HETEROCHROMATIN BANDING PATTERNS IN RUTACEAE-AURANTIOIDEAE— A CASE OF PARALLEL CHROMOSOMAL...

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American Journal of Botany 87(5): 735–747. 2000.

HETEROCHROMATIN BANDING PATTERNS IN

RUTACEAE-AURANTIOIDEAE—A CASE OF PARALLEL

CHROMOSOMAL EVOLUTION1

MARCELO GUERRA,2 KARLA GALVAO BEZERRA DOS SANTOS,2 ANA

EMILIA BARROS E SILVA,2 AND FRIEDRICH EHRENDORFER3,4

2Universidade Federal de Pernambuco, CCB, Departamento de Botanica, Recife, PE, Brazil; and3Institute of Botany and Botanical Garden, University of Vienna, Rennweg 14, A-1030 Vienna, Austria

The heterochromatin banding patterns in the karyotypes of 17 species belonging to 15 genera of Rutaceae subfamilyAurantioideae (5 Citroideae) were analyzed with the fluorochromes chromomycin (CMA) and 49-6-diamidino-2-phenylin-dole-2HCl (DAPI). All species were diploids, except one tetraploid (Clausena excavata) and two hexaploids [Glycosmisparviflora agg. (aggregate) and G. pentaphylla agg.]. There are only CMA1/DAPI2 bands, including those associated withthe nucleolus. Using recent cpDNA (chloroplast DNA) sequence data as a phylogenetic background, it becomes evident thatgenerally more basal genera with rather plesiomorphic traits in their morphology, anatomy, and phytochemistry exhibit verysmall amounts of heterochromatin (e.g., Glycosmis, Severinia, Swinglea), whereas relatively advanced genera from differentclades with more apomorphic characters display numerous large CMA1 bands (e.g., Merrillia, Feroniella, Fortunella).Heterochromatin increase (from 0.7 to 13.7%) is interpreted as apomorphic. The bands are mostly located in the largerchromosomes and at telomeric regions of larger arms. However, one of the largest chromosome pair has been conservedthroughout the subfamily with only very little heterochromatin. The heterochromatin-rich patterns observed in differentclades of Aurantioideae appear quite similar, suggesting a kind of parallel chromosomal evolution. In respect to the currentclassification of the subfamily, it is proposed to divide Murraya s.l. (sensu lato) into Bergera and Murraya s.s. (sensu stricto)and to place the former near Clausena into Clauseneae s.s. and the latter together with Merrillia into Citreae s.l. Thesubtribes recognized within Clauseneae s.s. and Citreae s.l. appear heterogeneous and should be abandoned. On the otherhand, the monophyletic nature of the core group of Citrinae, i.e., the Citrus clade with Eremocitrus, Microcitrus, Clymenia,Poncirus, Fortunella, and Citrus, is well supported.

Key words: chromosomal parallel evolution; Citrus; CMA/DAPI staining; heterochromatin banding patterns; Rutaceae-Aurantioideae; systematics.

The subfamily Aurantioideae (5 Citroideae) is an im-portant major clade of the family Rutaceae. It has beenextensively studied, mainly because it includes the genusCitrus with several species cultivated worldwide, such asthe oranges, lemons, and mandarins. Besides Citrus andFortunella (kumquat) with enormous economic impor-tance, some other closely related genera, like Poncirusand Eremocitrus, have received considerable attention,since they constitute a source of valuable new genes forcitri*culture (Barrett, 1977).

According to Swingle and Reece (1967), the subfamilycomprises 33 genera and ;200 species. The first generalreview of the subfamily was presented by Engler (1931)who recognized a single tribe Aurantieae with two sub-tribes, Hesperethusinae and Citrinae. Later, Swingle (up-

1 Manuscript received 3 December 1998; revision accepted 27 August1999.

The authors thank the following institutions for providing the plantmaterial used in this work: Botanical Garden of Vienna; Botanical Gar-den of Rio de Janeiro; Royal Botanic Gardens, Kew; National ResearchCentre of Cassava and Tropical Fruitculture of Cruz das Almas; CitrusExperimental Station of Campinas. This research was supported byBanco do Nordeste do Brasil (BNB), Conselho Nacional de Desenvol-vimento Cientıfico e Tecnologico (CNPq) e Fundacao de Amparo aCiencia e Tecnologia de Pernambuco (FACEPE), and by the Commis-sion for Interdisciplinary Ecological Studies at the Austrian Academyof Sciences. This paper constitutes part VI of a publication series onthe ‘‘Cytogenetics of Rutaceae.’’

4 Author for correspondence (FAX 1143-1-4277-9541; e-mail: [email protected]).

dated by Swingle and Reece, 1967, catalog: Carpenterand Reece, 1969), on the basis of morphological and an-atomical analyses, divided the subfamily into two tribes:Clauseneae (I), composed of subtribes Micromelinae(IA), Clauseninae (IB), and Merrilliinae (IC), and Citreae(II) with the subtribes Triphasiinae (IIA), Citrinae (IIB),and Balsamocitrinae (IIC). Since then, many authors haveused other approaches to clarify the relationships withinAurantioideae, mainly by analyzing secondary metabo-lites (e.g., Grieve and Scora, 1980; Waterman, 1983,1990; Da Silva, Gottlieb, and Ehrendorfer, 1988), but alsoisozymes and other proteins (Esen and Scora, 1977; Tor-res, Soost, and Mau-Lastovicka, 1982; Iwamasa and Nito,1988; Fang, 1993a, b; Fang, Zhang, and Xiao, 1993;Zhong and Ye, 1993). In spite of some new suggestionsthe concept of the subfamily by Swingle and Reece(1967) is still accepted and in current use. Nevertheless,recent analyses of cpDNA (Samuel et al., 1999a, b, andunpublished data) will necessitate considerable system-atic changes in the future.

Karyosystematic analysis of the Aurantioideae wasfirst discouraged by observations that nearly all its taxahave small chromosomes of similar size and morphology(Krug, 1943). Only subsequent karyotype analysis withC-banding revealed that most of the chromosomes hadheterochromatic blocks that allow a better differentiation(Guerra, 1985; Liang, 1988; Wei, Cheng, and Duan,1988). The distribution of C-bands was similar to the

736 [Vol. 87AMERICAN JOURNAL OF BOTANY

TABLE 1. List of species analyzed arranged according to the tribes (I 5 Clauseneae, II 5 Citreae), subtribes (IA-C, IIA-C), and nomenclature ofSwingle and Reece (1967) and Carpenter and Reece (1969), and with respective provenance, chromosome number, and cell source.

Species Provenance/numbers 2n Cell source

IB- ClauseninaeGlycosmis pentaphylla (Retz.) Corr. agg. BGRJ/RUT56, CESC/RUT947, BGV 54 root tips of adultsG. parviflora (Sims.) Little agg.

[5 G. citrifolia (Willd.) Lindl. agg.]BGV/RUT180 54 root tips of adults

Clausena lansium (Lour.) Skeels[as C. wampi (Blanco) Oliv.]

CESC/RUT941 18 root tips of seedlings

Clausena excavata Burm. f. BGRJ/RUT113 36 root tips of seedlingsMurraya koenigii (L.) Spreng.

[correct 5 Bergera koenigii L.]RBGK/RUT898 18 root tips of seedlings

Murraya paniculata (L.) Jack. BGV/RUT147, RBGK/RUT898, CESC/RUT937, PGR/RUT173

18 root tips of seedlings

IC- MerrilliinaeMerrillia caloxylon (Ridley) Swing. CNPMF/RUT841 18 root tips of seedlings

IIA- TriphasiinaeTriphasia trifolia (Burm. f.) P. Wils. PGR/RUT568 18 root tips of seedlings

IIB- CitrinaeSeverinia buxifolia (Poir.) Ten.

[as Atalantia buxifolia (Poir.) Oliv.]BGRJ/RUT55 (1), CNPMF/RUT694 (2),

BGV (3)18 root tips of seedlings

Atalantia monophylla (Roxb.) DC.[as A. monophylla (L.) Corr.]

CNPMF/RUT500 18 root tips of seedlings

Fortunella crassifolia Swing. CESC/RUT650 18 root tips of seedlingsEremocitrus glauca (Lindl.) Swing. RBGK 18 root tips of adultsPoncirus trifoliata (L.) Raf. CNPMF/RUT503 18 root tips of seedlings

BGV 27 root tips of seedlingsMicrocitrus australasica (F. Muell.) Swing. CNPMF/RUT502 18 young leavesCitrus reticulata Blanco CNPMF/RUT501 18 root tips of seedlings

IIC- BalsamocitrinaeSwinglea glutinosa (Blanco) Merr. CNPMF/RUT504 18 root tips of seedlingsFeroniella sp. CESC/RUT413 18 young flowers buds

Note: BGV, Botanical Garden of Vienna, Austria; BGRJ, Botanical Garden of Rio de Janeiro, Brazil; RBGK, Royal Botanic Gardens, Kew,England; CNPMF, National Research Centre of Cassava and Tropical Fruitculture, Cruz das Almas, Bahia, Brazil; CESC, Citrus ExperimentalStation of Campinas, Sao Paulo, Brazil; PGR, Private Garden in Recife, Pernambuco, Brazil.

pattern of heteropycnotic regions observed by conven-tional Giemsa or Feulgen staining in prometaphase chro-mosomes (Guerra, 1985). More recently, it was demon-strated for some representatives of Citrus that their chro-mosomes, when stained simultaneously with the fluoro-chromes chromomycin A3 (CMA) and 49-6-diamidino-2-phenylindol (DAPI), revealed a variable number of re-gions that appeared bright or positive with CMA, and faintor negative with DAPI. Chromomycin binds preferentiallyto sequences of repetitive DNA rich in cytosine-guanine,whereas DAPI has a reverse affinity, binding preferen-tially to adenine-thymine rich sequences (Schweizer,1976). The resulting fluorochrome banding allowed foran even better karyotype characterization than did pre-vious methods (Guerra, 1993).

Improved chromosomal analyses of Aurantioideae alsohave important implications for the citrus breeding pro-gram. Representatives of several wild relatives of Citrus,including such distantly related genera, as Murraya, Sev-erinia, Atalantia, and Swinglea, have been hybridizedwith cultivated Citrus species in order to introduce de-sirable traits, mainly resistance to pests and pathogens(Barrett, 1977; Motomura et al., 1995). Since many spe-cies of this group have potentials for nucellar embryonyand polyembryony (Cameron and Frost, 1968), the ge-netic identification of intervarieties or intergeneric hy-brids plays a fundamental role and is mainly done by

isoenzyme profiles (Torres, Soost, and Mau-Lastovicka,1982). The chromosomal identification of different ge-nomes may be an additional and simple method of iden-tifying citrus hybrids and is thus of importance for futurework on substitution lines.

In the present work we concentrate on the cytogenet-ical characterization of the most commonly used non-citrus species of Aurantioideae, mainly by CMA/DAPIstaining, and investigate the heterochromatin patterns of17 species from 15 genera, representing both tribes andfive of the six subtribes of the subfamily. Discussing ourresults against the background of available morphologi-cal, phytochemical, and DNA-analytical data we hope tocontribute to the chromosomal evolution of the Auran-tioideae and to improve the phylogenetic classification ofthe subfamily.

MATERIALS AND METHODS

Plant material—The species investigated are presented in Table 1,named and arranged taxonomically according to Swingle and Reece(1967) and Carpenter and Reece (1969), numbered as outlined in theintroduction, and listed with provenances, chromosome numbers, andcell sources. All tribes and subtribes of Aurantioideae are representedwith the exception of the monogeneric Micromelinae (IA). Seeds fromopen-pollinated mother plants were germinated in petri dishes, andseedlings were cultivated in our experimental garden at the FederalUniversity of Pernambuco, in Recife. Seeds of Poncirus and Citrus were

May 2000] 737GUERRA ET AL.—HETEROCHROMATIN BANDING PATTERNS IN AURANTIOIDEAE

TABLE 2. Haploid chromosome complement length (CCL), heterochro-matin length (HL), and heterochromatin proportion (H%) in someAurantioideae.

Species n CCL (mm) HL (mm) H%

Swinglea glutinosa 9 11.11 0.35 3.12Citrus reticulata 9 15.61 4.48 28.71Triphasia trifolia 9 16.37 4.99 30.50Clausena lansium 9 16.74 0.57 3.41Murraya paniculata 9 21.24 6.85 32.35Glycosmis pentaphylla agg. 27 54.51 2.38 4.36

Fig. 1–3. Interphase nuclei and prophase condensation patterns in representative species of Aurantioideae. 1. Swinglea glutinosa. 2. Eremocitrusglauca. 3. Glycosmis pentaphylla agg. Bars 5 2.5 mm.

highly polyembryonic, but since root tips of different embryos wereanalyzed and exhibited the same banding pattern we believe that inthose cases we have worked with nucellar rather than zygotic embryos.Seed samples of other genera were nearly exclusively monoembryonic,and their embryos were probably zygotic. Adult plants were grown inpots. For cytological analyses, mostly actively growing root tips fromseedlings and some adult individuals were collected. Voucher specimensare deposited in the herbarium of the respective institutions, and copiesof most of them are maintained in the herbarium of the Federal Uni-versity of Pernambuco (UFP).

Cytology—Somatic tissues were pretreated with 8-hydroxyquinoline(0.002 mol/L) at room temperature for 1 h followed by 23 h in therefrigerator (;108C). The material was fixed in Carnoy 3:1 for 3–5 hand stored at 2208C until required. Meiotic analyses were carried outonly when adequate mitotic metaphases were not found. In this case,the flower buds were fixed and stored like the root tips.

Slide preparation for fluorochrome staining follows Schweizer(1976), described in detail by Deumling and Greilhuber (1982). ForCMA/DAPI staining the meristems were washed twice in distilled water(10 min each), digested with a 2% cellulase-10% pectinase solution (1.5h), and squashed in 45% acetic acid. After coverslip removal the slideswere aged for 3 d, stained with CMA (1 h), counterstained with DAPI(30 min), and mounted in McIlvaine’s (pH 7.0) buffer-glycerol v/v 1:1.For conventional staining the root tips were hydrolyzed in 5mol/L HCl(20 min) and squashed in a drop of 45% acetic acid. The coverslip wasremoved in liquid nitrogen, and the slide was stained in 2% Giemsa inSorensen buffer (20 min) and mounted in Euparal (Guerra, 1983).

In order to estimate the variation in chromosome complement sizeand CMA1 heterochromatin proportion, a sample of six species (Table

2) were chosen for chromosome measurements. A hexaploid cytotypeof Glycosmis pentaphylla agg. and one diploid representative of eachsubtribe studied were included. Swinglea glutinosa and Murraya pani-culata were also chosen because they presented, respectively, the small-est and the largest chromosomes among all the species analyzed.CMA1 blocks and chromosome size were measured from chromosomedrawings of amplified projections of film negatives of the best fivemetaphases of each species. In a few species, with different chromo-some number and heterochromatin amount (Glycosmis pentaphylla agg.,Clausena excavata, Severinia buxifolia, Eremocitrus glauca, and Swin-glea glutinosa), the prophase condensation pattern was analyzed forcomparison with the CMA banding pattern.

Photomicrographs were taken on Kodak Tri-X Pan film for UV lightand Copex Pan Agfa film for visible light. For the purposes of discus-sion, the chromosomes are referred to as pair I, II, III, etc., in decreasingorder of size.

RESULTS

General observations—Most species analyzed werediploids with 2n 5 18, except the hexaploid cytotypesfrom Glycosmis parviflora agg. and G. pentaphylla agg.,both with 2n 5 54, and Clausena excavata with 2n 5 36(Table 1). Seeds of one plant of Poncirus trifoliata grownin the Botanical Garden of Vienna and apparently aga-mospermous produced only triploid embryos.

After conventional Giemsa staining, prophase chro-mosomes exhibited a very similar condensation patternin all species investigated. Proximal heteropycnosis wasobserved in most chromosomes and terminal heteropyc-notic blocks were present in those species that possessedterminal blocks of CMA1 heterochromatin (Figs. 1–3).The nuclear interphase structure in the whole subfamilywas always of the areticulate type, with a variable num-ber of chromocentres. Two types of chromocentres wereobserved: a smaller number of deeply stained, large,well-defined chromocentres and a larger number ofsmaller, less densely stained ones (Figs. 1–2); the latterwere more clearly observed in meristematic cells. Thenumber of larger chromocentres in each species was pro-portional to the number of heterochromatic blocks ob-served after CMA staining.


TABLE 3. Idiograms and banding patterns of 17 Aurantioideae species, arranged according to the subtribes as defined by Swingle and Reece (1967).Heterochromatic bands are shown according to their relative size and position on chromosomes I to IX (left to right). Differences in ploidy,chromosome size, and centromere position are not considered. Different provenances of Severinia buxifolia are illustrated: 1–2 (above) and 3(below).

All Aurantioideae species investigated exhibit veryclear and differentiated patterns of CMA1/DAPI2 blocks.Most bands were observed in telomeric regions. They arepreferentially located in the long arms, at least in themajority of chromosomes with clearly defined morphol-ogy. On the other hand, DAPI-positive blocks were neverobserved in prophase or metaphase chromosomes of thespecies investigated, although small chromocentres werealways present in the interphase nuclei. They seem tocorrespond to the small and less dense chromocentres ob-served with Giemsa.

Species differ widely in terms of chromosome size,number, and distribution of heterochromatin blocks pos-itive to CMA. The hexaploid G. pentaphylla exhibits amean chromosome size proportional to the chromosomesize of the diploid species. Size variation between thelargest and the smallest chromosomes of each karyotypewas very small in absolute values (one or two micro-metres), although proportionally this represents a varia-tion of 100–200%. Variation in chromosome complementlength also was relatively small and was far exceeded byvariation in heterochromatin amount (Table 2).

The chromosome banding with CMA appeared morepronounced in two or three of the largest pairs, wheregenerally a higher amount of heterochromatin was con-centrated. Medium and small-sized pairs showed an ap-parent greater similarity in banding pattern due to theirsmaller number of bands. A noteworthy feature was thepresence of a large chromosome pair common to practi-cally all species, independent of the heterochromatinamount within the karyotype. This largest or second larg-est pair, metacentric, without bands or only with very

small telomeric ones, is here referred to as FL (large chro-mosome of the F type described for Citrus by Guerra,1993).

In the schematic idiograms (Table 3) no differences inploidy, chromosome size, and centromeric position with-in and between the genomes of the various taxa areshown in order to facilitate comparison. Only the locationand relative size of CMA1 blocks are demonstrated.When two or more different banding patterns were ob-served for a single chromosome pair, only that with thehighest amount of heterochromatin was shown. The fol-lowing detailed specific presentation of our karyologicalfindings follow the taxonomic classification of Swingleand Reece (1967) and the (sub)tribal numbers outlined inthe introduction.

Tribe Clauseneae (I)—From the subtribe Clauseninae(IB) representatives of all four genera were investigated.Some species have a very low amount of heterochro-matin. The two populations of Glycosmis pentaphyllaagg. and one of Glycosmis parviflora agg. analyzed werehexaploids with very similar karyotypes, which seemedto be more symmetric than those observed in the re-maining species. They exhibit metacentric to submeta-centric chromosomes with very little variation in size.Only three of their 27 chromosome pairs had terminallylocated CMA1 blocks (Figs. 4–5), all of them associatedwith the nucleolus. The six chromosomes with hetero-chromatic bands corresponded in size to pairs IV to VI.Pairs I to III were large metacentrics without CMA1 het-erochromatin (Figs. 4–5). The interphase nuclei displayeda large number of small chromocenters when observed


Fig. 4–11. Patterns of Clauseninae species. 4, 5. Glycosmis pentaphylla agg. 6. Clausena lansium. 7. C. excavata. 8, 9. Murraya koenigii. 10,11. M. paniculata. Arrows in 7 and 10 point to secondary constrictions. Figs. 4, 6, 7, 8, and 10 were stained with CMA, and Figs. 5, 9, and 11were stained with DAPI. Bars 5 2.5 mm.


with DAPI fluorescence but only three to six large oneswith CMA staining, whereas both kinds of chromocenterscould be observed with conventional staining.

Among diploid species of this tribe, only in Clausenalansium (2n 5 18) was the heterochromatin restricted toa single chromosome pair (Fig. 6). The CMA1 block waslocated terminally on pair II and associated with the nu-cleolus in prophase and interphase cells. This karyotypeapparently is very similar to the monoploid complementof Glycosmis, with only one nucleolus-associated bandand the largest pair being wholly euchromatic.

Another species of Clausena, C. excavata, had 2n 536 and differed by a larger number of small CMA1bands, most of them very small. One chromosome pairhad a terminal band in each telomere region, six pairswere with a single terminal CMA1 block, and 11 pairswere without bands (Fig. 7), including the two largestones (FL type).

Two species of Murraya (M. koenigii and M. panicu-lata) were analyzed, both with 2n 5 18, but with verydistinct karyotypes. Murraya koenigii had less contrastedbands, terminally located on pairs II and either on IV orV (Figs. 8–9), which were associated with the nucleolusin prophase and interphase cells. In some cells, fine prox-imal bands in pair II and terminal ones in pair I (FL type)were observed.

On the other hand, Murraya paniculata was the specieswith the largest chromosomes (Table 2) and the highestnumber of bands in the tribe Clauseneae (Figs. 10–11).CMA1 blocks were found in all chromosomes except inone of the smallest pairs. The FL chromosome type cor-responded to one of the three largest pairs, showing asmall but always visible telomeric CMA1 block. The oth-er two large pairs were far richer in heterochromatin. Oneof them presented a very brilliant band in each telomericregion, whereas the other showed a large telomeric anda small proximal heterochromatic block. The latter some-times appeared decondensed like a secondary constric-tion. Each of the remaining five pairs exhibited a singlelarge terminal band. The analysis of four provenancesfrom far distant locations did not show any karyotypevariation.

Merrillia caloxylon, the only representative of subtribeMerrilliinae (IC), showed a heterochromatin-rich karyo-type. Bands were found in six of its nine chromosomepairs. Pair I (FL) with a small terminal band, pair II withlarge CMA1 blocks in both telomeres, and pair III witha large terminal block as well as a proximal band couldbe distinguished. The latter band exhibited heterozygoticvariation in size and strongly quenched fluorescence withDAPI; in some metaphases it appeared finer and distend-ed as a secondary constriction. In some cells, analyzedwith DAPI or Giemsa, pair III seemed to be split in two(Figs. 12–13). The other three larger chromosome pairsshowed single major terminal bands, whereas the threesmaller were wholly euchromatic.

Tribe Citreae (II)—All species analyzed were diploid(2n 5 18), but exhibited very diversified banding pat-terns. The subtribe Triphasiinae (IIA), composed of eightgenera, was only represented by Triphasia trifolia. It hasa distinct karyotype, with only single terminal bands inseven of its nine chromosome pairs. Among these chro-

mosomes, one of the shortest pairs was distinguished bya much fainter band, sometimes flanked by a secondaryconstriction on the proximal side. In the other pairs, thebands appeared large and bright, occupying one-fifth toone-third of the chromosome length (Figs. 14–15). Insome cells, pair I (FL) was observed with a CMA1 blockin one of its telomeres.

Seven of the 13 genera of subtribe Citrinae (IIB) wereanalyzed. The least amounts of heterochromatin werefound in Severinia and Atalantia, genera with rather sim-ilar banding patterns, both with pair I of the FL type. InS. buxifolia, CMA1 blocks were restricted to the threepairs II, III, and IV, always located at terminal positions(Figs. 16–17). When the nucleolus was visible in pro-metaphases, it was always associated with two or threeof these blocks. In some cells, pair I showed a very smallterminal CMA1 region. Two samples of S. buxifolia(BGRJ and CNPMF) did not exhibit karyotype differ-ences. A third, received as ‘‘Atalantia buxifolia’’ (BGV),differed by bands in both telomeric regions of pair II(Figs. 18–19). Atalantia monophylla had four pairs show-ing CMA1 blocks on the long arms of pairs II, III, IV,and V (Figs. 20–21).

The other genera analyzed from this subtribe (thegroup of ‘‘true Citrus fruit trees’’ according to Swingleand Reece, 1967; also called ’’Citrus clade’’ by Samuelet al., 1999a, b, and unpublished data) had karyotypeswith a much higher heterochromatin content. Eremocitrusglauca showed bands on all chromosomes except thesmallest pair. One of the largest pairs appeared withCMA1 blocks in both telomeres, whereas the others hadonly single terminal blocks (Figs. 22–23). This specieswas the only one in the tribe where the FL chromosomewas not identifiable.

The diploid sample of Poncirus trifoliata analyzed wasclearly heterozygous. Seven of the 18 chromosomes ofthe mitotic complement, including pair I (FL), had noCMA1 blocks. Three chromosomes had bands in bothtelomeres, three others with one terminal and one weakproximal band, and five with a single terminal CMA1

blocks (Figs. 24–25). Trying to pair the chromosomes onthe basis of similarities in size and banding patternsleaves at least two heteromorphic pairs: the first pair con-sisting of one chromosome without CMA1 blocks and itshomologue with a single terminal band and the secondpair consists of a chromosome with terminal heterochro-matin in both telomeres and its homologue with one telo-meric and one proximal band. Triploid embryos observedin a seed sample from the Botanical Garden of Viennahave a very similar banding pattern, but a detailed anal-ysis was not possible.

Citrus reticulata showed only three chromosome pairswithout bands, including one of the FL type. Five pairsexhibited a single terminal CMA1 block, conspicuouslylarge in three of them, and one pair with bands in bothtelomeres (Figs. 26–27). In some cells, one of the singlebanded chromosomes showed an additional heterochro-matic block in the opposite telomere.

Fortunella crassifolia exhibited the largest number ofbanded chromosomes per karyotype observed in thewhole subfamily, with CMA1 telomeric blocks in eachof its chromosomes. In two of them, the bands werefound in both telomeric regions, and in some cells one


Fig. 12–21. Banding patterns of Merrilliinae, Triphasiinae, and some Citrinae species. 12, 13. Merrillia caloxylon. Arrows show a DAPI2

secondary constriction. 14, 15. Triphasia trifolia. 16, 17. Severinia buxifolia (1–2). 18, 19. S. buxifolia (3). 20, 21. Atalantia monophylla. Figs. 12,14, 16, 18 and 20 were stained with CMA, and Figs. 13, 15, 17, 19 and 21 were stained with DAPI. Bars 5 2.5 mm.

of these pairs exhibited an additional proximal band,stained slightly more with CMA than the euchromatin.When observed with DAPI, this band was strongly neg-ative, giving the impression that each homologue chro-mosome was divided in two (Figs. 28–29). The otherchromosome pairs displayed terminal bands of very var-iable size. The largest chromosome pair was of the FL

type, showing a single, very fine but constant terminalband.

In Microcitrus australasica we were not able to obtainwell-condensed metaphases from the meristematic tissueof young leaves, although it was possible to identify thebanding pattern in many prometaphases (Figs. 30–31).One chromosome pair had terminal bands in both telo-meres, six pairs had single terminal bands of variablesize, whereas two pairs were wholly euchromatic, includ-

ing the largest one. In some cells, weak proximal or ter-minal bands were additionally found in some of the sin-gle banded chromosomes.

Of the seven genera that make up subtribe Balsamo-citrinae (IIC), only two, Swinglea and Feroniella, are rep-resented in this study. Swinglea glutinosa showed a verydistinct, heterochromatin-poor karyotype with CMA1

blocks restricted to one of the telomere regions of pairIII (Fig. 32). At interphase, these heterochromatic blockswere found associated with the nucleolus. This specieshad the smallest chromosomes known in the whole sub-family Aurantioideae (Table 2).

In Feroniella sp., on the other hand, there was a largeamount of heterochromatin. Although only meiotic anal-ysis was performed on this species, the banding patternobserved in metaphases I and II (Figs. 33–34) suggests


Fig. 22–34. Banding patterns of some Citrinae and Balsamocitrinae species. 22, 23. Eremocitrus glauca. 24, 25. Metaphase and interphasenucleus of Poncirus trifoliata. 26, 27. Citrus reticulata. Arrows show a telomeric CMA1/DAPI2 band. 28, 29. Fortunella crassifolia. 30, 31.Prophase and interphase nucleus of Microcitrus australasica. 32. Swinglea glutinosa. 33, 34. Metaphase I of Feroniella sp with bivalents. Figs. 22,24, 26, 28, 30, 32 and 33 were stained with CMA, and Figs. 23, 25, 27, 29, 31 and 34 were stained with DAPI. Bars 5 2.5 mm.

that CMA1 blocks were located in both telomeres and inthe centromeric region of five chromosome pairs, in asingle telomere of two pairs, and absent in two otherpairs. The meiotic behavior was regular in spite of theoccurrence of a few anaphase bridges.

DISCUSSION

Relative cytogenetic stability—The subfamily Auran-tioideae can be characterized by the dominance of dip-loids, stable basic chromosome number (x 5 9), and quite


uniform interphase nuclear structure, in sharp contrast tothe majority of the remaining subfamilies and tribes ofRutaceae (Krug, 1943; Stace, 1995). From ;60 speciesof Aurantioideae studied so far, the vast majority has 2n5 18 (2x) and infraspecific autopolyploids (3x, 4x. . . . )occur only occasionally. Proof for stabilized polyploidsis very limited: Glycosmis pentaphylla agg. (2x, 5x, 6x),G. parviflora agg. (5 G. citrifolia agg.) (4x), Clausenaexcavata (4x), C. dentata (5 C. willdenowii) (4x), andParamignya monophylla (4x). There is only one, obvi-ously erroneous indication for a deviation from x 5 9(Krug, 1943; Mehra, 1976; Guerra, 1984; Stace, Arms-trong, and James, 1993; Stace, 1995). Karyotype stabilityin Aurantioideae is apparently linked with a high capacityfor interspecific hybridization.

The areticulate nuclear structure of Aurantioideae isclearly correlated with the chromosome organization ob-served in prophase. During this stage, heteropycnoticblocks are visible after conventional Giemsa or Feulgenstaining in the proximal region of many chromosomes,but in some also in the terminal region (Guerra, 1985,1987). Such heteropycnotic regions may change depend-ing on the condensation stage and the conditions of fix-ation. However, CMA/DAPI staining reveals the true het-erochromatic nature of all of these terminal, but only ofvery few of the proximal prophase blocks, and helps todetect a large variation in banding patterns.

The maintenance of the areticulate nuclear structurethroughout the whole subfamily is not surprising sincethis kind of nuclear organization is commonly found inspecies with a low DNA amount and small chromosomesize (Barlow, 1977; Guerra, 1987). The chromosomecomplement size shows only limited variation (Table 2):Swinglea glutinosa seems to have the smallest chromo-some complement size (11.11 mm) of the subfamily (seealso Sharma and Bal, 1957; Banerji and Pal, 1957;Ghosh, 1966; Wei, Cheng, and Duan, 1988; Miranda etal., 1997), only about half of that observed in Murrayapaniculata (21.24 mm). This latter species has one of thelargest chromosome complement sizes among diploidspecies of the subfamily. Like other Aurantioideae withDNA amounts known (M. koenigii, Citrus sinensis, andAtalantia monophylla—Guerra, 1984; Ohri and Kumar,1986), M. paniculata, nevertheless, is among the angio-sperms with low DNA content (Bennett and Leitch,1995).

Heterochromatin quantity and banding—Relativeheterochromatin amounts per karyotype as measured byrelative length of bands vary largely among the speciesanalyzed (Table 2). The highest heterochromatin content(32.35%) was observed in Murraya paniculata, anamount tenfold higher than in Swinglea glutinosa(3.12%). However, the highest number of bands per kar-yotype was observed in Fortunella crassifolia. Mirandaet al. (1997) observed an identical CMA-banded karyo-type for this species and estimated a heterochromatin pro-portion of 34.25%, whereas for Citrus species they found20.58–22.74%. Liang (1988) also found higher hetero-chromatin proportions in Fortunella species (;20%) thanin Citrus (;16%). This variability is proportional to thatobserved in the satellite DNA percentage by Ingle, Pear-son, and Sinclair (1973) in Fortunella sp. (24%) and Cit-

rus spp. (19–23%), which were among the highest valuesobserved in a large sample of angiosperms.

The highly variable heterochromatin amounts andbanding patterns observed in different genera of Auran-tioideae make them very useful tools for identification ofintergeneric hybrids. For instance, the sexually obtainedhybrids of Citrus 3 Severinia (Medina-Filho, Bordignon,and Ballve, 1998) can be easily recognized by chromo-somal CMA staining, since the two genera have widelydifferent banding patterns. Similarly, zygotic and nucellarembryos of such hybrids would differ by at least threechromosome pairs. The heterozygosity observed in someAurantioideae species, mainly of Citrus and Poncirus, isprobably due to the natural and artificial hybridizationthat has occurred during their intensive breeding and cul-tivation. However, in most cases a few chromosomemarkers can distinguish the karyotypes of closely relatedspecies (Guerra, 1993; Miranda et al., 1997).

Different heterochromatin fractions—At least threetypes of heterochromatin can be recognized in species ofAurantioideae: (1) C-banding1/CMA1 heterochromatinassociated with the NORs (nuleolus organizing regions);(2) C-banding1/CMA1 heterochromatin not associatedwith the NORs, mostly terminal; and (3) C-banding1/CMA2 heterochromatin, observed in proximal regions ofsome species (Guerra, 1985, 1993). That the heterochro-matin type (1) is always associated with the nucleolus ismost easily seen in species of Aurantioideae with onlyone pair of CMA1 blocks per monoploid complement. Itsometimes appears partially decondensed and as a sec-ondary constriction. In several species this heterochro-matin was observed as a strongly DAPI-negative blockand as a CMA band brighter or fainter than the remainingones. In general, the chromatin associated to the nucle-olus is CMA1 and GC rich (Schweizer, 1976; Deumlingand Greilhuber, 1982) and represents a special kind ofheterochromatin.

On the other hand, in Aurantioideae species with alarge number of CMA1 blocks the majority of them arenot nucleolus-associated bands of type 2. Matsuyama etal. (1996) have shown by in situ hybridization that onlythree of the 16–18 CMA1 blocks of Citrus sinensis cor-respond to rDNA sites. These data suggest that the ma-jority of the CMA1 blocks are constituted by anotherDNA sequence involved in the heterochromatin amplifi-cation of Aurantioideae, which, although rich in GC(CMA1), is independent of the rDNA sequence. In someother angiosperms that have many CMA1 blocks, it hasalso been demonstrated that only a few are composed ofrDNA repeats (Deumling and Greilhuber, 1982; Parokon-ny et al., 1992).

Quantity and distribution of the C-banding1/CMA2

heterochromatin (3) within Aurantioideae still need fur-ther analysis. In most species the proximal region of pro-phase chromosomes is heteropycnotic after conventionalstaining and seems to correspond to small, DAPI-brilliantchromocenters observed in all genera and species ana-lyzed here (see also Guerra, 1993). This proximal chro-matin was detected in only a few chromosomes with theC-banding method (Guerra, 1985) but, at least in the‘Trovita’ orange, it has been clearly demonstrated to oc-


cur in every chromosome by HKG banding (Ito, Omura,and Nesume, 1993).

Constraints in banding patterns—A comparativeanalysis of the CMA1 heterochromatin within the kar-yotypes of Aurantioideae reveals remarkable constraints.Despite the high diversity found in representatives ofboth tribes, CMA1 blocks evidently are not distributed atrandom. Chromosome banding patterns of all species an-alyzed and of those previously described (Matsuyama etal., 1996; Miranda et al., 1997), correspond to one of thesix chromosome types reported for Citrus (Guerra, 1993)and suggest quite limited variation. Terminal bands dom-inate, whereas proximal ones are rare. Such proximalCMA1 bands have only been found in chromosomes withat least one telomeric band. Interstitial bands were neverobserved or have been misinterpreted as being proximalor terminal, due to the small size of chromosome arms.

In proximal positions, generally, CMA1 bands are lesscommon in plant chromosomes than DAPI1 bands (see,e.g., Schweizer, 1976; Deumling and Greilhuber, 1982;Moscone, Lambrou, and Ehrendorfer, 1996), althoughthey are known in some species (Roser, 1994). In general,when there is proximal and terminal heterochromatin ina banding pattern, single proximal bands also occur. Inthe tribe Anthemidae (Asteraceae), for example,Schweizer and Ehrendorfer (1983) have demonstrated abanding pattern very similar to that of Aurantioideae, butthere are also chromosomes with single proximal hetero-chromatic blocks.

The Aurantioideae banding patterns reveal a remark-able constraint of heterochromatin development in one ofthe three largest chromosomes of each set, designated asFL. This can be easily identified in nearly all species,independent of genome size and ploidy level. CMA1 het-erochromatin is usually absent in this chromosome, al-though in some species a small CMA1 band may be ob-served in one or both homologues. This was previouslyreported also for some Citrus species (see idiograms inGuerra, 1993, and Miranda et al., 1997). Obviously, thestructure of the FL chromosome has been strongly con-served during Aurantioideae evolution. Linkage maps ofCitrus species obtained through recombination of molec-ular markers also suggest high conservation of linkagegroups (Durham et al., 1992; Jarrel et al., 1992), anddetailed molecular maps in other plant groups suggestlinear conservation of genome structures even in spite ofvariable basic chromosome numbers (Gill, 1995; Mooreet al., 1995).

Another aspect of chromosome evolution in Auran-tioideae is that CMA1 heterochromatin is concentrated inthree of the largest chromosomes of each haploid com-plement. Such chromosomes display banding patterns oftypes A to D (according to the classification proposed forCitrus chromosomes by Guerra, 1993). Furthermore, themost commonly banded chromosome types had a singleterminal band (types D and E), preferentially located inthe long arm (see also, Guerra, 1993; Matsuyama et al.,1996; Miranda et al., 1997).

The above data partly disagree with the widely ac-cepted model of heterochromatin dispersion, proposed bySchweizer and Loidl (1987). This model predicts that‘‘telomeric bands tend to be preferentially associated with

short chromosomes or chromosome arms.’’ However, inAurantioideae the bands are preferentially distributed inlarge chromosomes and large arms. Furthermore, themodel assumes a coevolution of the C-band pattern innonhomologous chromosome arms of similar size. In Au-rantioideae this fits the preferential distribution of CMA1

blocks in the larger arms, but it does not apply to the FL

chromosomes with similar arm size, which remain het-erochromatin poor.

Phylogeny, systematics, karyotypes, and bandingpatterns—Current studies on cpDNA sequences and re-sulting trees (Samuel et al., 1999a, b, and unpublisheddata) clearly show the Aurantioideae to be monophyletic,but the resulting phylogeny of the subfamily is at vari-ance in several respects with the classical systematictreatment by Swingle and Reece (1967), which was basedon morphological and anatomical characters and mayneed considerable modifications. Earlier phytochemicalevidence had already been used to improve the Auran-tioideae classification and to calculate advancement pa-rameters for several biogenetic classes of compounds(e.g., Waterman, 1983, 1990; But et al., 1998; Da Silvaet al., 1988). This is supplemented by suggestions forcharacter progressions (plesiomorphic → apomorphic),both from phytochemistry (e.g., the stepwise replacementof anthranilic acid derived alkaloids by coumarins and/orlimonoids) and from morphology (e.g., development ofaxillary spines; pinnate → simple leaves; petioles and ra-chis roundish → winged; stamens 5 1 5 → more nu-merous; fruits many, small → few, large; exocarp thin,soft → thick, leathery, or woody; fruit locules without →with 6 specialized pulp-vesicles; etc.) (e.g., Tanaka,1936; Swingle and Reece, 1967; Da Silva et al., 1988).Against this background we propose to discuss the kar-yological findings presented in this study.

According to the cpDNA data the subdivision of Au-rantioideae into Clauseneae and Citreae (Swingle andReece, 1967) is justified, if the genera Murraya (withexception of the species segregated as Bergera, e.g., Mur-raya koenigii 5 Bergera koenigii) and Merrillia (Merril-liinae) are transferred to the Citreae. This narrower con-cept includes only genera within Clauseneae s.s., whichhave carbazoles and only a limited quantity of hetero-chromatin per haploid chromosome set (,5% in one tofour banded chromosomes). In contrast, the genera of Ci-treae s.l. lack carbazoles and have either little or moreoften considerable quantities of heterochromatin (.10%in up to eight banded chromosomes) (Tables 2–3).

One of the core genera of Clauseneae s.s. is Glycosmis,a large and widespread Australasian genus, morphologi-cally and phytochemically quite plesiomorphic, well cir-cumscribed, and relatively isolated. In no other genus ofAurantioideae have so many polyploids accumulated: G.pentaphylla agg. and G. parviflora agg. represent poly-ploid complexes (with 2x, 4x, 5x, and 6x populations),still very insufficiently understood (H. Greger, Inst. Bot.Univ. Vienna, personal communication). The bandingpattern of Glycosmis is simple, with only a single CMA1

block per monoploid complement on chromosome II.This results in the low heterochromatin value of 4.36%(Table 2).

Clausena forms a somewhat more advanced group of


Clauseneae s.s., but the diploid C. lansium still exhibitsa karyotype very similar to Glycosmis with only oneCMA1 block on chromosome pair II and a heterochro-matin value of 3.41%. The distinct tetraploid C. excavatahas small bands in seven of its 18 chromosome pairs, butonly a slightly higher amount of heterochromatin.

Morphological and phytochemical evidence (But et al.,1988; Waterman, 1990) as well as recent cpDNA data(Samuel et al., 1999a, b, and unpublished data) demon-strate that the genus Murraya s.l. in its present and widecircumscription (Swingle and Reece, 1967) is an ‘‘arti-fact’’ and should be split at least into Murraya s.s. andBergera. This suspicion is strongly supported by our kar-yological data: whereas the type species of Murraya, M.paniculata, exhibits an elaborate banding pattern withCMA1 blocks on eight of its nine chromosome pairs andthe highest heterochromatin index (.32%) in the Auran-tioideae, Bergera koenigii has bands on only two chro-mosome pairs (II, IV) and a low heterochromatin value(Table 3).

In conclusion, the Clauseneae s.s. are constituted bytwo sister clades, one formed by Glycosmis and the moredistantly related Micromelum (not studied here), and theother by Clausena and Bergera. These Clauseneae s.s.represent an assembly of genera with a dominance ofplesiomorphic characters. In contrast, Murraya s.s., to-gether with Merrillia, should be removed to the Citreaes.l. As described by Swingle and Reece (1967), the threesubtribes of Clauseneae s.l., Micromelinae (IA), Clausen-inae (IB), and Merilliinae (IC), appear heterogeneous andobsolete.

Within their Citreae s.s. (II) Swingle and Reece (1967)have differentiated three subtribes, i.e., Triphasiinae(IIA), Citrinae (IIB), and Balsamocitrinae (IIC). Avail-able cpDNA data (Samuel, 1999a, b, and unpublisheddata) demonstrate that these subtribes also are ‘‘artifi-cial.’’ The genera analyzed in the present study appar-ently belong to three major clades of Citreae s.l., the firsttwo have no or only plesiomorphic, broadly based pulp-vesicles and often lack limonoids, and the third, the Cit-rus clade, develops specialized pulp-vesicles with a slen-der stalk and mostly exhibits a variety of limonoids (DaSilva, Gottlieb, and Ehrendorfer 1988). Both of the firsttwo clades include taxa from the Clauseneae (i.e., IB andIC) and from all three of the Citreae subtribes (IIA, IIB,and IIC) as recognized by Swingle and Reece (1967).

In the first and most basal of the cpDNA-supportedclades we propose to include Swinglea (IIC), Severinia(IIB), Murraya s.s. (IB), and Merrillia (IC) as alreadymentioned, and possibly also Atalantia (IIB). Both Ta-naka (1936) and Swingle and Reece (1967) considerSwinglea as morphologically plesiomorphic and basalwithin Balsamocitrineae. The only species, S. glutinosa,has a single CMA1 block in its haploid chromosomecomplement and the lowest heterochromatin content yetrecorded for the Aurantioideae (3.12%). Severinia withinCitrinae (IIB) also exhibits relatively plesiomorphic fea-tures. Its banding pattern consists of three to four CMA1

bands in three chromosome pairs, and the heterochomatinamount is intermediate. Murraya s.s. (IB) with M. pani-culata with its extremely high heterochromatin value of32.35% has been discussed already. There are close re-lationships with the monotypic Merrillia (IC). Both gen-

era are former members of Clauseneae s.l., exhibit rela-tively apomorphic features, have quite similar karyotypeswith chromosome bands in six to eight of their chromo-some pairs, and should be transferred to the Citreae s.l.

Atalantia (IIB) was formerly considered close to Sev-erinia, and the two were even united into one genus (En-gler, 1931). However, Swingle and Reece (1967, p. 283)used the old generic name Atalantia to denominate thosespecies with more apomorphic characters (as larger flow-ers and well-formed conical pulp-vesicles, thus approach-ing the Citrus clade), whereas the species with more ple-siomorphic features were left in Severinia. As Atalantiamonophylla has bands in four of its nine chromosomepairs, the banding patterns and heterochromatin values ofthe two genera are similar. Nevertheless, the cpDNA se-quences suggest a separate position from the Citrus clade,in spite of the occurrence of several limonoids in Atalan-tia.

The second cpDNA-supported clade of Citreae s.l. in-cludes among others the genera Triphasia (IIA) and Fer-oniella (IIC). Nevertheless, the two genera are by nomeans close. Whereas the first is relatively plesiomorphicin its characters, the second exhibits quite apomorphicfeatures. Triphasia has a relatively high amount of het-erochromatin with single telomeric bands on seven of itsnine chromosome pairs. The genus was placed into Tri-phasiinae (IIA), a heterogeneous subtribe whose cytoge-netics still are poorly understood. The only other IIA-taxon karyologically investigated is the tetraploid Para-mignya monophylla (Mehra, 1976; Stace, 1995). The kar-yotype of Ferionella is also rich in heterochromatin, withbands in seven of the nine chromosome pairs, but thepattern differs considerably from Triphasia, because fiveof these pairs have two telomeric and one centromericband (Table 3).

The core of the Citrinae (IIB) are the ‘‘true Citrus fruittrees’’ of Swingle and Reece (1967; corresponding to theAurantieae of Tanaka, 1936). As the third clade of theCitreae s.l., this Citrus clade is well supported by theavailable cpDNA sequences (Samuel et al., 1999a, b, andunpublished data), and also quite coherent morphologi-cally, phytochemically, and in its crossing potentials. Atthe same time, this clade obviously is the most advancedin the tribe with the greatest number of apomorphic fea-tures. This corresponds well with its strongly banded kar-yotypes and the increased heterochromatin amount(28.71% in Citrus). There are one or more bands on sixto all nine chromosome pairs of Citrus, Poncirus, Micro-citrus, Eremocitrus, and Fortunella (Table 3). In spite ofsome clear-cut differences between species of Citrus andFortunella (the present data correspond to those in Guer-ra, 1993, and Miranda et al., 1997), the characteristicoverall banding pattern of the Citrus clade is not ob-scured.

In view of the possible links of the Citrus clade tomore basal members of the Aurantioideae with more ple-siomorphic characters (e.g., Clausena, Severinia, Atalan-tia), it would be important to analyze the banding patternsof other possibly related genera like Pleiospermium, Bur-killanthus, Limnocitrus, Hesperethusa, and Citropsis fora better resolution of their phylogeny.

In retrospect, the Citreae s.l. are the clearly more ad-vanced and diverse of the two Aurantioidae tribes. The


relationships within and between its two more basalclades with relatively plesiomorphic features need furtherstudy. In contrast, the third clade with Citrus and fiveother closely related genera is clearly circumscribed, ob-viously monophyletic, and strongly apomorphic in mostof its characters. Our results suggest an ‘‘artificial’’ natureof the subtribes Triphasiinae, Citrinae, and Balsamocitri-nae.

Chromosomal evolution—On the basis of the karyo-logical and other data available for Aurantioideae, onemay speculate about the ancestral karyotype and the evo-lution of banding patterns of the subfamily. The ancestralkaryotype was certainly composed of nine chromosomepairs, ranging in size from 1.0 to 3.5 mm. Concerning theheterochromatin pattern, there is an apparent correlationbetween heterochromatin-rich karyotypes and relativelyadvanced genera, more or less apomorphic in respect tomorphology, anatomy, and phytochemistry. What is note-worthy is the fact that all heterochromatin-rich karyo-types from different and unrelated clades within Auran-tioideae exhibit remarkably similar banding patterns, asshown in the foregoing discussion about ‘‘constraints.’’There are at least four instances of such heterochromatin-rich groups with similar banding patterns that are notclosely related, i.e., the Murraya s.s./Merrillia clade (IB/C), the genera Triphasia (IIA) and Feroniella (IIC), andthe Citrus clade (IIB).

The correlation of plesiomorphic/heterochromatin-poorand apomorphic/hetero-chromatin-rich karyotypes seemsto be rather general in plant groups at supra- and infra-generic levels and is commonly interpreted as hetero-chromatin accumulation during evolution (Ikeda, 1988;Morawetz and Samuel, 1989; Roser, 1994). In somegroups this trend does not result in a parallel evolutionof banding patterns. For example, Cyphomandra andCapsicum (tribe Solaneae of the Solanaceae) exhibit in-creased heterochromatin content but have completely dif-ferent banding patterns (Pringle and Murray, 1993; Mos-cone et al., 1993; Moscone, Lambrou, and Ehrendorfer,1996). On the other hand, there are cases like Aurantioi-deae with apparently independent lines showing hetero-chromatin increase following very similar parallel pat-terns, as in Scilla (Hyacinthaceae), where heterochro-matin amounts have increased independently in the spe-cies groups of S. vindobonensis and of S. luciliae. Here,remarkable similarities in banding patterns, except in theNOR-bearing chromosomes, have developed despitelarge differences in chromosome size and morphology(Greilhuber, 1979). Such parallelisms can only be a con-sequence of special structures in the ancestral karyotype,subsequent constraints, and karyotypic orthoselection(White, 1973). This author admitted three distinct waysin which selection might canalize structural changes: sim-ilar environmental pressure, similar cellular adaptation, orsimilar distribution of chiasmata. This kind of orthose-lection has frequently been correlated with heterochro-matin distribution (John and Miklos, 1979) and could bea reasonable explanation for the parallel evolution ofbanding patterns. Furthermore, if optimal regions for theoccurrence of heterochromatin and chiasmata exist, aspredicted by the chromosome field hypothesis (reviewedby Lima-de-Faria, 1983), heterochromatin would tend to

be selectively accumulated through amplification of pre-existing repeats (library hypothesis; Fry and Salser, 1977)in the same optimal regions of homeologous chromo-somes of different clades.

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HETEROCHROMATIN BANDING PATTERNS IN RUTACEAE-AURANTIOIDEAE— A CASE OF PARALLEL CHROMOSOMAL...

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