia example discussedabove, B. retama and B. chilensis, with the largest genomes,are presumed to show adaptation to extreme xeric environ-ments, while on the other hand B. sarmientoi, with thesmallest genome, is also considered to be derived due to itshighly specialized morphology (Poggio and Hunziker, 1986).An evolutionary gain has also been demonstrated in twospecies of Coreopsis related as progenitor and derivative. C.nuecensoides. E. B. Smith which is the progenitor has n = 9,10, 11 while the derived C. nuecensis Heller has n = 6, 7.However, the two populations of the derived species have12% more DNA than any population of the progenitor.This shows that DNA has increased despite aneuploidreduction therefore suggesting a gain in DNA contentduring speciation (Price, Crawford and Bayer, 1984).Similarly in Oxalis, DNA gain occurs during speciation (DeAzuke and Martinez, 1988). On the other hand, in Papaverthe phylogenetically advanced sections have less DNA thanthe primitive sections (Srivastava and Lavania, 1991) and inHelianthus the mean value (2C = 8-31 pg) of annual species(excluding that of H. agrestis) is significantly less than that(2C = 11 72 pg) of the perennial species. Even if the high

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FIG. 2. Frequency diagrams representing quantitative 2C DNAvariation pattern in various subgenera of genus Allium.

DNA value of H. agrestis is included with the other annuals,the mean value of annuals (2C = 9-41 pg) is still less thanthat of the perennials. In the genus Allium, measurement of4C DNA amounts in 85 species belonging to six subgenera,as proposed by Hanelt et al. (1992) shows a 835-folddifference, from 3560 (A. ledebourianum Roem & Schult, 2n= 16) to 29713 pg (A. validum Vats, 2n = 56) (Fig. 1).However, 2C DNA per genome varies 3-57-fold between A.ledebourianum and A. ursinum L. (2n = 14, 127-14 pg) (Fig.2). Analysis of the data shows that both increases anddecreases in DNA amount appear to have occurred duringthe evolution of the genus Allium (Ohri et al., 1998).

Bennetzen and Kellogg (1997), in an analysis of genomesize variation in relation to phylogeny in the Poaceae,suggested that evolution is accompanied by an increase ingenome size. Their approach allows inferences to be drawnon the genome size of ancestral species which are no longeravailable for direct measurement. Clearly this sort of analysisshould now be applied to some of the groups of plants thathave been discussed above where there appear to be bothincreases and decreases in genome size with evolution. Inthis way it should be possible to establish whether genomesgrow or shrink, or both, in particular groups of plants.

VARIABILITY IN WOODY ANGIOSPERMS

Woody angiosperms have been believed to possess a smalland relatively uniform genome size because of the impliedconstraints on maximum nuclear size by the small cambialcells which form wood fibres (Darlington, 1937; Stebbins,1950; Khoshoo, 1962). This is true of many hardwoodgenera such as Ficus which show low and uniform 2C DNAvalues (137-145 pg) in 14 species studied (Ohri andKhoshoo, 1987). Likewise, three species of Quercus alsohave similar 2C DNA values ranging from 158 to 161 pg(Ohri and Ahuja, 1990). Though the highest genome sizefound in woody species (Ohri and Kumar, 1986; Ohriunpubl. res.) is much smaller than that known in herbaceousspecies (Bennett and Smith, 1976; Bennett and Leitch,1995), some recent studies have shown remarkable inter-specific variation. Among shrubby and arboreal species ofCassia studied, the genome size of C. excelsa Schrad wasfound to be twice that of the other species (Ohri, Kumar andPal, 1986). Similarly, five diploid taxa of Leucena showed2C DNA variation from 135 pg [L. esculenta (Mocino &Sesse ex A.D.C.) Benth. subsp. esculenta] to 181 pg [L.diversifolia (Schldl.) Benth. subsp. diversifolia Schldl.] andthree tetraploid taxa varied between 266 pg [L. esculenta(Mocino & Sesse ex A.D.C.) Benth. subsp. paniculata(Britton & Rose) Zarate] to 331 pg (L. confertifolia Zaratesubsp. adenotheloidea Zarate) (Palomino, Romo and Zarate,1995).

The 2C DNA values of 12 Eucalyptus species studiedconform to their sectional classification. They range from077 pg (E. citriodora Hook.) to 147 pg (E. saligna Smith),therefore showing a -9-fold difference. The species ofsubgenus Symphomyrtus show 2C DNA values from 1 09 pg(E. globulus Labill.) to 1-47 pg (E. saligna), while those ofsubgenus Corymbia have much smaller 2C DNA valuesvarying from 077 in E. citriodora to 080 pg in E. torellianaF. Muell. (Grattapaglia and Bradshaw, 1994). A two-folddifference has also been observed in 12 diploid species ofCoffea. The 2C DNA amounts vary from 095 pg (C.racemosa Ruiz & Pav.) to 178 pg (C. humilis A. Chevalier).It has been deduced that the DNA amount has increasedduring evolution in subgenus Coffea, as the three species C.pseudozanguebariae, C. sessiliflora and C. racemosa with thelowest DNA amounts are endemic to East Africa which isthe centre of origin of this genus. Furthermore, speciesadapted to xeric conditions have a DNA content lower thanthat of species growing in evergreen forests of theGuinea-Congo region (Cros et al., 1995).

The genus Terminalia presents a rather interesting casewhere genome size variation reflects polyploidy and alsooccurs at the same ploidy level, as is clear from the 35-folddifference between T. oliveri Brandis (2n = 24, 360 pg) andT. bellirica (Gaertner) Roxb. (2n = 48, 1280 pg). Out of thesix species studied, T. oliveri, T. myriocarpa Heurck &Muell. and T. arjuna (Roxb.) Wight & Arn. are diploid (2n= 24), T. chebula (Gertner) Retz and T. bellirica are tetra-

ploid (2n = 48) and T. muelleri Benth. is triploid (2n = 36).Differences in DNA per basic genome or per chromosomeare greatest (197-fold) between T. oliveri and T. arjuna.Two species groups: (1) T. oliveri and T. chebula; and (2) T.

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myriocarpa, T. arjuna, T. muelleri and T. bellirica aretherefore well differentiated by their DNA per basic genomevalues, irrespective of ploidy levels. Terminalia species aretherefore classified in two distinct groups by DNA per basicgenome, the mean values of which are 181 and 334 pg,respectively. Within diploids and tetraploids there is 197-fold and 176-fold variation, respectively (Ohri, 1996).

CONCLUSIONS

(1) Present understanding of genome size variation showsthat it is fairly constant within a species (taking a narrowspecies concept). Therefore, the C-value is one of the salientfeatures defining a species; (2) remarkable variation ingenome size can be found even among closely relatedspecies, which may correlate with various adaptive featuresat nuclear, cellular, tissue and organismic levels. This showsthat genome size is under strong selection pressure and cantherefore be used as one of the basic factors demarcatinginfrageneric taxa; (3) the increase or decrease in genome sizecan be correlated with evolutionary advancement within agroup. The grouping of taxa according to C-values maysometimes correspond with taxonomic schemes based onother characters; (4) DNA variation in some genera occursin a discontinuous manner forming groups of taxa, themeans of which are separated by regular intervals. However,some genera such as Allium may show a continuousvariation; (5) there is no correspondence between the levelof genetic differentiation of species and their DNA amounts;in other words quantitative genome variation is not a pre-requisite of species divergence. However, in some cases thespecies C-values may coincide with their crossabilityrelationships; (6) these facts show that genome size can beappropriately used as corroborative evidence in systematics.

ACKNOWLEDGEMENTS

The author is grateful to the Director of the NationalBotanical Research Institute for facilities and to ProfessorM. D. Bennett for very useful suggestions and encour-agement.

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Genome Size Variation and Plant Systematics - Annals of Botany