Karyotype and C-Band Analysis in Two Species of Genipa L

C 1997 The Japan Mendel Society Cytologia 62: 81-90, 1997

Karyotype and C-band Analysis in Two Species of Genipa L. (Rubiaceae, Gardenieae Tribe)

Neiva I. Pierozzi1 and Sigrid L. Jung Mendacolli 2

1Secao de Citologia , Instituto Agronomico. Caixa Postal 28. Campinas, SP. 13.001-970 Brasil 2 Segdo de Botao de Botanica , Instituto Agronomico

Accepted November 28, 1996

Genipa L. (Rubiaceae, tribe Gardenieae) is a small neotropical genus which consist of G. americana L., G. infundibuliformis Zappi et Semir and G. williamsii Standley, all of them are dioecious perennial robust trees (Zappi et al. 1955). G. americana is a very popular species among tropical America indians who utilize the plant in different ways as medicine against some diseases. The fruits are a source of food and can also be used as a dye for body painting and as an insect repellent (Keeler 1964). G. infundibuliformis was described recently by Zappi et al. (1995). It had been wrongly considered as part of G. americana until chromosome studies developed by Pierozzi and Cruz (1989) who showed differences in these taxa. Chromosome number have been reported for G. americana as 2n = 22 and for G. infundibuliformis as 2n = 20 (Mendes 1941, Pierozzi and Cruz 1989). Preliminar studies with the use of Giemsa and fluorescent techniques on Genipa mitotic chromosomes have been demonstrated the possibility of a successful utilization of these techniques for chromosome characterization (Pierozzi and Cruz 1988, Guerra 1993). The present paper had the purpose of better G. americana and G. infundibuliformis chromosome characterization, the stablishment of a median idiogram for both species through acetic orcein and C-banding techniques for further comparative studies.

Materials and methods

Plant material

The plant studied here belong to Instituto Agronomico (IAC) collection. They were collected from native woody areas. Voucher specimens (both male and female) have been identified by S. L. Jung Mendacolli and were deposited in Botany Herbarium (IAC) and in

UNICAMP Herbarium of Botany Department (University of Campinas).

Pre-treatment, fixation and enzymatic treatment Seeds of ripe fruits of G. americana and G. infundibuliformis were collected and germi- nated in moist filter paper in Petri dishes at 26•Ž. Roots with approximately 1 cm long were treated with para - dichlorobenzene (p-DB) saturated solution for 3 hr at 16•Ž, fixed in 3 : 1 Carnoy solution (3 parts of absolute ethanol and 1 part of acetic acid p.a.) for 2 days, transferred to 70% ethanol and stored at - 20•Ž. Fixed roots were treated with an enzymatic solution mixture of 20% pectinase and 2% cellulase at 35•Ž for 2 or 3 hr for tissue softening.

Acetic orcein procedure Softened roots were stained as Medina and Conagin (1964) method, in a mixture of 9 parts

of 2% acetic orcein plus 1 part of 1 N HCl for 30 min at 40•Ž, and then transferred to 1% acetic orcein solution for 1 hr and squashed. 82 Neiva I. Pierozzi and Sigrid L. Jung Mendacolli Cytologia 62

Modified C-banding procedure

The C-banding technique applied here was a modification of Marks and Schweizer (1974) methodology. Softened roots were placed in 45% acetic acid solution for 30 min at 50•Ž and squashed. Laminas were placed in liquid nitrogen for the siliconized coverslips separation, briefly washed in 100% ethanol, dried and stored for the maturity. After this period of 2 or 3 weeks, slides were covered with barium hydroxide saturated solution film plus a coverslip and incubated for 15 min at 55•Ž in a Ba(OH)2 humidified chamber, rinsed in slightly acid distilated water (4 drops 1 N HC1/liter) and after in running distilated water. After dried, slides were covered by a pH 7.8 2 X SSC solution film plus a coverslip and incubated for 2 hr at 65•Ž in a 2 X SSC humidified chamber, rinsed in running distilated water, dried for 1 hr, stained in 1% Giemsa in SOrensen buffer pH 6.9 from 30 to 60 min at room temperature, rinsed again in distilated water, dried for 2 or 3 days and mounted in Permount (Fisher) media.

Draws and photographs Ten best metaphase of each species after the acetic orcein and seven metaphase after C- banding procedure were selected for drawing. Metaphasic chromosome draws were obtained from the shape of the chromosomes with the aim of a monocular camera bound in a ZEISS microscope. The schematization was done from the draws with the aim of a divider.

Photomicrographies were developed using Agfa Copex Pan film, ASA25.

Chromosome measures

The mean value, standard deviation and coeficient of variation for absolute lenght (um), relative lenght (%) and centromeric index (CI) were calculated for each chromosome of

Genipa species according to Aguiar-Perecin and Vosa (1985) and Guerra (1986). Karyotype asymmetry index (AsI) was calculated as done by Mugnier and Siljak-Yakovlev (1987). F-test was applied for absolute length mean values of the ten first metaphasic chromosome pairs and for ten metaphasic cells for AsI estimation.

C-band values for each chromosome were expressed as percentage of total chromosome lenght. The total heterochromatin amount (C-band) mean value was done for haploid set for both species and expressed as percentage too. T-test was applied to compare these values and also to compare total haploid karyotype length mean value.

Results

By using 2% acetic orcein staining it was possible to obtain good chromosome prepara- tions for measures and basic idiograms. It was confirmed the chromosome count for G. americana as 2n = 22 and for G. infundibuliformis as 2n = 20. Karyomorphometric data of metaphasic chromosomes of Genipa species were grouped in Tables 1, 2. Size variations within each species were a gradation of values. G. americana chromosome absolute length mean value varied from 1.98+0.10 gm to 4.22 +0.10 gm and the relative length mean values varied from 6.12 +0.28% to 13.08 +0.33%. Chromosomes 3, 7 and 11 were metacentric and the others were submetacentrics (Figs. 1, 4A, Table 1). Haploid karyotype formula proposed for this specie is n =11: 3m 7sm lsms. G. infundibuliformis chromosome absolute length mean values varied from 1.68 +0.8 um to 3.19 +0.13 um and the relative length mean values varied from 7.13 +0.22% to 13.58+ 0.49%. Chromosomes 3 and 5 were metacentrics and the others were submetacentrics (Figs. 2, 4B, Table 2). Haploid karyotype formula proposed for this species in n=10: 2m + 7sm + lsms. Chromosome absolute length mean values of both species were significatively differents at the level of 1% of probability (Table 3). G. americana had chromosomes larger than G. 1997 Karyotype and C-band Analysis in Two Species of Genipa L. 83

Table 1 . Morphometric data on G. americana metaphasic chromosomes

Fig . 1 . Chromosomes of G. americana (2n = 22) stained after 2% acetic orcein technique. A: mitotic metaphase; B: karyogram. infundibuliformis. Karyotype asymmetry index values were 62.67 for G. americana and 65.79 for G. infundibuliformis and this differences was significant at the level of 1% of probability. The mean values for total haploid karyotype lenght obtained were 64.38+1.66 gm for G. americana and 46.74+ 1.25 gm for. G. infundibuliformis and they differed significavively at 1% of probability. 84 Neiva I. Pierozzi and Sigrid L. Jung Mendagolli Cytologia 62

Table 2. Morphometric data on G. infundibuliformis metaphasic chromosomes

Fig. 2. Chromosomes of G. infundibuliformis (2n =20) stained after 2% acetic orcein technique. A: mitotic metaphase; B: karyogram.

It was not noticed the presence of B-chromosomes either in both species. Even though they have sex separation (dioecy), it was not observed any chromosome difference in size or heterochromatin amount between homologous that could be related to dioecy. Both Genipa species had appreciable constitutive heterochromatin amounts revealed after C-banding technique (Figs. 3A, C). This kind of heterochromatin was found in terminal position and near centromeric region. In some cases, the C-band width was almost the same the short arm length, such as chromosome 8 to 10 in both species, and also in chromosome 11 of 1997 Karyotype and C-band Analysis in Two Species of Genipa L. 85

Table 3 . F-values for absolute lenght (um) of the ten metaphasic chromosomes of G. americana and G. infundibuliformis

A B

C 0

Fig . 3 . Photomicrographies of G. infundibuliformis B-banded metaphasic (A-2500X) and acetic orcein prophasic chromosomes (B-1750X); and G. americana C-banded metaphasic (C-2500X) and acetic orcein prophasic chromosomes (D-1500X). Arrows = chromosomes 1 and 4; NU = nucleolus.

G. americana (Fig. 5). Except chromosome 4, all G. americana chromosomes short arms had terminal C-band and in G. infundibuliformis only chromosomes 1, 4 and 5 did not have the short arm C-banded. The NOR region of chromosome 4 was negative to C-band and satellited region was C-band positive. Chromosome 1 had secondary constriction but apparently not associated to NOR. Intercalary band appeared only in G. americana chromosome 1. Chromo- some 5 of G. infundibuliformis was unique in the karyotypes, possessing a centromeric/pericen- tromeric C-band that formed an entire heterochromatic block. This has problably caused by the band intensity in this region or by degree of chromosome contraction (Fig. 5B). Total heterochromatin relative mean value per haploid set, as revealed by C-banding was 35.45+1.83% for G. americana and 27.61 +2.23% for G. infundibuliformis and they were significantly different at 2% level. The C-band relative length mean value in each chromosome varied from 1.01 +0.27% (chromosome 5) to 2.45 + 0.28% (chromosome 8) in G. americana and from 1.18 + 0.8% (chromosome 1) to 3.02 +0.33 (chromosome 3) in G. infundibuliformis (Table 4). 86 Neiva I. Pierozzi and Sigrid L. Jung Mendacolli Cytologia 62

Fig . 4 . Idiogram of G. americana (A) and G. infundibuliformis (B) metaphasic chromosomes after acetic orcein technique. *= NOR bearing chromosome.

Fig . 5 . Idiograms of G. americana (A) and G. infundibuliformis (B) metaphasic chromosomes after C-band technique. *= NOR bearing chromosome.

Discussion

Submetacentric chromosome types prevailed in Genipa karyotypes as can be seen through haploid karyotype formula and Tables 1, 2. Chromosomes 1 to 4, 6, and 8 to 10 had the same classification in both species. Chromosomes 1 and 4 showed the same basic pattern, e.g., they were submetacentric, with secondary constriction and a satellite segment in the long arm. Cytological analysis have showed that only chromosome 4 is associated to the nucleolar organizer region (Fig. 3). These observations differed from those found in G. americana mitotic chromosomes by Guerra (1993) who concluded that chromosome 11 was associated to 1997 Karyotype and C-band Analysis in Two Species of Genipa L. 87

NOR, based on the chromomicin positive signal in a terminal segment. This kind of fluorescence is often found in NOR and denotes the presence of G-C rich sequences, but it is not exclusive for this region (Cerbah et al. 1995). In some Vigna specie, for example, Galasso et al. (1993) observed chromomicin positive fluorescence in many non-satellited chromosomes. The use of silver staining and also in situ hybridization techniques may help solving the question of r-DNA region in Genipa. On the other hand, the localization and extention of C-banded segments in G. americana mitotic chromosomes seemed to coincide with the great majority of those chromomicin fluorescent signals obtained by Guerra (1993). This led to the conclusion that C-heterochromatin in G. americana might be rich in G-C sequences. Khuong and Schubert (1990) also saw a high percentage of C-band segments coincidents with those positive chromomicin ones in maize mitotic chromosomes. Karyotype comparative analysis also showed up some differences. G. americana had chromosomes lenght, haploid karyotype lenght and also heterochromatin amount larger than those of G. infundibuliformis. Inversely, G. infundibuliformis had the karyotype more asymmetric than G. americana. The increasing of karyotype asymmetry in angiosperms seems to be a predominant evolutive tendency (Sybenga 1972). In the well-known genus Crepis and also in some Hypochoeris species the general karyotype evolution trends have been towards increasing asymmetry, decreasing chromosome number and DNA content accompained by morphological and ecological changes (Stebbins 1971, Cerbah et al. 1995). At this viewpoint, G. infundibuliformis could be considered more advanced than G. americana since the former has karyotype more asymmetric, lower chromosome number and lesser karyotype lenght than the latter. In addition to, G. infundibuliformis has heterophylly, a new morphological character in neotropical Rubiaceae (Zappi et al. 1995). The karyological informations about Neotropical Rubia- ceae are limited (Kiehn 1985, 1995) but the basic number n =11 is the commonest found among Rubiaceae species studied suggesting that it is the basic primary number and from this other numbers have been derived by aneuploid (Lee and Rink 1985, Selvaraj 1987). So, it seems probable that G. infundibuliformis has derived from G. americana or from a common ancestor of both of them, by an aneuploid mechanism, that led to the chromosome elimination followed by chromosome lenght and heterochromatin amount reduction. In some Lathyrus species, a progressive reduction in constitutive heterochromatin (C-bands) has taken place from perennial outbreeding to annual forms. (Lavania and Sharma 1980) and the tendency to chromosome number decreasing associated to restriction in geographical occurence areas was proposed for Crepis negleta (n = 4) and for its derived specie C. fuliginosa (n = 3). This last specie has a very limited geographical distribution than the former (Stebbins 1966), resembling the species studied in this work. G. americana has a larger distribution through tropical America, occuping humidy areas that suffer periodical flood. The G. infundibuliformis distribution is more restricte, viz., in southern and easthern part of Brasil occuring in areas that vary from very humid coastal Atlantic forest to dry forest surrounding the northern of Minas Gerais state (Zappi et al. 1995). The heterochromatin decreases observed in G. infundibuliformis (Table 4) may be also explained, besides unequal translocations, as a consequence of pericentric and paracentric inversions that have changed the eu- and heterochromatin segments positions, leading to the lost of some of heterochromatic DNA amount and stablishing new gene groups adapting the plant to explore dry areas. Some chromosomes might be involved in this hypothetical scheme (Fig. 6). According to Greilhuber (1984) differences in gain or lost of heterochromatin C-band may reflect C-value adjusts of a specie. An association of heterochromatin gain with telomeric C-band amount, life form and evolutive degree have been seen in some groups of related species such as Anacyclus (Schweizer and Ehrendorfer 1983), or Capsicum (Moscone et al. 1993) or 88 Neiva I. Pierozzi and Sigrid L. Jung Mendagolli Cytologia 62

Table 4. C-band mean values in percentage (%) for each chromosome and standard deviation in relation to chromosome haploid set of G. americana and G. infundibuliformis

A . G . americana

B . G . infundibuliformis

Fig . 6 . Hypothetical explanatory scheme for some C-band changes and euchromatin reduction that have probably occurred involving some G. infundibuliformis chromosome, such as 4 and 5. *= eu- or heterochromatin reduction; >=break points; C = inversion of segment position . G.A. =G . americana; G.I. =G. infundibuliformis.

Orchis (D'Emerico et al. 1996). The most considered advanced species possess high amount of heterochromatin and also predominant telomeric C-bands. Thinking in this way, G. americana might be considered a derivative form from G. infundibuliformis based on its higher heterochromatin amount and telomeric C-bands. But this trend seems less probability having occurred in Genipa species, e.g., a more asymmetric karyotype specie giving raise to a less asymmetric karyotype one, and it have not been seen any evidence of Robertsonian chromo- 1997 Karyotype and C-band Analysis in Two Species of Genipa L. 89 some fission bringing about two acrocentric ones in G. americana. The Genipa species relationships is still an open question and futher cytological studies in the other specie, G. williansii, as well as meiotic behavior of these three Genipa species will be necessaries to elucidate the phylogenetic relationships in the genera.

Summary

Genipa L. (Rubiaceae) is a small genera with only three neotropical species. Karyological studies were developed in two of these species, G. americana (2n = 22) and G. infundibuliformis (2n = 20), by acetic orcein 2% and Giemsa C-band techniques. Haploid karyotype formulas were n =11: 3m + 7sm + 1sms and n =10: 2m + 7sm + 1sms, respectively. Chromosomes 3, 7 and 11 of G. americana and 3 and 5 of G. infundibuliformis were metacentrics and the others were submetacentrics. Chromosomes 1 and 4 had secondary constriction and according to the cytological observations only chromosome 4 had the NOR. Even so both species are dioecious, it have not been seen any chromosome or heterochromatin heteromorphism that could be associated to sexual differences. Both species had a high amount of heterochromatin revealed by C-band technique. G. infundibuliformis had the more asymmetric karyotype but the lesser chromosome sizes, heterochromatin amount and karyotype length. Preliminar comparative studies were done in both species and it were discussed the possible phylogenetic relationships.

Key words : Genipa, Mitotic chromosomes, C-band, Karyotype.

Aknowledgements

The authors wish to thank Ms. Antonia A. M. Ferreira, Ms. Sonia M. O. Falivene and Angela M. C. Silva for technical works.

References

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