CARYOLOGIA Vol. 53, no. 3-4: 205-212, 2000

Cytogenetic Analysis of seven species ofEucalyptus L'Her. (Myrtaceae)SILVIA TAMIE MATSUMOTO1, MARIA A. MARIN-MORALES2,*, CLAUDETE DE FATIMA RUAS3 and PAULOMAURICIO RUAS3

1 Departamento de Ciencias Biologicas, Universidade Federal do Espirito Santo, Av. Marechal Campus n° 1468, Cep. 29040-090.Vitoria-ES, Brazil. 2 Departamento de Biologia, UNESP, Av. 24A n° 1515, Cep. 13506-900. Rio Claro-SP, Brazil. 3 Departamento deBiologia Geral, Universidade Estadual de Londrina, Campus Universitario, Cx. Postal 6001. Cep. 86051970. Londrina-PR, Brazil.

Abstract — Seven species of the genus Eucalyptus were studied cytogenetically (E.deanei, E. dunni, E. grandis, E. maculata E. propinqua, E. saligna and E. tereti-cornis). The species showed a symmetrical karyotype with 2n=22 chromosomes,with chromosome length ranging from 0.58 �m to 1.39 �m. Karyotypic analysis in-dicated homogeneity of morphology and of chromosome number for most of thespecies of this genus studied here, although casual disploid species with 2n=24 havebeen found in previous studies. According to these data, a basic number of x=11was established for this genus. The evolutionary tendency probably occurred bystructural alterations (deletions, duplications, additions and translocations) and insome cases by aneuploid chromosome alterations.Key words: Chromosome, Eucalyptus, Karyotype, plants cytogenetics.

INTRODUCTION

The family Myrtaceae comprises 140 generawith about 2400 species (CRONQUIST 1981). Eu-calyptus is one of the most important genera inthis family, with wide ecological and geographi caldistribution (from northern Australia to southernTasmania) and with about 700 species.

Cytogenetic studies on the genus Eucalyptuscan provide valuable information about the sys-tematic and evolution of the group, being of helpin the solution of taxonomic problems. Accordingto GUERRA (1986), Cytogenetic data are being usedby many investigators in plant systematic for theestablishment of kinship relations and for theunderstanding of genetic and evolutionarymechanisms. The determination of chromosomenumbers plays an important role in speciescharacterization and, according to SENN (1938),can be useful for he study of the

* Corresponding author: fax ++55 211 9534 0009, e-mail:mamm@rc.unesp.br

phylogenetic relations within and betweengroups. STEBBINS (1971) stated that species pre-senting the same chromosome number can becompared on the basis of the morphologicalvariations between their chromosomes. Theirvariations can be detected both on the basis ofdata referring to chromosome length, which re-flect the variation in nuclear DNA content, andof the variations in centromere position(BRANDHAN and BENNET 1983).

Chromosome studies on the genus Eucalyptus,with few exceptions, have been limited tochromosome counts and such counts are avail-able for about 10% of the species (SUCIURA1931, 1936; ATCHISON 1947; McAULAY et al.1937; SMITH-WHITE 1942,1948; RUGGERI 1960, 1961;MOUSSEL 1965; RYE 1979; BOYLAND and KLEINING1983; VIJAYAKUMAR and SYBRAMA-NIAN 1985).

According to HAQUE (1984), the chromosomenumber and morphology of the genus Eucalyptusare little known. This lack of information aboutthe karyology of this group is due to the fact thatthe chromosomes are small, thus preventing thedistinction of the different pairs.

206 MATSUMOTO, MARIN-MORALES, RUAS and RUAS

Fig. 1 — Metaphases of the Eucalyptus species: A) E. deanei, B) E. dunni and C) E. grandis.

On the basis of the cytogenetic characterizationof four species, the author observed a remark-able similarity in chromosome morphologyamong the species studied.

We report here a cytogenetic study of sevenspecies from the plantation of a Brazilian paperand cellulose factory — Klabin, located inTelemaco Borba, Parana, Brazil.

MATERIALS AND METHODS

In the present investigation we studied seven speciesof the genus Eucalyptus: E. deanei, E. dunni, E.grandis, E. maculata, E. propinqua, E. saligna and E.tereticornis, kindly donated by the paper and cellu-lose industry Klabin-Parana-Brasil. The origin of thematerial analyzed is given in Table 1.

TABLE 1 — Origin of the samples utilized in the cytogeneticanalyses.

Root tips were collected for mitotic analysis,preferably from 11:30 h to 12:30 h, as a greater mi-totic incidence was observed at this time. The meris-tems were pretreated with 0.002 M 8-hydroxyquino-line (8-HQ) and 0.5 ml dimethylsulphoxide (DMSO)per 100 ml of 8HQ solution over a period of 4:30 h,involving 1 h at room temperature and 3:30 h at 8°C.The roots tips were then fixed in Carnoy solution(three parts ethyl alcohol: one part ace-

KARYOTYPES OF EUCALYPTUS 207

Fie. 2 — Metaphases of the Eucalyptus species: A) E. maculata, B) E. propinqua, C) E saligna and D) E. tereticornis.

tic acid) for 12 h at room temperature and later con-served in the same fixer at - 20°C.

The staining methodology described by Feulgenand Rossembeck, 1924 was used, with modifications(MELLO and VIDAL 1978). The material was hydrolyzedin 1 N HC1 at 60°C for 11 min, and then stained withthe Schiff reagent for 90 min. The material was thensquashed on slides containing a drop of 1 % aceticcarmine.

Eucalyptus cells in mitotic metaphases were ana-lyzed to establish the chromosome number and size.The values obtained for chromosome morphologywere: absolute length of each chromosome pair,length of haploid chromosome and arm ratio (AR)calculated by the following formula:

AR = long arm length/short arm length The relativelength was calculated by the following formula:

CR= absolute length x 100/total haploid lot length

The values obtained were used to determine thechromosome type, as described by LEVAN et al.

(1964). The HUZIWARA (1962) test was used to assess thekaryotypic symmetry, using the following formula:

TF = � short arm length x 100/totalchromosome length

RESULTS AND DISCUSSION

Most of the studies conducted on the genusEucalyptus only discuss the chromosome number,with few karyomorphological investigationsbeing available for the genus. According toRUGGERI (1960, 1961), approximately 62 Eucalyptusspecies are known in karyomorphological terms.HAQUE (1984) stated that few or no studies havebeen conducted on the chromosome morphologyof this genus, probably due to the smallchromosome size, which impairs theidentification of the different pairs as well as

208 MATSUMOTO, MARIN-MORALES, RUAS and RUAS

Fig. 3 — Karyotype of the Eucalyptus species with 2n=22 chromosomes: A) E. deanei, B) E. dunni, C) E. grandis, D) E. maculata, E) E.propinqua, F) E. saligna and G) E. tereticornis.

the localization of primary and secondary con-strictions. The karyomorphological informationreported here has not been reported previouslyfor the species E. deanei, E. dunni, E. grandis, E.maculata, E. propinqua and E. saligna.

The karyomorphological data of the speciesstudied (Tables 2 and 3) showed that the chro-mosome complement has a similar pattern in allseven species. The species are characterized by2n=2x=22 chromosomes of relatively small size,ranging from 0.6±00.1 to 1.4+0.01 �m (Table 2,

Figures 1-4). A comparison of haploid chromo-some length (Table 2) showed that E. deaneidiffered slightly from the other species. The armratio was also similar for all species, with only E.saligna showing a pair of subterminal (sm)chromosomes. The other six species have onlyMedium (M) and median (m) type chro-mosomes, with the arm ratios varying from1.0±0.01 to 1.4+0.07 �m. Such homogeneity inchromosome morphology was also observed byHAQUE (1984) in four species of Eucalyptus.

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TABLE 2 — Chromosome numbers and Tukey test for chromosome length, symmetry index (TF%), ratio of longest/shortest chromosome(L/S), and karyotype formula of Eucaliptus spp.a

The similarities observed in chromosome mor-phology explain why so many hybrids occur innature and why artificial crossing between taxo-nomically related species easily produces Fl hy-brids.

In a study of four Eucalyptus species, HAQUE(1984) observed variation in chromosome sizeof the order of 2 to 6 �m. According to this in-vestigator, in the species E. tereticornis the

chromosome length was 2 to 3 �m for non-sat-ellited chromosomes and 6 �m for satellitedones. Our data for the same species differ fromthose reported by HAQUE (1984) since E.tereticornis presented a variation of 0.75 to 1.39�m, with no satellites being observed in any ofits chromosomes. The remaining speciesanalyzed here also presented no satellites in anychromosomes. Results obtained by chromosomecount

Fig. 4 — Ideogram of the Eucalyptus species with 2n=22 chromosomes: A) E. deanei, B) E. dunni, C) E. grandis, D) E. maculata, E) E.propinqua, F) E. saligna and G) E. tereticornis.

210 MATSUMOTO, MARIN-MORALES, RUAS and RUAS

KARYOTYPES OF EUCALYPTUS 211

in the present study and others suggest that theevolution of this genus must have occurredmore by gene alterations than by chromosomealterations since few species present a chromo-some number differing from 2n=22. The chro-mosomes showed homogeneous morphologyfor all the species analyzed in the present study.Our data agree with those reported by MEHRA andKHOSLA (1972) who cited speciation for the genus,probably at the ecospecific level, due to genemutations with some chromosome reor-ganizations.

The relative length, with the exception ofpair 6, showed differences in the other chromo-some pairs for at least one species (Table 3).However, the highest difference among specieswas observed for pair 11.

Our data and data from the literature (RUG-GERI 1960, 1961; BOYLAND and KLEINING 1983; HAQUE1984; VIJAYAKUMAR and SUBRAMANIAN 1985)showed that 80.6% of the 90 Eucalyptus speciesstudied until now presented 2n=2x=22chromosomes, 15.2% of the species showed2n=2x=24 chromosome and 4.2% showed2n=2x=20 chromosomes. These results showthat the number 2n=2x=22 is conservative inthe genus, with the infrequent occurrence ofdiploid species with 2n=2x-20 and 2n=2x=24.More than one chromosome number was foundin three species. E. citriodora showed 2n=20(SUGGIURA 1931, 1936) and 2n=22 (ATCHISON1947). In E. globules, SUGGIURA (1931, 1936)described a population with 2n=20 andMCAULAY and CRUICKSHANK (1937) found 2n=22.RUGGERI (1960) found 2n=24 for E. huberiana, andSMITH and WHITE (1942) described 2n=22chromosomes. Thus, on the basis of the presentinvestigation and of previous information fromchromosome studies of the genus, it appearsthat chromosome evolution occurred in aconservative manner in Eucalyptus, with mostspecies having 2n=2x=22 chromosomes (x=11)of the metacentric type. To a small extent, somespecies derived from the species with x=11 bychromosome aneuploidy.

The seven species analyzed here (Table 2)were characterized by a TF% ranging from46.0±0.37 to 48.6±0.17. These seven speciesand the four species reported by HAQUE (1984)presented a symmetrical karyotype. The analysisof TF% showed agreement in the most sym-metrical and asymmetrical karyotypes. On thebasis of the TF%, E. maculata showed the most

symmetrical karyotype (TF=48.6±0.17%) andE. saligna the most asymmetrical one(TF=46.0±0.37%).

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Received 10 July 2000; accepted 14 September 2000