Genetic variation relevant for the conservation of thenarrow endemic Senecio carbonensis (Asteraceae) fromthe southern Andespsbi_316 145..157

MARTINA FERNÁNDEZ, CECILIA EZCURRA, MARIA PAULA QUIROGA and ANDREA C. PREMOLILaboratorio Ecotono, Centro Regional Universitario Bariloche, Universidad Nacional del Comahue, INIBIOMA—CONICET,Quintral 1250, 8400 Bariloche, Argentina

Abstract

We studied morphological variation, reproductive biology and genetic structure ofSenecio carbonensis, a narrow endemic from high elevations of the southern Andes, andcompared it to Senecio peteroanus, a closely related widespread congeneric species. UsingANOVAs and discriminant analysis we showed that populations of S. carbonensis weremore similar to each other in their morphology, had comparatively limited reproductivecapacity and presented lower plant density and more reduced plant cover than popula-tions of S. peteroanus. Similar high genetic variation was found at the population andspecies level in both species, based on isozyme variation at 14 and 11 putative gene locithat were resolved in S. carbonensis and S. peteroanus, respectively. The two species weregenetically distinct. However, the small genetic distance between populations of eachspecies suggests that either they were recently founded and that genetic drift did not havetime to promote divergence, or that its effects are masked by significant current gene flow.These results show that ecological and reproductive characteristics of S. carbonensis maylimit its extension and abundance. In contrast, high genetic variation at the populationlevel is probably favored by self-incompatibility. Despite the restricted geographicaldistribution and low plant density of S. carbonensis populations, genetic characteristicsdo not seem to be limiting their long-term persistence. This information suggests thatpopulations of S. carbonensis are not at risk of extinction as a result of genetic factors,although it is important to study and monitor population dynamics to further assess thedegree of recruitment through time.

Keywords: alpine, genetic diversity, isozymes, morphology, rare species.

Received 8 June 2010; accepted 17 November 2010

Introduction

Species with restricted geographic distributions and lowdensities, and that grow in relatively specific habitats arepotentially more vulnerable in terms of their long-termpersistence than those with wide distribution, high den-sities, and whose populations are less isolated (Rabinow-itz 1981; Rabinowitz et al. 1986). Range-restricted plantsare particularly vulnerable to habitat loss, which mayresult not only in local population extirpation, butalso, under extreme situations, in species extinction. In

particular, demographic and genetic factors associatedwith reductions in population size may affect populationviability if such modifications alter historical populationdynamics and patterns of mating and gene flow. In addi-tion, reproductive and genetic factors often act synergis-tically because extinction is fundamentally a demographicprocess influenced by genetic and environmental factors(Lande 1988; Booy et al. 2000). Therefore, demographicstudies that take into account reproductive rates andpopulation densities provide important information withregard to the processes that can limit the size and stabilityof populations (Schemske et al. 1994; Bevill & Louda1999).

In rare species, the amount of genetic variation withinand among populations can contribute to knowledge on

Correspondence: Martina FernándezEmail: mfernandez@comahue-conicet.gob.ar

Plant Species Biology (2011) 26, 145–157 doi: 10.1111/j.1442-1984.2011.00316.x

© 2011 The AuthorsJournal compilation © 2011 The Society for the Study of Species Biology

the distribution of genetic polymorphisms that may influ-ence their evolutionary potential (Hamrick & Godt 1996;Ge et al. 1999). Genetic characteristics are important in thatrare species often consist of small and isolated popula-tions that tend to lose genetic variation and divergegenetically as a result of random drift and limited geneflow (Wright 1931, 1951). This, together with inbreeding,can produce fixation of deleterious alleles and increaseinbreeding depression (Charlesworth & Charlesworth1999), thus influencing the fecundity and survival of smallpopulations (Young et al. 1993, 1999; Couvet 2002). Inaddition, severe bottlenecks and founder events in thepast can have a large effect on the current level of geneticdiversity (e.g. O’Brien 1994; Sun 1997).

Several surveys of isozyme variation in plants haveshown that rare, endemic or narrowly distributed plantstend to maintain lower isozyme variation than wide-spread species (Karron 1987; Hamrick & Godt 1989, 1996;Premoli 1997; Gitzendanner & Soltis 2000), and havelimited ability to adapt to changing environmental condi-tions (Rymer & Ayre 2006). However, there are also casesin which genetic variation is relatively high for rarespecies (Linhart & Premoli 1993), even within extremelynarrowly distributed taxa (Soltis & Soltis 1991; Cosner &Crawford 1994; Lewis & Crawford 1995). Nevertheless,results may differ depending on the criteria used to deter-mine rarity, which is generally based on local abundance,geographic range and habitat specificity (Rabinowitz1981; Rabinowitz et al. 1986), and life-history attributes ofthe species (Premoli 1997; Premoli et al. 2001; Rymer &Ayre 2006). Given that phylogenetic relatedness of speciesmay have important effects on the outcome of compara-tive studies (Harvey & Pagel 1991), growing interest existsin conservation genetic studies comparing phylogeneti-cally close congeneric species that differ in abundance/forms of rarity (e.g. Karron et al. 1988; Soltis & Soltis 1991;Linhart & Premoli 1993; Lewis & Crawford 1995; Premoli1997; Ge et al. 1999; Gitzendanner & Soltis 2000; Premoliet al. 2001; Quiroga 2009).

High mountain environments of the southern Andesconsist of a number of microhabitats with different envi-ronmental conditions determined by varying factors, suchas precipitation, altitude, slope exposure, solar radiationand soil type (Ferreyra et al. 1998b). These microhabitatscontribute to great environmental diversity that hasallowed the evolution and establishment of a highnumber of species, including several restricted endemics(Ezcurra et al. 1995; Ferreyra et al. 1998a). It has generallybeen found that the number of rare species at a site tendsto be positively correlated with species richness (Gaston1994). In contrast, these high elevation environments arecurrently characterized by their vulnerability. Currentglobal climate change can drastically reduce the area ofthese ecosystems, and growing tourism activities and the

introduction of exotic plant species and cattle grazing cancontribute to the deterioration and fragmentation of thesehabitats (Ferreyra et al. 2006).

We evaluated the hypothesis that the degree of rarity ofa species is related to its ecological, reproductive andgenetic characteristics. We analyzed the morphologicalvariation, reproductive biology, and the amount and dis-tribution of genetic diversity in two closely related speciesof Senecio from high elevation areas of the southernAndes. These species are the narrow endemic Senecio car-bonensis Ezcurra and its widespread congener Seneciopeteroanus Phil. We were particularly interested inwhether the rare endemic S. carbonensis is genetically dep-auperate compared with its widespread congener, whichin combination with different ecological and reproductivecharacteristics may affect the viability of its populations.Such information can contribute to a better understandingof the causes of the rarity of S. carbonensis and may be usedin developing recommendations for its conservation andmanagement. Together with other rare endemic species(e.g. Úbeda et al. 2002), S. carbonensis is a focal species of acritical area of conservation priority of Nahuel HuapiNational Park, Argentina. Continued study of the causesof rarity will also improve the theoretical and practicalfoundations of conservation programs of other threatenedspecies (Gaston 1994).

Materials and methods

Plant species and study area

Senecio carbonensis is a narrow endemic plant restricted tothe high mountain habitats of the semiarid region in theeast of Nahuel Huapi National Park in southern Argen-tina, at elevations of 1550–1750 m a.s.l. (Ezcurra et al.1995). Although the mountains of this area have beenthoroughly explored, particularly over the past few years(Ferreyra et al. 1998a), only two populations of this specieshave been found to date (Fig. 1). In contrast, the wide-spread and sympatric S. peteroanus is widely distributed inhigh-elevation areas of the southern Andes from 35° to42°S in Chile and Argentina (Cabrera 1971). The twospecies are very similar morphologically, which suggeststhat they are phylogenetically related (Ezcurra et al. 1995).Both species are herbaceous perennials that reproducevegetatively by rhizomes as well as by seed, and producewind-dispersed fruits (achenes) with hairy pappus.

The study was carried out in 2007 in two populations ofeach species approximately 5–10 km apart from eachother. Senecio carbonensis was studied on Mt. Carbón andMt. Ñireco, and S. peteroanus was studied on Mt. Chall-huaco and in the valley of Río Ñirihuau. All populationsare located at eastern Nahuel Huapi National Park, Argen-tina (Fig. 1; Table 1). At these sites the weather is cold most

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of the year, with precipitations that largely fall duringwinter in the form of snow. The first three mountain sitesare characterized by treeless alpine environmentsbetween 1500 and 1750 m a.s.l. The site of S. peteroanus atRío Ñirihuau, although just above 1000 m a.s.l, occupies acold valley bottom that favors the establishment of high-mountain species in combination with species from thenearby Patagonian steppe.

To determine whether characteristics of the species andpopulations were related to soil properties, edaphic analy-ses were conducted from soil samples taken in the fourpopulations. These consisted of approximately 200 cm3 ofsoil collected from a depth of 10 cm from three randomlychosen points in each population. Soil samples were ana-lyzed at the Chemistry Department, Universidad delComahue, Bariloche, Argentina. The variables measuredwere pH in H2O (soil : water = 1:2.5) (Thomas 1996), pH insodium fluoride solution (soil : solution NaF = 1:50) after2 and 60 min of solid–liquid contact, and electrical con-ductivity (soil : water = 1:5) (Blakemore et al. 1987). Thesecharacteristics reflect important soil properties in relationto plant growth, such as the degree of acidity or alkalinityand the concentration of salts and macronutrients.

Morphological and reproductive study

In each population a plot measuring 15 m ¥ 15 m wasestablished. Within each plot, we randomly selected 10individuals separated from each other by at least 10 cm toavoid sampling the same genet. These plants were labeledwith a permanent aluminum tag and several morphologi-cal and reproductive variables were measured. For labo-ratory analysis, we collected one leaf from the middle partof the stem and one flower head from each individual.These were mounted on paper and scanned using aCanoscan N340P (Canon Inc., Japan). Digital images wereused to measure leaf traits using the program SigmaScanPro v. 5 (SPSS Science, Chicago, IL). Leaf size was quanti-fied by length, width, area and perimeter. Leaf shape wasdescribed using the length to width ratio and the degreeof incision by dividing the perimeter by the square root ofthe area (Steinke et al. 2008). We also measured andcounted the number of teeth on the leaf margins, and thelength of cilia or mucros homologous of the cilia in thetips of the teeth of the leaf margins of S. carbonensis and S.peteroanus, respectively. With regard to the reproductivevariables, in the laboratory we measured the length andwidth of the flower heads, number of bracts, number oftotal fruits and number of healthy and insect-damagedfruits in each flower head.

In the field, we estimated plant density in each popu-lation by directly counting all individuals within eachplot. Plant cover for each species and population was esti-mated using the mean of the variables minimum andmaximum diameter of the 10 individuals measured ineach population, and then multiplying this value by thetotal number of individuals. To analyze the capacity ofsexual reproduction of populations we measured the fre-quency of flowering ramets per individual and fruit pro-duction per flower head. Fruit production was evaluatedas the mean number of healthy fruits collected in 10flower heads from plants selected at random in eachpopulation. Based on these variables we calculated theamount of fruit produced annually by each of the popu-lations per plant using the following calculation: numberof flowering ramets per individual ¥ number of fruits perflower head. We also calculated fruit production per m2

for each population. Using these parameters, we esti-mated the opportunities for population growth and plantdescent via sexual reproduction.

Isozyme characterization

In January 2007, fresh leaf samples were collected from 50randomly selected individuals from each population; 10 ofthese individuals were the same as those used in themorphological analysis. Samples were kept refrigerateduntil enzyme extraction in the laboratory. Enzymes

Fig. 1 Location of the four sampled populations of Senecio.Senecio carbonensis at Mt Carbón ( ) and Mt Ñireco (�) andSenecio peteroanus in the valley of Río Ñirihuau (�) and at MtChallhuaco (�) within Nahuel Huapi National Park, Argentina.

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were extracted using the buffer of Mitton et al. (1979).Homogenates were stored at -80°C until horizontal elec-trophoresis on 12% v/v starch gels (Starch Art Corpora-tion, Smithville, TX, USA) was conducted. A total of 14loci in S. carbonensis and 11 loci in S. peteroanus wereresolved using two buffer systems. These were: (i)morpholine–citrate buffer system (Ranker et al. 1989),which ran for 5 h at 25 mA and was used to resolveglycerate-2-dehydrogenase (G2d), isocitrate dehydroge-nase (Idh), phosphoglucoisomerase (Pgi1, Pgi2) and shiki-mate dehydrogenase (Skdh); and (ii) tris–citrate buffersystem (King & Dancik 1983), which ran for 5:5 h at 35 mAand resolved malate dehydrogenase (Mdh1, Mdh2), malicenzyme (Me), menadione reductase (Mnr1, Mnr2),cathodic peroxidase (Per1, Per2) and phosphoglucomu-tase (Pgm1, Pgm2). Electrophoresis was carried out at 4°Cuntil the bromophenol blue marker had moved 10 cmfrom the origin towards the anode. The anodic portion ofthe gels was sliced horizontally in four sections, each1 mm thick. Stain procedures for the studied enzymesfollowed commonly used protocols reported elsewhere(Conkle et al. 1982; Soltis et al. 1983). The alleles werenumbered sequentially, with the lowest number indicat-ing the fastest moving towards the anode. The loci areconsidered putative because we did not carry out quanti-tative genetic analysis in segregating individuals of thesespecies. However, the observed banding patterns weretypical of the same enzymatic systems reported in speciesfor which formal analyses have been conducted (Murphyet al. 1996).

Morphological and reproductive data analyses

The vegetative and reproductive variables and leaf mor-phological traits of the four populations were analyzedusing one-way ANOVAs. We assessed the degree towhich a given population differed from others by ana-lyzing each variable using a Tukey’s post-hoc test formultiple comparisons of means. In cases when the datadid not fit a normal distribution, a non-parametricKruskal–Wallis test was carried out, with pairwise com-parisons using Dunn’s method for post-hoc multiplecomparisons. In both cases, the probability levels wereadjusted by Bonferroni (Rice 1989). We also carried out adiscriminant analysis that classified individuals into dif-ferent a-priori established groups from the combinationof populations and species. A Wilk’s l (lambda) test wasused to determine whether the groups present signifi-cant differences in the position of their centroids.Squared Mahalanobis distances between the group cen-troids were calculated to study the discriminatory powerbetween any four groups (Legendre & Legendre 1983).Variables that did not fit a normal distribution were notincluded in this analysis.

Isozyme data analyses

We calculated genetic variability at the species and popu-lation level including: the mean number of alleles perlocus (A), number of alleles per polymorphic locus (AP),effective number of alleles (AE), total number of alleles(AT), number of unique alleles (AU) (i.e. those present inonly one population), number of rare alleles (AR) withfrequencies <5%, number of diagnostic alleles (AD) (i.e.those present in only one species), percentage of polymor-phic loci using the sensu stricto criterion (PSS), andobserved and expected heterozygosity under Hardy–Weinberg equilibrium (HO and HE, respectively) usingPOPGENE v1.32 (Yeh et al. 1999). Heterogeneity in genefrequencies across populations was evaluated using ac2-test following Workman and Niswander (1970).

Departures from Hardy–Weinberg expectations wereevaluated for each polymorphic locus in each populationby calculating Wright’s (1922) fixation index (F = 1 - [HO/HE]). Significant deviations of F from zero were testedusing a c2-test (Li & Horvitz 1953). Population geneticstructure was measured using F statistics: FIS is the within-population inbreeding coefficient and FST indicates thedegree of among-population divergence, which is also anindirect estimator of gene flow (Wright 1965). The degreeof significance of such estimations was analyzed by meansof 95% confidence intervals that were computed by per-mutations over all loci following Weir and Cockerham(1984) using FSTAT version 2.9.1. (Goudet 2002). Totalgenetic diversity (HT) and the proportion of the totaldiversity found within populations (HS) were analyzedaccording to Nei (1973). A matrix of between-populationsand species genetic distance was calculated using Nei’s(1978) unbiased index. Similarly, a Unweighted PairGroup Method with Arithmetic Mean (UPGMA) multi-variate cluster analysis was used to generate a phenogramof genetic distance among populations and species usingSTATISTICA version 6.1. The isozyme loci Mnr1 and Pgmwere removed from genetic distance calculations becausethey did not present activity in S. peteroanus.

Results

Morphological and reproductive study

The ANOVAs carried out on vegetative and reproductivevariables in the studied populations of Senecio resulted instatistically significant differences for 18 of the 20 vari-ables measured (Table 1). Most variables differed betweenspecies: S. carbonensis had plants of smaller diameter withmore toothed and shorter leaves, and produced a lowernumber of flowering ramets per plant. In contrast, the sizeof flower heads and the number of fruits per head washigher in S. carbonensis. Other vegetative variables such as

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Table 1 Environmental and morphological characteristics of populations of Senecio carbonensis (Sc) and Senecio peteroanus (Sp)

TraitsSpecies/population

F HSc-Carbón Sc-Ñireco Sp-Ñirihuau Sp-Challhuaco

Environmental variablesLatitude (S) 41°12′10.4″ 41°13′38″ 41°17′37.4″ 41°16′10.8″Longitude (W) 71°17′25.6″ 71°20′11.4″ 71°15′12.9″ 71°16′56″Elevation (m a.s.l.) 1538 1720 1067 1557Precipitation (mm) 1600 1900 1700 1700Vegetation cover (%) 12,0 16,0 50,5 8,0

Vegetative variablesHeight (cm) 4.8a 7.5b 11.1c 7b 22.11*

(1.68) (1.58) (2.37) (1.15)Minimum diameter (cm) 5.2a 6.9a 9.3ab 12.4b 8.11*

(1.87) (2.76) (4.11) (4.47)Maximum diameter (cm) 10.3a 10.7a 18.7b 23.6b 9.69*

(3.16) (3.36) (8.43) (8.94)Height/diameter 0.68ab 0.96b 0.88ab 0.45a 3.98*

(0.37) (0.45) (0.34) (0.23)No. ramets 13.4 21.5 22.7 21.8 0.99

(6.81) (8.84) (20.18) (14.87)Leaf morphology

Length (cm) 0.69a 0.82a 1.09b 0.9b 7.57*(0.07) (0.22) (0.23) (0.19)

Width (cm) 0.32ab 0.39b 0.26a 0.31ab 7.53*(0.04) (0.06) (0.05) (0.06)

Length/width 2.18a 2.11a 4.27c 2.92b 23.74*(0.33) (0.38) (1.01) (0.65)

Perimeter (cm) 1.7a 2.05ab 2.42b 2.04ab 4.73*(0.24) (0.53) (0.43) (0.45)

Area (cm2) 0.11a 0.16ab 0.18b 0.15ab 2.84*(0.02) (0.06) (0.07) (0.05)

Perimeter/√Area 4.9a 4.9a 5.59b 4.98a 4.77*(0.37) (0.45) (0.62) (0.39)

Length of cilia or mucrones 1.86a 1.62a 0.74b 0.54b 28.15*(0.63) (0.37) (0.1) (0.16)

No. teeth on leaf margins 7.90a 8.50a 1.60b 2.10b 45.69*(1.79) (1.43) (1.42) (2.13)

Reproductive variablesNo. flowered ramets 1.60a 2.60a 8.00b 7.50b 13.89*

(0.69) (2.11) (12.35) (6.11)Length of flower head (cm) 0.87a 0.86a 0.63b 0.77b 19.45*

(0.12) (0.11) (0.08) (0.09)Width of flower head (cm) 0.80a 0.86a 0.51b 0.57b 28.16*

(0.18) (0.09) (0.05) (0.08)No. bracts 19.70a 20.40a 14.90b 14.40b 30.25*

(1.76) (1.07) (0.99) (1.07)No. fruits per flower head 58.10a 68.80a 41.00b 42.30b 14.92*

(15.41) (8.37) (12.07) (4.78)No. healthy fruit per flower head 45.60a 66.10b 28.60a 37.10a 9.47*

(23.69) (9.35) (20.15) (6.00)No. eaten fruit per flower head 12.50 2.70 12.40 5.20 3.69

(19.64) (3.97) (12.00) (7.43)

Standard errors are in parentheses. F and H indicate the results of a one-way ANOVAs and a non-parametric Kruskal–Wallis test(* P < 0.05) for each variable, respectively (after Bonferroni correction * P < 0.05). Within each row different letters indicate significantdifferences (Tukey’s or Dunn’s post-hoc tests for multiple comparisons of means).

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characteristics of leaf shape, area and width showed atendency to differentiate populations within the twospecies. Larger plant height and leaf shape metrics of S.peteroanus from the Ñirihuau population appeared to beassociated with physical characteristics of the site(Table 1). Soil analyses did not show any differencesbetween species and among populations (data notshown).

The multivariate discriminant analysis separated thefour populations that were used as a priori grouping vari-able (Wilks’ l 0.0111, F(27, 82) = 11.158, P = 0.0001). Nine of15 variables entered in the forward stepwise model. Wechose the first two discriminant functions, which togetheraccount for approximately 95% of the total variance of thedata. The first discriminant function explained 82% of thetotal variance and significantly separated the two species(Wilks’ l 0.01, c2 = 145.9, P = 0.000) and the second dis-criminant function explained 12% of the total variance andsignificantly separated the populations of each species(Wilks’ l 0.16, c2 = 58.03, P = 0.000) (Fig. 2). Significantsquared Mahalanobis distances between group centroidswere obtained for the four populations (P < 0.01 in allcases). A correct classification into a priori groups was100% and 80% for S. carbonensis populations from Carbónand Ñireco, respectively, and 100% for both S. peteroanuspopulations from Ñirihuau and Challhuaco, thus showinghigher morphological similarities between the popula-tions of S. carbonensis.

Population density was higher in populations of S.peteroanus (Ñirihuau and Chalhuaco: 0.34 individuals/m2

and 0.28 individuals/m2, respectively) than in popula-tions of S. carbonensis (Carbón and Ñireco: 0.26

individuals/m2 and 0.22 individuals/m2, respectively).Plant cover was also higher in populations of S. peteroanus(10.8% and 11.7%, in Ñirihuau and in Challhuaco, respec-tively) than in populations of S. carbonensis (4.6% inCarbón and 4.4% in Ñireco, respectively), which in turnhad smaller diameters (Table 1). Moreover, despite S. car-bonensis having larger flower heads than S. peteroanus anda greater number of fruits per head, the species presentedless flowering ramets and therefore a lower number offruits per plant (Table 1). Therefore, S. carbonensis popu-lations produce less fruits per plant and m2 each year(Carbón 92.96 fruits/plant and 24.16 fruits/m2 and Ñireco178.9 fruits/plant and 39.35 fruits/m2) than S. peteroanuspopulations (Challhuaco 328 fruits/plant and 88.83fruits/m2 and Ñirihuau 317.25 fruits/plant and 111.52fruits/m2). Although seed production was higher for S.peteroanus, we did not observe seedlings growing undernatural conditions in any of the studied populations of thetwo perennial species.

Isozyme characterization

At the species level, S. carbonensis and S. peteroanus werehighly polymorphic. Twelve of the 14 (86%) resolved lociwere polymorphic in S. carbonensis, and nine of the 11(82%) resolved loci were polymorphic in S. peteroanus, inat least one population (Table 2). We found two diagnosticalleles (Per1-1 and Skdh-4) that were only present in S.carbonensis, and three (Idh-1, Per1-2 and Pgi1-7) in S.peteroanus (Table 2). The mean number of alleles per poly-morphic locus and the effective number of alleles perlocus were similar in both species (Table 3). Gene diver-sity measured by expected heterozygosity (HE) wasslightly higher in S. carbonensis (0.341) than in S. peteroanus(0.300), which in turn did not differ significantly from theobserved heterozygosity (HO) (0.317 and 0.264 for S. car-bonensis and S. peteroanus, respectively) (Table 3).

At the population level, both species maintained com-parable levels of isozyme variation (Table 3). The analyzedpopulations were highly heterogeneous in allelic frequen-cies of polymorphic loci (P < 0.05; c2-test). Allelic frequen-cies differed significantly in six (G2d, Idh, Mdh2, Mnr1,Pgm1 and Skdh) of the 12 (58%) possible tests in S. carbon-ensis populations, and three (Mnr2, Pgi2 and Skdh) of thenine (33%) in S. peteroanus populations.

Overall, heterozygous deficiencies were detected in68% of the possible tests for S. carbonensis and in 47% of S.peteroanus (Table 4). However, both species had averagevalues of inbreeding within populations (FIS) not signifi-cantly different from zero. In contrast, FST values, althoughrelatively low (FST = 0.05 in S. carbonensis and FST = 0.03 inS. peteroanus) were significantly different from zero. TheFIS and FST values did not differ between the two speciesbecause the confidence intervals overlapped (Table 4).

Fig. 2 Multivariate discriminant analysis of morphological vari-ables (vegetative and reproductive) of Senecio using a prioripopulations as the classifying factor. Sc, Senecio carbonensis; Sp,Senecio peteroanus. Black diamonds represent the populationmeans.

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Table 2 Allelic frequencies for 14 and 11isozyme loci resolved in Senecio carbonensis(Sc) and Senecio peteroanus (Sp),respectively

LocusSpecies/population

Sc-Carbón Sc-Ñireco Sp-Ñirihuau Sp-Challhuaco

G2dN 50 50 50 501 0.930 0.540 0.250 0.3702 0.070 0.460 0.750 0.630

IdhN 50 50 50 501 0.000 0.000 0.830 0.9102 0.920 0.740 0.160 0.0903 0.080 0.260 0.010 0.000

Mdh1N 50 50 50 501 0.030 0.000 0.270 0.2702 0.220 0.210 0.520 0.4403 0.600 0.710 0.210 0.2604 0.150 0.080 0.000 0.030

Mdh2N 50 50 50 501 0.000 0.100 0.000 0.0202 0.250 0.320 0.350 0.4203 0.570 0.580 0.650 0.5504 0.180 0.000 0.000 0.010

MeN 50 50 50 501 1.000 1.000 1.000 1.000

Mnr1(N) 50 501 0.080 0.1302 0.040 0.0003 0.780 0.6004 0.080 0.2305 0.010 0.0406 0.010 0.000

Mnr2N 50 50 50 501 0.110 0.000 0.050 0.1502 0.890 1.000 0.950 0.850

Per1N 50 50 50 501 1.000 1.000 0.000 0.0002 0.000 0.000 1.000 1.000

Per2N 50 50 50 501 0.080 0.080 0.140 0.1102 0.920 0.920 0.860 0.890

Pgi1N 50 50 50 501 0.000 0.030 1.000 0.9802 1.000 0.970 0.000 0.020

Pgi2N 50 50 50 501 0.000 0.030 0.050 0.0302 0.050 0.020 0.050 0.0303 0.330 0.290 0.280 0.1004 0.240 0.250 0.050 0.1905 0.330 0.410 0.430 0.6006 0.050 0.000 0.090 0.0407 0.000 0.000 0.020 0.0108 0.000 0.000 0.030 0.000

Pgm1N 50 501 0.250 0.4102 0.540 0.4803 0.130 0.1104 0.080 0.000

Pgm2N 50 501 0.160 0.2002 0.840 0.800

SkdhN 50 50 50 501 0.000 0.080 0.250 0.0002 0.350 0.340 0.640 0.8703 0.500 0.530 0.110 0.1304 0.150 0.040 0.000 0.0005 0.000 0.010 0.000 0.000

In bold are unique alleles for each species. Rare alleles with frequencies <5% are underlined. Diag-nostic alleles are in italics. Isozyme loci Mnr1, Pgm1 and Pgm2 were not resolved in populations of S.peteroanus. N, sample size.

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Overall total genetic diversity measured by Nei’s statisticswas higher in S. carbonensis (HT = 0.341) than in S. peteroa-nus (HT = 0.300), most of which was distributed withinpopulations of both species (HS = 0.332 in S. carbonensisand HS = 0.295 in S. peteroanus).

The genetic distance between populations of S. carbon-ensis was 0.028, and it was 0.014 between populations of S.peteroanus. In contrast, the average distance betweenspecies was as high as 0.535 (ranging from 0.485 to 0.586),which was an order of magnitude higher than thebetween–population distance values for any of the twospecies (Fig. 3), evidence that S. carbonensis populationswere genetically differentiated from those of S. peteroanus.

Discussion

In the present study we analyzed genetic and morpho-logical variation in the rare S. carbonensis and its wide-

spread congener S. peteroanus. Surprisingly we foundsimilar high genetic diversity in both species, generallyhigher than the diversity reported in Gitzendanner andSoltis (2000) based on a review of the patterns of geneticvariation in rare and widespread plant congeners. Thevalues were also higher than the averages reported forother long-lived perennials (Hamrick & Godt 1989). Wealso found that the rare high-elevation S. carbonensis, inassociation with its narrow distribution, had relativelyminor morphological variability together with low plantdensity and limited sexual reproduction.

Several factors can cause species to have low abundancesand small range sizes when contrasted with closely relatedtaxa. Range size can be limited mainly by environmentalfactors, such as climate, by reproductive characteristics ofthe species or by historical events (Gaston 1994). In certaincases some of these factors can be more important thanothers, but they can also act in combination.

Environmental factors

Between-species comparisons show that S. carbonensis, aspecies restricted to higher altitudes, differed from S.

Table 3 Genetic diversity parameters of Senecio carbonensis (Sc) and Senecio peteroanus (Sp)

Population N A AE AT AP PSS HO HE

Sc-Carbón 50 2.7 (0.4) 1.7 (0.24) 38 3.18 78.57 0.293 (0.08) 0.315 (0.07)Sc-Ñireco 50 2.6 (0.4) 1.7 (0.6) 36 3 78.57 0.341 (0.096) 0.349 (0.069)Sc 100 3.1 (0.5) 1.7 (0.17) 43 3.41 85.71 0.317 (0.078) 0.341 (0.067)Sp-Ñirihuau 50 2.5 (0.6) 1.5 (0.21) 28 3.125 72.73 0.244 (0.077) 0.303 (0.079)Sp-Challhuaco 50 2.6 (0.5) 1.6 (0.22) 29 3 81.82 0.284 (0.082) 0.287 (0.073)Sp 100 2.9 (0.6) 1.6 (0.16) 32 3.33 81.82 0.264 (0.074) 0.300 (0.075)

Standard errors are in parentheses. N, sample size; A, mean number of alleles per locus; AE, effective number of alleles; AT, total numberof alleles; AP, number of alleles per polymorphic locus; PSS, percentage of polymorphic loci; HO, observed heterozygosity; HE, expectedheterozygosity.

Table 4 Fixation indices (F) for each locus and a population andhierarchical analysis of inbreeding coefficients of each species

LociSc Sp

Carbón Ñireco Ñirihuau Challhuaco

G2d -0.075 -0.691 -0.333 -0.502Idh 0.457 0.480 0.439 -0.099Mdh1 -0.373 0.057 0.380 -0.083Mdh2 -0.172 -0.234 0.165 0.040Mnr1 0.363 0.578Mnr2 0.285 -0.053 -0.176Per2 -0.087 -0.087 -0.163 -0.124Pgi1 -0.031 -0.020Pgi2 -0.195 -0.287 -0.111 0.255Pgm1 0.582 -0.697Pgm2 0.256 0.875Skdh 0.074 0.563 0.845 0.912FIS 0.041 (-0.145–0.265) 0.107 (-0.124–0.360)FST 0.050 (0.011–0.120) 0.032 (0.006–0.064)

FIS is the inbreeding within-population and FST the among-population divergence. Confidence intervals are in parentheses.Results significantly different from zero are depicted in bold. Sc,Senecio carbonensis; Sp, Senecio peteroanus.

Fig. 3 Phenogram of the genetic distance among the four popu-lations studied based on Nei’s (1978) unbiased index and anUPGMA algorithm. The cophenetic correlation coefficient is0.997.

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peteroanus by the smaller diameter of its plants and largersize of its flower heads. In mountain plants, as the altitudeincreases, vegetative portions appear proportionally morereduced in relation to floral parts. It has been suggestedthat in high mountain plants, the scarcity of pollinatorsselectively favors the evolution of large and attractiveflowers (Körner 1999). For example, in Cerastium arvense(Caryophyllaceae) from the Patagonian Andes, popula-tions at higher altitudes are low plants with largerflowers, whereas those that grow below 1000 m a.s.l. aretaller plants with smaller flowers (Quiroga et al. 2002). Inaddition, within-species comparisons show that popula-tions of the range-restricted S. carbonensis were moresimilar to each other in their morphology than the studiedpopulations of S. peteroanus.

Although the Challhuaco population of S. peteroanus isfound in high elevations, the Ñirihuau population, locatedin a protected valley at lower altitudes with higher plantcover, is characterized by increased plant height andlarger leaves, which may be reflecting adaptation to com-petition for light. As a result, greater morphological diver-sification and wider niche conditions characterize thewidespread S. peteroanus. In contrast, the niche width ofrare species is generally more limited in nature (i.e. lessavailable) than that of widespread species (Gaston 1994).Therefore, the scarce morphological variation of the rare S.carbonensis may be reflecting environmental extremes athigh elevations. Nevertheless, more studies are neededalong the range of the common S. peteroanus to confirmthese results.

Reproductive characteristics

Senecio carbonensis is a ‘rare’ species according to the cri-teria proposed by Rabinowitz (1981), owing to its smallgeographical distribution, low local abundance andrestriction to very specific habitats. The present studyshowed lower reproductive capacity in S. carbonensisbecause the amount of fruit produced annually per plantand per m2 in its populations was smaller than in S.peteroanus. This, coupled with lower population densityand plant cover in S. carbonensis, gives limited opportuni-ties for population growth via sexual reproduction. Thesize and spatial structure of a population can affect theeffective dispersal of pollen between individuals by influ-encing the behavior of pollinators (Schemske & Lande1985; Sih & Baltus 1987). As a result, reduced populationsof S. carbonensis may decrease in size over time, as in thecase of Eupatorium resinosum (Asteraceae) compared withits widespread congener Eupatorium perfoliatum (Byers &Meagher 1997). These authors emphasize the fact that rarespecies and their common close relatives may differ dras-tically in their reproductive strategies (e.g. sexual repro-duction vs vegetative propagation). Other authors (Gaston

1994; Schmazel et al. 1995, Esparza-Olguín et al. 2002)indicate that rare species have low recruitment rates thatare generally associated with stressful environmental con-ditions (e.g. intense radiation, low precipitation, low pro-ductivity). In the case of S. carbonensis, both factors, that is,low levels of sexual reproduction together with a stressfulenvironment, could be interacting to produce low recruit-ment rates. However, because our study was of relativelyshort duration we do not know at this time whether S.carbonensis populations are at demographic risk. It hasbeen observed that for some long-lived species, favorableconditions for reproduction only arise occasionally(Gaston 1994). Nevertheless, reproductive characteristicsof rare species, such as those measured in S. carbonenis,may indicate that they are potentially vulnerable to long-term persistence and thus are of priority conservationconcern (Drury 1980).

Genetic characteristics and historical events

The isozyme study provides evidence for the genetic dis-tinctiveness of S. carbonensis and S. peteroanus, which canbe differentiated electrophoretically by the presence ofdiagnostic alleles and by a multivariate cluster analysis ofgenetic distances. In addition, the study shows that therange-restricted S. carbonensis maintains geneticallydiverse populations with negligible effects of inbreeding.Typically, species with restricted distributions have lessgenetic diversity than more widespread congeners owingto greater effects of drift and isolation in the former(Karron 1987; Hamrick & Godt 1989; Gitzendanner &Soltis 2000). Thus, our study contributes to the growingbody of evidence that narrow endemics may maintain asmuch genetic variation as their common congeners(Linhart & Premoli 1993; Lewis & Crawford 1995; Godt &Hamrick 1998; Ge et al. 1999; Neale et al. 2008). Relativelyhigh genetic diversity in rare plant species has beenexplained by several factors. Hybridization, recent specia-tion from a widespread species, multiple origins, com-paratively recent bottlenecks and maintenance of geneticdiversity within refugial populations recently severedfrom continuous distributions have all been suggested ascauses of relatively high genetic diversity in rare plantspecies (Lewis & Crawford 1995; Neale et al. 2008). Recenthistorical events related to these factors could be deter-mining the high genetic diversity of S. carbonensis. Forexample, the current restricted distribution on the semi-arid easternmost parts of the north Patagonian Andesof this species suggests that it may be a relic of a formermore extended range. Its present narrow environmentalrequirements suggest that in the past S. carbonensis mayhave attained its maximum distribution during thecoldest and most arid periods of the last glaciations,including late Holocene glacial advances (Glasser 2002;

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Masiokas et al. 2009). As a result, during these cold phasesthe now isolated populations of S. carbonensis may havebecome periodically into contact at lower elevations.However, recent warming trends have probably resultedin refugial populations currently restricted to highelevations.

Insect pollination and self-incompatibility mechanismsappear to be important characteristics of the reproductivesystem of some species that allow demographic persis-tence and high heterozygosity even at low populationdensities (Ward et al. 2005). Seeds dispersal by wind canalso have important consequences for recruitment and forthe genetic structure of populations (Nathan & Muller-Landau 2000; Nathan et al. 2000). High genetic diversitywithin these populations of Senecio may also be related totheir self-incompatible reproductive systems as reflectedin their not significantly different from zero values ofwithin-population inbreeding. This may be favored by therole of insects as pollinators in the Senecioneae (Funk et al.2005), which increase the rates of cross-fertilization.Although both species also propagate vegetatively by rhi-zomes, isozyme data suggest that they may have mecha-nisms that maintain diversity within populations. Thiscould be because of the establishment of new individualsunder already-established genets from fruit dispersed bywind.

Although our sampling scheme was designed to avoidsampling spatially near and thus closely related geno-types, we nevertheless were able to evaluate levels ofclonality of the two species. For this, we selected a set ofapproximately seven individuals in five randomly locatedsubplots of 5 m ¥ 5 m within each population, and calcu-lated the average number of genotypes that were foundwithin each population at this quadrat scale (followingSteinke 2004). If clonal spread was the driving propaga-tion force at any given population, we would haveexpected a reduced average number of genotypes withineach quadrat. We found that spatially close individuals ofS. peteroanus had on average 21.2 distinct genotypes, andS. carbonensis was similarly diverse with 30.5 genotypes(P > 0.05, ANOVAs), suggesting a lack of differences inclonal behavior between species.

In the long run, continuous recruitment via seedwould lead to a high degree of interdigitation amongdifferent genotypes as previously suggested for high-elevation populations of the perennial herb Alstromeriaaurea (Souto et al. 2002), which would lead to an increasein genetic diversity. In addition, individual longevity asa result of vegetative reproduction in these perennial rhi-zomatous species may also explain the high geneticdiversity, as the same genotype can be maintained in apopulation for several reproductive events, reducing theinfluence of genetic drift (Ellstrand & Roose 1987;Azevedo et al. 2007).

Under the assumption that isozyme loci are neutral, thelow genetic divergence among populations of the samespecies (FST <5%) suggests that they were recentlyfounded and that genetic drift did not have time topromote divergence, or that its effects are masked by animportant amount of current gene flow. In high altitudepopulations of the anemophyllous and anemochoroustree Nothofagus pumilio (Nothofagaceae) the amount ofgenetic divergence between these plants from differentmountains is similarly reduced (FST = 2%) (Premoli 2003).Nevertheless, another study carried out at the same high-elevation locations in the insect pollinated with relativelylocal seed dispersal Alstroemeria aurea yielded an order ofmagnitude greater divergence at isozyme loci (FST = 15%),and thus higher limitations for gene flow between distinctpopulations within a given mountain range (Souto &Premoli 2003). The low divergence found between popu-lations of Senecio is evidence that in high elevations geneflow can occur by insect pollination and/or wind-dispersed seeds, and/or that these environments havesuffered recent fragmentation as a result of the beginningof the final warm Holocene trend. The presence of rarealleles in the four studied populations could result fromgenetic drift that may have occurred after the foundationof these populations.

Conservation implications

The information obtained in the present study suggeststhat the rare high elevation S. carbonensis, despite itsnarrow distribution and relatively minor morphologicalvariability together with its low density and limitedsexual reproduction, is not genetically at risk of extinc-tion as a result of genetic stochasticity. However, it willbe important to study and monitor the dynamics of itspopulations including seedling recruitment as to furtherunderstand the causes of its rarity. Some researchershave argued that populations and species more often goextinct for ecological and demographical reasons ratherthan for a lack of genetic variation (Schemske et al. 1994;Hamrick & Godt 1996). This is most likely the case withS. carbonensis. Therefore, for the long-term survival ofthis species habitat protection and in situ conservationare of particular importance. In addition, ex situ effortsneed to be undertaken for these small populations of S.carbonensis, which seem particularly vulnerable to demo-graphic stochasticity.

The achenes of S. carbonensis are small and conservetheir germination power even after several months ofstorage (M. Ferreyra, 2008, pers. com.). This has also beenobserved in other Asteraceae (e.g. Helianthus annuus,Vranceanu 1977; Zarbakhsh et al. 1999), although germi-nation rates decrease significantly after 2 or 3 years andare completely lost after 4 or 5 years. These promising

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results in germination trials suggest that achenes of S.carbonensis are suitable for ex situ conservation, and thusstress their potential use in restoration practices.

Conclusions

The present study shows that the rare S. carbonensis, knownonly from two populations restricted to semiarid highelevation areas of the southern Andes, presents relativelylimited morphological variation and reproductive capac-ity, together with low plant density and reduced plantcover. However, high genetic variation is found at thepopulation level in this species. These results show that itspopulations are not genetically depauperate as a result ofdrift after severe bottlenecks or strong directional selec-tion, but that reproductive characteristics, including selfincompatibility, insect pollination and anemochory,together with recent environmental restrictions as a resultof Holocene climatic change, may be limiting the extensionand abundance of this species. This information shows theimportance of ecological observations and long-termstudying and monitoring of population dynamics tofurther assess the viability of rare species.

Acknowledgments

We thank Delegación Regional Patagonia, Administraciónde Parques Nacionales Argentina for allowing us to workin Nahuel Huapi National Park, Mauro Tammone forimportant help with the fieldwork, and Marina Arbetmanfor helpful comments on the manuscript. This work waspartially supported by a grant from Administración deParques Nacionales to M. Fernández and by AgenciaNacional de Promoción Científica y Tecnológica, Fondopara la Investigación Científica y Tecnológica (ANPCYT-FONCYT), Argentina (BID 1728 OC/AR) to projects PICT25833 to A. C. Premoli and 38148 to C. Ezcurra. M. P.Quiroga, C. Ezcurra and A. C. Premoli are scientificresearch members of Consejo Nacional de InvestigacionesCientíficas y Técnicas of Argentina (CONICET).

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