_??_1987 by Cytologia, Tokyo Cytologia 52: 213 -222, 1987

Cytogenetics of IV. Structure and systematic significance of interphase nuclei

M. dos S. Guerra

Departamento de Biologia Geral, Universidade Federal de Pernambuco , 50.000-Recife, PE, Brazil

Accepted November 6, 1985

The chromatin of interphase nuclei in exhibits a very variable structure which depends on a series of nuclear and chromosomal parameters, the most important of which appear to be the amount and density of nuclear DNA and the nature and quantity of hetero chromatin (Barlow 1977, Nagl 1982, Guerra 1985a). If we assume that these parameters are relatively constant within each species, especially in root-tip cell nuclei, we should expect the nuclear structure also to be constant and definite for each species. When viewed with the light microscope, the nucleus can be characterized on the basis of chromocenter number, size and shape and of the general appearance of non-chromocentric chromatin. Some chromatin remains condensed practically throughout the entire nuclear cycle, forming heterochromatic chromocenters or the heterochromatic portion of the chromo centers. The remaining chromatin may have the appearance of a chromatic reticule of variable density in the interphase nucleus forming aggregations which constitute the condensed euchro matin (Nagl 1979). This chromatin fraction may be associated with heterochromatic chromo centers or may form separate euchromatic chromocenters. Generally, the latter can be dis tinguished by more heterogeneous and less dense staining and by their irregular shape, frequent ly resembling projections of the heterochromatic chromocenters (Guerra 1985a). Several authors have established different classifications of types of interphase nuclei (Eichhorn 1934, Delay 1946-48, Tschermack-Woess 1963, Nagl and Fusenig 1979). In the present paper, we shall consider three fundamental types: areticulate, semi-reticulate and re ticulate, according to the classification proposed by Delay (1946-48). The same author also considered a fourth type ("eureticule") which was not found among Rutaceae. Considering that the nuclear structure, in contrast to other cytogenetical parameters such as DNA amount of chromosome number, cannot be quantified, and that the variation of the nuclear structure is very wide and continuous, the fundamental types must be divided into subtypes, as done by other investigators for large numbers of species and genera (Delay 1946-48, Tanaka 1971). In the present study we shall consider eight types of structural organization of interphase chro matin, although, if we wanted to be more rigorous in classifying each type, this number would certainly be higher. The main objective of the present study was to determine the potentiality of the structure of the interphase nucleus as a cytotaxonomic parameter for Rutaceae. At the same time, several observations were made on the origin and nature of chromocenters and on their relationship with the organization of prophase chromosomes. The results were interpreted in the light of previous information on chromosome number and size, DNA amounts and heterochromatin distribution patterns obtained for these species (Guerra 1984a, b, 1985b) which will permit better characterization of nuclear types.

Material and methods

The material investigated consisted of 61 species, whose exact origin and chromosome 214 M. dos S. Guerra Cytologia 52

numbers were published in the first article of this series (Guerra 1984a), plus the following 5 species collected and supplied by Prof. F. Ehrendorfer, Institute of Botany of Vienna: Correa laurenciana Hook., Australia, S. Qld., Brisbane, central part of Springbrook Plateau, Ehren dorfer 600-23. 14; Esenbeckia spec., State of Sao Paulo, Botucatu, Mata de Butignolli, Ehren dorfer and Gottsberger 73912-33. 102; Flindersia bourjotiana F. Muell., Australia, N. Qld., E. of Atherton, 1 mile South of Lake Eacham, Ehrendorfer 6600-31.02; Lunasia amara Blanco, New Guinea, Port Moresby, Brown River Forest Station, Ehrendorfer 6600-56.14; Toddalia asiatica (L.) Lam., Japan, Okinawa, Naha, Ehrendorfer 6900-17.03, and Ceylon, Central Province, slopes NE above Nuwara-Eliya, Ehrendorfer 6600-189.10. Table 1 presents a complete listing of the species analyzed, ordered by the classification of Engler (1931).

Table 1. List of the analysed species (arranged according to Engler 1931)

The material was fixed in 3:1 Carnoy and stored in the deep freezer for an indeterminate period of time. For most species, cytological examination was limited to root tips. In some species, nuclei from different tissues such as roots, anthers, petals , leaf buds, ovaries etc. were analyzed. Exceptionally, in Eriostemonpallidus, only anther tissues were investigated. Slides were prepared as described by Guerra (1984a), using Carmin, Feulgen or Giemsa staining . 1987 Cytogenetics of Rutaceae IV 2 15

Results

Although interphase nuclei can be classified into three fundamental types, variation among the different types is continuous and some times difficult to establish limits. Within the same root tip, chromatin can vary in structure from the stage in which most or all of the chromatin is evenly distributed throughout the nucleus with a homogeneous level of condensation (phase Z) to the phase in which the nucleus differentiates and the contrast between chromocenters and diffuse chromatin is highest. A small variation can also be observed between nuclei from dif ferent tissues. Despite these features , however, the nuclear structure of each species can be perfectly characterized. Among individuals of the same species, variation is nil and within each genus is minimal or at times imperceptible. Each type was characterized on the basis of the following parameters: a) density or staining of the chromatin reticule; b) nature, size, shape, number and density of the chromo centers, and c) amount and shape of condensed euchromatin . On the basis of these observa tions, the nuclei of the genera analyzed can be classified into the following types: 1. Areticulate: Invisible or very fine and weakly stained chromatin reticule. Well-delineat -ed chromocenters. Condensed euchromatin practically absent . 1.1. Generally simple, regularly-shaped chromocenters (A1). Boenninghausenia, Haplo phyllum, Vepris, Ruta, Atalantia, Citrus, Fortunella, Glycosmis, Micromelum, Mur -raya, Poncirus. 1.2. Compound, irregularly-shaped chromocenters (A2). Erytrochiton. 2. Semi-reticulate: Generally simple or occasionally fused chromocenters. Very variable amounts of condensed euchromatin. 2.1. Weakly stained reticule. Regularly-shaped chromocenters, rarely showing prolon gations of condensed euchromatin (S1). Agathosma, Barosma, Coleonema, Me licope, , , Toddalia, Acronychia, Ptelea. 2.2. Medium-stained chromatin reticule. Chromocenters of very variable density and generally irregular shape (S2). Correa, Eriostemon, Evodia, Fagara, Flindersia, Zanthoxylum, Zieria. 2.3. More deeply stained chromatin reticule. 2.3.1. Medium-sized, sparse, frequently compound and irregularly-shaped chro mocenters (S3). Orixa, Lunasia. 2.3.2. Compound, large and irregularly-shaped chromocenters. Slightly polarized chromatin reticule (S4). Skimmia. 2.3.3. Simple, numerous and small chromocenters. Appearance generally granular and homogeneously distributed chromatin (S5). Phellodendron, Esenbeckia, Pilocarpus, Pelea. 3. Reticulate: Intensely stained chromatin reticule. Small chromocenters of difficult visuali zation. Polarized chromatin (R). Dictamnus. In the species with Al nuclei (Fig. 1), the chromocenters are formed of perfectly disting uishable heterochromatin blocks at any phase of the nuclear cycle. Small portions of condens ed euchromatin associated with heterochromatic chromocenters and forming short projections were detected in Citrus and Glycosmis. In Ruta, Haplophyllum, Boenninghausenia and Vepris, each prophase chromosome has a single proximal heteropycnotic block which permits easier quantitative identification during interphase. In the species of the tribe Aurantieae, the pro phase chromosomes show large and dense terminal blocks, which are generally restricted to one of the chromosome arms, as well as small proximal blocks frequently fused during in terphase. 216 M. dos S. Guerra Cytologia 52

In general, the number of chromocenters increases with increased chromosome numbers. In Ruta, when the tetraploid species are compared with the octaploid R. graveolens, duplication of chromocenter number is observed. In all species, metaphase chromosomes are always very small, generally measuring less than 1.5ƒÊm or exceptionally reaching 2 to 3ƒÊm. In the A2 types (Fig. 2), diffuse chromatin is very difficult to detect. The fact that the number of chromocenters is much smaller than the number of chromosomes, the chromo centers are larger in size than the chromosomes and the structure of prophase chromosomes is apparently identical in the entire chromosome complement clearly indicates that the chromo centers are of compound origin ("Sammelchromozentren"). Prophase chromosomes exhibit

Figs. 1-3. Prophase and interphase nuclei with areticulate (1-2) and semi-reticulate (3) structure . 1, Al type, a, b.-Vepris undulata; c-Ruta graveolens; d-Poncirus trifoliata. 2, A2 type , Erytrochiton brasiliensis. 3, S1 type. a-Coleonema album; b-Ptelea trifoliata. la, b, c, 3b Giemsa staining. Id, 2, 3a Feulgen staining . 1987 Cytogenetics of Rutaceae IV 217

small eupycnotic terminal portions, whereas their central portion is clearly denser, even though they do not stain as deeply as type Al blocks. Metaphase chromosomes are all approximately the same size, varying around 2ƒÊm. The semi-reticulate nuclei have the most variable structure and therefore they are the most difficult to classify. The main factor determining this variation is the amount and distribution

Figs. 4-8. Prophase and interphase nuclei with semi-reticulated (4-7) and reticulated (8) struc ture. 4, S3 type, Oriza japonica. a-Prophase; b-Interphase. 5, S2 type. a-Evodia danielli; b-Correa virens; c-Flindersia bourjotiana. 6, S4 type, Skimmia japonica. a-Interphase with fused (left) and isolated (right) chromocenters; b-Prophase. 7, S5 type, Phellodendron japonicum. 8, R type, Dictamnus albus. 4a, b, 5a, b, 7, 8 Feulgen staining. 5c, 6a Giemsa staining. 6b O rcein staining. 218 M. dos S. Guerra Cytologia 52

of condensed euchromatin. In some genera with S 1 type nuclei, such as Petlea and Coleonema,

the structure of the nucleus may be confused with the areticulate one, especially when Feulgen staining is used. However, when a more intense stain such as Giemsa is used, it is possible to

perceive the presence of larger fractions of condensed euchromatin (Fig. 3). On the other hand, the dense proximal blocks are not clearly visible throughout the complete nuclear cycle, a fact that distinguishes then from the areticulate ones. In these species and in type S3 ones

(Fig. 4), prophase chromosomes exhibit proximal heteropycnosis, although the discontinuity between this region and the eupycnotic one is not as evident as in the areticulate type. In type

S2, prophase chromosomes may present themselves in two different manners. In Correa, Eriostemon, Zieria and Flindersia, they are similar to those of the S1 and S3 types, whereas in Evodia, Fagara and Zanthoxylum they often exhibit more and less dense regions throughout their length (Fig. 5). In the latter genera, fully euchromatic chromocenters appear frequently, which are probably related to the denser interstitial regions of prophase chromosomes. In type S4 (Fig. 6), prophase chromosomes are intermediate between those of type S3 and those of the reticulated type. The metaphase chromosomes are the largest among the species in

vestigated here, varying in length between 2.1 and 5.4ƒÊm. The S3 type is quite distinct from the others and is characterized by prophase chromosomes with small variations in density throughout their entire length (Fig. 7). At metaphase, they measure about 2.0 to 3.0ƒÊm. In the species having reticulate nuclei, the chromosome appears to be equally decondensed throughout its length (Fig. 8), although at the end of prophase the terminal regions are slightly less condensed than the proximal ones, with small terminal heterochromatin blocks being some times visible. At metaphase, the chromosomes have a mean size of 3.5ƒÊm. Photographs of

prophase or prometaphase chromosomes of Boenninghausenia albiflora, Haplophyllum ob tusifolium, Erytrochiton brasiliensis, Ruta chalepensis, Murraya paniculata and Dictamnus albus, and interphase nuclei of Skimmia japonica, R. chalepensis, M. paniculata, Citrus hystrix, Coleonema pulchrum and D. albus have also been published elsewere together with the C banding patterns (see Guerra 1985b).

Discussion

The structure of the interphase nucleus is the consequence of a series of cytogenetical parameters and represents, by itself, a good example of what Bennett (1971) called "nucleo type". At the light microscope level, with the use of conventional staining techniques, the most important parameter seems to be the structure of prophase chromosomes. Except for some heterochromatic terminal blocks, prophase chromosomes show decreasing condensation in the centromere-telomere direction. These chromosomes may show: a) a sharp or not so sharp limit between the eupycnotic and the heteropycnotic region in species with type Al; b) a proximal heteropycnotic region without a sharp limit with the eupycnotic one in type A2 and semi-reticulate except S5; c) numerous and fine denser zones in type A5; and d) almost uniform condensation in the species with reticulated nuclei. Correlation between the pattern of prophase condensation and nuclear structure has been frequently observed (see, for example, Kurabayashi et al. 1962, Tanaka 1971, Merritt 1974). This correlation is of particularly difficult interpretation in species having the second type of prophase organization, which is related to five different nuclear types. Additional studies on the nature of heteropycnosis in these nuclei (C-banding, organization of nucleotide sequences, pattern of DNA replication) may possibly help us to understand why some species characteristically form compound chro mocenters and others simple chromocenters, or to discover what basically distinguishes con densed from diffuse chromatin. Chromosome size is also somehow correlated with nuclear type. The species with Al 1987 Cytogenetics of Rutaceae IV 219

areticulate nuclei always have small chromosomes less than 3ƒÊm-long , even though species with 2 to 3ƒÊm-long chromosomes, such as those of the genera Ptelea , Coleonema, Acronychia and others, may exhibit type Sl semi-reticulate nuclei. Favarger (1946) and many other authors have observed also a tendency for species with larger chromosomes to exhibit reticulate nuclei . No species with large chromosomes appear to occur among Rutaceae . Even so, only genus with a reticulated nucleus (Dictamnus) has relatively large chromosomes, whose size is only smaller than that of S. japonica chromosomes. Lafontaine (1974) proposes that this relation

ship can be better analyzed as the mean content of chromosomal DNA, which may be propor tional to the mean extension of the chromosomes. For this author, mean chromosomal DNA content may be the factor that determines nuclear structure. Barlow (1977), however, con siders nuclear DNA content to be the parameter that best correlates with nuclear type.

Table 2. Relationship between nuclear DNA amount (4C), mean chromosomal DNA amount (1C/n) and interphase nucleus structure (I.N. S.). Additional data from Guerra (1984a, b)

Table 2 shows the amount of nuclear DNA, the mean amount of chromosomeal DNA and the chromosome numbers from 17 of the species analyzed here, data from Guerra 1984a, b. The data available for Rutaceae suggest that; a) species with areticulate Al nuclei have an amount of DNA of 4C_??_3pg;b) species with S1 nuclei (semi-reticulate with the smallest amount of condensed chromatin) have nuclear DNA amounts close to those of Al types and of less than 5pg; c) species with 4C_??_5pgdo not have a clear relationship with the nuclear interphase type; and d) the mean chromosomal DNA content is only weakly correlated with the nuclear type, although the density of the chromatin reticulate tends to intensify with increasing chromo somal DNA content. The small amount of condensed euchromatin observed at interphase and prophase in some Aurantieae may be the consequence of the relatively high mean chromosomal DNA content found in these species. On the other hand, the difference in nuclear type between D. albus and S. japonica is more probably related to their differences in the nature, amount and distribution of heterochromatin (Guerra 1985b) than to the DNA content. Table 2 also shows that the A2 type profoundly differs from the Al type. The classification of this nucleus as an areticulate one is due to the absence of a visible chromatin reticule and to 220 M. dos S. Guerra Cytologia 52 the presence of well-delineated chromocenters. However, on the basis of nuclear or chromo somal DNA content, of the structure of prophase chromosomes and of the essentially compound nature of the chromocenters, this nucleus could be classified as a special semi-reticulate type (Guerra 1985a). The structure of interphase nuclei, although sometimes difficult to classify, appears to be a good cytotaxonomic parameter in Rutaceae. No interindividual variation was observed at the species level. The intraspecific variation of the nuclear structure is probably related to the occurrence of strong numerical or structural chromosome polymorphism, such as that observed in Scilla (Vosa 1973), Claytonia (Lewis et al. 1967) or Wolffia (Urbanska-Worytkiewick 1980) species. Actually, in studies on radishes, it was possible to characterize different lines by the nuclear structure (Dayal 1975). However, intense structural or numerical polymorphisms are not recorded to the Rutaceae and appear to be the exception rather than the rule among an giosperms, especially the tropical ones (Mehra 1972). Intrageneric variation is very small too. In none of the 16 genera with more than one species investigated here did we find variation that would justify separation into one or more nuclear types, although some differences in reticulate density were evident in Zanthoxylum species with very different chromosome numbers as Z. piperitum (2n=70) and Z. alatum (2n= 136). Intrageneric variation, although infrequent, has been reported for some genera of other families (see, for example, Gauthe 1965, Gross 1965 and Merritt 1974). In very close genera, such as Barosma and Agathosma, Fagara and Zanthoxylum or Boen ninghausenis and Haplophyllum, the nuclear structure is practically identical. It is interesting to note that some systematists have proposed fusing these genera on the basis of the strong morphological identity (see Pillans 1950, Waterman 1975a and Engler 1931, respectively). From a cytogenetical point of view they could be fused too. Among distinct genera of the same tribe, the differences tend to be more evident. Thus, S1 types (Melicope, Comptonella, Platydesma), S2 types (Fagara, Zanthoxylum, Evodia), S3 types (Lunasia, Orixa), and S5 types (Pelea) occur in the tribe Zanthoxyleae. Similarly, the tribe Toddalieae contains type Al (Vepris), Sl (Ptelea, Toddalia, Acronychia), S4 (Skimmia) and S5 (Phellodendron). In the tribe Cusparieae, the S5 type characterizes the genera of the subtribe Pilocarpinae analyzed here, whereas Erytrochiton, a representative of the subtribe Cuspariinae, has a totally different nuclear structure (A2). The same occurs in the tribe Ruteae, where the subtribe Rutinae (Haplophyllum, Boenninghausenia, Ruta) is characterized by a type (Al) completely different from that of Dictamnus (R), the only genus in the subtribe Dictamninae. This diversification of nuclear types may reflect intense diversification that occurred within these tribes or suggest an artifi cial grouping of these genera. Waterman (1975b), in a review of the distribution and systematic importance of alkaloids in Rutaceae, found that his data were in strong conflict with the classifi cation made by Engler (1931) especially for the tribes Toddalieae, Zanthoxyleae and Cuspari eae. These results support Moore's statement (1936) that "The present classification of Ruta ceae is one which runs directly across the lines of specialisation in floral anatomy" and call for a much needed global and multidisciplinary revision of the family. On the other hand, the uniformity in nuclear type observed in the tribe Aurantieae seems to reflect great closeness among its genera, as shown by the weak barriers of reproductive isolation. In this tribe, species belonging to morphologically well distinct genera can hy bridize and produce fully or partially fertile descendants. Furthermore, when sterile hybrids are formed, they can reproduce by adventitious embryony. While this situation contributes to the difficulty in classifying species and genera (Stebbins 1969), it also contributes to the main tenance of nuclear structure in the group. 1987 Cytogenetics of Rutaceae IV 221

Abstracts The interphase nuclei of 68 species of Rutaceae representing the eight major tribes in the family were analyzed. Nuclei were classified as areticulate (two types), semi-reticulate (five types) and reticulate (one type). No eureticulate nuclei were detected. Variation of the struc ture among different tissues, individuals or species of the same genus was minimal and mainly associated to tissue differentiation or ploidy levels. The differences in chromatin organization among nuclear types are mainly correlated with the pattern of prophase condensation and the amount of nuclear DNA. The structure of the interphase nucleus appears to be particularly important in the cytogenetical characterization of tribes and sub-tribes of the Rutaceae . The tribe Aurantieae is the only one to be characterized by a single nuclear type (areticulate) whereas the remaining tribes show varied nuclear structure for the different subtribes. Some tribes, as Rutineae and Cusparieae, appear to have a characteristic nuclear type for each sub tribe. The systematic significance of these findings is discussed.

References

Barlow, P. W. 1977. Determinants of nuclear chromatin structure in angiosperms. Ann. Sci, Nat. Bot. 18: 193-206. Bennett, M. D. 1971. The duration of meiosis. Proc. Roy. Soc. Lond. B. 178: 277-299. Dayal, N. 1975. Genotypic control of chromocenters in radish (Raphanus sativus L. var. radicola Persoon). Caryologia 28: 427-435. Delay, C. 1946/48. Recherches sur la structure des noyaux quiescents chez les phanerogames. Rev. Cytol. et Cytophysiol. Veg. 9: 169-222; 10: 103-229. Eichhorn, A. 1934. Types definis et types intermediaires dans la mitose des vegetaux. Cytologia 5: 253-268. Engler, A. 1931. Rutaceae. In: "Die naturlichen Pflanzenfamilien" Band 19a, pp. 187-359. Engelmann, Leipzig. Favarger, C. 1946. Recherches caryologiques sur la sous-famille des Silenoidees. Bull. Soc. Bot. Suisse 56: 365-446. Gauthe, Z. 1965. Contribution a 1'etude caryologique des Tillandsiees. Mem. Mus. Natl. Hist. Nat., Ser. B., Bot. 16: 39-59. Gross, J. P. 1965. Contribution a 1'etude cyto-taxonomique des Pittosporacees. Mem. Mus. Natl. Hist. Nat., Ser. B., Bot. 16: 61-90. Guerra, M. S. 1984a. Cytogenetics of Rutaceae I. New chromosome numbers in Rutaceae. P1. Syst. Evol. 146: 13-30.- 1984b. Cytogenetics of Ruatceae II. Nuclear DNA content. Caryologia 37: 219-226. Guerra, M. S. 1985a. Estrutura e diversificacao dos nticleos interfasicos em plantas. In: Topicos de Citogene tica e Evolufao de Plantas (eds. Aguinr-Perecin, Martins and Bandel), pp: 137-153. Soc. Brasil. Genet., Ribeirao Preto, SP. - 1985b. Cytogenetics of Rutaceae III. Heterochromatin patterns. Caryologia 38: 335-346. Kurabayashi, M., Lewis, H. and Raven, P. H. 1962. A comparative study of mitosis in the Onagraceae. Amer. Jour. Bot. 49: 1003-1026. Lafontaine, J. G. 1974. Ultrastructural organization of cell nuclei. In: Bush, H. (Ed.): The Cell Nucleus Vol. 1: 149-185. New York, Academic Press. Lewis, W. H., Oliver, R. L. and Suda, Y. 1967. Cytogeography of Claytonia virginica and its allies. Ann. Missouri Bot. Gard. 54: 153-171. Mehra, P. N. 1972. Cytogenetical evolution in tropical hardwoods. The Nucleus 15: 64-83. Merritt, J. F. 1974. The distribution of heterochromatin in the genus Nicotiana (Solanaceae). Amer. Jour. Bot. 61: 982-994. Moore, J. A. 1936. Floral anatomy and phylogeny in Rutaceae. New Phytologist 35: 318-322. Nagl, W. 1979. Condensed interphase chromatin in plant and animal cell nuclei: fundamental differences. Pl. Syst. Evol. Suppl. 2: 247-260.- 1982. Condensed chromatin: species-specificity, tissue-specificity and cell cycle-specificity, as monitored by scanning cytometry. In: Cell Growth (ed. Nicoline, C.) pp. 171-218, Plenum Press, New York.- and Fusenig, H. P. 1979. Types of chromatin organization in plant nuclei. Pl. Syst. Evol., Suppl. 2: 221 - 223. 222 M. dos S. Guerra Cytologia 52

Pillans, N. S. 1950. A revision of Agathosma. Jour. S. Mr. Bot. 16: 55-183. Stebbins, G. L. 1969. The effect of sexual reproduction on higher plant genera with special reference to Citrus. Proc. Int. Citrus Symposium 1: 455-458. Tanaka, R. 1971. Types of resting nuclei in Orchidaeae . Bot. Mag., Tokyo 84: 118-122. Tschermak-Woess, E. 1963. Strukturtypen der Ruhekerne von Pflanzen and Tieren. Wien, Springer Verlag. Urbanska-Worytkiewick, K. 1980. Cytological variation within the family of Lemnaceae. Veroff. Geobot . Inst. ETH, Stiftung Rilbel 70: 30-101. Vosa, C. 1973. Heterochromatin recognition and analysis of chromosome variation in Scilla sibirica. Chro - mosoma 43: 269-278. Waterman, P. G. 1975a. New combinations in Zanthoxylum L. Taxon 24: 361-366. - 1975b. Alkaloids of the Rutaceae: Their distribution and significance . Biochem. Syst. Ec. 3: 149-180.