SYSTEMATIC STUDIES ON THE MEXICAN ZAMIACEAE. I. CHROMOSOME NUMBERS AND KARYOTYPESI Instituto Nacional de Investigaciones Sobre Recursos Bi6ticos, Apdo. Postal 63, Xalapa, Veracruz, Mexico Chromosome numbers and karyotypes are reported for seven species in three genera, of Mexican cycads. Karyotypic analysis of Cycads is made difficult by differential contraction, of their large chromosomes but idiograms are presented. Satellite variation is reported for three species of Ceratozamia. No satellites are reported for the three species of Zamia. THECYTOTAXONOMILITC ERATUREon Mexican Francisco Javier Clavijero' (INIREB) at Xal- cycads has been rather sporadic. Sax and Beal apa. Watering the plants a day or two before (1934) reported 2n = 16for Ceratozamia mex- collection gave reasonably good results al- icana Brongn. and 2n = 18 for Dioon spinu- though cell divisions seemed to be variable. losum Dyer. They pointed out that chromo- Two different methods of pretreatment were some number and morphology may differ in tried in order to obtain adequate contraction different genera but the genomes of the species of the large cycad chromosomes. These con- within each genus seem to be similar. Marchant sisted of: 1) 1% aqueous solution of 1 ml ABN (1968) reported chromosome numbers of2n = (alpha-Bromonapthalene) dissolved in 100 ml 16 for C. mexicana, 2n = 18 for D. edule Dyer, of absolute ethanol (Dyer, 1979); 2) 0.05%, and 2n = 16 for Zamia fischeri Miq. He de- 0.1 %, and 0.2% aqueous solutions of colchi- scribed karyotypes and noted that the presence cine. The pretreatment in all cases was carried of telocentrics is apparently correlated with out for 5-6 hr in a refrigerator at ca. 5 C. The primitiveness, basic numer, and nucleolar or- material was then fixed in Farmer's solution ganization. Storey (1968) reported 2n = 18 for (absolute ethanol and glacial acetic acid 3: 1) D. spinulosum and reported somatic reduction for 3-4 hr, and then transferred into 90% eth- in the apogeotropic roots of cycads. These root anol in the refrigerator until use. Hydrolysis cells may be reduced to two or even to one was carried out in 1N HCl at 60 C. for 10 min single chromosome. Most recently, Norstog in a water bath. Staining was done using the (1980) reported 2n = 18and 27 for Zamia lod- Feulgen reaction technique (Darlington and La digesii Miq. and suggested, in accord with Mar- Cour, 1976; Sharma and Sharma, 1980). chant (1968), that the trend in karyotype evo- Counter staining with a 2% lacto-propionic or- lution in Zamia has been toward increasing cein solution during maceration in 45% acetic symmetry caused by the fusion oftelocentrics. acid helped to improve contrast and to reduce This paper presents documented chromo- stain fading in permanent preparations (Brigh- some numbers and karyotypes of Mexican cy- ton, pers. commun.). cad species not previously reported. These re- Root tip sizes were variable and it was found ports include counts and idiograms of three that the medium to larger tips (1-1.5 mm di- newly described species. ameter) gave the best results. The dry-ice tech- nique (Sharma and Sharma, 1980), was used MATERIALSANDMETHODS-Root tips were to make permanent preparations. 'Histoclad' collected in the morning between 9.00 and proved to be a satisfactory mounting medium. 11.00 A.M. from potted plants kept in an un- Photomicrography was done with an Amer- heated g,reenhouse at the 'Jardin Botanico ican Optical Microstar One-Twenty photo- microscope fitted with planachromatic objec- tives. Measurements were made using an I Received for publication 2 August 1982; revision ac- cepted 22 December 1982. eyepiece graticule. Diploid idiograms were pre- My sincere thanks are given to the following persons pared from the complements using the mean whose revision and criticism of the manuscript was found arm lengths of chromosomes from three meta- to be most helpful; Dr. Arturo G6mez-Pompa, Mrs. Nan- phase cells (see Fig. 1). cy Moreno, Miss Laura Snook and Dr. Spencer Tomb. This research was funded by the Flora de Mexico program Vouchers of the species investigated have of the lnstituto Nacional de Investigaciones sobre Recursos been deposited at the INIREB herbarium Bi6ticos (l IREB). (XAL). The specimens examined are listed by 10 1 1' II111111 II II II I I JIIIIIII il ii II DIIIIIIII IIIII1 IIII II II II 'I II II I! II EIIIIII II I I II •... II II 1IIII111 B II II IIii IIii I I I I I I 1 1 IIII11I11111 II II JIIIIIIIIIIIII .. 1111111111111111 cl I II II I i I I Ii I I 11 II II 1.1.II Gill II II II II II I I ; i III1I1 111I11111111111 Fig. IA-G. A. Ceratozamia hildae (2n = 16). B. C. zaragozae (2n = 16). C. C. microstrobila (2n = 16). D. Dioon purpusii (2n = 18). E. Zamia loddigesii var. angustifolia (2n = 18). F. Z. inermis (2n = 16). G. Z. purpurea (2n = 16). their botanic garden accession nurn ber and col- other authors: Dioon 2n = 18, (Marchant, 1962; lector's number (Table I). Storey, 1968); Ceratozamia 2n = 16, (Mar- chant, 1968; Vovides and Rees, 1980; Vovides RESULTS-The ABN pre-treatment gave sat- and Rees, 1983); Zamia 2n = 16, 18 (Mar- isfactory results but the best contractions were chant, 1968; Norstog, I980-Norstog also re- obtained by using 0.2% colchicine. ported Z. foddigesii Miq. 2n = 27 from Yu- Chromosome numbers of the seven species catan). Unlike Zamia, the Dioon and are listed in Table I. In agreement with Mar- Ceratozamia seem constant in their diploid chant (1968), it was found that pairs of ho- number (2n = 18 and 16, respectively). mologues of a complement did not always All the Ceratozamia species examined have match in length (Fig. I). This was most prob- six mesocentric pairs of chromosomes, one ac- ably due to unequal contraction or stretching rocentric pair and one telocentric pair. How- of the arms during preparation, a difficulty cre- ever the chromosomes appear to vary in the ated by the large size of cycad chromosomes. number of satellites or, possibly, terminal het- Counts for the genera examined agree with erochromatic regions and their placement. TABLEI. List of cycad specimens examined No. individuals Species 2/7 Accession no. Voucher examined Origin Ceratozamia hildae Landry & Wilson 16 77-181,77-197 AV-312 4 San Luis Potosi, Queretaro Ceratozamia microslrobila Vovides & Rees 16 79-047,78,404 R-1613 5 San Luis Potosi Ceratozamia zaragozae Medellin-Leal 16 79-127 AV-435 5 San Luis Potosi Dioon purpusii Rose 18 79-128 AV-739 I Oaxaca Zamia inermis Vovides, Rees & Vazquez Torres 16 79-049 AV-666 2 Veracruz Zamia loddigesii var angustifolia Regel 18 80-474 AV-600 2 Oaxaca Zamia purpurea Vovides, Rees & Vazquez Torres 16 78-639 AV-734 2 Veracruz Fig. 2-5. 2. Ceratozamia hildae (2n = 16). 3. Zamia purpurea (2n = 16).4. Z. loddigesii var. angustifolia (2n = 18). 5. Dioon purpusii (2n = 18). Seven satellites have been observed in C. hil- both arms, as in D. edule, a phenomenon not dae (Fig. 1A, 2), eight in C. zaragozae (Fig. 1B, observed in any of the other two genera ex- 8) and six in C. microstrobila (Fig. 1C, 6). Their amined. karyotypes are remarkably similar in other re- In Zamia inermis (Fig. 1F, 7) and Z. pur- spects. A more detailed study of Ceratozamia purea (Vovides et aI., 1983) there are six me- is in progress. socentric pairs and two acrocentric pairs. How- In Dioon purpusii, there are four mesocentric ever Z. purpurea shows a heterochromatic band pairs, four acrocentric pairs and one telocentric on both the short arms of the last acrocentric pair of chromosomes (Fig. 1D, 5). This karyo- pair (Fig. IG, 3). This is similar to the karyo- type is similar to that of D. edule reported by type of Z.fischerireported by Marchant (1968). Marchant (1968) but there are differences in Zamia loddigesii var. angustifolia has five me- number and position of what appear to be het- socentric pairs, three acrocentric pairs and one erochromatic terminal regions. The telocentric telocentric pair (Fig. IE, 4). This karyotype pair of chromosomes of the D. purpusii com- differs from the one reported for Z. loddigesii plement seem to be heterochromatic at the cen- (2n = 18) by Norstog (1980) mainly in having tromere region and some of the chromosomes only one pair of telocentrics instead of 2 pairs. show 'heterochromatic' terminal regions on It is interesting to note that none of the Za- mia species examined have satellites. Their karyotypes seem to differ in the number of telocentric chromosomes present, thus show- ing increasing asymmetry with increase in chromosome number. DISCUSSION-Satellites- The presence of satellites can be of taxonomic value, but care must be taken in recording them as they are often very variable in appearance and can give inconsistent results (see Stace, 1980). There also have been reports on possible sex differ- entiation in Cycads involving satellites, see Abraham and Mathew (1962) and Marchant (1968). During this study, satellite heteromor- phy ofthe acrocentric pair of chromosomes in Ceratozamia hildae was seen to be apparently correlated with sex, but further work is needed in order to verify whether this satellite hetero- morphy is sex linked. Sex genes may not be associated with visible differences in these chromosomes. Karyotype variation- Fusion of telocentrics could explain the dissimilarity in the Zamia karyotypes, although fission could also be a possibility, as mentioned by Norstog (1980). Jones, (1977) mentions that on the level of speciation in cycads, and more so in the Podo- carpaceae, Robertsonian changes have taken place, a process well known and frequent in animals but thought to be rare in plants.