Phylogenetic and phylogeographic evidence for a Pleistocene disjunction between Campanula jacobaea (Cape Verde Islands) and C. balfourii (Socotra

Molecular Phylogenetics and Evolution xxx (2013) xxx–xxx

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Molecular Phylogenetics and Evolution

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Phylogenetic and phylogeographic evidence for a Pleistocene disjunctionbetween Campanula jacobaea (Cape Verde Islands) and C. balfourii(Socotra)

1055-7903/$ - see front matter � 2013 Elsevier Inc. All rights reserved.http://dx.doi.org/10.1016/j.ympev.2013.06.021

⇑ Corresponding author. Fax: +34 932890614.E-mail address: [email protected] (M. Alarcón).

Please cite this article in press as: Alarcón, M., et al. Phylogenetic and phylogeographic evidence for a Pleistocene disjunction between Campanula ja(Cape Verde Islands) and C. balfourii (Socotra). Mol. Phylogenet. Evol. (2013), http://dx.doi.org/10.1016/j.ympev.2013.06.021

Marisa Alarcón a,⇑, Cristina Roquet b, Alfredo García-Fernández a, Pablo Vargas c, Juan José Aldasoro a

a Institut Botànic de Barcelona (IBB-CSIC-ICUB), Passeig del Migdia s.n., Parc de Montjuïc, E-08038 Barcelona, Spainb Laboratoire d’Écologie Alpine, CNRS UMR 5553, Université Grenoble 1, BP 53 F-38041 Grenoble Cedex 9, Francec Real Jardín Botánico de Madrid (CSIC), Plaza de Murillo 2, E-28014 Madrid, Spain

a r t i c l e i n f o

Article history:Received 1 April 2013Revised 24 June 2013Accepted 27 June 2013Available online xxxx

Keywords:BiogeographyPhylogenyColonisersClimatic fluctuationsMacaronesiaCampanula bravensis

a b s t r a c t

Our understanding of processes that led to biogeographic disjunct patterns of plant lineages inMacaronesia, North Africa and Socotra remains poor. Here, we study a group of Campanula speciesdistributed across these areas integrating morphological and reproductive traits with phylogenetic andphylogeographic data based on the obtention of sequences for 4 highly variable cpDNA regions and AFLPdata. The phylogeny obtained shows a sister relationship between Campanula jacobaea (endemic to CapeVerde Islands) and C. balfourii (endemic to Socotra), thus revealing a striking disjunct pattern (8300 km).These species diverged around 1.0 Mya; AFLP and haplotype data suggest that no genetic interchange hasoccurred since then. Their closest taxon, C. hypocrateriformis, is endemic to SW Morocco. The archipelagos ofMacaronesia and Socotra have probably acted as refugia for North-African species, leading to speciationthrough isolation. Although C. balfourii has a restricted distribution, its genetic variability suggests thatits populations have suffered no bottlenecks. C. jacobaea is also genetically rich and its distribution acrossCape Verde Islands seems to have been influenced by the NE–SW trade winds, which may also havefavoured the admixture found among the populations of the three southern islands. Floral features ofthe morphologically hypervariable C. jacobaea were also measured to assess whether the taxonC. bravensis, described for some of the southeast populations of C. jacobaea, corresponds to a differentevolutionary entity. We show that morphological variation in C. jacobaea does not correspond to anygenetic or geographic group.

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1. Introduction

Islands have long been used as natural laboratories for the studyof species dispersal, diversification and extinction. Their discretegeography makes them especially suitable for studying how selec-tion, gene flow and genetic drift have shaped patterns of currentbiodiversity (e.g. Caujapé-Castells et al., 2010; Franks, 2010). Thestudy of genetic diversity of endemic island taxa is also of particu-lar relevance to understand the evolutionary processes that havelead to current biogeographic species patterns. An enigmatic dis-junct floristic pattern, linking the Atlantic Macaronesian archipela-gos and Morocco with the Indian Ocean archipelago of Socotra andEast Africa, has been suggested for many plant groups (Le Brun,1971; Monod, 1971; Quézel, 1978). Some molecular phylogeneticstudies have provided evidence of this biogeographical connection,but further research is needed to confirm it for additional taxa

(Andrus et al., 2004). This intriguing distribution between closelyrelated species present on distant islands and/or the African conti-nent may be explained as the result of dispersal during favourableperiods followed by isolation during harsh periods, and differenti-ation in situ in refugial distribution centres (Francisco-Ortega et al.,1999; Carine, 2005; Galley et al., 2007; Sanmartín et al., 2010; Thu-lin et al., 2010). To date, no study has attempted to explore this dis-junct pattern with phylogeographic tools, which would allow for afiner-scale research.

The biogeographic region of Macaronesia (which is constitutedby five Atlantic archipelagos: the Azores, Madeira, the SalvageIslands, the Canary Islands, the Cape Verde Islands; plus acontinental enclave on North West Africa) displays a high degreeof plant endemism (Sunding, 1979). Macaronesia is thought tohave constituted a refugium for plant groups during Tertiary andQuaternary climatic fluctuations (Vargas, 2007). A high numberof evolutionary studies have focused on Macaronesian endemics,improving our understanding on the processes that enhancedbiodiversity on this region (Emerson, 2002; Kim et al., 2008).

cobaea

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However, there are patterns of diversity that are still not wellunderstood due to the low number of studies that integrate infor-mation on reproductive traits, population structure and geneticdiversity, together with a biogeographical perspective. As previ-ously said, some Macaronesian taxa are thought to be close rela-tives of species occurring in distant areas such as Socotra, anIndian Ocean archipelago with a striking proportion of endemics(37%, Miller and Morris, 2004) whose flora has only been poorlystudied with molecular phylogenetic tools.

The present work focuses on a group of related species that be-long to the clade Campanula s. str. (Roquet et al., 2009). The groupstudied here includes species distributed in Macaronesia, the Med-iterranean Basin, east Africa, Socotra, southern Arabia and centralAsia (the Azorina group, Borsch et al., 2009; Roquet et al., 2009).Though heterogeneous in habit, morphology, breeding systemand life history, the species belonging to the Azorina group forma monophyletic subclade (Mansion et al., 2012). This subclade in-cludes east–west disjunctions across Africa, e.g. species of Macaro-nesia and western Africa sister to species of eastern Africa andSocotra (Borsch et al., 2009; Haberle et al., 2009; Roquet et al.,2008, 2009; Mansion et al., 2012). The bellflowers are thereforean interesting group for the study of dispersal and speciation inMacaronesia, Northern Africa and Socotra.

In this study we first analyse in detail the phylogenetic relation-ships of this group of species; second, we conduct a phylogeograph-ic analysis of two sister species of the Azorina subgroup found ontwo widely separated archipelagos: Campanula jacobaea in CapeVerde Islands, and C. balfourii in Socotra. Campanula jacobaea growsin crevices on north and northeast-facing cliffs, where moisture isavailable from the trade wind fogs, while C. balfourii grows in occa-sionally wet places on Socotra mountains. Campanula jacobaea is

Table 1Material sampled from populations, plus the number of polymorphic fragments, exclusive

Species/Island Population n Number ofpolymorphicfragments

C. jacobaea,Cape Verde,Santo Antâo

A1: Cova, 17�60N25�40200W, 1375 m, Aldasoro& Alarcón 9291

10 604

A2: Corda, 17�70500N 25�502200W, 1145 m,Aldasoro & Alarcón 9307

10 536

A3: Pico da Coroa, 17�40N25�130W, 957 m,Aldasoro & Alarcón 9309

10 566

C. jacobaea,Cape Verde,São Nicolau

N1: Tarrafal, 16�3605700N24�1904700W, 699 m,Aldasoro & Alarcón 9707

10 394

N2: Monte Gordo, 16�3704500N 24�2102200W,1020 m, Aldasoro & Alarcón 9704

9 358

C. jacobaea,Cape Verde,Santiago

S1: Pico da Antonia, 15�110N 23�400W,900 m, Aldasoro 9510

9 571

S2: Sierra da Malagueta, 15�100N 23�410W,806 m, Aldasoro 9509

10 586

C. jacobaea,Cape Verde,Fogo

F1: Ponta Verde, 14�590N 24�270W, 431 m,Aldasoro 9506

10 549

F2: Cha das Caldeiras, 14�590N 24�270W,1730 m, Aldasoro 9500

10 539

C. jacobaea,Cape Verde,Brava

B: Fontainhas, Vila Nova de Sintra, 14�510200N24�420000W, 859 m, Aldasoro 9503

10 558

Total number of private fragments for C. jacobaeaTotal number of accessions and fragments for C. jacobaea 98 895

C. balfourii,Socotra

Q1: Qalansiyah, 12�3902900N 53�2602300W,Aldasoro & Susanna 184

5 370

Q2: East of Hadibo, Haggiher Mts, 751 m,12�3503000N 54�104700W, 751 m, Aldasoro &Susanna 14643

11 443

Total number of private fragments for C. balfouriiTotal number of accessions and fragments for C. balfourii 16 496

Total number of accessions and fragments for C. jacobaea plusC. balfourii

114 1005

Please cite this article in press as: Alarcón, M., et al. Phylogenetic and phylogeog(Cape Verde Islands) and C. balfourii (Socotra). Mol. Phylogenet. Evol. (2013), h

fairly variable and has been subdivided into four varieties. One ofthis varieties, C. jacobaea var. bravensis Bolle, was later given speciesrank by Chevalier (1935) as C. bravensis (Bolle) A. Chev, based ondifferences on corolla shape and colour (campanulate and usuallyblue or violet corolla in C. jacobaea, tubular corolla usually green-ish-white in C. bravensis) as well as ovary shape and pubescence(glabrous and flat in C. jacobaea, pubescent and conical in C. braven-sis). We have included populations attributed to Campanula bravensisin our phylogeographic analysis, in order to assess whether thepopulations of C. bravensis correspond to a different evolutionaryentity from C. jacobaea. Here, we generate and analyse a compre-hensive data set, including genetic data and morphological andreproductive traits, to address the following questions: (1) Doesthe taxon Campanula bravensis correspond to a distinct evolution-ary entity and thus is the species rank justified? (2) Do the CapeVerdean C. jacobaea and the Socotran C. balfourii constitute sisterspecies? (3) Which factors could explain the disjunct distributionof the bellflowers clade here studied?

2. Materials and methods

2.1. Study group and taxon sampling

To clarify the phylogenetic positions and relationships ofCampanula jacobaeaand C. balfourii, 15 species were sampled: tenspecies of section Medium subsect. Rupestres belonging to the Azorinaingroup (which is constituted at least by 14 species according toMansion et al. (2012); Table S1), plus four species of the subgenusRoucela as closest outgroup and C. sibirica as a second outgroupaccording to Olesen et al. (2012) and Mansion et al. (2012). One indi-vidual per species was studied except for C. balfourii (12 individuals),

fragments and Hj values obtained in AFLP analysis.

Percentage ofpolymorphicfragments

Hj S.E.(Hj) Exclusivefragments /population

Exclusivefragments/island or species

67.48 0.1975 0.00557 1 26

59.88 0.1944 0.0059 2

63.24 0.1895 0.00573 1

44.0 0.1417 0.00600 1 7

40.0 0.1430 0.00611 1

63.8 0.2101 0.00580 3 8

65.5 0.1958 0.00560 0

61.3 0.2048 0.00613 1 5

60.2 0.1955 0.00589 0

62.3 0.1868 0.00574 1 1

509

74.6 0.3116 0.00766 7

89.3 0.2777 0.00688 17

110

raphic evidence for a Pleistocene disjunction between Campanula jacobaeattp://dx.doi.org/10.1016/j.ympev.2013.06.021


M. Alarcón et al. / Molecular Phylogenetics and Evolution xxx (2013) xxx–xxx 3

C. jacobaea (67individuals), C. hypocrateriformis (2 individuals),C. kremeri (3 individuals) and C. dichotoma (3 individuals) (TablesS1 and S2).

For AFLPs analyses, ten populations of C. jacobaea and two ofC. balfourii were sampled in the field. Three of the populations col-lected of C. jacobaea correspond to populations attributed toC. bravensis: one in Brava and two in Fogo. We also collected twopopulations in Santiago, the only island where both species aresupposed to be present. For each population, when it was possible,leaves from 5 to 11 plants some 7–15 m apart along a number oftransects were collected, numbered, dried and conserved in silicagel. Voucher specimens of all sampled species or populations weredeposited in the herbarium of the Institut Botànic de Barcelona(BC) (Table 1, S1, and S2).

2.2. DNA sequencing

Total DNA was extracted from silica gel-dried plant tissue orherbarium material using the ‘‘DNeasy Plant Mini Kit’’ (QIAGENInc., California, USA) according to the manufacturer’s instructions).Four highly variable cpDNA regions were sequenced:petB1365–petD738 (24 sequences), rpl32-trnLUAG (96 sequences),trnSGCU–trnGUCC (96 sequences) and trnLUAA–trnFGAA (18 se-quences), producing a total of 234 new sequences (Appendix S1).Six trnL–trnF sequences were obtained from Roquet et al. (2008).Voucher information and GenBank accession numbers for taxaused in this study are detailed in Appendix S2 (Tables S1 and S2).

2.3. Phylogenetic analyses

The sequences of each region were aligned with MAFFT (Katohet al., 2005) and checked by eye using Bioedit (Hall, 1999). Phylo-genetic analyses were performed on a concatenated dataset com-prising the four sequenced regions. MrModeltest v.2.2 (Nylander,2004) was used to determine the best fitting model of sequenceevolution for each data partition, employing the Akaike Informa-tion Criterion (AIC) (GTR + C for each dataset). Bayesian inference(BI) analyses under the GTR + C model were conducted usingMrBayes v.3.1 (Ronquist and Huelsenbeck, 2003). These analysesconsisted of two independent runs with 4 chains each over 10 mil-lion generations, with one tree sampled each 100 generations. Thefirst 25,000 trees were eliminated before summarising the poster-ior tree distribution. The convergence of both runs was checkedusing Tracer 1.4.1 software (Drummond and Rambaut, 2007).

We analysed the relationships among island and African lin-eages through a haplotype network approach using 1–6 individualsof different populations/islands with the two most variable re-gions: trnS-trnG (96 sequences) and rpl32-trnL (96 sequences).Genealogical relationships among haplotypes were inferred withthe statistical parsimony algorithm implemented in TCS 1.21(Clement et al., 2000). The maximum number of differences result-ing from single substitutions among haplotypes was calculatedwith 95% confidence limits, treating gaps as missing data.

A relaxed clock method with uncorrelated rates drawn from alognormal distribution was used to reconstruct divergence timesusing BEAST v.1.6.0. (Drummond and Rambaut, 2007). Analyseswere run using GTR + C model applying the auto-optimisation op-tion with random starting trees and Yule process as tree prior, withfour runs of 5 � 107 generations each, sampling every 1000th gen-eration. The resulting posterior distributions were checked usingTracer 1.4.1 software (Drummond and Rambaut, 2007). Four analy-ses were combined using LogCombiner 1.4.8. and the maximumcredibility trees from the BEAST analysis were calculated using TreeAnnotator v.1.6 after removing a burn-in of 20%. A secondary cali-bration value of 12 ± 2 Mya was assigned to the split between the


Roucela and Azorina groups (root node), based on the dating analy-ses of Olesen et al. (2012) and assuming a normal distribution .

2.4. AFLP genotyping

AFLP analysis (Vos et al., 1995) was performed to study the ge-netic variability and structure of the populations of C. jacobaea andC. balfourii. We used the AFLP Plant Mapping Kit (Applied Biosys-tems) following manufacturer’s recommendations. We tested 32combinations of selective primers, and selected the 4 pairs that pro-duced the most polymorphic and clear profiles: 1-EcoRI6-FAM-ACC/MseI-CCT; 2-EcoRI6-FAM-ACT/MseI-CAC; 3-EcoRIVIC-AGG/MseI-CAA,and 4-EcoRIVIC-AGG/MseI-CAC. We combined 0.3 lL of 6-FAM-labelled and 0.3 lL of VIC-labelled selective PCR products with0.5 lL of GeneScan 500 LIZ size standard and 13.5 lL of formamide.Fragment electrophoresis was performed at the Parque Científico deMadrid (Spain) using ABI 3730 capillary sequencer (AppliedBiosystems).

Amplified fragments were analysed using GeneMapper 3.7 soft-ware (Applied Biosystems), and peaks ranging between 100 and500 base pairs were recorded. AFLP Scorer software (Whitlocket al., 2008) was employed to carry out a reproducibility test. Themaximum acceptable error rate was fixed at 5% for each primercombination (Bonin et al., 2004). The AFLPdat R package (Ehrich,2006) was used to reformat the data. The number of private frag-ments found considering populations, pairs of populations and is-lands were determined. Only unambiguous markers and fullyrepeatable among duplicates were scored. Data reliability was as-sessed by comparison of duplicates, using one or two individualsper population (23 tests). The reproducibility obtained was 92–98% with a mean of 95.6%.

2.5. AFLP data analysis

The resulting AFLP presence/absence matrix was analysed usingAFLPSURV v.1.0 software (Vekemans, 2002) to estimate Nei’s genediversity, Hj, FST, the percentage of polymorphic fragments perindividual (P), and the bootstrapped Nei’s genetic distance matrixbetween individuals and populations (Nei and Li, 1979; Lynchand Milligan, 1994). The dataset was analysed assuming both par-tial self-fertilisation and Hardy–Weinberg equilibrium. A Bayesianmethod was used to estimate allelic frequencies, employing non-uniform prior distribution (Zhivotovsky, 1999). Ten thousand per-mutations were performed to calculate the FST.

Genetic distances between individuals, populations and geo-graphic groups of C. jacobaea and C. balfourii were then calculated.Neighbour-nets of AFLP data were also estimated for individualsand populations with SplitsTree v.4.10 software (Huson and Bry-ant, 2006) (Fig. S1). To quantify the amount of genetic differentia-tion attributable to geographic and population subdivision, ahierarchical analysis of molecular variance was performed (Excof-fier and Lischer, 2010) using ARLEQUIN v.3.5 software.

We analysed the structure of populations with the Bayesianmethods implemented in STRUCTURE v.2.2 (Pritchard et al.,2000; Falush et al., 2007) and BAPS (Corander and Marttinen,2006), which allow for clustering genotypes into K populationswith no a priori knowledge of individual’s origin. For the STRUC-TURE analysis, an admixture model and uncorrelated allele fre-quencies between groups were assumed. We evaluated thelikelihood of K values (from 1 to 7) with 10 runs performed foreach K. For each run, we sampled 500,000 MCMC replicates aftera burn-in of 100,000 replicates). For each K value, only the run withthe highest maximum likelihood value was considered. The LnP(D)for the successive decomposition of groups was used in all STRUC-TURE analyses (Evanno et al., 2005; Figs. S2 and S3). A second anal-ysis with the same assumptions was performed for only C. jacobaea




(Fig. S3). An analysis with the same parameters was carried outwith BAPS.

Mantel tests for correlation between matrices of genetic andgeographic distances were performed on the entire dataset andfor groups (using mean values) using NTSYS v. 2.1 software (Rohlf,1998). The genetic distance was based on the presence/absencematrix; the geographic distance was based on absolute distancesbetween geographic coordinates for each collected population. Tocomplement this, the kinship multilocus coefficient (FIJ) was alsoestimated using SPAGeDi software (Hardy and Vekemans, 2002)to determine the spatial structure of the examined populations.The inbreeding coefficient (FIS) was set at 0.1 (other values wereused but the results were similar and are therefore not shown),as suggested by Hardy (2003), with 20,000 permutations. In addi-tion, BARRIER v.2.2 software (Manni and Guérard, 2004) was usedto identify possible geographic locations acting as major geneticbarriers among C. jacobaea populations, the significance of thoseidentified was examined by means of 1000 bootstrapped distancematrices obtained with AFLPsurv. Assignment tests for C. jacobaeawere performed to show whether the number of miss-assignedindividuals in populations varied with respect to the different is-lands or groups of islands. In STRUCTURE analysis it was assumedthat the sample consisted of 11 populations and that prior migra-tion between populations was rare (m = 0.05). Known informationfor the potential source populations was incorporated for all indi-viduals. Individuals were then assigned to populations using pos-terior probabilities.

2.6. Pollinators and floral morphology of Campanula jacobaea

Corolla length and width, sepal length and fruit length weremeasured in 11 populations of C. jacobaea from five islands tosearch for morphological differences that might be related to anydifferentiated genetic group. The corolla length and width weremeasured in first and late blossoms, in eight plants per populationand in two–three flowers per plant. The corolla width was mea-sured in the middle and the outer part of the tube.

Fig. 1. Phylogeny based on cpDNA sequences, biogeographic areas, lifespan and chronColumns to the right of the taxon names indicate distribution areas (letters) and lifespaneastern Mediterranean, WM: western Mediterranean, M: Mediterranean, MA: Macarone


Observations of insects arriving to white and blue flowers weremade in two populations of C. jacobaea in the islands of SantoAntâo (Cova and Corda), and São Nicolau (Monte Gordo). In eachcensus the number of open flowers on each plant and the numberof insects that landed on them were recorded over 15 min periods.The total amount of time spent observing each species was 8 h.Flower-visiting insects were collected and identified in thelaboratory.

To test for self-compatibility, around five inflorescences from 8to 10 cultivated plants were bagged and manually self-pollinated,and the number of seeds in the mature mericarps counted. A sim-ilar number of field non-bagged plants were used as controls.

3. Results

3.1. Dated phylogeny of Azorina group

The entire cpDNA dataset resulted in a sequence alignment of2934 bp (petB-petD, rpl32–trnL, trnS–trnG, and trnL–trnF). In theAzorina group, C. polyclada is sister to the other eight species andthen Azorina vidalii is sister to the remaining species. A sister grouprelationship exists between C. kremeri, and the clade formed byC. filicaulis, C. mollis, C. embergeri, C. dichotoma, C. hypocrateriformis,C. jacobaea and C. balfourii (Fig. 1). The split between C. polyclada,distributed across central Asia, and the rest of the Azorina subgroupis estimated at 5.7 Mya (2.9–8.5). C. hypocrateriformis-C. jacobaea-C. balfourii clade was well supported. Separation of C. hypocrateri-formis–C. balfourii–C. jacobaea and the remaining species is datedat 3.4 Mya (1.8–5.4). Finally, the ancestor of the African C. hypo-crateriformis, C. jacobaea and C. balfourii dates back to 1.4 Mya(0.59–2.41), while the separation of the last two species wouldhave occurred 1.0 Mya (0.39–1.7).

3.2. Genetic diversity and haplotypes of C. balfourii and C. jacobaea

Few differences were detected between C. hypocrateriformis,C. jacobaea and C. balfourii for the two cpDNA regions used in hap-

ogram of the Azorina group. Numbers above branches are posterior probabilities.(squares). Distributions: A: Arabia (Socotra), CA: central Asia, WA: western Asia, EM:sia, N: northern Africa. Lifespan: open squares: annual, filled squares: perennial.



Fig. 2. (a) Geographic distribution, genetic clusters inferred from Bayesian clustering of the AFLP data, number of private fragments for each group/species, and number ofprivate fragments found in pairs of groups/species. The species/group is indicated by a colour dot. Each population includes (1) the assignment of individual species/groups tothe two clusters detected in STRUCTURE analysis; the number on each cluster indicates the population, and (2) a histogram where: i the first bar on the left indicates thenumber of private fragments for each group/species (number in red), and ii the following bars represent the number of private fragments found in pairs of groups/species,represented by their colours (numbers in black). They are also indicated by a letter in the histogram: A: Santo Antâo, N: São Nicolau, S: Santiago, F: Fogo, B: Brava, Q: Socotra.(b) The inset shows the haplotypes network (pink: C. hypocrateriformis, blue: C. jacobaea and yellow: C. balfourii).

Fig. 3. Geographic distribution and genetic clusters inferred from Bayesianclustering of the AFLP data for each population of C. jacobaea. The population isindicated by a number (the same as those used in Table 1). The figure shows theassignment of the populations to the three clusters detected in STRUCTUREanalysis. Dotted lines in black indicate significant BARRIER boundaries with theirbootstrap values.


lotype analyses (Table S3). Differences between C. hypocraterifor-mis and C. jacobaea were found only for eight nucleotide positionsin total; nine between C. hypocrateriformis and C. balfourii, andseven between C. jacobaea and C. balfourii. Fig. 2b shows the result-ing haplotype network.

A total of 1005 AFLP fragments from 114 individuals belongingto 12 populations were scored, 110 for C. balfourii, 509 for C. jaco-baea, and 386 fragments for both species. Since only two samplesof C. hypocrateriformis were collected, this taxa was not includedit in the AFLP study. A high number of AFLP fragments werepolymorphic for C. balfourii (370–443) and C. jacobaea (358–604)(Table 1). The highest Hj and P were recorded for C. balfourii, whilethe lowest were those of the C. jacobaea populations of São Nicolau(Table 1). Both populations of C. balfourii contained seven or moreprivate AFLP fragments, while only two populations of C. jacobaeacontained more than one (Table 1).

The number of private fragments per island and species washigh in C. balfourii (110) and C. jacobaea of Santo Antâo (26), andlow in C. jacobaea of Brava (1) (Table 1; Fig. 2a). All islands, except-ing Brava, returned more private fragments than fragments exclu-sive to any pair of islands of the same archipelago (Table 1 and S4;Fig. 2a). Considering separately the populations within islands, acomparison of the number of private fragments found in pairs ofpopulations from the same island returned a higher figure forC. balfourii (populations of the Qalansiyah and the Haggiher moun-tains had 62 private fragments) than for C. jacobaea, with only twoto five fragments found in the populations of Santo Antâo, and fivein those of São Nicolau (Table S5).

Considering both species together, we found a low number ofprivate fragments found in pairs of populations composed by onepopulations of C. balfourii plus one population of C. jacobaea; con-cretely, there was three private fragments in Socotra and Santo


Antâo, two in Socotra and São Nicolau or Santiago, and one in Soco-tra and Fogo (Table S4; Fig. 2a).




3.3. Genetic structure and population differentiation

STRUCTURE analysis of both species gave the highest support toK = 2, separating C. balfourii and C. jacobaea (Fig. 2a and S2). Afterexcluding the two populations of C. balfourii from the analysis,the highest support was for K = 3; the three groups were SantoAntâo, São Nicolau and the southern Islands (Fig. 3 and S3). Similarresults were obtained with BAPS (K = 3, data not shown) and BAR-RIER analyses of C. jacobaea, which detected three major bound-aries separating four islands or groups of islands: Santo Antâo,São Nicolau, Fogo plus Santiago, and Brava (Fig. 3). The neigh-bour-net diagrams obtained via AFLP analysis (Fig. 4 and S1)showed four main clusters: a first one corresponding to C. balfouriipopulations; a second one corresponding to São Nicolau popula-tions; a third one composed by Santo Antâo populations, and afourth one corresponding to the populations of southern islands(Brava, Fogo and Santiago). Only two nodes had high bootstrap val-ues (>90%): one connecting both populations of C. balfourii and an-other one linking the three populations of C. jacobaea of SãoNicolau. Individual membership assigned by STRUCTURE analysisand prior population information showed the highest number ofmiss-assigned individuals to occur for the southern islands, andthat the proportion of individuals assigned to a different island de-pended on the island in question and was significantly different tothat expected (v2 = 14.884, p < 0.005) (Table S6). In agreementwith these results, the AMOVA analyses showed the greatest ge-netic variance among groups when taking into account two or four

Fig. 4. Neighbour-net of AFLP data for individuals of C. jacobaea and C. balfourii. Colouneighbour-joining analysis using Nei-Li distances over 1000 bootstrap replicates. Coloursda Antonia (S1), Sierra da Malagueta (S2), and Brava (B); green, Santo Antâo: Cova (A1)yellow: C. balfouri of Socotra: Qalansiyah (Q1), Haggiher Mts. (Q2). Pictures show the sh


groups, i.e., either C. balfourii and C. jacobaea, or C. balfourii (Soco-tra), C. jacobaea (Santo Antâo), C. jacobaea (São Nicolau) and C. jac-obaea (Santiago, Fogo and Brava) (Table S7).

The Mantel test performed on C. jacobaea to search for correla-tions between genetic and geographic distances returned a non-significant result (r = �0.085, p = 0.62). However, the first intervalanalysed (0–35 km) using the SPAGeDi software (Fig. S4) gave asignificant positive result, suggesting populations to be more sim-ilar at short distances than expected by chance. This distance inter-val includes the comparisons between populations of the sameislands.

3.4. Morphological variability in Campanula jacobaea

C. jacobaea exhibits perennial lifespan and larger flowers than C.balfourii which is an annual that looks like their sister C. hypocrat-eriformis. Regarding C. jacobaea,a wide variability was observed indiagnostic characters used to separate this species from C. braven-sis among populations and islands. Individuals on Brava showedshorter corollas than the rest, while those of Santiago and São Nic-olau were wider (Fig. S5). The colour of the corolla was also vari-able: it was always greenish-white on Fogo and Brava, blue onSantiago and São Nicolau, and sometimes blue or white on SantoAntâo (Table S8; Fig. 4). To investigate whether these colour andshape differences were related to pollinator species, the visitorsto white and blue flowers on Santo Antâo and São Nicolau were re-corded. They were always visited by small solitary Halictidae bees,

rs indicate species or groups of populations. Numbers are bootstrap values fromand acronyms are: red, Fogo: Ponta Verde (F1), Cha das Caldeiras (F2), Santiago: Pico, Corda (A2), Coroa (A3); blue: São Nicolau: Tarrafal (N1), Monte Gordo (N2) , andape of representative flowers in each island.




and in some populations (those of Cova and Corda in Santo Antâo,and Tarrafal in São Nicolau), the visitation rate was so low that self-pollination would seem more likely. However, this observationcould be biased by the fact that the total census time was relativelyshort. On the other side, the hypothesis of self-pollination is sup-ported by the results of a greenhouse experiment involving plantsfrom Tarrafal in São Nicolau. Self-pollination produced a seed set of50% compared to artificial crosspollination (Table S9). In contrast,C. balfourii is predominantly autogamous while the rest of speciesof this group may be autogamous, facultative or allogamous(Tables S1 and S9; Nyman, 1991, 1992; Olesen et al., 2012).

4. Discussion

4.1. Recent disjunction between C. jacobaea and C. balfourii: CapeVerde archipelago and Socotra as Pleistocene refugia

This study shows a striking pattern of disjunction between twosister bellflower species distributed on two distant archipelagosseparated by an entire continental landmass (8300 km): C. balfouriiendemic to Socotra (a small archipelago in the Indian Ocean) and C.jacobaea, restricted to a few islands of the mid-Atlantic ocean. Thedating analysis and the AFLP data suggest that they underwentstrong divergence in the Lower Pleistocene. The presence of manyprivate fragments per species, their clear division into two cpDNAclades, and the seven nucleotidic differences found in the trnS-trnGplus rpl32–trnL matrix, provides strong evidence of this diver-gence. Both species have likely been disconnected since the sepa-rating event.

Interestingly, the annual species Campanula hypocrateriformis,distributed in southwestern Morocco where there is a Macarone-sian type of climate (Dobignard, 2002) was found to be a sister tax-on of C. jacobaea and C. balfourii. It would have diverged from theancestor of C. jacobaea and C. balfourii some 1.4 Mya. It is plausiblethat C. hypocrateriformis descended from an ancestral species thatmay have been distributed in North Africa during the Pleistoceneand expanded at some point to Cape Verde islands and Socotra.Consequently, the disjunction between C. jacobaea and C. balfouriimay be the result of dispersal from Africa, while the ancestral con-tinental populations may have later retreated due to a successionof arid periods. The fact that the sister species of C. jacobaea andC. balfourii is a northern African species is consistent with thehypothesis of ancient fragmentation.

The long history of climatic shifts in North Africa led to a wan-ing-and-waxing process in many plant groups across the Sahara(Maley, 2000). Consequently, many taxa might have expandedtheir ranges during favourable periods, with populations becomingfragmented and differentiated during unfavourable ones (Besnardet al., 2009). Unfortunatelly, only a few taxa with east–west ornorth–south disjunctions in the African continent and/or Macaro-nesia or Socotra have been studied using phylogenetic, phylogeo-graphic and dating analyses.

Pan-African disjunct distributions are commonly explained asthe result of colonisation during favourable periods followed bydivergence or differentiation in situ in certain refugial distributioncentres (Sanmartín et al., 2010; Thulin et al., 2010). According tothe definition of Médail and Diadema (2009), refugia are ‘areaswhere distinct genetic lineages have persisted through a series ofTertiary–Quaternary climate fluctuations’ (see also Vargas, 2007).The archipelagos of Macaronesia and Socotra meet this definitionof refugia since they harbour many different lineages. Refuge lin-eages rarely show high levels of genetic diversity compared tothose found in neighbouring areas (but see Désamoré et al.,2011; Fernández-Mazuecos and Vargas, 2011). However, a com-parison between Socotra and Africa is unfeasible since most


African relatives of Socotran plants have never been studied orare currently extinct.

The present distribution of C. hypocrateriformis, C. jacobaea andC. balfourii agrees with the refuge hypothesis. Campanula jacobaeais not found in the drier islands (Boavista, Sal and Maio), and isvery rare on Sao Vicente. Similarly, C. balfourii is absent on the xericislands of Samha, Darsa and Abd-al-Kouri, and is only present onthe main island of Socotra. C. balfourii had the highest percentageof polymorphic fragments (74.6–89.3%; Table 1), but the totalnumber of polymorphic fragments was generally higher in C. jaco-baea (358–604; Table 1). The high number of private fragments inC. balfourii may be explained by the existence of a long and stableisolation period for the island, while the high number of polymor-phisms indicates that the size of the populations never reached ex-tremely low values (Charlesworth, 2009). Currently, while thepopulations of C. jacobaea are generally large, those of C. balfouriiare smaller, as seen for other annuals, and population sizes fre-quently fluctuate among years and localities. It seems to be welladapted to occasionally wet and disturbed mountain habitats,quite similar to those described for other African annuals (C. hypo-crateriformis and C. kremeri) related to C. jacobaea and C. balfourii.

4.2. Population genetic diversity in C. jacobaea and C. balfourii

For C. jacobaea, the number of genetic polymorphisms and pri-vate fragments per island was highest on Santo Antâo, the largestand westernmost island of the Cape Verde archipelago which is of-ten covered by fog. Polymorphism was high (and similar in extent)across the three southern islands; the lowest level was seen in SãoNicolau.

The question remains as to how the seeds of Campanula reachedthe isolated archipelagos where they are now found, and how theydispersed from one island to another. In northeast Africa, strongnorth-westerly winds (summer) produced by the subtropical highpressure cells of Africa flow towards Arabia. These may have per-mitted Campanula seeds to reach the Arabian Peninsula from EastAfrica (Van Campo, 1991). Northwest Africa is characterised by apronounced atmospheric circulation system, with the major windbelts being the Northeast Trade Winds, the January Trade Winds,the Southerly Trade Winds (all surface winds), and the AfricanEasterly Jet (Hooghiemstra and Agwu, 1988). The distribution ofgenetic diversity for Campanula and the genetic distances amongthe islands suggest that the dispersal of seeds between the CapeVerde islands is performed by the Northeast Trade Winds, as forother Macaronesian islands (Marzol, 2008; Rognon and Coudé-Gaussen, 1996). In the Cape Verde archipelago, the lowest FST val-ues occur between the islands Santiago, Fogo and Brava which aresituated NE–SW to one another. The Northeast Trade Winds maymore frequently carry seeds and increase the genetic flow amongthe southern islands than between northern and southern isles,or between the two northern islands. The directions of the tradewinds, along with the differences in the islands’ surface reliefand distance, might explain the genetic structure of C. jacobaeain three main clusters.

No general patterns can be inferred for plants from the CapeVerde islands or Socotra because of the low number of speciesstudied to date. The only study performed to date on another groupof plant species endemic to Cape Verde islands focused on threespecies of Echium (Romeiras et al., 2007). This work showed thesetaxa differed in terms of genetic diversity depending on the island.The more widely spread E. stenosiphon (Santo Antâo, São Vicenteand São Nicolau) showed greater diversity than the restricted E.vulcanorum (Fogo) and E. hypertropicum (Santiago). Low diversitywas also reported for the São Nicolau populations of Echium steno-siphon compared to the remaining islands (Romeiras et al., 2007).This low genetic diversity may be explained by São Nicolau’s




smaller mountainous area compared to the other Cape Verde is-lands (except for Brava), and its fewer sites moistened by the tradewind mists. The only other pair of species to have been studied interms of genetic diversity in the Socotra archipelago (Hughes et al.,2003) also showed an interesting pattern. A lack of genetic varia-tion was recorded for Begonia samhaensis, a very rare species foundon Samha Island. This is likely due to the small size and low alti-tudes of the island where the mist zone habitat is very narrowand altitudinal migration in dry periods is not possible, unlikeB. socotrana on Socotra (Hughes et al., 2003).

4.3. Floral morphological variability in C. jacobaea

The lack of a trade wind connection between the northern andsouthern groups of islands of Cape Verde may explain the slightmorphological differentiation of C. jacobaea (the flowers are widerin Santiago and São Nicolau, and generally blue in all islandsexcepting Fogo and Brava). As a consequence of this variability,C. jacobaea was split into two different species by Chevalier(1935): C. jacobaea corresponded to the populations in the north-ern islands plus some populations in the southern island of Santi-ago, while the name C. bravensis was given to those found in thesouthern islands of Brava and Fogo plus some of the populationsin the south of Santiago. Thus, the two species were supposed tocoexist in Santiago (Leyens and Lobin, 1994; Brochmann et al.,1997). However, our phylogeographic data does not agree with thisdelimitation; the populations sampled in Santiago (which at leastone, S2, would clearly correspond geographically and morphologi-cally to C. jacobaea) appear in the southern genetic cluster with thepopulations attributed to C. bravensis. Moreover, the morphologicaldata obtained in this study shows a wide variability in the diagnos-tic features of corolla colour, shape and width, and this variabilitydoes not correspond to any geographic or genetic groupings. Fur-ther, neither the colour nor shape differences seem to be relatedto pollinator species, and different flower types may be found inclose localities (e.g. on Cova, Santo Antâo).

4.4. Concluding remarks

Many disjunct distributions may be explained as the result ofthe isolation of lineages in refugial distribution centres. The archi-pelagos of Macaronesia and Socotra have probably acted as refugiasince they contain lineages with high levels of genetic diversitycompared to neighbouring areas. The present study shows that C.jacobaea and C. balfourii are closely related species despite thestriking disjunction between their widely-separated archipelagos.The fact that their closest known taxon (C. hypocrateriformis) isfound in northern Africa, which is also sister to a lineage includingnorthern African species, and that the three species were estimatedto have diverged in recent periods of the Pleistocene, suggests thatrecent climatic shifts in northern Africa played a major role in theirevolutionary history. More studies in different groups would benecessary to detect any generality of such a pattern, and to deter-mine the processes of differentiation that occurred in these refugia.Reproductive and population analyses of C. hypocrateriformiswould help to further our understanding of the evolution of theAfrican Campanula species.

Acknowledgements

The authors thank I. Sanmartín and J. M. Olesen for their kindsupport and helpful discussions and comments. Special thanksare owed to the Ministry of Water and the Environment, the SCDPand EPA of Yemen (Socotra) for their authorization to collect C. bal-fouri on the Socotra Archipelago. Ahmed Saleh was a most valuableguide. In Cape Verde we thank the directors of Monte Gordo (Saô


Nicolau), Serra da Malagueta (Santiago) and Fogo National Parksfor permissions to conduct fieldwork. We also thank B. Vigalondofor her helpful collaboration during the AFLP analyses. E. Cano, G.Andreu, G. Sanjuanbenito and F. Durán (Real Jardín Botánico de Ma-drid) provided excellent technical assistance. J. Caujapé-Castells,L.Saéz, J. Calleja, J. Molero, A. Susanna, S. Scholz, A. Marrero, K. Alp-inar, R. Rodrigues, and M. Sequeira are thanked for their help withfield collecting. Gratitude is also owed to the following herbaria:BC, BCB, BCN, ISTE, LE, MA, MO, P, UPS and W. This work was partlyfinanced by the Spanish Dirección General de Investigación Científicay Técnica through the research Projects DGICYT N�CGL2006-09696,DGICYT N�CGL2009-13322-C03-01 and DGICYT N�CGL2010-18631/BOS, and via grants from the Consejería de Educación de laComunidad de Madrid. M. Alarcón was funded by the JAE-Doc pro-gramme (CSIC/FSE) (Ministerio de Economía y Competitividad) andA. García-Fernandez by a postdoctoral contract (CGL2010-22234-C02/BOS).

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.ympev.2013.06.021.

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Phylogenetic and phylogeographic evidence for a Pleistocene disjunction between Campanula jacobaea (Cape Verde Islands) and C. balfourii (Socotra