Ecology, 81(6), 2000, pp. 1554±1564 ᭧ 2000 by the Ecological Society of America

ASSESSMENT OF PARASITE-MEDIATED SELECTION IN A HOST±PARASITE SYSTEM IN

RODRIGO MEDEL Departamento de Ciencias EcoloÂgicas, Universidad de Chile, Casilla 653, Santiago, Chile

Abstract. A two-year ®eld study was conducted to evaluate the potential of two , Echinopsis chilensis and acida, to evolve defensive traits against the parasitic aphyllus (). The adaptive value of host traits against parasitism was inferred through: (1) identi®cation of the relevant characters of cacti to prevent infection, (2) evaluation of the ®tness impact of parasitism on cacti, and (3) estimation of the linear and nonlinear selection coef®cients on the relevant characters. Different lines of experimental and correlative evidence indicated that spine length was important in preventing individuals of the two cactus species from becoming parasitized. However, the impact of the mistletoe on cactus fecundity was contingent on the species involved. Even though parasitism decreased fruit production, seed number per fruit, and the total seed output in E. chilensis, low and nonsigni®cant linear and nonlinear gradients of selection were prevalent in this species, indicating absence of directional and stabilizing/ disruptive selection for spine length. Additional analysis based on logistic regression, how- ever, revealed that long-spined E. chilensis had a higher probability of reproduction than did short-spined individuals. Unlike its effect on E. chilensis, the mistletoe had no ®tness impact on E. acida, and the maintenance and evolution of spines in this species could not be attributed to parasite-mediated selection. Even though spines act as a ®rst line of defense against parasitism in the two cactus species, selection was detected only on E. chilensis. These results indicate that inferences on the adaptive value of host traits based only upon their role in preventing infection run the risk of overestimating parasite-mediated selection, and thus the potential for host±parasite coevolution. Key words: Cactaceae; cactus spines; Chile; coevolution; deserts; Loranthaceae; Mediterranean ecosystem; mistletoe; parasite; phenotypic selection.

INTRODUCTION 1989, Schluter and Nychka 1994), the adaptive value Host±parasite relationships have long attracted the of host traits against parasites has not been previously attention of ecologists because of suggestions that re- tested using this perspective. There is an important con- ciprocal adaptive responses may evolve as a result of ceptual distinction between the evolutionary response the antagonistic interaction (e. g., Price 1980, May and to natural selection and phenotypic selection. While Anderson 1982, Minchella et al. 1985, Toft et al. 1991, the evolutionary response to natural selection requires Thompson 1994, Yan and Stevens 1995). In spite of assessment of the genetic change that occurs from one its apparent simplicity, this idea has been extremely generation to the next, phenotypic selection describes dif®cult to evaluate empirically, and coadaptation of the immediate within-generation effects of natural se- host and parasite traits is usually assumed rather than lection on the statistical distribution of phenotypes, re- demonstrated. Implicit in most models of host±parasite gardless of the genetic basis and inheritance of char- coevolution is the idea that host characteristics pro- acters (Lande and Arnold 1983). In this paper I take viding defense against parasitism are adaptive and advantage of this methodology to inquire into the adap- evolve under parasite-mediated selection. However, a tive signi®cance of a putative defensive trait in two rigorous demonstration that a defensive trait is adaptive sympatric host species against a shared parasite. against parasitism requires, at least, (1) the existence Although parasitism by angiosperms is a widespread of a functional value for the trait in preventing para- phenomenon with ϳ1% of ¯owering plants being par- sitism, (2) that parasites have a negative impact on host asitic (Kuijt 1969, Musselman and Press 1995), studies ®tness, and (3) that variation in the defensive trait in- on the evolutionary consequences of parasitism in ¯uences host ®tness. Although recent methodological ±plant interactions have been only recently initi- ated (Norton and Carpenter 1998). Most studies as- advances for the analysis of phenotypic selection offer sessing adaptive traits in plants have focused on char- us a powerful set of tools with which to evaluate this acteristics relevant to seed dispersal (e. g., Jordano postulate (e. g., Lande and Arnold 1983, Mitchell-Olds 1995), pollination (e. g., Galen 1989, Johnston 1991, and Shaw 1987, Schluter 1988, Phillips and Arnold Eckhart 1993, Herrera 1993), and herbivory (e. g., Manuscript received 2 July 1998; revised 7 April 1999; ac- Rausher and Simms 1989, Simms and Rausher 1989, cepted 12 May 1999; ®nal version received 4 June 1999. Rausher 1992, NunÄez-FarfaÂn and Dirzo 1994, Mauricio 1554 June 2000 PARASITE-MEDIATED SELECTION IN PLANTS 1555 and Rausher 1997). Even though parasitic plants are vations made during three consecutive years indicate commonly thought to cause dramatic impacts on host that the only bird species responsible for disseminating plant ®tness, surprisingly few studies have assessed the seeds of T. aphyllus is the Chilean mockingbird their real impact in natural populations (but see GoÂmez Mimus thenca (Mimidae). The bird swallows the ripe 1994, Gomes and Fernandes 1994, Silva and Martinez fruits whole and defecates the seeds intact, no more del RõÂo 1996), and therefore the importance of parasite- than three seeds at a time. Mimus thenca tends to defend mediated selection in plants is unknown. feeding territories and favors the use of some cacti as This paper presents the results of a two-year ®eld perches most of the time, which results in large masses study on the potential of parasite-mediated phenotypic of mistletoe seeds being deposited on the cactus col- selection by the mistletoe, (Loran- umns. Once defecated by M. thenca, seeds turn from thaceae), on the columnar cacti Echinopsis chilensis white to pale brown in 2±3 d, and to red after ϳ10 d. and (Cactaceae). The investigation re- The naked seeds of T. aphyllus are covered entirely by ported here was designed to examine the adaptive value a viscid layer that adheres most of the time to the cuticle of the spines of cacti in preventing parasitism through and spines of cacti. Seeds germinate within a day after evaluation of its role in preventing bird perching and defecation and the bright red radicle grows up to 8 wk thus seed transmission to cacti. The adaptedness of or until it makes contact with the epidermis of the spines will be inferred through assessment of: (a) the cactus. After that, an haustorial plate is developed from functional role of spines, (b) the impact of T. aphyllus which several ®laments penetrate into the cactus tissues on cactus fecundity, and (c) the potential correlation (Mauseth et al. 1984, 1985). between spine length and cactus ®tness. Consideration Echinopsis chilensis and Eulychnia acida are colum- of these relationships will help elucidate the ecological nar cacti that inhabit north-facing slopes in north-cen- and evolutionary signi®cance of spines as a defensive tral Chile. Their reproduction is relatively synchronous, trait. More speci®cally, this study addresses the fol- with the ¯owering season extending from early Sep- lowing questions: (1) Does spine length affect individ- tember to mid-November, and the fruiting season from ual susceptibility to infection?; (2) What is the effect mid-October to late December or mid-January. The two of T. aphyllus on cactus fecundity?; (3) Does within- cactus species are characterized by broad variability in population variation in spine length translate into var- spine length both within and among populations (Run- iation in relative ®tness? Because all analyses were del 1976, Hoffmann 1986). Areoles of E. chilensis con- carried out separately for each host species, it is pos- sist of 1±2 long central scleri®ed spines that project sible to address comparative questions such as: (4) Do outward, and 8±12 short lateral spines that point in all cactus species exhibit similar responses to parasitism, directions. Areoles of E. acida consist of 1±2 long cen- and how comparable is the magnitude, sign and form tral spines and 10±13 short lateral spines. of the phenotypic selection on spine length? The an- This study was conducted from March 1994 to March swers to questions 1±4 relate to the more general one: 1996 in the Reserva Nacional Las Chinchillas (31Њ30Ј (5) What is the potential of cacti to evolve adaptations S, 71Њ06Ј W, AucoÂ, IV RegioÂn, Chile), ϳ300 km north in response to parasitism by T. aphyllus? Santiago. The climate is of a semiarid Mediterranean type with most rainfall concentrated in the winter sea- METHODS son. Mean annual precipitation is 185.0 mm with large variations from year to year, and frequent droughts (di Natural history and study system Castri and Hajek 1976). The total annual precipitation Tristerix aphyllus is an unusual lea¯ess holoparasitic in the Reserve was 43.0 mm in 1994 and 77.1 mm in mistletoe endemic to the arid and semiarid ecosystems 1995. The topography of the 4229-ha reserve is rugged of northern Chile that infects only species of the family and disrupted by several ravines. All measurements Cactaceae (Follman and Mahu 1964, Kuijt 1969, 1988). were made on populations located on a north-facing Unlike most Loranthaceae, this species has leaves that slope of Quebrada Torca (slope 25Њ±30Њ). are reduced to minute scales, and the only portion that emerges from the stems of cacti is its red in¯orescence. Sampling procedure The vegetative portion exists as an endophyte within I studied 286 individuals (218 E. chilensis and 68 the tissues of the cacti it parasitizes, and unlike most E. acida) exceeding 1 m in height, including almost hemiparasitic that tap the xylem of their every individual present on the slope. Each cactus was hosts, T. aphyllus parasitizes the phloematic vessels of tagged with a white plastic plate 14 cm in diameter the cacti (Mauseth et al. 1984, 1985). The fruiting sea- before the initiation of the seed dispersal season of T. son of the parasitic plant extends from mid±late March aphyllus in 1994. I measured the height of the tallest to early November, and the peak of fruit production column of each individual (precision 1 cm), and count- occurs in July±September, depending upon the year. ed the number of columns exceeding 10 cm in length. Fruits are single-seeded pseudoberries containing 80% Because the bird M. thenca tends to perch on the top water, that turn from green and red-opaque when unripe of columns (Martinez del RõÂo et al. 1995), I calculated to pale pink or translucent white when ripe. Obser- a mean spine length from ten apical spines measured 1556 RODRIGO MEDEL Ecology, Vol. 81, No. 6 with precision 0.1 mm. The individual status of cacti tion of long-lived species over years may more real- (parasitized or not parasitized) was recorded at the be- istically re¯ect differences in maternal ®tness than ginning of the study, and checked periodically for po- would a series of ®tness estimates from different re- tential emergence of in¯orescences of T. aphyllus. The productive episodes (Herrera 1993). Because the two parasite load supported by each individual was mea- approaches provide different perspectives on the same sured as the number of discrete in¯orescences of T. phenomenon, a more complete picture of phenotypic aphyllus emerging from the columns of cacti. The ac- selection may be achieved by performing the two anal- tivity of M. thenca was quanti®ed during 18 d in 1994 yses simultaneously. Character values were log-trans- and 16 d in 1995, always from sunrise to noon, from formed and standardized to have zero mean and unit a35Њ polar-facing slope ϳ200 m from the plot. This variance before analysis. This transformation provides distance proved to be appropriate for recording the standardized selection gradients, thus making compar- identity of the visited cacti as well as the time spent ison of phenotypic selection among years and species by the bird on each tagged individual. Because the possible. ␤i represents the average slope of the selection seeds of T. aphyllus are white for ϳ5 d after defecation, surface in the plane of the character i, and it is com- turning reddish after 10 d, it was possible to calculate puted as the partial regression coef®cient in a linear the daily infection rate (DIR hereafter) on each indi- regression of ®tness for the character. The magnitude vidual by recording the number of white seeds depos- of ␤i describes how much standardized ®tness changes ited on cacti every 20±25 d, and dividing the total of for a unit change in character i, holding all other char- white seeds recorded in the year by the days comprising acters constant. The sign of ␤i indicates the direction the seed dispersal season of each year. This method of change expected from selection acting directly on may underestimate the real DIR but avoids repeated character i. For ␥, the sign of the coef®cient indicates sampling of the same seed over time. The seed dispersal the curvature of the relation between the character and season of T. aphyllus comprised 171 d in 1994 and 180 ®tness. When the sign is negative, it re¯ects downward d in 1995. The fruit production of cacti was assessed concavity and stabilizing selection in¯uencing the through monthly censuses during the entire fruiting character i. When the sign is positive, it re¯ects upward season. The maximum number of ripe fruits recorded concavity and disruptive selection. ␥ is obtained by each year was considered to be an estimate of the year's regressing standardized ®tness on the characters and fruit production. This was a reasonable assumption, all pairwise products among characters. In addition to because most fruits in the same cactus develop in rel- selection gradients, I estimated the opportunity for se- ative synchrony up to maturity. A sample of ripe fruits lection, from the population variance in relative ®tness. was removed from cacti and maintained in the labo- This coef®cient describes the upper limit of the inten- ratory at Ϫ20ЊC prior dissection for seed counting sity of selection that can act on any character, and al- (mean number of fruits removed ϭ 3.43, range: 1±13). lows comparison of the potential of cacti to evolve in I calculated a mean seed number per fruit that allowed response to parasite-mediated selection. Linear and estimation of the total seed output per cactus from (fruit nonlinear standardized selection differentials for spine production) ϫ (mean seed number per fruit). length were also calculated for each year and for cu- mulative data. The standardized directional selection Statistical procedure differential, siЈ, re¯ects the extent to which selection The number of fruits, number of seeds per fruit, and shifts the mean of the character i between the actual total seed output were considered as indicative of ®t- and potential parents within a generation. siЈ was cal- ness. Fitness values were normalized to one by dividing culated as si /␴i, where si ϭ Cov(w, xi), w is the relative individual ®tness by the mean ®tness of the population. ®tness, and xi is the character i. The standardized non-

I performed multivariate analysis of phenotypic selec- linear selection differential, CiЈ, indicates the stan- tion as suggested by Lande (1979) and Lande and Ar- dardized change in the variance of character i between nold (1983), by considering height, number of columns, actual and potential parents that is attributable to se- and spine length as morphological characters of cacti. lection, excluding the effects of directional selection. 2 2 I calculated directional (␤) and quadratic (␥) selection CiЈ was calculated as Ci/␴ i, where Ci ϭ Cov(w, xi ). gradients separately for 1994 and 1995, and for cu- mulative ®tness over the 2 yr. I decided to assess phe- RESULTS notypic selection in separate and cumulative analyses Effects of spine length on the infection process for two reasons. First, patterns of phenotypic selection in plants usually vary among years depending of the Three lines of evidence corroborated the importance selection regime (e. g., Campbell 1989, Schemske and of spines as a defensive device against parasitism by Horvitz 1989, Herrera 1993). Because of high between- T. aphyllus. First, the number of visits and the time year variation in precipitation in the study site, separate spent by M. thenca perching on cacti were dependent annual analyses may help to identify singular selection upon spine length in the two cactus species. Except for events attributable to the prevailing selection regime the number of visits to Echinopsis chilensis in 1995, on a year-by-year basis. Second, cumulative reproduc- and the time spent on Eulychnia acida in the cumulative June 2000 PARASITE-MEDIATED SELECTION IN PLANTS 1557

TABLE 1. Effect of spine length on the infection process.

Echinopsis chilensis Eulychnia acida Year Visits Time DIR Visits Time DIR 1994 Ϫ0.155* Ϫ0.155* Ϫ0.152* Ϫ0.246* Ϫ0.308* Ϫ0.198* 1995 Ϫ0.119² Ϫ0.130* Ϫ0.152* Ϫ0.224* Ϫ0.214* Ϫ0.239* Pooled Ϫ0.161* Ϫ0.188** Ϫ0.143* Ϫ0.246* Ϫ0.221 Ϫ0.244* Notes: Numbers are standardized partial regression coef®cients of spine length on the activity of M. thenca and daily infection rate (DIR). Separate multiple regression analyses were performed on each dependent variable using cactus height and number of columns as covariates. ² P Ͻ 0.1; * P Ͻ 0.05; ** P Ͻ 0.01. data, all partial regression coef®cients were negative without spine removal, as similar as possible in height and signi®cant (Table 1). The negative effect of spines and number of columns to experimental individuals. on bird activity translated into a reduced DIR on the No individual presented external evidence of parasit- long-spined individuals. This result was consistent ism by T. aphyllus at the beginning of the experiment. among years (Table 1). Univariate contrasts for cu- Risk was calculated from the ratio (I/N), where I ϭ mulative data revealed that the spines of the unvisited number of experimental (or control) cacti that received E. chilensis and E. acida were 1.29 and 2.32 cm longer seeds, N ϭ number of experimental (or control) cacti. on the average than spines of the visited individuals Tests of the risk ratio among groups were carried out (Table 2). Similarly, the spines of E. chilensis and E. by Stata (1997) software for epidemiological analysis. acida without seed deposition were 0.84 and 3.05 cm Individuals were checked for deposited seeds of T. longer on the average, respectively, than spines of in- aphyllus after 156 d, at the end of the seed dispersal dividuals with deposited seeds. Because seed deposi- season of T. aphyllus. Control individuals of the two tion tends to occur on the previously parasitized cacti species did not receive seeds. Five experimental E. chi- (Martinez del Rio et al. 1995, 1996), most DIR values lensis, however, received a total of 15 seeds of T. aphyl- were null in the not parasitized individuals (Fig. 1). lus (range 1±5), thus indicating a higher risk of be- Pooling cacti in 1-cm spine length intervals allowed coming infected in comparison to control individuals comparison of the observed time spent by M. thenca (experimental ϭ 0.278, control ϭ 0; ␹2 ϭ 5.81, df ϭ perching on cacti against an expected time based on 1, P ϭ 0.016). Although two experimental E. acida random perching. Mimus thenca spent more time than received a total of 14 seeds, risks did not differ sig- would be predicted by the null expectation in the ®rst ni®cantly among groups (experimental ϭ 0.154, con- intervals of spine length in the two cactus species, trol ϭ 0, ␹2 ϭ 2.17, df ϭ 1, P ϭ 0.141). Pooling data showing a reversed trend for longer spines (G test, E. from the two species revealed that cacti with excised chilensis: G ϭ 14 642, df ϭ 8, P Ͻ 0.001; E. acida: spines had a higher overall risk becoming infected than G ϭ 21 786, df ϭ 7, P Ͻ 0.001, Fig. 2). Second, logistic did individuals with intact spines (experimental group regression of spine length on the presence and absence risk ϭ 0.226, control group risk ϭ 0, ␹2 ϭ 7.89, df ϭ of T. aphyllus, with cactus height and number of col- 1, P ϭ 0.005). umns as covariates, revealed that long spines decrease the probability of E. chilensis and E. acida being par- Fitness impact and parasite-mediated selection asitized (model II logistic regression, E. chilensis: odds Echinopsis chilensis.ÐParasite load had a clear and ratio, OR ϭ 0.813, b ϭϪ 0.207, z ϭ 3.035, P ϭ 0.007; consistent detrimental impact on the fruit production, E. acida:ORϭ 0.571, b ϭϪ0.560, z ϭ 2.744, P ϭ the seed number per fruit, and the total seed output as 0.006, Fig. 3). Third, in order to test experimentally revealed by the signi®cant partial regression coef®- the correlative evidences described above, in June 1995 cients, on every fecundity component (Table 3). Cu- I excised the apical spines of 18 E. chilensis and 13 E. mulative seed output of parasitized individuals was acida leaving only 1±2 cm of each spine. The risk of 67.2% lower on the average than for not parasitized these cacti receiving seeds of the mistletoe was com- cacti (Fig. 4). Regarding the opportunity for selection, pared with that of an equivalent number of control cacti E. chilensis consistently showed a higher variance than

TABLE 2. Mean spine length (cm; Ϯ SE) of cacti in relation to bird visits and seed deposition.

Visits by M. thenca Seed deposition by M. thenca Species Visited Not visited F Deposited Not deposited F Echinopsis chilensis 10.42 Ϯ 0.23 11.71 Ϯ 0.25 14.90*** 10.56 Ϯ 0.26 11.40 Ϯ 0.19 7.42** Eulychnia acida 11.86 Ϯ 0.67 14.18 Ϯ 0.29 16.83*** 10.93 Ϯ 0.88 13.98 Ϯ 0.29 16.86*** Note: Degrees of freedom are (1, 217) in Echinopsis chilensis and (1, 66) in Eulychnia acida. ** P Ͻ 0.01; *** P Ͻ 0.001. 1558 RODRIGO MEDEL Ecology, Vol. 81, No. 6

FIG. 1. Associations between spine length and daily infection rate in the parasitized and not parasitized individuals of Echinopsis chi- lensis and Eulychnia acida. Arrows indicate mean spine length. Polynomial equations: E. chilensis (parasitized): y ϭ 0.751 Ϫ 0.123x; E. chilensis (not parasitized): y ϭ 0.011 Ϫ 0.002x ϩ 0.0001x2; E. acida (parasitized): y ϭϪ2.086 ϩ 0.492x Ϫ 0.023x2; E. acida (not parasitized): y ϭ 0.005 Ϫ 0.001x.

E. acida in the seed number per fruit and the seed gression of parasite load and spine length on the pres- output ®tness components (Table 4). In spite of the high ence (1) and absence (0) of reproduction as expected variance in relative ®tness, no signi®cant linear (␤Ј) ®tness (see Brodie and Janzen 1996). Unlike least and nonlinear (␥Ј) gradients of selection were detected, squares regression, this procedure may only reveal di- indicating an absence of directional and stabilizing/ rectional selection, but says nothing regarding nonlin- disruptive selection on spine length (Table 5). Direc- ear selection (Price and Boag 1987). The probability tional and quadratic regression models accounted for of E. chilensis becoming reproductive tended to de- a small amount of the variance in the standardized ®tness crease with a unit increment in parasite load (Table 6). of E. chilensis (range of R2, linear model: 0.004%± These results were consistent among years and in cu- 0.041%, range of R2, nonlinear model: 0.012%± mulative data. Regarding spine length, positive and sig- 0.093%) (Table 5). Similarly, linear and nonlinear stan- ni®cant logistic regression coef®cients were observed dardized differentials were low and nonsigni®cant, im- in 1995 and in the cumulative analysis, indicating that plying that no substantial change in the mean and var- the probability of reproduction increased signi®cantly iance of spine length is expected between actual and with a unit increment in the spine length (Table 6). potential parents in this species. These results were Graphical depiction of cumulative data in E. chilensis consistent among years and in cumulative data on every revealed an asymptotic increase in the probability of ®tness component (Table 5). reproduction up to a spine length of ϳ12 cm, followed Even though parasitism had an important impact on by a slight decrease toward longer spines (Fig. 5). the fecundity of E. chilensis, this effect was not trans- Eulychnia acida.ÐUnlike the impact on Echinopsis lated into statistically signi®cant gradients and differ- chilensis, T. aphyllus had no signi®cant effect on any entials of selection. This discrepancy may be explained fecundity component of E. acida (Table 3, Fig. 4). Most by the small fraction of the within-population variation directional gradients of selection were low and nonsig- in the fecundity of this species that is attributable to ni®cant except in 1994, where long-spined cacti pro- T. aphyllus. For instance, T. aphyllus accounted for duced less fruits than did short-spined individuals (Table 15.5%, 11.4%, and 8.4% of the variation in the cu- 5). Borderline signi®cance was detected for a reduction mulative fruit production, seed number per fruit, and in fruit production and seed output with increasing spine seed output, respectively. These ®gures did not change length (fruits 1995: ␤Ј ϭ Ϫ0.204, t ϭ 1.74, P ϭ 0.087; substantially when data were examined on a yearly cumulative fruit data: ␤Ј ϭ Ϫ0.226, t ϭ 1.96, P ϭ basis. However, the absence of effect on the continuous 0.054; seeds 1994: ␤ЈϭϪ0.206, t ϭ 1.77, P ϭ 0.082) variation of ®tness does not necessarily imply that T. (Table 5). The linear regression models for directional aphyllus had no impact in suppressing E. chilensis re- selection accounted for a higher fraction of variance in production. For instance, systemic parasites such as the annual standardized ®tness in comparison to E. chi- endophytes, rusts, and smuts often cause complete sup- lensis (range R2: 0.159±0.279), and even though the ®t pression of reproduction in host plants (see reviews in was improved in the nonlinear models (range R2: Clay 1991, Clay and Kover 1996). Because consider- 0.255±0.431), all quadratic gradients were nonsigni®- ation of such a qualitative effect may reveal phenotypic cant, indicating the absence of stabilizing/disruptive selection otherwise undetectable in least squares re- selection (Table 5). Most linear and nonlinear selection gression, I explored such a possibility in more detail differentials were statistically signi®cant indicating an by using a generalized linear model in a logistic re- important total selection to reduce the mean and var- June 2000 PARASITE-MEDIATED SELECTION IN PLANTS 1559

iance of spine length (Table 5). However, because T. aphyllus had no impact on E. acida fecundity, I cannot suggest parasitism as the factor responsible for the de- creasing spine length in this species. No signi®cant logistic regression coef®cients were observed, indi- cating that parasite load and spine length did not affect the probability of individuals becoming reproductive (Table 6). The absence of impact of T. aphyllus on the fecundity of E. acida and therefore the lack of signi®cant selec- tion coef®cients attributable to parasitism may be ex- plained, in part, by the low frequency of parasitism on this species. A small number of individuals of E. acida are parasitized in the Reserve in comparison to E. chi- lensis (mean frequency of parasitism from 10 popu- lations, E. acida ϭ 14.7%, E. chilensis ϭ 46.6%). Be- cause the parasite prevalence on E. acida was 17.6%, it is unlikely that T. aphyllus could have a big selective impact on E. acida in the study site. Although the ques- tion why E. acida is less parasitized than E. chilensis was not the focus of this paper, it is possible that in comparison to E. chilensis, ϳ2.5 cm-longer spines found in this species represent a more ef®cient barrier against bird perching (mean spine length [cm] Ϯ SE, E. acida, 13.53 Ϯ 0.31, N ϭ 68; E. chilensis, 11.09 Ϯ 0.15, N ϭ 218).

DISCUSSION Different lines of correlative and experimental evi- dence indicate that spines were a relevant trait pre- venting parasitism in Echinopsis chilensis and Euly- chnia acida. Spines in¯uenced the perching behavior of M. thenca and therefore the daily infection rate on cacti. The impact of parasitism on cactus fecundity was FIG. 2. Observed and expected time spent by M. thenca on cacti (1994 and 1995 combined). The observed time on contingent on the host species involved. While every each spine-length interval was calculated by summing the fecundity component of E. chilensis tended to decrease time spent by the bird on every cactus included in the interval. with increasing parasite load, T. aphyllus had no de- Because intervals included a variable number of individuals, monstrable effect on any fecundity component of E. the expected time per interval was estimated from the product between the total time spent by the bird on cacti and the acida. In spite of these differences, parasitism contrib- fraction of the total population of cacti included in the in- uted to a small proportion of the within-population var- terval. iation in the fecundity of the two host species. This resembles conclusions from studies designed to ex- amine the importance of pollinator-mediated selection on ¯oral traits (Herrera 1996). The contribution of pol- lination to the variance in plant ®tness usually does not

FIG. 3. Cubic spline estimate (␭ϭ100) of the probability of Echinopsis chilensis and Eu- lychnia acida becoming parasitized in relation to spine length. Dots represent raw data values for the presence (1) and absence (0) of repro- ductive structures of T. aphyllus on cacti. 1560 RODRIGO MEDEL Ecology, Vol. 81, No. 6

TABLE 3. Impact of parasitism on the fecundity components of Echinopsis chilensis and Eulychnia acida.

Echinopsis chilensis Eulychnia acida Fecundity component 1994 1995 Pooled 1994 1995 Pooled Fruit production Ϫ0.430*** Ϫ0.262** Ϫ0.347** Ϫ0.097 Ϫ0.069 Ϫ0.125 Percentage 5.9 10.9 15.5 0.6 0.7 4.4 Seeds per fruit Ϫ0.423*** Ϫ0.260** Ϫ0.325** Ϫ0.068 Ϫ0.138 Ϫ0.164 Percentage 10.1 10.1 11.4 3.8 5.2 4.8 Seed output Ϫ0.412*** Ϫ0.252** Ϫ0.256* Ϫ0.064 Ϫ0.122 Ϫ0.088 Percentage 9.7 9.3 8.4 3.2 9.8 4.9 Notes: Numbers are standardized partial regression coef®cients of the parasite load (number of in¯orescence of T. aphyllus) on fecundity. Separate multiple regression analyses were performed for each fecundity component per year and for cumulative data using cactus height and number of columns as covariates. Percentage indicates the proportion of the within-population variation in fecundity that is attributable to parasitism. Percentage values were calculated from the variance component in a random effect model. * P Ͻ 0.05; ** P Ͻ 0.01; *** P Ͻ 0.001. exceed 8% (e.g., Schemske and Horvitz 1988, Herrera stricted set of environmental conditions. Parasitic 1993), a percentage similar to that documented in this plants may affect host ®tness by absorbing resources study. that are essential for host growth and reproduction. In spite of the higher opportunity for selection shown However, the way hosts are affected may depend not by E. chilensis relative to E. acida (Table 4) and the only on how much of their resources are captured by comparable coef®cients of variation in spine length parasites but also upon the supply available in the among species (E. chilensis CV ϭ 20.5%, E. acida CV environment, which may constrain the evolutionary ϭ 18.7%), parasite-mediated selection on E. chilensis was only detected in the context of logistic regression as a consequence of the suppression of reproduction in the parasitized and short-spined individuals (Table 6). Because nonsigni®cant gradients may not only indicate weak selection but also result from a small sample size, I calculated the minimum sample size required to achieve statistical signi®cance at the ␣ϭ0.05 level as (t␴/␤)2, where ␤ is the gradient of selection, ␴ is the standard deviation of ␤, and t ϭ 1.96 (Johnston 1991). Results indicated that the minimum sample size for detecting directional selection in pooled data was 1197 individuals in E. chilensis (pooled data, ␤ϭ1.238, ␴ ϭ 21.85). This implies that increasing sample size in E. chilensis may not only reveal phenotypic selection in logistic regression analysis but also in least squares regression. Unlike E. chilensis, phenotypic selection for de- creasing spine length in E. acida was detected in mul- tivariate linear regression, but not in logistic analysis. The minimum sample size required to detect a sig- ni®cant selection gradient was 68 individuals (pooled data, ␤ϭϪ6.188, ␴ϭ26.02), which indicates that the sample size considered in this study was suf®cient to detect statistical signi®cance. However, even though the observed selection coef®cients are real, improved ®t may be achieved by increasing sample size. The reason why spines of E. acida, even though functional in preventing infection, tend to evolve to- ward shorter states is intriguing. Three, not mutually exclusive, potential explanations can account for such phenomenon. FIG. 4. Mean seed output of parasitized and not parasit- First, it is possible that T. aphyllus has a negative ized individuals of Echinopsis chilensis (F ϭ 10.88) and Eu- ®tness impact only in exceptionally dry years, with lychnia acida (F ϭ 2.03) in 1994 and 1995. Bars represent parasite-mediated selection limited only to a very re- 1 SE. June 2000 PARASITE-MEDIATED SELECTION IN PLANTS 1561

TABLE 4. Opportunity for selection (variance in relative ®t- at present due to the lack of impact of T. aphyllus on ness) in Echinopsis chilensis and Eulychnia acida. cactus fecundity. The last important glacial period in Year Fitness component E. chilensis E. acida F semiarid Chile ended ϳ14 000 years ago (Mercer 1972), followed by an intense warming trend at the 1994 Fruit production 5.69 5.07 1.12 Seeds per fruit 6.60 0.99 6.64*** Pleistocene/Holocene transition that culminated ϳ6500 Seed output 11.36 5.63 2.02*** years ago, when temperatures were at least 1Њ±3ЊC 1995 Fruit production 4.92 5.97 0.82 above current values (Solbrig et al. 1977, Ortlieb et al. Seeds per fruit 6.94 1.29 5.40*** Seed output 11.78 6.04 1.95*** 1996, Veit 1996). Current spine length in E. acida may Pooled Fruit production 3.76 5.51 0.68² thus represent the product of intense selection that oc- Seeds per fruit 4.94 0.67 7.32*** curred in the past rather than in present conditions. The Seed output 11.59 6.53 1.77** lack of adaptive value of long spines in E. acida sug- Note: F values result from Bartlett's test for homogeneity gests this trait might be better considered anachronic of variance. to parasitism, if demonstration of its adaptive value ² P Ͻ 0.1; * P Ͻ 0.05; ** P Ͻ 0.01; *** P Ͻ 0.001. against parasitism in the past is provided. In the same vein, herbivory may also be invoked as responsible for response of the plant hosts. Water availability is an the evolution of cactus spines. Janzen (1986) suggested obvious factor affecting the fecundity of cacti (Gul- that herbivorous species were the main selective agents mon et al. 1979, Gibson and Nobel 1986, Silva and for the evolution of spines in nopaleras (Opuntia). It Acevedo 1995). Even though cacti are better protected is possible that the spine length shown by E. acida at against desiccation than most other plants in arid and present evolved as a defensive device against a Chilean semiarid zones, availability of summer water often megafauna that disappeared around the late Pleistocene sets limits to photosynthesis which may ultimately (Marshall 1981). These two ``anachronism'' hypothe- translate into limitations in fruit and seed production ses, although dif®cult to test unequivocally, advocate (Mooney 1983, Lechowicz 1984, Tenhunen et al. not only a lack of adaptation of spines to parasitism 1985). Because T. aphyllus represents a sink for water but also a cost of maintenance for long-spined indi- and inorganic carbon nutrients otherwise available to viduals. The absence of impact of T. aphyllus on the cacti, its contribution to the population variation in fecundity of E. acida suggests that the maintenance of cactus fecundity may be contingent on the water avail- long spines may represent an unnecessary or even mal- ability in the study site. Moreover, long-lasting adaptive investment of resources that otherwise could droughts and short pulses of high precipitation de®ne be allocated to growth and reproduction. It is known the between-year climatic conditions in north-central that investment in defense against herbivory and path- Chile as a consequence of El NinÄo events (di Castri ogens represents an important component of the life- and Hajek 1976, Ortlieb 1994). If current ®tness of time resource budget of plants (Bazzaz et al. 1987). cacti comes from resources stored in past years, then Most models of the evolution of resistance assume that current phenotypic selection may not necessarily re- defensive traits have a ®tness cost, because the pro- ¯ect the prevailing selective regime imposed by T. duction and maintenance of such structures often de- aphyllus, but rather the environmental conditions of mands energy that could be used for vegetative or re- previous years. productive tissue (e. g., Gulmon and Mooney 1986, Second, long spines may be a consequence of par- FagerstroÈm et al. 1987, Simms and Rausher 1987). Al- asite-mediated selection that occurred in the past under though ®tness costs have been demonstrated in a va- more stringent conditions for cacti, but are unnecesary riety of species (e. g., Coley et al. 1985, Coley 1986,

TABLE 5. Gradients and differentials of selection for spine length (cm) in Echinopsis chilensis and Eulychnia acida.

Echinopsis chilensis Eulychnia acida Year Fitness component ␤Ј SЈ CЈ ␤Ј SЈ CЈ 1994 Fruit production 0.050 Ϫ0.048 Ϫ0.761 Ϫ0.237* Ϫ0.815** Ϫ8.045** Seeds per fruit 0.022 Ϫ0.059 Ϫ0.935 Ϫ0.031 Ϫ0.159 Ϫ1.749 Seed output 0.086 0.046 0.007 Ϫ0.206² Ϫ0.785** Ϫ7.716* 1995 Fruit production 0.027 0.062 0.366 Ϫ0.204² Ϫ0.800** Ϫ7.911* Seeds per fruit 0.065 0.177 1.704 0.146 Ϫ0.025 Ϫ0.339 Seed output Ϫ0.002 Ϫ0.019 Ϫ0.373 Ϫ0.135 Ϫ0.656* Ϫ6.954* Pooled Fruit production 0.059 0.068 0.393 Ϫ0.226² Ϫ0.826** Ϫ8.138** Seeds per fruit 0.051 0.060 0.397 0.082 Ϫ0.096 Ϫ1.082 Seed output Ϫ0.009 Ϫ0.094 Ϫ1.296 Ϫ0.178 Ϫ0.778** Ϫ7.627* Notes: ␤Ј indicates standardized directional gradients of selection; SЈ and CЈ indicate standardized linear and nonlinear differentials of total selection, respectively. P values for selection differentials were calculated from Pearson's product- moment correlation. ² P Ͻ 0.1; * P Ͻ 0.05; ** P Ͻ 0.01. 1562 RODRIGO MEDEL Ecology, Vol. 81, No. 6

TABLE 6. Logistic regression coef®cients (Ϯ SE) of parasite load and spine length (cm) on the cumulative probability of cacti becoming reproductive.

Species Variable Coef®cient Odds ratio z Echinopsis chilensis Parasite load Ϫ0.038 Ϯ 0.009 0.963 4.34*** Spine length 0.151 Ϯ 0.066 1.163 2.30* Eulychnia acida Parasite load Ϫ0.061 Ϯ 0.054 0.941 1.13 Spine length Ϫ0.164 Ϯ 0.170 0.848 0.96 Notes: Cactus height (m) and number of columns were used as covariates in analyses. P levels were estimated from Wald's test. * P Ͻ 0.05; *** P Ͻ 0.001.

Simms and Rausher 1989, AÊ gren and Schemske 1993), co-opted functional role to prevent parasitism (ex- it is becoming increasingly clear that allocation of re- aptations in the terminology of Gould and Vrba 1982, sources to defense is expected to occur until the bene®t Reeve and Sherman 1993). Because spines of E. acida of investment becomes limited by costs (e. g., Sagers are signi®cantly longer than those exhibited by non- and Coley 1995, Mauricio and Rausher 1997, Mauricio parasitized congeneric species (R. Medel, unpub- 1998). lished data), it is unlikely that actual spines result Third, long spines in E. acida may result from phy- from phylogenetic inertia. logenetic inertia rather than having evolved by natural In this paper I assessed the potential of two cactus selection for its defensive function. Although ques- species to evolve adaptations under parasite-medi- tions pertaining to the origin of the character can be ated selection. The adaptive value of spines against better assessed by phylogenetic studies of adaptation parasitism was inferred through examination of the (Reeve and Sherman 1993, Losos 1994, Larson and following three conditions: (a) the importance of Losos 1996), spines perform important physiological spines in preventing infection, (b) the impact of par- functions in cacti. For example, they protect the stems asitism on cactus fecundity, and (c) correlation from damaging wavelengths of solar radiation (Gib- among spine length and relative ®tness. Results from son and Nobel 1986) and also create a boundary layer this study revealed a picture far more complex than between stems and the external environment that re- previously thought. The adaptedness of spines was duces evapotranspiration as well as the risks of over- not similar in the two host species, and conclusions heating and freezing damage from extreme tempera- about parasite-mediated selection are necessarily tures (Nobel 1978, Gibson and Nobel 1986, Nobel contingent upon the host species involved. Even 1988). Although I cannot rule out the importance of though different lines of evidence indicate that these abiotic factors for the evolution of spines in spines prevented infection in E. chilensis and E. aci- cacti, such hypotheses are more concerned with the da (condition ``a'' veri®ed for the two cactus spe- original adaptive value of spines rather than with their cies), T. aphyllus had a detrimental impact only on the fecundity of E. chilensis (condition ``b'' veri®ed for E. chilensis but not for E. acida). While parasite- mediated selection on the relative ®tness could not be detected, suppression of reproduction attributable to parasitism, and thus parasite-mediated selection for long spine length was observed in E. chilensis (condition ``c'' veri®ed for E. chilensis but not for E. acida). But why, if spines are important to prevent infection in E. acida, are they not only inconse- quential in terms of ®tness advantage but even mal- adaptive at present? The answer to this question may require a long-term study where the various envi- ronmental factors that directly and indirectly affect the impact of parasites on host ®tness can be quan- ti®ed during several host reproductive events. The extent to which parasite-mediated selection on cacti varies over time needs to be assessed in future stud- ies. In the absence of such information, inferences on the adaptive value of host traits against parasitism FIG. 5. Cubic spline estimate (␭ϭ100) of the ®tness that rest only upon their ecological function to pre- function for Echinopsis chilensis in relation to spine length. Dots represent raw data values for the cumulative probability vent infection may ignore the real evolutionary sig- of reproduction (1), and absence of reproduction (0). ni®cance of host characters, and thus may lead to June 2000 PARASITE-MEDIATED SELECTION IN PLANTS 1563 erroneous conclusions about host-parasite coevolu- Gould, S. J., and E. S. Vrba. 1982. Exaptation: a missing tion. term in the science of form. Paleobiology 8:4±15. Gulmon, S. L., and H. A. Mooney. 1986. Costs of defense ACKNOWLEDGMENTS and their effect on plant reproduction. Pages 681±698 in T. J. Givnish, editor. On the economy of plant form and I thank R. Bustamante, P. GarcõÂa-Fayos, C. Hernandez, M. function. Cambridge University Press, Cambridge, Mas- Mendez, L. Pacheco, C. Ramirez, E. Rivera, A. Silva, C. sachusetts, USA. Smith, and E. 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