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The role of macrozamin and cycasin in cycads (Cycadales) as antiherbivore defenses1

Posted on: Friday, 12 December 2003, 06:00 CST

CASTILLO-GUEVARA, C. AND V. RICO-GRAY (Departamento de Ecologia Vegetal, Instituto de Ecologia, A. C., Apdo. 63, Xalapa, Veracruz 91070, Mexico). The role of macrozamin and cycasin in cycads (Cycadales) as anliherbivore defenses. J. Torrey Bot. Soc. 130:206- 217. 2003.-Macrozamin and cycasin are very toxic azoxyglycosides of the Cycadales. Caterpillars (Lepidoptera) and weevils (Coleoptera) feed on cycad root, stem, leaf, and reproductive tissues. Azoxyglycosides may have played an important ecological role as antiherbivore defenses. We evaluated the association between herbivory and the amount of azoxyglycosides in the Cycadales using phylogenetic independent contrasts. We hypothesized that herbivory types should be related to the presence of macrozamin and cycasin, thus herbivory should be lower in species with higher concentrations of azoxyglycosides. We gathered information available on the literature of these two characters as well as life form, geographic distribution, height, and seed volume for the majority of cycad species, in order to assess correlated evolution and control for possible allometric effects. Herbivory types and macrozamin were negatively correlated, suggesting a possible defensive function for macrozamin against herbivores. No significant correlation was observed between cycasin percent and herbivory type. However, when analysed using phylogenetic independent contrasts and thus removing the historical effect, the association did not hold. This suggests that the presence of metabolites in plants may have evolved for some other reason, and has been mantained among cycads perhaps by phylogenetic inertia. The presence of macrozamin should then be explained as an exaptation, playing today an important role in defense against herbivores. Furthermore, this analysis showed that macrozamin has independently and repeatedly (Bowenia, Macrozamia, Stangeria) increased over evolutionary time.

Key words: association of characters, azoxyglycosides, cycads, cycasin, herbivores, herbivory, independent contrasts, macrozamin.

Macrozamin and cycasin are characteristic and exclusive azoxyglycosides of the Cycadales (De Luca et al. 1980; Siniscalco 1990). This ancient gymnosperm group was abundant and widespread during the Mesozoic; however, it is currently isolated and confined to the tropics (Norstog and Nicholls 1997). They have remained relatively unchanged morphologically since the Mesozoic time (Norstog and Nicholls 1997; Jones 2000) and, according to most authors, extant cycads represent the end-points of a number of different lines, doubtless cognate with others now extinct (Siniscalco 1990).

The azoxyglycosides are mutagenic and carcinogenic only when deglucosylated. Its principal metabolite, methylazoxymethanol (MAM) (Matsumoto and Strong 1963; Whiting 1963; Kobayasi and Matsumoto 1965; Yagi et al. 1980; Morgan and Hoffmann 1983; Hoffmann and Morgan 1984) is released and responsible for their toxicologie properties. MAM induces genetic alterations in various test systems in bacteria, yeast, plants, Droshopila and mammalian cells. Tn adult mammals, the deglucosylation of cycasin is catalyzed only by enzymes of the gut microflora and, therefore, active when administered orally (Hoffmann and Morgan 1984). Further, the azoxy group is antibacterial and antifungal (Siniscalco 1990). These chemical characteristics may have played an important ecological role as antiherbivore defenses during the long evolutionary history of cycads (Moretti et al. 1983).

Today caterpillars and weevils feed on roots, stems, leaves, reproductive cones, and seeds of cycads. Classifying these herbivores by their feeding modes (Thompson 1982), caterpillars (Lepidoptera) can be considered parasites, feeding mainly on leaf tissues, although they may also feed on reproductive cones, in which case they would be seed predators (DeVries 1976, 1977, 1983; Clark and Clark 1991). Whereas weevils (Coleoptera) can be considered seed predators (Jones 2000). Other insect groups also feed on cycads, members of the families Cecidomyiidae, Pseudococcidae, Coccidae, Diaspididac, Aphidae (Jones 2000). Experimental and observational studies suggest that cycasin confers a defensive quality to caterpillars against predators (Rothschild et al. 1986; Bowers and Larin 1989; Bowers and Parley 1990; Nash et al. 1992; DeVries 1994; Trigo 2000), but little is known on the role of macrozamin as antiherbivore defenses.

Most studies have addressed ecological aspects of these interactions. However, analysing this information for the Cycadales jointly with azoxyglycosides concentrations and the evolutionary history of the group may offer a possible evolutionary scenario of the association of these characters. Comparative methods allow testing evolutionary explanations based on ecological and morphological information. Recent advances in systematics and evolutionary biology have shown that incorporating phytogenies into comparative studies is critical for understanding and developing hypotheses on evolutionary processes, the evolution of characters, and adaptation (Martins and Hansen 1996; Ornelas 1998). We used published information of percent macrozamin and cycasin by De Luca et al. (1980) and Moretti et al. (1981, 1983), and throughout the nomenclature follows Moretti et al. (1983) and Vovides et al. (1983). Using phylogenetic independent contrasts (Purvis and Rambaut 1995) we present a comparative analysis of the evolutionary association between herbivory and the amount of azoxyglycosides in the Cycadales. We hypothesized that herbivory types should be related to the presence of macrozamin and cycasin, thus herbivory should be lower in species with higher concentrations of azoxyglycosidcs.

Materials and Methods. DISTRIBUTION OF AZOXYGLYCOSIDES AMONG CYCADS. All cycads produce secondary metabolites, such as cycasin, macrozamin and neocycasin (De Luca et al. 1980; Moretti et al. 1983; Siniscalco 1990). The exclusive occurrence of azoxyglycosides in cycads supports the view that they form a monophyletic group (Siniscalco 1990). Furthermore, structural features such as simple ovulate cones and girdling leaf traces, a specialized pattern of vascular bundles in the petiole (omega), the presence of mucilage canals, and distinctive meristems, as well as cycasin, are unique synapomorphies that distinguishing cycads from other seed plants (Crane 1985). These metabolites are members of a family of azoxyglycosides (Hoffmann and Morgan 1984). The axoxy-group is very rare in organisms (Moretti et al. 1983) and it is known only in three other naturally occurring compounds which have been isolated from two species of bacteria (Streptomyces spp.) (Schoental 1969) and une species of fungi (Calvatia lilacina) (Gasco et al. 1974). The molecule is made of two main components, a carbohydrate and an aglycone named methylazoxymethanol (MAM) (Morgan and Hoffmann 1983; Hoffmann and Morgan 1984) (Fig. 1A, 1B). The three compounds have the same aglycone but differ in their sugar, e.g., cycasin contains - D-glucose (a monosaccharide) (Nishida et al. 1955), macrozamin contains primeverosa (a disaccharide formed by glucose and xylose) (Lythgoe and Riggs 1949), while neocycasin contains different sugar components (Nagahama 1964). Macrozamin probably represents the most primitive azoxyglycoside, whereas cycasin and neocycasin can be considered derived (Siniscalco 1990). Neocycasin has only been isolated from Cycas, where it was found in small quantities (Nagahama 1964; Yagi et al. 1985; Yagi and Tadera 1996).

Macrozamin concentrations vary at the genus level: Cycas 0.20- 0.45%, Bowenia 4.33-5.04%, Lepidozamia 1.1%, Macrozamia 2.41-3.88%, Microcycas 0.13%, Encephalartos 2.09-2.86%, Stangeria 4.70%, Ceratozamia 1.01-1.05%, Dioon 0.64% and Zamia 1.01-1.02% (Siniscalco 1990). Based on macrozamin content, the Zamiaceae is a heterogeneous family and its tribal subdivisions are not well established (Siniscalco 1990).

We used the macrozamin and cycasin data lor 30 species obtained by De Luca et al. (1980) and Moretti et al. (1981, 1983). Plant height, and seed measurements were based on Vovides et al. (1983), Norstog and Nicholls (1997) and Jones (2000). Seed measurements were used to obtain their volume (V = [([pi]/6) (L) (W^sup 2^)], where L is length and W is width). We did simple linear regressions between plant characteristics (height, seed and cone volume) and azoxyglycosides content to assess possible allometric effects between plant size and azoxyglycosides per species. One-way ANOVA's were computed in order to compare the percent of azoxyglycosidcs among life form, shrubby (0.50-1.99 m) and arborescent (2.00-10.0 m), and KolmogorovSmirnov tests to compare percent azoxyglycosides with distribution of cycads (Old World or New World species) (Zar 1999). Variables were transformed (natural logarithmic for height and volume, and arcsin [the square root of] x for percent azoxyglycosides) for statistical analyses (Zar 1999).

HERBIVORY TYPES AMONG CYCADS. There is a general lack of information on specific interactions in cycads; most are on herbivory by lepidopteran caterpillars (Appendix 1). We used the following criteria to categorize cycad species based on (1) whether herbivores consumed most of the cones and leaves and their damage was reported as infrequent in litera\ture, (2) whether they also consumed cones and leaves but damage was reported twice a year particular in new leaves, and (3) they consumed the whole cone and damaged individual plants to their subsequent death. Kruskal-Wallis tests with tied ranks were computed in order to compare herbivory type with height of plants, and a 2 3 contingency table was constructed in order to compare herbivory type with life form (shrubby, arborescent) and distribution of cycads (Old World or New World species) (Zar 1999). Herbivory type 3 species are from the New World (C. mexicana, D. edule, Z. furfuracea, Z. integrifolia) (Table 1), and they are mainly herbivorized by lepidopteran caterpillars of the American genus Eumaeus (Appendix 1).

Table 1. Data base used in comparative analysis. Macrozamin and cycasin (%) in seeds, taken from De Luca et al. (1980) and Moretti et al. (1983). Other data calculations from Vovides et al. (1993), Norstog and Nicholls (1997), Jones (2000). NA = Not available. The genus Cycas does not produce cones (-). Nomenclature follows Moretti et al. (1983) and Vovides et al. (1983).

CYCAD PHYLOGENY. Few studies have addressed phylogenetic relationships in cycads (Stevenson 1990). Efforts to resolve cycad phylogeny at a generic level using cladistic methods have been reported by Crane (1988), Stevenson (1990), and Schutzman and Dehgan (1993). These analyses have stimulated discussions on character states and evolutionary trends, but differ in their results and conclusions (Bogler and Francisco-Ortega in press). However, the formal classification of cycads proposed by Stevenson (1992), based on cladistic analysis of morphological and anatomical characters, is a useful rcference. The Order Cycadales is considered a monophyletic group, divided into three families (Cycadaceae, Stangeriace and Zamiaceae), 11 genera (Cycas, Dioon, Bowenia, Macrozamia, Lepidozamia, Ecephalartos Cerafozamia, Stangeria, Microcycas, Zamia, and Chigua) and 190-230 species (Stevenson 1990; Norstog und Nicholls 1997; Jones 2000).

The first molecular systematic study of cycads showed the potential of using molecular markers to solve relationships among genera in this group (Caputo et al. 1991; Caputo et al. 1993). Bogler and Francisco-Ortega (in press) made an initial molecular systematic study using ITS2 and fmL intron markers in 25 taxa, providing a single most parsimonious tree.

We constructed a phylogenetic hypothesis based on the most parsimonious tree of Bogler and Francisco-Ortega (in press), which includes 30 taxa. Major clades are maintained up to genus level. The species Bowenia serrulata, B. spectabilis, Macrozamia moorei, Lepidozamia peroffskyana, Encephalartos ferox, Stangeria eriopus and Microcycas calocoma were maintained in the same position. For Dioon, species were placed according to Moretti et al. (1993). Due to the absence of molecular information, Zamia furfuracea, Z. integrifolia, Z. latifolia, Ceratozamia mexicana, C. matudae, Encephalartos umbeluziensis, E. villosus, E. lebomboensis, E. altensteinii, Macrozamia heteromera, M. diplomera, M. fawcettii, M. miquelii, Cycas revoluta, C. pruinosa, C. cairnsiana, C. lane-poolei, C. basaltica, C. thouarsii and C. circinalis, were left as polytomies (Fig. 1).

ASSOCIATION BKTWBBN AZOXYGLYCOSIDES AND HERBIVORES. In order to assess the evolutionary association between percent azoxyglycosides and herbivory, and to test the hypothesis that higher concentrations of azoxyglycosides in plants will result in less damage to plants, we used a comparative analysis by independent contrasts (CAIC; Purvis and Rambaut 1995). Statistical methods that treat species values as statistically independent points are not valid, because closely related species will tend to share many characters through common descent rather than through independent evolution (Harvey and Pagel 1991). Such phylogenetic inertia may result in two related characteristics among species, however this correlation is an artifact of the nonindependence of species. CAIC avoids problems in the results by considering independent evolutionary events. CAIC is a method derived from Felsenstein (1985) and it can be used whenever comparative data including continuous and categorical variables are to be analyzed, and CAIC deals with soft polytomies so it can be used to analyze non resolved phytogenies. The independent contrast method is based on the comparison between pairs of sister species. Each comparison produces a new variable termed a "contrast", which results from the difference between the values of the variable measured on the species within the pair. Contrasts may be standardized if divided by the square root of the length of the branches under comparison. These contrasts are considered independent among pairs of sister species, because they result from the evolutionary divergence that occurred after the origin of each pair. Homogeneity of variance of standardized contrasts was confirmed using the method proposed by Purvis and Rambaut (1995). In order to compute contrasts we used the Crunch and Brunch algorithms; Crunch is suggested when all the variables are continuous and Brunch when the predictor variable is categorical.

Results. DISTRIBUTION OF A/.OXYGLYCOSIDES AMONG CYCADS. Macrozamin is generally more abundant than cycasin, ranging from 0.13% in Microcycas calocoma to 5.04% in Bowenia spectabilis', whereas cycasin quantities are usually lower (e.g., 0.020% to 0.72% in Cycas lanepoolei; Table 1). Mean amounts of macrozamin are usually significantly larger (mean = 1.774 SE = 0.299, N = 26) than mean quantities of cycasin (mean = 0.134 SE = 0.027, N = 30; t = 7.794, DF = 54, P = > 0.0001).

The highest cycasin and macrozamin contents are in Old World species (Table 1; Fig. IA, B). The mean (mean SE) of macrozamin content was significantly different (Kolmogorov-Smirnov d^sub max^ = 0.650, DF = 2, P = 0.0405) between Old World (2.063 0.363, N = 20) and New World (0.810 0.150, N = 6) species. New World species had low macrozamin content (Table 1; Fig. 1A). The mean of cycasin content was not significantly different (KolmogorovSmirnov, d^sub max^ = 0.397, DF = 2, P = 0.2750) between Old World (0.142 0.035, N = 21) and New World (0,1 14 0.043, N = 9) species (Table 1; Fig. 1B). There is not one species with high macrozamin and cycasin contents, except for B. spectabilis, which is the only species with high macrozamin and medium cycasin content (Table 1, Fig. 1A, B).

Azoxyglycoside content was significantly different (ANOVA, F, 24 = 14.650, P = 0.0008) between plant life forms, macrozamin being higher in shrubby (2.615 0.405, JV = 13) than in arborescent (0.933 0.300, TV = 13) life forms (Table 1; Fig. 1A). However, cycasin was not significantly different (ANOVA, F^sub 1,28^ = 0.137, P = 0.7141) between shrub (0.125 0.033, N= 15) and arborescents (0.143 0.045, N = 15; Table 1; Fig. 1B). Plant height determines life form, therefore, the results were consistent with subsequent analysis with and without CAIC. The highest macrozamin contents were also found in Old World species with arborescent life forms B. spectabilis, B. serrulata, M. moorei, E. altensteinii, E. lebomboensis; and the shrubby M. miqueHi, M. fawcettii, M. diplomera, E. villosus, E. umbeluziensis, E. ferox, S. eriopus (Fig. 1A). High cycasin contents were found in Old World basal species with arborescent life forms (C. lane-poolei, C. revoluta and B. spectabilis), and appeared after in New World apicales species with shrubby life forms (Z. integrifolia and Z. furfuracea; Fig. 1 B). In spite of the fact that the American genus Dioon is arborescent and related with Cycas, Dioon does not present high cycasin content.

Table 2. Regression analyses with and without CAIC.

ASSOCIATION BETWEEN AZOXYGI.YCOSIDES AND HERBIVORES. Cone volume data was not used due to the fact that the genus Cycas does not produce cones and the sample was thus reduced. We then used seed volume as an indicator of cone size, and found a positive significant association between plant height and seed volume (r^sup 2^ = 0.414; Table 2). To control for this allometric relationship we regressed the residuals of that association with percent of macrozamin and cycasin. In both cases, the relationship was not significant (P > 0.05; Table 2). We then directly compared herbivory type with percent of cycasin and macrozamin; the former was not significant and the latter resulted in a negative significant association (Table 2). Using independent contrasts analysis (CAIC) and linear simple regressions, we found that the associations between herbivory types and percent of macrozamin and cycasin in cycads were not significant (Table 2).

HERBIVORES AND OTHER VARIABLES. We did not find any significant differences (H = 5.225, DF = 2, P = 0.0734) between plant height and herbivory types (type 1 = 3.004 3.176,W = 14; type 2 = 3.750 0.684, N = 12; type 3 = 1.662 0.786, N = 4). We found that herbivory type is independent of life form (shrubby or arborescent; [chi]^sup 2^^sub 0.05,2^ = 5.142), but herbivory type is not independent of distribution (Old or New World species; [chi]^sup 2^^sub 0.05.2^ = 14.692).

Discussion. Herbivory types and macrozamin were negatively correlated, suggesting a possible defensive function for macro/amin against herbivores. However, when analysed using independent contrasts and thus removing the historical effect, the association was not present. This suggests that the presence of metabolites in plants evolved for some other reason, and is maintained, perhaps, by phylogenetic inertia (i.e., closely related species share characters through common descent). The presence of macrozamin could be an exaptation, playing today an important role in defense against herbivores. Also, this analysis showed that there is no relationship between cycasin and geographic distribution, life form, and herbivory type and the percent macrozam\in was high among Old World, shrubby species.

The results must be taken with caution. First, we recognize that the data on azoxyglycoside concentration were taken from phytochemical studies that do not necessarily correlate herbivory and chemical content. For example, it is common that material derived from a few plants at one site (sometimes from a garden) is processed to purify and characterize the secondary metabolites. Considering that quantitative variation in chemical content within and among populations is a common feature, the sampling of few individuals in one site may not be representative for a species. Also, the methods used to isolate and characterize secondary metabolites frequently underestimate the concentration of the chemicals in the plant. second, the use use of CAIC had some restrictions. Branch lengths were not available and CAIC generated default branch lengths (every branch in the phylogeny is the same length). This is an explicitly punctuated view of evolution. Also, our phylogenetic hypothesis did not include all extant cycad species, Bank et al. (2000) and Treutlein and Wink (2002) describe new studies of molecular phylogeny cycads that can be used in future analysis. More species can be included in the phylogeny as more data on azoxyglycosides are available, in particular more New World species.

Cycads are among the most ancient of living seed-bearing plants and still retain clearly primitive features, such as motile sperm (Crane 1985). Cycads evolved in the Carboniferous or early Permian, about 280 million years ago, and reached their peak abundance and diversity in the Mesozoic. They were among the earliest seed plants, and there is reason to believe that insect-pollination systems (e.g., pollination by beetles) and relationships with pollinators may also have originated with cycads considerably earlier than such relationships with the laterevolving flowering plants (Norstog and Nicholls 1997; Jones 2000). Herbivory in this group (parasitism, prdation) could be equally ancient, although extant herbivores are probably totally different. Historical patterns of host shifts strongly correspond to patterns of host chemical similarity, indicating that plant chemistry has played a significant role in the evolution of host shifts by phytophagous insects (Becerra 1997).

Lepidopteran caterpillars as well as weevils, although sporadic in some years, can appear in large numbers causing infestations and severe damage (Jones 2000). Nevertheless, some weevil species are effective cycad pollinators (Norstog and Fawcett 1989; Vovides 1991; Vovides et al. 1993), exemplifying the array of different interaction outcomes (mutualistic, antagonistic) within an insect order. For example, Z. furfuracea and Z. pumila are pollinated by Rophalotria mollis and R. slossoni (Curculionidae), respectively (Norstog and Fawcett 1989); Dioon edule is pollinated by R. bicolor (Vovides 1991); E. villosus is pollinated by Porthetes sp. (Curculionidae), while Antliarhinus zamiae (Brentidae) is a important parasite (Donaldson 1997); members of the Crysomelidae (Aulacoscelis sp. and A. costaricensis) feed of Zamia fairchildiana leaves (Windsor et al. 1999); while the Cuban cycad Microcycas calocoma is endangered because its pollinators are extinct (Vovides et al. 1997).

Macrozamin has not really evolved as a defense mechanism against insect herbivores; interspecific interactions change in space and time (Thompson 1994), and cycads have endured severe environmental changes throughout their evolutionary history. A major assumption behind the hypothesis of azoxyglycoside concentration and herbivory is that two of those chemicals are the only or the major variables that determine reduced herbivory. Other factors might have affected herbivores in cycads including intrinsic plant characteristics such as leaf age (Bogacheva 1994) that is related with leaf toughness, number and leaf size (Freitas et al. 1999), abundance and quality of plants (Price et al. 1990; 1991), resource availability as the turnover of leaves in time and space. Also, because a number of specialist insects that feed on cycads sequester azoxyglycosides, a neutral or even a positive correlation between herbivory and these chemicals could be expected.

Macrozamins probably are the most primitive azoxyglycosides. Their structure is complex, including two types of carbohydrates (Siniscalco 1990; Norstog and Nicholls 1997). Cycasins and neocycasins contain only one carbohydrate (glucose) and are considered derived (Siniscalco 1990). The presence of macrozamin could be considered an exaptation, a characteristic which currently increases fitness but originally evolved for a different purpose, in contrast with an adaptation, a characteristic that increases fitness and that evolved and was selected to play its current role (Could and Vrba 1982; Losos and Miles 1994). Azoxyglycosides could represent the products of a primitive biochemical pathway designed to store nitrogen (Siniscalco 1990), and cycads retained these products probably due to their toxic effects, which may have played an important ecological role during the long evolutionary history (Siniscalco 1990).

The cycads have often serious competition from other plants, they are particulary vulnerable in this respect, especially in enviroments such as rainforests, because they are so slow-growing that other forms tend to cover them rapidly (Norstog and Nicholls 1997). The large and long-lived leaves of Zamia skinneri in rainforests accumulate over the years a heavy and no doubt light- intercepting layer of small epiphytic bryophytes and ferns. The growth and reproductions of a similar species (Z. neurophyllidia) are also greatly retarded by the low levels of available light under the closed canopy of rainforest trees (Clark et al. 1992). It is perhaps surprising that toxic chemicals are not released from the leaf surfaces or by the roots into the surrounding soil, which might inhibit the growth of potential competitors. There is some evidence, however, that root extracts of Cycas circinalis can inhibit the germination and seedling development in other plants (Shirmal and Prasad 1977), but no evidence as yet that the inhibitory substance, whatever it is, is released into the enviroment. We found macrozamin being higher in shrubby than in arborescent life forms, and a relationship could exist between this high concentration of azoxyglycoside in shrubby plants in rainforests as inhibitory substance to avoid competititon with other plants, these need further testing. An alternative explanation is that high concentration of azoxyglycosides is maintained among shrubby rainforest cycads as antibacterial and antifungal defenses.

Conclusions. Our results represent the first comparative analysis of the association between herbivory types and azoxyglycosides content in cycads. The results of this study show a negative relationship between herbivory types and percent macrozamin, but this relationship disappeared when phylogeny was accounted for. A non-significant association was found in either case for cycasin counted and herbivory types. An increase in macrozamin content was mainly observed among shrubby, old world species. The presence of macrozamin could be an exaptation, playing today an important role in defense against herbivores, but more studies are needed on azoxyglycoside content in order to test it, to evaluate and quantify the effect of herbivory in cycad fitness (for example by guild herbivores), quantify concentration of azoxyglyxosides in more species, to include enviromental factors, like vegetation, in order to compare the percent of azoxyglycosides.

Appendix I.

List of cycad herbivores: Caterpillars of lepidoptera, beetles, weevils, sap suckers, scale insects and other insects.

Appendix I.

List of cycad herbivores: Caterpillars of lepidoptera, beetles, weevils, sap suckers, scale insects and other insects.

1 This research was supported by Instituto de Ecologia, A.C. (902- 16), and CONACyT (112677) graduate, scholarship to CCG.

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Citlalli Castillo-Guevara2 and Victor Rico-Gray

Departamento de Ecologia Vegetal, Instituto de Ecologia, A. C., Apdo. 63, Xalapa, Veracruz 91070, Mexico

2 We thank J. Francisco-Ortega for providing the phylogeny of Cycads, J.F Ornelas for his help throughout the study, L. Jimenez for her help with the computer program, and C. Lara for his comments to an earlier version of the manuscript.

Received for publication September 10, 2002, and in revised form March 30, 2003.

Copyright Torrey Botanical Society Jul-Sep 2003

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