American Journal of Botany 92(2): 272±278. 2005.

CLONAL DIVERSITY AND DISTRIBUTION IN ERUCA (CACTACEAE), A NARROW ENDEMIC OF THE SONORAN DESERT1

RICARDO CLARK-TAPIA,2 CECILIA ALFONSO-CORRADO,3 LUIS E. EGUIARTE,4 AND FRANCISCO MOLINA-FREANER2,5

2Instituto de EcologõÂa-UNAM, Departamento de EcologõÂa de la Biodiversidad, EstacioÂn Regional del Noroeste, Apartado Postal 1354, C.P. 83000, Hermosillo, Sonora, Mexico; 3Instituto de EcologõÂa-UNAM, Departamento de EcologõÂa Funcional, Apartado Postal 70-275, MeÂxico, D.F. C.P. 04510, Mexico; and 4Instituto de EcologõÂa-UNAM, Departamento de EcologõÂa Evolutiva, Apartado Postal 70-275, MeÂxico, D.F. C.P. 04510, Mexico

Stenocereus eruca (Cactaceae), a prostrate cactus endemic to the Sonoran Desert, is thought to be highly clonal. We examined its clonal diversity and distribution: (1) at the population level, in four distinct populations along its distribution range; and (2) at a micro scale level, within a single population. Our objective was to evaluate the importance of sexual versus clonal recruitment through the use of RAPD markers. Contrary to previous ®eld observations, clonal diversity was relatively high across the distribution range. This ®nding suggests that sexual recruitment is an important regeneration mechanism. The proportions of distinguishable genotypes (G/N ϭ 0.83) and genotypic diversity (D ϭ 0.987) were greater than in other clonal cacti, suggesting that clonal propagation is not the major regeneration mechanism. Autocorrelation analyses revealed a spatial genetic structure that may be the result of restricted gene ¯ow (via pollen or seeds) and clonal propagation. A molecular variance analysis (AMOVA) indicated that most of the variation (66.3%) was found within and not across populations. Future studies on pollen and seed dispersal are needed to understand the role of the clonal habit in the mating system of S. eruca.

Key words: Baja California; Cactaceae; clonal diversity; RAPD; spatial genet structure; Sonoran Desert; .

Many perennial combine sexual reproduction and tures and unpredictable rainfall events (Polis, 1991). In these clonal propagation as population regeneration mechanisms environments, seedling recruitment of succulent species may (Abrahamson, 1980; Richards, 1997). In some clonal species occasionally be restricted to pulses during rare heavy rainfalls the success of sexual vs. clonal recruitment often varies geo- (Jordan and Nobel, 1979; Turner, 1990; Pierson and Turner, graphically in response to ecological and genetic factors that 1998), even when viable seeds are consistently produced limit one regeneration mechanism or the other (Eckert, 2002). (Fleming et al., 1996; Mandujano et al., 1998). In contrast, The demographic balance between sexual and clonal recruit- clonal propagation is often a successful mechanism of regen- ment is likely to have important consequences for the clonal eration in populations of succulent species (Parker and Ham- diversity and genetic structure of populations (Ellstrand rick, 1992). Nonetheless, our knowledge about the clonal and Roose, 1987; Eckert and Barrett, 1993). Seedling recruit- structure and the relative importance of clonal vs. sexual re- ment strategies in clonal species range from repeated seedling cruitment in clonal cacti is poor. recruitment, or the establishment of seedlings on a regular ba- Stenocereus eruca, a clonal columnar cactus of the Sonoran sis, to initial seedling recruitment, their establishment only Desert (Gibson, 1989; Turner et al., 1995), is an extremely during the initial colonization of a site (Eriksson, 1993, 1997). narrow endemic species restricted to coastal areas of the Mag- The pattern of seedling recruitment in¯uences the maintenance dalena Plains in Baja California Sur, Mexico (Gibson, 1989), of genotypic diversity, as clonal diversity rapidly declines in as shown in Fig. 1. The ¯owers of S. eruca are self-incom- the absence of new seedling input and selection (Watkinson patible. Fruit set is generally low and highly variable in space and Powell, 1993). In contrast, even low rates of seedling re- and time because of pollinator limitation associated with var- cruitment are enough to maintain relatively high levels of ge- iations in sphingid abundance (Clark-Tapia and Molina-Fre- notypic variation within plant populations (Soane and Watkin- aner, 2004). Because seedling recruitment has seldom been son, 1979; Watkinson and Powell, 1993). For this reason, stud- observed in natural populations of S. eruca, clonal propagation ies that document clonal diversity can be used to draw infer- is thought to be the major regeneration mechanism (Gibson, ences about the role of sexual as compared to clonal 1989; Turner et al., 1995). A 3-yr demographic study of S. recruitment in the regeneration of plant populations. eruca suggests that sexual recruitment's relative contribution In arid environments, the population dynamics of many to population growth rate (␭) is less important than that of plant species vary considerably because of extreme tempera- clonal propagation (Clark-Tapia et al., in press). Stenocereus eruca is considered the ``most extreme case of clonal propa- 1 Manuscript received 2 March 2004; revision accepted 21 September 2004. gation in the cactus family'' (Gibson and Nobel, 1986). Clonal The authors thank Teresa Valverde, Daniel PinÄero, and two anonymous re- propagation occurs by detachment of branches from the major viewers for critical comments on earlier versions of the manuscript, and Dan- shoot as the base of the branch dies and rots. In S. eruca, the iel Morales, RocõÂo Esteban, and Oscar RodrõÂguez for their lab and ®eld as- ramets (vegetatively produced modular units, Harper, 1977) of sistance. Financial support was provided by SEMARNAT-CONACYT (0665/ A1), PAPIIT-DGAPA-UNAM (IN-211997 and IN-205500), and a CONACYT a genet (set of all products from a particular zygote) thus tend and DGEP scholarship to Ricardo Clark-Tapia. to detach and fragment. Therefore the genetic similarity among 5 Author for correspondence (E-mail: [email protected]). ramets can only be studied using genetic markers, namely iso- 272 February 2005] CLARK-TAPIA ET AL.ÐCLONAL DIVERSITY IN STENOCEREUS ERUCA 273 zymes or DNA based markers. A previous isozyme study on random DNA samples from each population. Six informative and reproducible the clonal diversity of S. eruca suggested that both sexual and primers (A01, A10, A15, G03, G16 and G18) were identi®ed, and only strong clonal recruitment are important (mean G/N ϭ 0.53) in the polymorphic bands between 350 and 2036 bp were used for data analysis. regeneration of its populations (Clark-Tapia, 2000). Isozyme Control samples containing all the reaction materials except DNA were used to ensure that no self-ampli®cation or DNA contamination had occurred. In evidence also showed moderate levels of genetic variation (He ϭ 0.158, P ϭ 46.2); substantial deviation from Hardy-Wein- the construction of the data matrix, all RAPD bands were scored as present berg expectations (f ϭ 0.739); low genetic differentiation (1) or absent (0). among populations (␪ϭ0.069); and no evidence of isolation by distance (Clark-Tapia, 2000). DNA-based markers usually Data analysisÐClonal diversityÐBased on the scored RAPD banding pat- have higher resolution power than isozymes. DNA-based terns, putative genets were identi®ed. To estimate clonal diversity, the follow- markers thus allow a more precise characterization of geno- ing parameters were calculated for both the population-level and Estero Sa- typic diversity in clonal plant populations (Peakall et al., 1995; linas surveys. (1) The proportion of distinguishable genotypes (Ellstrand and Roose, 1987) was measured as G/N, where G is the number of genets and N Ayres and Ryan, 1997). To analyze S. eruca's clonal diversity is the total number of individuals (ramets) sampled. (2) Simpson's diversity and genetic structure, the present study used RAPD markers index (D) modi®ed for ®nite sample size by Pielou (1969), measures the to: (1) evaluate the importance of sexual versus clonal recruit- probability that two ramets selected at random from a population of N plants ment in four populations of this clonal columnar cactus; and will be from different multilocus genotypes. This index thus yields a measure (2) describe the spatial distribution of genotypes in a single of multilocus genotype diversity. D ranges from 0 to 1, with 1 being the population and explore whether they have a random or aggre- maximum diversity. Each index value was calculated as: gated distribution; and (3) describe the genetic structure of [n (n Ϫ 1)] four populations. D ϭ 1 Ϫ ͸ ii Ά·[N(N Ϫ 1)]

MATERIALS AND METHODS where ni is the number of individual ramets of genotype i, and N is the sample size. (3) Genotypic evenness (Fager, 1972) was measured as: Collection of genetic materialÐTo investigate the importance of sexual vs. (D Ϫ D ) clonal recruitment across populations, we selected four populations in the E ϭ obs min Ϫ Magdalena Plains of Baja California. These speci®c populations were chosen (DmaxD min) in an effort to cover the entire distribution range of S. eruca (Fig. 1). For where population-level surveys, we sampled 30 ramets along a 1200 m long transect Ϫ Ϫ Ϫ ϭ [(G 1)(2N G)]ϭ [(G 1)(N)] in each of these four sites, collecting tissue samples every 30±40 m in order Dmin, and D max [N(N Ϫ 1)] [G(N Ϫ 1)] to cover each population in its entirety. To assess the spatial distribution of clones within a single population, we selected the Estero Salinas site because where G is the number of clones, and N is the sample size. Index values can it had by far the largest population density (ca. 4500 ramets/ha), much than range from 0 for a population dominated by one genotype, to 1 for a popu- at the other sites. For a demographic study, all ramets within a 10 ϫ 60 m lation in which all genotypes are represented by the same number of ramets. plot at Estero Salinas have been tagged, numbered and mapped (Clark-Tapia et al., in press). All ramets within this plot (N ϭ 282) were sampled and their Spatial genetic structureÐThe data concerning band presence/absence in exact locations recorded by measuring their distances from the plot margins. the RAPD analysis were used to determine genetic identity and examine the With a cork borer, small (2±3 cm3) samples of rib chlorenchyma were spatial distribution of genets at Estero Salinas. The spatial distribution in this extracted from each selected ramet. Tissue samples were collected during population was evaluated by constructing a detailed map that identi®ed the November 2001 and were stored on ice for two d and then in a Ϫ70ЊC freezer genotype of each ramet and enabled the distribution and spatial extent of for 30 d prior to DNA extraction. genets to be determined. Spatial autocorrelation analysis was performed on the basis of genetic similarity/dissimilarity in the RAPD banding patterns RAPD analysisÐTotal genomic DNA was extracted using a modi®cation found among single ramets. The spatial autocorrelation was examined with a of the Qiagen Plant Minikit method (Qiagen, Inc., Valencia, CA, USA; M. distance measure, namely the geographical distance, and Tanimoto's genetic

SaÂnchez-HernaÂndez and R. Esteban-JimeÂnez, Instituto de EcologõÂa UNAM, distance (DG), which is typically used with binary data such as ®ngerprints. unpublished data). Tissue from rib chlorenchyma was frozen brie¯y in liquid This analysis was performed on 10 geographical distance classes (see Results nitrogen and ground with a mortar and pestle to a ®ne powder. Ampli®cation section), using Spatial Genetic Software v.1.0c (Degen, 2001). To assess sta- reactions of the target DNA were carried out in a total volume of 23.3 ␮L tistical signi®cance and control the overall probability that we would mistak- per sample. The reaction mixture consisted of 16 ␮L puri®ed water enly declare DG signi®cant, we used the 99% con®dence intervals generated (GibcoBRL, Invitrogen Life Technologies, Carlsbad, CA, USA); 2 ␮L 10- from 5000 permutations (Degen et al., 2001). We visually examined the spa- base primer (Operon Technologies, Inc., Alameda, CA, USA); 1.5 U Taq tial autocorrelation in the form of a correlogram that plotted DG values, mean DNA polymerase (GibcoBRL); 10 ng template DNA; and 5.07 ␮L master genetic distances, and the 99% con®dence intervals as a function of geograph- stock (a mixture of 1.38 ␮L puri®ed water, 2.6 ␮L GibcoBRL buffer minus ic distance. If DG (observed) is greater than the DG (99% CI), then there is a ϭ ␮ ␮ Mg at pH 8.4, 0.05 L of 1 mol/L MgCl2, and 0.26 L [0.1 mmol/L] of signi®cant autocorrelation at that distance class and therefore signi®cant ge- each Pharmacia (Uppsala, Sweden) dNTP). PCRs (polymerase chain reac- netic structure (Degen et al., 2001). In addition, we analyzed the spatial ge- tions) were performed in a Techgene (Bethesda, Maryland, USA) thermal netic structure within Estero Salinas as a result of clonal propagation or iso- cycler programmed for one cycle of 5 min at 94ЊC, followed by 44 cycles of lation by distance through limited gene ¯ow. Spatial Genetic Software v.1.0c 1 min at 94ЊC, 1 min at 38ЊC, 30 s at 54ЊC and 2 min at 72ЊC. After the last (Degen, 2001) was used to perform spatial autocorrelations at both the genet cycle, a ®nal extension step of 13 min at 72ЊC was carried out. Ampli®cation and ramet levels (Reusch et al., 1999; Hangelbroek et al., 2002). At the ramet products were separated electrophorectically on a 1.4% agarose gel with 0.5X level, all 282 samples were included, while at the genet level only one ramet TBE running buffer at 120 V for 2.5 h. Gels were stained with ethidium of each genet was taken at random from the ®eld plot. bromide and visualized under UV light. A digital camera recorded the images. Using DNA test samples from the four different populations of S. eruca, Genetic variationÐThe following estimates of variation were obtained us- 40 primers were screened with Operon Technologies, Inc. Kits A and G for ing Tools for Population Genetic Analysis (TFPGA) software v.1.3 (Miller,

reproducible ampli®cation patterns. For each primer, the reproducibility and 2000): percentage polymorphic loci (Pp), and expected heterozygosity (Hs). repeatability of the ampli®cation pro®les were tested three times with four Because co-dominant markers (isozymes) revealed signi®cant deviations from 274 AMERICAN JOURNAL OF BOTANY [Vol. 92

Fig. 1. Distribution range (shaded area) of Stenocereus eruca in Baja California Sur, Mexico and locations of the four populations sampled in this study. Modi®ed from Turner et al. (1995).

Hardy-Weinberg equilibrium predictions (Clark-Tapia, 2000), a molecular var- n is the number of subpopulations (Crow and Aoki, 1984). The TFPGA im- iance analysis, or AMOVA (Excof®er et al., 1992), was used to describe plementation of the Mantel test (Mantel, 1967; Miller, 2000) was used to population structure where the variance was partitioned within and among correlate genetic and geographic distances among the four cacti populations ␾ populations. AMOVA determined the pair-wise genetic distances ( st) among in order to test for isolation by distance. the four populations, as well as the signi®cance levels of these distances. All the molecular variance analyses were performed with AMOVA-PREP v. 1.01 RESULTS (Miller, 1998) or WINAMOVA v.1.55 (Excof®er et al., 1992; Excof®er, ␾ Clonal diversityÐThe population-level survey detected 1993). WINAMOVA generates st, a parameter analogous to Wright FST (an indicator for the degree of differentiation among populations), and thus fa- high clonal diversity. In the 30 ramets that we examined from ␾ cilitates comparison of results with those from other studies. st was used to each population, we obtained RAPD banding pattern varia- calculate the number of migrants per generation entering each population tions that indicated a large number of distinct genets. The ϭ ␾ Ϫ ␣ ␣ϭ Ϫ 2 (Nm), using the formula Nm ((1/ st) 1)/4 , where (n/n 1) , and number of putative genets within each population ranged from February 2005] CLARK-TAPIA ET AL.ÐCLONAL DIVERSITY IN STENOCEREUS ERUCA 275

TABLE 1. Parameters of genetic and clonal diversity in four populations of Stenocereus eruca. (A) Among the populations. (B) Within a single population.

Population NHsPpGm G/ND E A) Population-level survey La Poza Grande 30 0.253 61.90 24 0.800 0.986 0.864 Santo Domingo 30 0.279 71.43 25 0.833 0.986 0.743 San Carlos 30 0.272 71.43 25 0.833 0.986 0.743 Estero Salinas 30 0.301 77.78 26 0.867 0.991 0.791 Mean 0.277 70.64 25 0.833 0.987 0.785 SD 0.020 6.55 0.027 0.003 0.057 B) Micro scale survey Estero Salinas 282 0.359 98.0 109 0.387 0.940 0.853 Note: N ϭ number of individuals sampled per population; Hs ϭ expected heterzygosity; Pp (99% criteria) ϭ percentage of polymorphic loci; ϭ ϭ ϭ ϭ Gm observed number of genotypes; G/N proportion of distinguishable genotypes; D genotype diversity; and E genotype evenness.

24 to 26 (Table 1). The proportions of distinguishable genets of S. eruca (Table 2). The average migration per generation

(G/N), genotypic diversity (D), and evenness (E) were gener- (Nm) was estimated to be 0.30. The Mantel test detected no ally high as well as similar across populations. evidence of isolation by distance, as genetic and geographical In the microscale survey at Estero Salinas, the clonal di- distances (in kilometers) among populations were not signi®- versity was lower than in the population-level survey (Table cantly correlated (r ϭ 0.808, P ϭ 0.158). 1). The 282 sampled ramets yielded 109 different putative gen- ets. The mean proportion of distinguishable genets (G/N) with- DISCUSSION in this site was 0.39. Estimates of Simpson's diversity index (D) and evenness (E) were 0.940 and 0.907, respectively (Ta- This study has shown that populations of S. eruca are com- ble 1). posed of a number of different genets (that is, they are mul- ticlonal) and are characterized by high levels of clonal diver- Spatial distributionÐThe spatial distribution of genets at sity. In general, ramets sharing the same genotype were spa- Estero Salinas is shown in Fig. 2. These genets had from 2 to tially aggregated (Ͻ20 m), while only a few genets were 41 ramets, with a mean number of 2.4 Ϯ 5.1 ramets (Ϯ1 SD), spread over larger distances. Signi®cant levels of genetic var- and 32% of the ramets had a different and unique putative iation were found within a single population, while substantial genet. Most of the sampled ramets from a particular genet differentiation was observed among populations. Overall, the were growing close to each other in a clumped distribution available evidence indicates that S. eruca combines sexual re- (Fig. 2). However, we found several genets with an extensive cruitment and clonal propagation as mechanisms of regener- distribution. The largest genets (numbers 15 and 17) had ation. spread throughout the entire plot (600 m2). The greatest num- Contrary to our expectations, which were based on ®eld bers of ramet pairs with identical RAPD band patterns were observations (Gibson, 1989; Turner et al., 1995), clonal di- separated by distances between 0 and 20 m and the number versity was relatively high across the distribution range of S. of identical genets decreased as distance among pairs in- eruca. This is a surprising result, as seedling recruitment has creased. not been detected in the ®eld and a demographic study cur- The correlogram of the 282 ramets from Estero Salinas re- rently in progress suggests that clonal propagation is the most vealed a signi®cant positive spatial autocorrelation over dis- important mode of regeneration (Clark-Tapia et al., in press). tances Ͻ7 m and negative spatial autocorrelation Ͼapproxi- However, our ®ndings of high clonal diversity levels are con- mately 29 m (Fig. 3a). The correlogram and the 99% con®- sistent with results from other short-term studies of plant spe- dence intervals of DG for the 109 genets show a similar pattern cies in which seedling recruitment has not been observed (Fig. 3b). Although the correlograms show similar spatial pat- (Jonsson et al., 1996; Verburg et al., 2000; Hangelbroek et al., terns at both the ramet and genet levels, with positive corre- 2002). Without seedling recruitment, clonal diversity is ex- lations at smaller spatial scales, it is important to note that the pected to rapidly decline and populations to become dominat- degree of relatedness (for example, the magnitude of DG) dif- ed by a few large genets (Eriksson, 1993; Watkinson and Pow- fered markedly between the two levels. Compared to the genet ell, 1993). Thus our evidence for high clonal diversity suggests level, the ramet level had relatively small signi®cant values of that sexual recruitment is a very important mechanism of re- Ͻ DG for the ®rst two distance classes ( 11 m, Fig. 3). generation in S. eruca populations. Seedling recruitment, how- ever, may be restricted to ``narrow windows of opportunity'' Genetic variationÐFrom a total of 75 reproducible bands (Jelinski and Cheliak, 1992; Eriksson and FroÈborg, 1996) analyzed, 57 (76%) were polymorphic at the species level. The when favorable conditions occur during rare heavy rainfalls. average percentage of polymorphic loci (Pp) among the four Those pulses might be rare temporal and spatial events that populations was 71.98 Ϯ 7.09 (ranging from 63.79 to are dif®cult to detect on the ecological time scale of demo- 81.03%), whereas in the single population at Estero Salinas it graphic studies. was 98%. The average genetic variation (Hs) among popula- The parameters of genotypic diversity and evenness found tions was 0.27 Ϯ 0.02 (ranging from 0.25 to 0.30), while in in S. eruca were within the value ranges detected in isozyme the plot at Estero Salinas the average Hs value was 0.36 (Table studies of other clonal species (Ellstrand and Roose, 1987; ␾ ϭ 1). Pair-wise AMOVA showed highly signi®cant ( st 0.337, WideÂn et al., 1994) and RAPDs (Hangelbroek et al., 2002). P Ͻ 0.002) genetic differentiation among the four populations Our estimates of population-level clonal diversity were greater 276 AMERICAN JOURNAL OF BOTANY [Vol. 92

Fig. 3. Correlograms plotting the Tanimoto distance (DG) for all Steno- cereus eruca individuals as a function of geographic distance (in m) at Estero Salinas: (a) genet level (N ϭ 109), and (b) ramet level (N ϭ 282). Open

circles are the observed DG values estimated using 5000 permutations. Closed circles represent the expected values, and the dashed lines represent the upper and lower 99% con®dence intervals. Note that the scales of the y-axes differ between the two graphs.

than those obtained from isozyme studies of Lophocereus schottii (Cactaceae) (Parker and Hamrick, 1992). Thus we sus- pect that S. eruca is not really the ``most remarkable case of clonal propagation in the cactus family'' (Gibson and Nobel, 1986). These differences in clonal diversity may be attribut- able to the differences in the resolution power of isozyme vs. RAPD analysis, such as those we have detected for S. eruca (G/N ϭ 0.53 in Clark-Tapia, 2000, compared to G/N ϭ 0.83 in the present study). The difference in clonal diversity ob- served between our population level survey (G/N ϭ 0.83) and our smaller-scale analysis (G/N ϭ 0.39) revealed a scaling effect associated with the sampling scheme. Clonal diversity usually shows an association with the sampling scale and the sampling grid's size. This is because collecting larger numbers of ramets at ®ner spatial scales increases the probability that the same genets will be sampled repeatedly (Cheliak and Pitel, Fig. 2. Spatial distribution of Stenocereus eruca genets in the Estero Sa- 1984; WideÂn et al., 1994). linas population, using 57 polymorphic RAPD markers. Repeated numbers Our data on the distribution of genotypes at Estero Salinas represent ramets from the same genet. Squares without numbers represent unique putative genets. Distances between genets in the map are proportional suggest that, in general, the ramets sharing the same genotype to distances measured in the ®eld. Genets represented by a large number of show a clumped distribution, while a few genets are widely ramets are shown in bold numbers. distributed. Most ramet pairs with identical genotypes were February 2005] CLARK-TAPIA ET AL.ÐCLONAL DIVERSITY IN STENOCEREUS ERUCA 277

␾ TABLE 2. Molecular variance analysis (AMOVA) summary for four population of Stenocereus eruca. The signi®cance (P)ofthe st test was based on 5000 permutations.

Variance % of total ␾ Source d.f. SS component variance st P Among populations 3 264.05 2.75 33.71 0.337 Ͻ0.002 Within populations 116 628.13 5.41 66.29 n/a n/a P: signi®cance; n/a: not applicable.

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