AFLP and breeding system studies indicate vicariance origin for scattered populations and enigmatic...

Molecular Phylogenetics and Evolution 53 (2009) 13–22

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Molecular Phylogenetics and Evolution

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AFLP and breeding system studies indicate vicariance origin for scatteredpopulations and enigmatic low fecundity in the Moroccan endemic Hypochaerisangustifolia (Asteraceae), sister taxon to all of the South AmericanHypochaeris species

Anass Terrab a,b, María Ángeles Ortiz a, María Talavera a, María Jesús Ariza a, María del Carmen Moriana a,Juan Luis García-Castaño a, Karin Tremetsberger a,b, Tod F. Stuessy b, C. Marcelo Baeza c, Estrella Urtubey d,Claudete de Fátima Ruas e, Ramón Casimiro-Soriguer a, Francisco Balao a, Peter E. Gibbs f,Salvador Talavera a,*

a Departamento de Biología Vegetal y Ecología, Facultad de Biología, Universidad de Sevilla, Apdo. 1095, 41080-Sevilla, Spainb Department of Systematic and Evolutionary Botany, Institute of Botany, University of Vienna, Rennweg 14, A-1030 Vienna, Austriac Departamento de Botánica, Universidad de Concepción, Casilla 160-C, Concepción, Chiled Instituto Darwinion, Labardén 200 (y Estanislao del Campo), B1642HYD. C.C. 22. San Isidro, Prov. de Buenos Aires., Argentinae Departamento de Biología Geral, Universidade Estadual de Londrina, Londrina, Paraná, Brazilf School of Biology, University of St. Andrews, Scotland, UK

a r t i c l e i n f o a b s t r a c t

Article history:Received 30 September 2008Revised 9 June 2009Accepted 12 June 2009Available online 18 June 2009

Keywords:Breeding systemGenetic diversityHypochaerisPhylogeographyMorocco

1055-7903/$ - see front matter � 2009 Elsevier Inc. Adoi:10.1016/j.ympev.2009.06.008

* Corresponding author. Fax: +34 954557059.E-mail address: stalavera@us.es (S. Talavera).

We used Amplified Fragment Length Polymorphism markers (AFLP) and breeding system studies toinvestigate the population structure and reproductive biology of Hypochaeris angustifolia (Asteraceae:Cichorieae). This species is endemic to altiplanos of the Atlas Mountains (Morocco) where it occurs inscattered populations, and it is the sister species to c. 40 species of this genus in South America. PCoA,NJ, and Bayesian clustering, revealed that the populations are very isolated whilst AFLP parameters showthat almost all populations have marked genetic divergence. We contend that these features are more inaccord with a vicariance origin for the scattered populations of H. angustifolia, rather than establishmentby long-distance dispersal. The breeding system studies revealed that H. angustifolia is a self-incompat-ible species, with low fecundity in natural and in experimental crosses, probably due to a low frequencyof compatible phenotypes within and between the populations.

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1. Introduction

Species (or groups of related taxa) that present fragmented ordisjunct distributions usually evoke either a vicariance or long-distance dispersal explanation. The former implies the establish-ment of dispersal barriers within an ancestral geographical area,whilst the latter involves propagules overcoming such barriers topermit colonisation outwith the original area of distribution. Tra-ditionally, because long-distance dispersal hypotheses have beendifficult to falsify, most studies have involved detailed analysesof vicariance models using morphological parameters (Estabrook,2001; Sanmartín, 2003), but since vicariance vs. long-distancedispersal should result in different patterns of genetic diversity,molecular markers have been increasingly used to study such

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phylogeographic situations (Schönswetter and Tribsch, 2005;Kropf et al., 2006).

Many of the latter studies have involved European mountainplant species of the Alps, Pyrenees, Apennines, Betic, Balkan or Car-pathian systems (e.g. Zhang et al., 2001; Despres et al., 2002;Schönswetter et al., 2004) but, to our knowledge, there are no stud-ies of vicariance vs. long-distance dispersal events for species en-demic to North Africa. Here we present such a study forHypochaeris angustifolia (Litard. & Maire) Maire. This species com-prises perennial, rhizomatous herbs that are endemic to the altipl-anos of the Atlas Mountains where it occurs over an extensive area,but in rather isolated populations restricted to the margins ofwater courses and wet meadowlands between 1000 and 2900 m.Plants flower in summer, and are pollinated by solitary bees. H.angustifolia is a singularly interesting species since molecular stud-ies (Tremetsberger et al., 2005) have shown that it is more closelyrelated to the South American species of Hypochaeris than with theMediterranean or Macaronesian species of this genus. Indeed, it is

14 A. Terrab et al. / Molecular Phylogenetics and Evolution 53 (2009) 13–22

very similar morphologically to many of the South American spe-cies, and shares with these taxa a bimodal karyotype (Oberprielerand Vogt, 2002; Tremetsberger et al., 2005). According to Tremets-berger et al. (2005), H. angustifolia is the sister species to thearound 40 South American species of the genus Hypochaeris thatdiversified during the Pliocene-Pleistocene, around 3.5 mya, froma common ancestor that arrived in that subcontinent by long-dis-tance dispersal.

The altiplano of the Atlas mountains of Morocco presents avast area that constitutes the biogeographical province knownas Atlasica, that includes all of the Middle Atlas, High Atlasand Anti Atlas above 1000 m, and comprises the largest suchprovince in Morocco (Benabid and Fennane, 1994; Deil and Galá-n de Mera, 1999). In large part, the modern oreography of theAtlasica region was modelled by volcanic activity during theQuaternary (1.8–0.5 mya; Piqué, 1994; Duggen et al., 2003),especially in the central Middle Atlas region. Both before andafter this period, these uplands probably consisted of a treelessherb-rich steppe, since pollen data indicate that trees invadedthe area only recently, at some 8500 BP for oaks, and some4000 BP for cedars (Lamb et al., 1989; Terrab et al., 2008). How-ever, the volcanic activity disrupted the original topography ofthe Atlas uplands to give rise to numerous isolated depressionsin the altiplano that accumulate snow in winter and water inspring to summer, creating an area of fragmented marshlandsthat extends for some 100,000 km2 (i.e. about the size of Hun-gary). The plants of the herbaceous community of these wet-lands flower and fruit in the short alpine summer (July–

Fig. 1. Localities for the 10 sampled populations of H. angustifolia (for details of numbereshown at 1000 and 3000 m a. s. l. (map modified from Deil and Galán de Mera, 1999). Theto the different clusters as detected in an admixture analysis of AFLP data conducted w

August), a season that coincides with the annual transhumanceof flocks of sheep and goats moving from lower altitudes, wherethey are unable to find sufficient pasture, to congregate in themontane plateau area. Ruiz (1984) estimated the number ofsheep and goats in Morocco at over 22 million animals. The con-sequent grazing overload has a marked impact on the sexual cy-cle of all of the non-spiny and palatable herbaceous species thatlive in these upland communities, especially those of the marsh-land/wetland and so verdant areas, since flowers and youngfruits are removed, and as a consequence, the regeneration ofthe populations is impeded.

These scattered marshlands provide the habitat for the dis-junct distribution of H. angustifolia, and the history of the arearaises the question: did H. angustifolia once have a more wide-spread distribution on the altiplanos, which was subsequentlyfragmented by volcanic activity? Or did this species achieve itsscattered distribution by invading suitable sites in a sequenceof dispersal events?

We used Amplified Fragment Length Polymorphism (AFLP), asin previous studies with this genus (Muellner et al., 2005; Ortizet al., 2006, 2007, 2008; Stuessy et al., 2003; Tremetsbergeret al., 2003a,b, 2004) to address this question. And, given the factthat an immediate ancestor of H. angustifolia gave rise to a SouthAmerican cohort of some 40 species that are known to compriseself-incompatible and self-compatible taxa (Talavera, unpublisheddata; Tremetsberger et al., in press), we also investigated thebreeding system of this species by means of in situ and ex situstudies.

d circles see Table 1). Grey area indicates the Atlasica region, in Morocco. Elevationsgraphs next to each population indicate the proportional assignment of individuals

ith the program Bayesian Analysis of Population Structure (BAPS).

A. Terrab et al. / Molecular Phylogenetics and Evolution 53 (2009) 13–22 15

2. Materials and methods

2.1. Plant material for AFLP sampling

We obtained plant material from 10 natural populations of H.angustifolia from the three disjunct range areas of the species:the northern Middle Atlas (3 populations), central Middle Atlas(5 populations) and High Atlas (2 populations) (Fig. 1). This sam-pling effectively represents the total area of distribution of the spe-cies. Table 1 provides details of the sampling localities andpopulation parameters. For each locality we estimated the areaoccupied by the population and collected leaf samples from 20 to28 individuals (a total of 246 individuals). Samples were storedin silica gel until DNA extraction. Vouchers of all sampled popula-tions are deposited in the Herbaria of the University of Seville (SEV,Spain) and/or University of Vienna (WU, Austria).

2.2. DNA isolation and AFLP analyses

Total genomic DNA was extracted from dry leaf material follow-ing the CTAB protocol (Doyle and Doyle, 1987) with modifications(Ortiz et al., 2007, 2008; Tremetsberger et al., 2003b, 2004), andthe AFLP procedure followed established protocols (Vos et al.,1995) also with modifications (Tremetsberger et al., 2003b, 2004;

Table 1Populations of Hypochaeris angustifolia included in this study.

Populations Coordinates(N, W)

Col.No.

Pop. size(m2)

No. individualsanalysed

No. AFLP(error ra

North Middle Atlas

Pop. 1. N.P. Tazzeka 1,1550 m (M36)

34�040 ,4�070 ST 782/05M

74 23 19

Pop. 2. N.P. Tazzeka 2,1488 m (M54)

34�030 ,4�080 ST 633/03M

32 23 23

Pop. 3. Jbel Bou-Iblan,1987 m (M37)

33�37,4�090 ST 53/06M

25 20 1

Centre Middle Atlas

Pop. 4. Timahdite,1815 m (M55)

33�090 ,5�030 ST 676/03M

50 26 18

Pop. 5. Larais, 1608 m(M38)

33�010 ,4�520 ST 73/06M

150 26 20

Pop. 6. Bekrite 1,2016 m (M42)

33�010 ,5�050 ST 693/03M

195 22 22

Pop. 7. Bekrite 2,1883 m (M56)

32�580 ,5�080 ST 703/03M

120 26 26

Pop. 8. Boumia,1590 m (M41)

32�410 ,5�050 ST 270/03M

850 27 27

High Atlas

Pop. 9. Jbel Azourki,2234 m (M40)

31�470 ,6�210 ST 158/06M

42 25 20

Pop. 10. Oukaimeden,2160 m (M39)

31�080 ,7�530 ST 134/06M

56 28 28

Populations, coordinates, collection number, population size (m2), number of individuanumber of fragments per population (Fragtot), number of polymorphic fragments (Fragpoly

index (DW), Neis gene diversity, in brackets the 0.95 confidence interval (HD), and distbrackets the 0.95 confidence interval.

a The diversity indices were calculated with a randomly selected subset of 18 individusee Section 2).* P < 0.00001.

Ortiz et al. 2007). An initial screening of selective primers using36 primer combinations with three selective nucleotides was per-formed on 16 individuals of eight populations (two individuals perpopulation). The three primer combinations chosen for the selec-tive PCR were EcoRI (FAM)-ACC/MseI-CAT, EcoRI (HEX)-ACG/MseICAG, and EcoRI (NED)-AGC/MseI-CAG. Twenty four individualswere replicated in order to exclude non-reproducible bands andto calculate the error rate according to Bonin et al. (2004). The fluo-rescence-labelled selective amplification products were separatedon a sequencer (Applied Biosystems 3130xl Genetic Analyzer) withan internal size standard (GeneScan�-500 ROX, PE Applied Biosys-tems). Amplified fragments from 60 to 500 base pairs were scored,and exported as a presence/absence matrix using ABI Prism Gene-Scan� Analysis Software 2.1 (PE Applied Biosystems) and Genogra-pher (v. 1.6.0 � Montana State University 2001; available at http://hordeum.oscs.montana.edu/genographer/). Criteria for choosingAFLP bands were (1) visual clarity, (2) straightforward interpret-ability, (3) similar fluorescence intensity, and, most importantly,(4) reproducibility between independent replicates.

2.3. AFLP data analyses

The presence/absence matrix originated with the three primercombinations was imported into Paup* (v. 4.0b10; Sinauer Associ-

phenotypeste 2%)

Fragtota Fragpoly

a Fragpriva

(fixed)DWa HD

a (0.95C.I.)

FSTa (0.95

C.I.)

0.5347*

(0.46–0.58)243 132 8 35 0.102

(0.09–0.12)

237 142 3 31 0.102(0.09–0.12)

186 29 5 (4) 23 0.015(0.01–0.02)

0.2809*

(0.24–0.31)226 140 7 31 0.105

(0.09–0.12)

283 180 23 58 0.133(0.12–0.15)

261 179 11 43 0.128(0.11–0.14)

262 179 14 46 0.122(0.10–0.14)

264 187 8 42 0.136(0.12–0.15)

0.3954*

(0.31–0.45)248 144 11 41 0.104

(0.09–0.12)

248 133 18 47 0.097(0.08–0.11)

ls analysed, number of different AFLP phenotypes founded at error rate 2%, total), number of private fragments, in brackets if they are fixed (Fragpriv), rare fragmentsance among populations, (FST) within each of the three regions considered here, in

als per population which showed different phenotypes, but pop. 3 (for more details

16 A. Terrab et al. / Molecular Phylogenetics and Evolution 53 (2009) 13–22

ates). To represent overall genetic relationships among all the ana-lysed individuals of H. angustifolia, we constructed a dendrogramapplying the neighbour-joining method (NJ) in conjunction withNei and Li (1979) genetic distances. Support for each node wastested by 10,000 bootstrap replicates. The upgma algorithm in con-junction with Nei and Li (1979) genetic distances was also appliedto the data matrix and resulted in a similar dendrogram (data notshown). A Principle Coordinates Analysis (PCoA) was performed inFAMD (Schlüter and Harris, 2006) using Nei & Li’s coefficient (Neiand Li, 1979), in conjunction with SPSS v. 14.0 (SPSS Inc.), to illus-trate populations grouped according to the AFLP fragment similar-ity pattern.

Within-population genetic diversity was assessed for each pop-ulation using the total number of AFLP fragments present (Fragtot),the number of polymorphic fragments (Fragpoly), and the numberof private fragments (Fragpriv), calculated with the program FAMDv. 1.1 (Schlüter and Harris, 2006). The Neis gene diversity (HD) wascalculated using the formula D = n/(n � 1) � [1 – (freq(1)2 + -freq(0)2)] for each marker and then taking the average (Nei,1987), with the program AFLPdat (Ehrich, 2006). We also used thisprogramme to calculate the rare fragments index (DW; ‘frequency-down-weighted marker values’ (Schönswetter and Tribsch, 2005)).Since H. angustifolia is a rhizomatous plant, we also checked forclones, taking into account the 2% error rate, i.e. considering onlyphenotypes that differ in more than 9 markers, using AFLPdat (Eh-rich, 2006; Ehrich et al., 2008). In order to compare equal numbersof samples to calculate the diversity indices, we selected at random18 individuals in each population, since this was the number of dif-ferent phenotypes in population 4 (Timahdite), that with thesmallest number of different phenotypes. For population 3, whichshowed only one phenotype, we selected 18 individuals.

The amova derived fixation index FST (arlequin v. 3.01) de-scribes the reduction in heterozygosity within populations relativeto the total population (Wright, 1951) and is an indirect approachto estimate gene flow. With the total dataset we also assessed thenumber of fragments that were shared exclusively between pairsor groups of populations (shared exclusive fragments, i.e. frag-ments that where shared exclusively between two populations).We also tested whether there was a correlation between the aver-age genetic diversity and the population size using a Spearmannonparametric correlation; conducted with JMP v. 5.0.1a software(SAS Institute Inc., 1989).

We assessed patterns and levels of population genetic differen-tiation using analyses of molecular variance (AMOVA), conductedwith arlequin v. 3.01, (Excoffier et al., 2005) that we undertookwith three different groupings of the H. angustifolia populations.The first grouping (a) has two hierarchical levels and describes dif-ferentiation among all populations of the species. Groupings b, c,and d have three hierarchical levels and each describes the differ-entiation among two geographical areas: between northern MiddleAtlas versus the rest of the populations (grouping b); all Middle At-las populations versus High Atlas populations (grouping c); and be-tween the Centre Middle Atlas versus the northern Middle Atlasand the High Atlas populations (grouping d). In this way, we aimedto test which potential geographical barrier (represented by group-ings b, c and d), if any, had the largest effect on genetic differenti-ation in H. angustifolia. To test for isolation by distance, wecompared the populations’ pairwise FST values with their geo-graphical distance using Mantel tests based on Spearman correla-tions (on 10,000 random permutations); this analysis wasperformed on the entire sample and also on the subgroups, andXLSTAT-PRO 2007 software (Addinsoft) was used.

The overall population structure was further explored usingmodel-based Bayesian assignment, as implemented in BAPS v.5.1 (available at http://www.abo.fi/fak/mnf/mate/jc/software/baps.html) (Corander and Marttinen, 2006; Corander et al., 2004,

2003). The simulation was run from K = 2 to K = 16 as the maxi-mum number of diverged groups (i.e. larger than the number ofsampled populations), with five replicates for each K, and the op-tion ‘‘clustering of individuals” which was performed with the fol-lowing settings: minimal size of clusters at five individuals; 100iterations to estimate the admixture coefficients for the individu-als; 200 simulated reference individuals from each population;and 20 iterations.

2.4. Plant material for reproductive biology studies

We uplifted a minimum of 10 individuals separated by at least1 m from eight of the 10 populations analysed for AFLPs (pops. 3–10), and these plants were cultivated in the glasshouse of the Uni-versity of Seville during 2004–2006. Environmental conditions,with a photoperiod (16 h light/8 h darkness), temperature (18–22 �C), and watering (every 4 h) were controlled. Of the original115 transplants, 81 survived until fruiting (4, 14, 11, 23, 8, 14and 7 ramets from populations 4, 5, 6, 7, 8, 9 and 10, respectively).When these plants began to flower, we bagged capitula, and car-ried out the following hand pollinations: geitonogamous selfs(1153 capitula), and crosses within the population and also be-tween populations (a total of 270 capitula). Moreover, with 10plants from the population at Larais a full diallel was undertaken.Each population was crossed with at least five different popula-tions, except for population 4, which was crossed with only two(6 and 7). Pollinated capitula were re-bagged until fruit collection(around 30 days). We counted withered flowers with undevelopedovules, and fruits with embryos, and estimated the fruit/flowerratio.

In addition, with two populations in the field that had escapedloss of flowers due to grazing (pop. 6 and pop. 7), we collected onemature capitulum from 17 individuals, again at least 1 m apart.Subsequently, for each capitulum we again counted the numberof (withered) flowers and the number of fruits with an embryo,and we calculated the fruit/flower ratio for these capitula. Withthese data we were able to compare the probable breeding systemin these two natural populations with that established for them,and also the other populations, with the transplants cultivated inthe glasshouse.

We checked pollen viability with samples from flowers of twoindividuals of all transplant populations using the fluorescein diac-etate technique (Heslop-Harrison and Heslop-Harrison, 1970).

2.5. Data analyses

General Linear Models (GLMs) were used to assess differencesamong treatments and populations, considering the individual asa random effect nested within the population effect, and assessedwith the method of moments (EMS). The relationship betweenHD and the reproductive success (estimated through the intra-pop-ulation fruit/flower ratio) was assessed with the Spearman correla-tion coefficient. Statistical analyses were performed with JMP v.5.0.1a (SAS Institute Inc., 1989).

3. Results

3.1. AFLP data

The three AFLP primer combinations generated 443 unambigu-ous DNA fragments: EcoRI (NED)-AGC/MseI-CAG: 139; EcoRI (HEX)-ACG/MseI-CAG: 123; EcoRI (FAM)-ACC/MseI-CAT: 177, and all but30 of these were polymorphic within each of the 10 populationsinvestigated. Based on phenotypic comparisons, there were 24 rep-licated individuals, which gave an error rate of 2% according to Bo-

A. Terrab et al. / Molecular Phylogenetics and Evolution 53 (2009) 13–22 17

nin et al. (2004). Consequently, although all of the 246 individualsanalysed were different, if we take in account the 2% error rate (i.e.including only individuals which differed in more than 9 markers),then only 209 distinct phenotypes were found.

Fig. 2. Neighbour-joining analysis of AFLP phenotypes of H. angustifolia, based on Nei &(based on 10,000 replicates). For details of populations see Table 1.

3.2. Population structure

Seven of the 10 populations of H. angustifolia formed well-sup-ported clusters in the NJ analysis (Fig. 2), whilst the populations 4,

Li’s genetic distance. Bootstrap values higher than 50% are indicated at each node

Fig. 3. Principal Coordinate Analysis (PCoA) based on a matrix of Nei & Lisimilarities among the 246 investigated individuals of H. angustifolia. For populationdetails see Table 1.

18 A. Terrab et al. / Molecular Phylogenetics and Evolution 53 (2009) 13–22

7 and 8 were separated in different groups but without bootstrapsupport. Similar results were obtained in the Bayesian (Fig. 1) withnine different groups inferred, and also the Principal CoordinateAnalysis (Fig. 3). The Bayesian analysis revealed that population4 includes 17 distinct individuals that genetically belong to thispopulation, and nine that were genetically similar to the individu-als of the populations 7 and 8, but the PCoA (Fig. 3) indicates thatthey are in fact related with population 7.

The Mantel test, which compared the pairwise distances be-tween populations (in direct line) with their respective FST values,indicated that there is a positive correlation for these parameters(r = 0.324, P = 0.029). However, this correlation is not significant(r = 0.175, P = 0.302) when the population 10, which is very distantfrom the remainder, was removed from the analysis.

The results of the analyses of molecular variance (AMOVA; Ta-ble 2) reveal that: (1) most of the variation (58.57%) resides withinpopulations, while a very small proportion the variation is ex-

Table 2Results of three analyses of molecular variance (AMOVA) of AFLP data (Squared Euclidean dGroupings b, c and d were used to test the effectiveness of geographical barriers in H. angupopulations and regions see Table 1; d.f. degrees of freedom; SS, mean sum of squares; gene(FSC) level are shown.

Grouping N Source of variation d.f. SS Va

a [1–10] 10Among populations 9 3787 16Within populations 236 5403 22

b [1–3][4–10] 2Among regions 1 473 0Among populations 8 3313 15Within populations 236 5403 22

c [1–8][9–10] 2Among regions 1 775 4Among populations 8 3012 14Within populations 236 5403 22

d [1–3][4–8][9–10] 3Among regions 2 1241 3Among populations 7 2546 13Within populations 236 5403 22

* P < 0.05.** P < 0.01.*** P < 0.00001.

plained by between-group differences: northern Middle Atlas ver-sus the rest of the populations (grouping b) only 2.25%, MiddleAtlas populations versus High Atlas populations (grouping c)10.73%; and between northern Middle Atlas and the High Atlaspopulations versus Centre Middle Atlas populations (grouping d)8.40%. This is in agreement with all the other analyses (NJ, Bayesianinference, PCoA and Mantel test) that populations are geneticallywell differentiated, and that there is no other spatial structurefor the genetic variation. With respect to fragment parameters,the majority of the populations do not have any shared exclusivefragments (data not shown). The exceptions were: population 5,which had most shared fragments with other populations (onefragment with pops. 2, 3 and 7, two fragments with pop. 9, fourfragments with pop. 10, and six fragments with pop. 8); popula-tions 6 and 7 shared five exclusive fragments; population 4 sharedthree exclusive fragments with pop. 7; population 1 shared theleast exclusive fragments with other populations (one fragmentwith pop. 8), and the two populations in the High Atlas (pops. 9and 10) shared six exclusive fragments.

The values of the pairwise fixation index (FST) between popula-tions (Table 3) indicate that the pops. 7 and 8 (FST = 0.144), and 4and 7 (FST = 0.195) are the most related, whilst the populations 3and 10 are the least related.

3.3. Genetic diversity

Results of the genetic diversity analyses are shown in Table 1.These indices were calculated in each population with 18 individ-uals with different phenotypes, with the exception of pop. 3, whichshowed only one phenotype, and in this case we selected 18 indi-viduals at random. Populations contained between 186 (pop. 3)and 283 (pop. 5) fragments. All of the 10 populations containedat least three private fragments. The largest population(8:850 m2), presented the largest gene diversity, and the smallest(3:20 m2), was the least diverse (Table 1). Over all populations,there is a positive correlation between size and average gene diver-sity (Spearman’s q = 0.8182, P = 0.0038, n = 10).

3.4. Reproductive biology data

The means for the fruit/flower ratio from hand self pollinationsvaried between 0.02 (pop. 9) and 0.06 (pop. 7), and the means for

istance) from 10 populations of Hypochaeris angustifolia, considering 0, 2 or 3 regions.stifolia by maximising the percentage of variation among regions. For abbreviations ofral fixation index (FST), fixation index for the region (FCT), and population within region

riance components Percentage of variance Fixation index (95% CI)

FST = 0.414*** [0.391–0.438].19 41.43.90 58.57

FST = 0.422*** [0.397–0.445].89 2.25 FCT = 0. 0.022 [0.001–0.045].80 39.92 FSC = 0.408*** [0.383–0.434].89 57.83

FST = 0.453*** [0.425–0.482].50 10.73 FCT = 0.107* [0.072–0.144].50 34.61 FSC = 0.388*** [0.363–0.412].90 54.65

FST = 0.430*** [0.405–0.454].37 8.40 FCT = 0.084** [0.060–0.109].89 34.58 FSC = 0.378*** [0.352–0.403].90 57.02

Table 3Pairwise geographical distance (in direct line in km) between populations (values above diagonal), pairwise fixation index (AMOVA-derived FST values) (values below diagonal) inHypochaeris angustifolia based on analysis of 443 AFLP fragments.

Populations North Middle Atlas Centre Middle Atlas High Atlas

1 2 3 4 5 6 7 8 9 10

Pop. 1. Tazzeka 1 — 2 50 133 136 147 154 178 328 480Pop. 2. Tazzeka 2 0.293 — 48 131 133 144 152 175 326 478Pop. 3. J. Bou-Iblan 0.614 0.620 — 98 94 109 116 135 289 446Pop. 4. Timahdite 0.447 0.363 0.664 — 23 15 22 52 194 348Pop. 5. Larais 0.362 0.384 0.602 0.450 — 20 25 42 195 353Pop. 6. Bekrite 1 0.356 0.368 0.544 0.383 0.372 — 7 37 181 337Pop. 7. Bekrite 2 0.369 0.285 0.547 0.195 0.388 0.264 — 32 174 329Pop. 8. Boumia 0.293 0.217 0.527 0.236 0.350 0.272 0.144 — 155 315Pop. 9. J. Azourki 0.429 0.426 0.656 0.504 0.423 0.349 0.409 0.338 — 162Pop. 10. Oukaimeden 0.414 0.443 0.674 0.550 0.424 0.418 0.465 0.404 0.400 —

Fig. 4. Fruit to flower ratio for H. angustifolia populations studied in the glasshouse(geitonogamy and xenogamy), and the reproductive success in the wild (onlypopulations 6 and 7). The number of hand pollinated and field sampled heads isindicated above each bar. For details of populations see Table 1.

A. Terrab et al. / Molecular Phylogenetics and Evolution 53 (2009) 13–22 19

both types of cross pollinations varied between 0.02 (pop. 9) and0.19 (pop. 7). These differences were significant whereas the differ-ences between intra-populational and inter-population crosses,with means for fruit/flower ratios for the former that varied be-tween 0 (pop. 9) and 0.19 (pop. 8), and for the latter between0.02 (pop. 9) 0.19 (pop. 7), (data not shown, but see Fig. 4), werenot significant (F2,1330 = 36.1615, P < 0.0001; F96,1330 = 8.652,P < 0.0001, R2 = 0.339994 for the model; post-hoc Tukey HSD test:q* = 2.43633, a = 0.05). The results of the diallel with the Laraispopulation are shown in Fig. 5. They encapsulate the results overallfor the intra- and inter-population crosses: most yielded 0 or veryfew seeds, whilst relatively few, e.g. 4 � 16 (45%), 6 � 12 (49%),7 � 12 (39%), 7 � 16 (91%), 16 � 1 (54%) and 16 � 12 (91%), gavemoderate to good seed set; moreover plants nos. 4 and 7 showedmoderate partial self-compatibility (PSC).

The fruit/flower ratios for the only two populations (6 and 7)studied in the field did not show any marked differences withthe means obtained with the hand cross pollinations in the glass-house (field with 0.09 vs. 0.10 in hand crosses for the pop. 6, andfield 0.13 vs. 0.19 for hand crosses for those from pop. 7). Pollenviability of all analysed flowers was between 95% and 100%.

4. Discussion

4.1. Vicariance or long-distance dispersal?

One important aspect that can be examined in the light of ourAFLP data is whether the sequence of isolated populations of H.angustifolia that occur across the high mountain altiplano marsh-lands of the Atlas most likely arose by fragmentation of a previ-ously much more extensive population system (i.e. a vicarianceorigin), or whether they were established progressively by dis-

persal. It is our contention that the AFLP data clearly indicate avicariance origin for the widespread but now fragmented popula-tions of this species.

First, the neighbour-joining dendrogram, PCoA, and Bayesiananalysis indicate that most of the populations of H. angustifoliaare genetically isolated from the others, despite the lack of anytopographical features (barriers) that might cause such isolation.The only exceptions are the populations at Boumia, Bekrite 2 andTimahdite, for which there are no significant differences, indicatingthat they have, or have had in the recent past, some gene flow be-tween them (see further discussion below). The fact that even geo-graphically close populations of H. angustifolia were geneticallywell differentiated, indicates an absence of gene flow even overshort distances: the genetic distance (FST = 0.293) between Tazzeka1 and Tazzeka 2, which are 2 km apart, is the same as that betweenTazzeka 1 and Boumia (FST = 0.293), which are 178 km apart, and ishigher than that between Tazzeka 2 and Boumia (FST = 0.217),which are 175 km apart.

The Mantel test with the populations of the Middle Atlasshowed that there is no correlation between genetic distance andactual distance between populations. This lack of correlation againsupports a vicariance hypothesis, with a system of ancient isolatedpopulations that maintain their genetic variability except in caseswhere the population size is so reduced (see below) that driftand stochastic factors, such as herbivory, have a role in their genet-ic structure. A similar absence of isolation by distance has beenfound in European alpine populations of Carex atrofusca (Schöns-wetter et al., 2006), and also with the Japanese alpine Arctericanana (Ikeda and Setoguchi, 2006).

The exceptional populations at Boumia, Bekrite 2 and Timahditethat do exhibit relationships almost certainly have a differentexplanation. Those at Boumia and Bekrite 2 belong to the samecluster (Fig. 1) and the occurrence of identical or almost identicalgenotypes at Timahdite, derived from the population at Bekrite 2(Fig. 3), probably reflects a one-way dispersion via historic sheeptranshumance. Bekrite 2 and Timahdite are the only two sampledpopulations that occur at the margins of streams that have abun-dant water throughout the year. These localities are consequentlyused as watering places for the flocks, and it is possible that theoccasional fruits that are produced (see below) can adhere to thebody of sheep, as in other Hypochaeris species (Baker and ÓDowd,1982), thus transporting diasporas from Bekrite 2 to Timahdite.

A vicariance hypothesis is also supported by the fact that ingeneral, the average gene diversity is high or moderately high inH. angustifolia (HD = 0.097 – 0.136), with the exception of the pop-ulation from Jbel Bou-Iblan (HD = 0.015) (see further discussion be-low). Likewise, parameters of genetic divergence, such as thenumber of private fragments (mean = 11), and the index of rarefragments (mean = 40), are also high. In contrast, with long-dis-tance dispersal, there is likely to be a founder effect, such that ge-

Fig. 5. Results of diallel crosses between 10 transplants from the population at Larais. Values = percentage fruit set for each pollinated head; where repeated pollinations gave0 aquenes, the number of heads is indicated in parentheses; C = bagged unpollinated heads used as controls; crosses not attempted = –.

20 A. Terrab et al. / Molecular Phylogenetics and Evolution 53 (2009) 13–22

netic impoverishment is likely to occur unless the newly estab-lished population is capable of rapidly re-establishing geneticdiversity. Even in the latter situation, we would expect to finddiminished genetic diversity in the most recently established pop-ulations (Hewitt, 1996, 2000; Kropf et al., 2002). These parametersfor high genetic diversity also indicate that these populations arelikely to be old. This kind of vicariant scenario has been reportedin European alpine populations of species such as Kernera saxatilis,Gentiana alpina, Silene rupestris, and Papaver alpinum (Kropf et al.,2006).

The exceptional population at J. Bou-Iblan is that with the leastgenetic diversity. This situation is typical of that expected in a rel-atively newly established population showing founder effects, as inAvena barbata, in which the populations in California (which ar-rived along with the cereals from Spain in the 16–17th centuries)contained only half the number of rare alleles present in the pop-ulations in SW Spain (García et al., 1989). But although the popu-lation at Bou-Iblan probably comprises a single genotype, thepresence of five private fragments and the absence of a relation-ship with any other population, indicates that this is not a ‘young’population, but rather an old one that due to its now reduced sizeexhibits severe genetic erosion, and is most likely heading forextinction.

We cannot trace the original immigration of H. angustifolia intothe Atlas Mountains region because of the strong genetic differen-tiation of all the populations of this species. Moreover, the fact thatthe sister group to this species are the South American speciesmeans that there is no outgroup taxon to which we can refer forinsights on the origin or migration of H. angustifolia. However,

the congruence of the AFLP data with a vicariance origin of thenow scattered populations of this species supports the hypothesisthat H. angustifolia previously occupied a much more widespreaddistribution across the humid grasslands of the Middle and HighAtlas altiplano. As the high mountain wetland habitats becamescarcer following volcanic activity, we assume that the once exten-sive H. angustifolia populations became isolated in different alpineregions.

4.2. Reproductive biology

The data resulting from this aspect of the study clearly indi-cate that: (1) H. angustifolia is a self-incompatible species, andas with other Asteraceae, we assume this is the homomorphicsporophytic mechanism (SSI); (2) in hand cross pollinationsthe fruit production is very low; and, (3) the overall level offruit production in the two natural populations sampled (Bek-rite 1 and Bekrite 2) was also very low. The presence of self-incompatibility in H. angustifolia is supported by the high genet-ic diversity found in these isolated populations. However, thelow values for reproductive success in H. angustifolia are in con-trast to those reported in other SI species in the genus Hypo-chaeris in the Mediterranean: In Hypochaeris arachnoidea, anannual species from NW Africa, the five populations studied(also from the Atlas Mountains region of Morocco) had afruit/flower ratio >0.7, and in Hypochaeris radicata, a perennialspecies, eight populations studied from Morocco and Spain pre-sented fruit/flower ratios between 0.7 and 0.85 (Ortiz et al.,2006).

A. Terrab et al. / Molecular Phylogenetics and Evolution 53 (2009) 13–22 21

Low fecundity in the populations of an SI species may be duepollination limitation, or to a low diversity of the S alleles that con-trol the incompatibility mechanism, or rarely, to pollen fertilityproblems. However, in H. angustifolia, we also encountered lowfecundity in all intra-population hand cross pollinations, so thatwe can exclude pollen limitation as factor, and moreover, no prob-lems of pollen viability were encountered. Consequently, we mustsuppose that a loss of S alleles has occurred in this species. The tra-ditional view holds that with a diminished number of S alleles inthe population, particularly in a species with SSI, the availabilityof compatible mates will be reduced, leading to a lack of fruitingsuccess (Byers and Meagher, 1992). In various studies on speciesof Asteraceae with low fruiting success, the authors have assumedsuch low S allele frequency as the cause, e.g. Hypochaeris maculata(Wells, 1976), Aster furcatus (Reinartz and Les, 1994), Scalesia affinis(Nielsen et al., 2003), and Hymenopsis acaulis var. glabra (DeMauro,1993).

However, some caution is necessary in making this assumption:in one of the very few studies that have estimated the number of Salleles in populations of an SSI species of the Asteraceae, Brennanet al. (2006) estimated only 6–7 alleles in all populations of thevery successful colonising Senecio squalidus in Britain. The low al-lele number is unsurprising, given that this species, native to Sicily,passed through a major genetic bottleneck under cultivation in theOxford Botanical Garden before its escape. What is remarkable isthat despite such allelic impoverishment, cross-compatibility isrelatively little impaired, since wild populations around Oxfordshowed some 70% cross-compatibility (Brennan et al., 2002). Nev-ertheless, in H. angustifolia the limited S allele hypothesis is furthersupported by the fact that in the diallel between 10 plants com-prising the transplant population from Larais, the majority (70%)of the reciprocal crosses were inter-incompatible, with onlyaround 30% showing marked cross-compatibility. With similardiallel crosses between 12 plants of H. radicata, 80% of the plantswere cross-compatible (Ortiz et al., 2006).

If the lack of fertility in the populations of H. angustifolia is dueto a scarcity of compatible phenotypes, it might be expected thatinter-populational crosses would be more successful. But our datashow that both intra- and inter-population crosses are equallypoor in fruit production, with no significant difference in the re-sults for these treatments. Generally, poor fertility between indi-viduals of distant populations has been attributed to‘‘outbreeding depression” caused by the breakdown of coadaptedgene complexes or unfavourable epistatic relationships (Fensterand Galloway, 2000; Fontdevila and Moya, 1999; Lynch, 1991).However, an alternative explanation is that maybe the low fecun-dity of intra- and inter-populational crosses in H. angustifolia is dueto the fact that all populations of this species share a similar lownumber of the same S alleles, possibly because S alleles were lostin this species during ancient bottleneck events. There is a generalconsensus (Uyenoyama, 1995) that S alleles, once lost, are difficultto replace. Interestingly, there is some evidence that the SouthAmerican cohort of Hypochaeris species also show low fecundity:Tremetsberger et al. (in press) report low fecundity in populationsof Hypochaeris incana (mean 0.25, range 0.01–0.56), and Talaveraet al. (unpublished data) have also found low inter-compatibilityin other South American taxa. Thus, it is possible that the commonancestor for H. angustifolia and the South American species alreadypossessed very low frequencies of S alleles.

5. Conclusions

The different genetic parameters indicate that the isolated pop-ulations of H. angustifolia are old, and the genetic relations betweenthem are very low. These data are congruent with the hypothesis

that this species once occupied a wide distribution across the alti-plano of the Atlas Mountains but that these extensive populationssubsequently became fragmented during volcanic activity in theQuaternary. On this view, the currently isolated populations of H.angustifolia are the result of vicariance events. However, whilstthe AFLP parameters indicate relatively high levels of geneticdiversity, the in situ and ex situ data on the reproductive biologyof this species indicate very low fecundity following intra- andinter-population crosses, implying loss of S alleles. High geneticdiversity but low S allele frequency presents an enigma, andwe can only hypothesise that due to ancient bottleneck eventsthat occurred before or accompanying its expansion across theplanalto, the species lost S allele diversity which it has neverrecovered. Thus, it seems that the breeding system of H. angust-ifolia presents a curious equilibrium: the low level of inter-com-patibility that occurs in all populations, aggravated by the lowsurvival of fruiting heads due to grazing impact, neverthelessseems to provide sufficient outbreeding to maintain geneticdiversity, whilst the constraints on inter-populational outcross-ing have permitted the accumulation of marked genetic diversitybetween individuals.

Acknowledgments

This work was supported by a grant from Fundacion BBVA (BIO-CON 04 to S.T.), a postdoctoral contract to A.T. (Consejería de Inno-vación, Ciencia y Empresa, Junta de Andalucía, Proyecto deExcelencia 2005/RNM 484 to S.T.), a predoctoral grant to M.Á.O.(BES-2003-1506), a Juan de la Cierva grant to K.T., and grants toS.T. (REN2002-04634-C05-03 and CGL 2006-00817), M. Arista(REN2002-04354-C02-02 and CGL 2005-01951) from the Ministe-rio de Educación y Ciencia (Spain), and the Austrian Science Foun-dation (FWF P-15225 to T.S.), and Junta de Andalucía (group RNM-204). We thank staff of the Glasshouse and Bioinformatic Labora-tory of the University of Seville General Services (CITIUS), andthe Molecular Laboratory of the Institute of Botany, University ofVienna.

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