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Syst. Biol. 57(5):732–749, 2008
Copyright c
Society of Systematic Biologists
ISSN: 1063-5157 print / 1076-836X online
DOI: 10.1080/10635150802302450
Multiple Colonizations, In Situ Speciation, and Volcanism-Associated Stepping-Stone
Dispersals Shaped the Phylogeography of the Macaronesian Red Fescues
(Festuca L., Gramineae)
ANTONIO D´
IAZ-P ´
EREZ,1MIGUEL SEQUEIR A,2ARNOLDO SANTOS-GUERRA,3AND PILAR CAT AL ´
AN1
1Department of Agriculture (Botany), High Polytechnic School of Huesca, University of Zaragoza, Ctra. Cuarte km 1, 22071 Huesca, Spain;
E-mail: adiape@cantv.net (A.D.-P.); pcatalan@unizar.es (P.C.)
2Department of Biology (CEM), Universidade da Madeira, Alto da Penteada, 9000 Funchal, Portugal; E-mail: sequeira@uma.pt
3Botanic Garden of La Orotava (ICIA), Retama 2, 38400 Puerto de la Cruz, Tenerife, Spain; E-mail: asantos@icia.es
Abstract.—Whereas examples of insular speciation within the endemic-rich Macaronesian hotspot flora have been docu-
mented, the phylogeography of recently evolved plants in the region has received little attention. The Macaronesian red
fescues constitute a narrow and recent radiation of four closely related diploid species distributed in the Canary Islands
(F. agustinii), Madeira (F. jubata), and the Azores (F. francoi and F. petraea), with a single extant relative distributed in mainland
southwest Europe (F. rivularis). Bayesian structure and priority consensus tree approaches and population spatial correla-
tions between genetic, geographical, and dispersal distances were used to elucidate the phylogeographical patterns of these
grasses. Independent versus related origins and dispersal versus isolation by distance (IBD) hypotheses were tested to
explain the genetic differentiation of species and populations, respectively. Genetic structure was found to be geographi-
cally distributed among the archipelagos and the islands endemics. The high number of shared AFLP fragments in all four
species suggests a recent single origin from a continental Pliocene ancestor. However, the strong allelic structure detected
among the Canarian, Madeiran, and Azorean endemics and the significant standardized residual values obtained from
structured Bayesian analysis for pairwise related origin hypotheses strongly supported the existence of three independent
continental-oceanic colonization events. The Canarian F. agustinii, the Madeiran F. jubata, and the two sister F. francoi and
F. petraea Azorean species likely evolved from different continental founders in their respective archipelagos. Despite the
short span of time elapsed since colonization, the two sympatric Azorean species probably diverged in situ, following eco-
logical adaptation, from a common ancestor that arrived from the near mainland. Simple dispersal hypotheses explained
most of the genetic variation at the species level better than IBD models. The optimal dispersal model for F. agustinii was a
bidirectional centripetal stepping-stone colonization pattern, an eastern-to-western volcanism-associated dispersion was fa-
vored for F. francoi, whereas for the recently derived F. petraea a counterintuitive direction of colonization (west-to-east) was
suggested. The population-based phylogeographical trends deduced from our study could be used as predictive models
for other Macaronesian plant endemics with similar distribution areas and dispersal abilities. [Bayesian genetic analyses;
colonization of oceanic islands; dispersal models; Festuca sect. Aulaxyper; Macaronesia; phylogeography.]
Oceanic islands have been considered natural labora-
tories for the study of colonization and evolutionary ra-
diation processes (Darwin, 1859; Mayr, 1942; Carlquist,
1965) and for investigating biogeographical issues re-
lated to the origins and evolution of their respective
biotas (MacArthur and Wilson, 1967; Emerson, 2002).
Evolutionary theories about these islands that are often
young in a geological context suggest that the potential
for colonization is inversely related to isolation and that
niche preemption often precludes multiple colonizations
of congeneric taxa from the mainland (Whittaker, 1998;
Silvertown, 2004; Carine et al., 2004). Modeling studies
further suggest that species that locate few propagules in
the dispersal medium with high survivorship for a given
distance will ultimately reach the island(s) and undergo
founder speciation (Paulay and Meyer, 2002).
The Macaronesian islands are characterized by a high
level of plant endemism (Humphries, 1979; Bolos, 1996;
Santos-Guerra, 1999) and the region represents one of
the best-studied geographical settings of oceanic plant
speciation (Francisco-Ortega et al., 1996; Moore et al.,
2002; Carine et al., 2004). Most endemic Macarone-
sian lineages are derived clades and have sister clades
that are distributed mainly in the western Mediter-
ranean region (Emerson, 2002; Comes, 2004; Carine et al.,
2004; Carine, 2005). The relatively short distance to the
African and European continents would have allowed
for the colonization from this area with the influence of
oceanic currents (North Atlantic and Canary streams)
and northeasterly trade winds likely to have fostered
the dissemination of plant germplasm from the con-
tinent. For this reason, it has been proposed that fre-
quent colonizations from the continent have contributed
to the high number of species endemic to the Canary
Islands (Kim et al., 1999; Francisco-Ortega et al., 2000).
For islands extremely isolated from continental sources,
vacant ecological spaces are filled through adaptive radi-
ation (Gillespie and Roderick, 2002). The origins of sev-
eral Macaronesian plant groups have been interpreted as
the results of single colonization events followed by in
situ speciation (i.e., B¨ohle et al., 1996; Kim et al., 1996;
Francisco-Ortega et al., 1997a, 1997b, 2002; Jorgensen
and Frydenberg, 1999; Helfgott et al., 2000; Jorgensen
and Olesen, 2001; Mort et al., 2002; reviewed in Silver-
town, 2004, and Carine et al., 2004), though an increas-
ing number of studies indicated the likely existence of
multiple colonization events from the near continent
(i.e., Francisco-Ortega et al., 1996; Panero et al., 1999;
Vargas et al., 1999; Hess et al., 2000; Bohs and Olmstead,
2001; Percy and Cronk, 2002; Fuertes-Aguilar et al., 2002;
Molero et al., 2002; reviewed in Silvertown, 2004, and
Carine et al., 2004) or even from more distant regions
(Carine, 2005). Genetic surveys that have explored the
level and structure of genetic diversity of Macaronesian
plant endemics have demonstrated greater levels of ge-
netic variation than in Pacific oceanic endemic species
732
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EREZ ET AL.—PHYLOGEOG RAPHY OF THE MACAR ONES IAN RED FES CUES 733
FIG URE 1. (a) Geographical distances (in km) among the three Macaronesian archipelagos containing the four native Macaronesianred fescues
(Festuca sect. Aulaxyper) and (b–d) geographical location of their studied populations: (b) F. agustinii (western Canary isles, 15 populations); (c) F.
petraea (Azores, 7 populations preceded by the letter p, triangles) and F. francoi (Azores, 7 populations preceded by the letter f, crosses); and (d)
F. jubata (Madeira, 7 populations). Numbers are abbreviations from populations codes indicated in Table 1 (i.e., in the Canary Isles 1 identifies
Fagus1).
(Frankham, 1997; Francisco-Ortega et al., 2000; Oliva
et al., 2004), adding support to the evolutionary theory of
higher feasibility of colonization from less distant main
lands. However, recent molecular studies have found
contrasting patterns of genetic diversity and genetic di-
vergence among populations of different Macaronesian
plant species (Francisco-Ortega et al., 2000; Batista and
Sosa, 2002; S´anchez et al., 2004; Kim et al., 2005; Prohens
et al., 2007).
Three successive stages of volcanic activity have been
generally recognized in the geological evolution of Mac-
aronesian islands: an initial shield-stage of high volcan-
ism, followed by a quiescent stage, and ending with a
stage of posterosional reduced volcanism (Carracedo,
1999; Garc´ıa-Talavera, 1999). This pattern is reproduced
in the western Canary Isles where the oldest easternmost
island of Gran Canaria (14.5 Ma) is presently in the final
stage, the central Gomera (12 Ma) and Tenerife (7.5 Ma)
islands are in the second, and the youngest westernmost
islands of La Palma (2 Ma) and El Hierro (1.12 Ma) are in
the first (Carracedo, 1999). The island of Madeira (5.3 Ma)
is considered to be in a posterosional stage, which started
approximately 0.7 Ma (Geldmacher et al., 2000). In con-
trast, most of the nine islands of the young archipelago
of Azores (all except the oldest easternmost island of
Santa Maria that is 8.12 Ma) show recent volcanic ac-
tivity (Valad˜ao et al., 2002). This occurs in all eastern
(S˜ao Miguel, 4.01 Ma), central (Terceira, 3.52 Ma; Gra-
ciosa, 2.5 Ma; Faial, 0.73 Ma; S˜ao Jorge, 0.55 Ma; Pico,
0.23 Ma), and western (Flores, 2.16 Ma, Corvo, 0.71 Ma)
subarchipelagos (Fig. 1). Explosive volcanic eruptions in
the Macaronesian islands have been linked with catas-
trophic demographic events that virtually eliminated all
living organisms (Emerson, 2003). Massive landslides
also affected the population genetic pools after important
losses of islands volumes (Masson et al., 2002). Volcanic
events have joined islands (e.g., Tenerife; S˜ao Miguel),
favoring secondary contacts of previous allopatric pop-
ulations, though more commonly they led to fragmen-
tation of populations, resulting in new vicariant species
(G ¨ubbitz et al., 2005). All these phenomena, alone or in
concert, have the potential to affect the phylogeography
of the Macaronesian biotas.
The Macaronesian red fescues comprise four related
species: Festuca agustinii Linding, F. jubata Lowe, F. fran-
coi Fern. Prieto, C. Aguiar, E. Dias & M. I. Gut, and
F. petraea Guthnick ex Seub. With the exception of F.
petraea, which is found in coastal halophytic soils of the
Azores, all species grow on medium- to high-altitude
laurisilva cliffs in the central-western Canary Islands
(Gomera, Gran Canaria, El Hierro, La Palma, Tener-
ife), Madeira, and Azores, respectively. Independent
sources of evidence have suggested that these diploid
species are of relative recent origin, as their lineages
collapse in a basal polytomy, together with the only
diploid continental species (F. rivularis) and an other-
wise more recently evolved clade of highly polyploid
cosmopolitan taxa, within the well supported Festuca
sect. Aulaxyper (F. rubra group) clade (Catal´an, 2006). A
relaxed-clock analysis of nuclear ITS and plastid trnTF
sequences of Loliinae suggest that the radiation occurred
ca. 2.5 ±0.9 Ma, assuming that the divergence between
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734 SYSTEMATIC BIOLOGY VOL. 57
Triticeae and Aveneae-Poeae ocurred ca. 21 Ma (Inda
et al., 2008).
The simplest hypothesis for the colonization of Mac-
aronesian archipelagos is that of a stepping-stone model
of colonization (Emerson, 2002). A plausible scenario for
the Canary Islands would involve the colonization of
the older eastern islands with successive colonizations of
more westerly islands in an east-to-west direction. How-
ever, long-distance dispersals among islands and extinc-
tion might have obscured the direction of past dispersal
routes, generating other intricate patterns of colonization
(Kim et al., 1996; Francisco-Ortega et al., 1996).
The aim of this study was to investigate the colo-
nization and speciation patterns among recent Pliocene
colonists of the Macaronesia region through (i) tests of in-
dependent versus related origin hypotheses, and (ii) tests
of isolation by distance (IBD) and dispersal hypotheses.
More than a decade of research has produced a sub-
stantial number of evolutionary studies of Macaronesian
plants (see Carine et al., 2004, for a review). However,
most correspond to presumably older Tertiary groups
and have been based on a limited sample of sequences ac-
cessions. We were interested in testing whether the Mac-
aronesian red fescues resulted from a single colonization,
with subsequent, among-archipelago dispersal or re-
sulted from multiple insular colonization events. The
group is distributed in three out the four main archipela-
gos of Macaronesia (all except Cape Verde) and occupies
an island land area of 450 km2embedded within a to-
tal spatial area of 450,000 km2. The group therefore also
serves as a model to investigate the potential rapid dis-
persal among Atlantic archipelagos separated by more
than 800 km from each other. To our knowledge this is
one of the first attempts to resolve the phylogeography
of a recently radiated Macaronesian angiosperm plant
group based on a large population sampling.
Analyses of AFLP markers are used to detect phylo-
geographical patterns among species and populations
working at the interface of population differentiation
and speciation. This technique has been successfully em-
ployed for resolving phylogenetic relationships in plant
groups with low plastid and nuclear sequence variability
(Koopman et al., 2001; Ogden and Thorpe, 2002; Despres
et al., 2003; Koopman, 2005; Pimentel et al. 2007) and
for characterizing the genetic diversity among individ-
uals, populations, and species (Bensch and ˚
Akesson,
2005; Althoff et al. 2007). Koopman (2005) concluded
that AFLP markers produced reliable phylogenetic infor-
mation when nuclear sequences are too conserved and
are especially useful in the range of 0.016% to 0.05% se-
quence divergence, as observed in these otherwise un-
resolved Macaronesian red fescues (e.g., ITS: Catal´an,
2006; Inda et al., 2008). In contrast to the patterns of
rich species radiation observed in other Macaronesian
endemic plant groups (e.g., Argyranthemum: Francisco-
Ortega et al., 1996; Echium:B¨ohle et al., 1996; Sonchus: Kim
et al., 1996;Aeonium: Jorgensen and Frydenberg, 1999;
Crambe: Francisco-Ortega et al., 2002), the Macarone-
sian red fescues comprise four morphologically similar
species that are almost archipelago specific. This could
facilitate the testing of IBD and colonization hypotheses
from mainland ancestors that might have been affected
by glaciations and the dynamic volcanic environment
within old and newly arising archipelagos.
MATERIALS AND MET HOD S
Sample Collection and AFLP Analysis
We analyzed 36 populations of the four species of
Macaronesian red Festuca:F. agustinii (15 populations),
F. jubata (7 populations), F. francoi (7 populations), and F.
petraea (7 populations). Sampling covered the geograph-
ical distribution of each species, totaling 215 individuals
(Table 1, Fig. 1).
DNA was isolated following a modified CTAB pro-
tocol (Doyle and Doyle, 1987). The extraction DNeasy
Plant Mini Kit of Qiagen was employed for small quan-
tities of sample tissue in some cases. The concentra-
tion of each DNA sample was checked on 1% (0.5 ×
TBE) agarose gel using samples of known concentra-
tion. Approximately 200 ng of DNA was used for AFLP
analysis following the instructions of Invitrogen manu-
facturers with slight modifications. EcoRI and MseIre-
striction enzymes and their respective double-stranded
adaptors were employed in three successive steps: di-
gestion at 37◦C for 2 h, heat inactivation of restriction
enzymes at 70◦C for 15 min, and ligation at 20◦C for 2
h. Preselective amplification was performed after dilut-
ing the ligated DNA 10-fold with EcoRI+A and MseI+C
primers. PCR products were diluted 33-fold and used
for selective amplification with EcoRI and MseI primers
plus three additional selective nucleotides. Seven combi-
nations of EcoRI-MseI primers were evaluated with two
individuals of each of the four species. Two combinations
(M-CAA/E-ACC and M-CAG/E-AAG) that provided a
higher number of polymorphic markers were selected
and used for the analysis of all samples. Amplifications
were performed in PCT-100 MJ Research, Inc., and Ge-
neAmp System 9700 Applied Biosystem thermocyclers.
Preheated (50◦Cto55
◦C) 6% polyacrylamide gels were
run in 5×TBE electrophoresis buffer at 80 W for 21
/
2h.
Gels were subjected to silver staining for visualization of
bands following Bassam et al. (1991).
Accuracy of the AFLP markers was tested by recon-
ducting the whole AFLP protocol in one individual per
population in the most representative populations of
each species (approximately 5% of the total sample size)
and checking for consistency of recorded bands. As the
calculated error rate (4.83%), which corresponded to the
number of phenotypic differences related to the total
number of phenotypic comparisons, was below the crit-
ical bound of 5% indicated in previous reports (Bonin
et al., 2004, 2007; Pompanon et al., 2005; Pi˜neiro et al.,
2007), the obtained AFLP patterns were considered to be
highly reproducible. In order to increasethe quality of the
data, potentially unreliable bands that showed slight size
differences among putative homologous bands across
individuals, low-intensity bands, and either high (i.e.,
>410 bp) or low (i.e., <50 bp) molecular weights were
discarded from the final data matrix.
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TABLE 1. Data on sampled localities and AFLP variability of the four species of Macaronesian red Festuca: population codes, island location,
number of individuals (N), number of unique fragments (UFr), and number of shared fragments among species. agus =F. agustinii; juba =F.
jubata; juba* =F. jubata Fjuba6; fran =F. francoi; petr =F. petraea. Letters represent shared bands observed in at least two individuals per species.
Total number of shared bands between a single pair of species is indicated in parentheses.
Population Location N UFr agus fran (12) agus juba (3) agus petr (2) juba fran (2) juba petr (2) fran petr (1)
F. agustinii Canary Islands 126 (7)
Fagus1 Gran Canaria 3 ad j
Fagus2 Gran Canaria 5 abg j
Fagus3 Tenerife 10 adefg hj k
Fagus4 Tenerife 6 adeg ij k
Fagus5 Tenerife 12 acefg hij k
Fagus6 Tenerife 10 abdefg hj k
Fagus7 Tenerife 10 aceg hij k
Fagus8 La Gomera 7 ace hij
Fagus9 La Gomera 10 ae j k
Fagus10 La Palma 3 ae j k
Fagus11 La Palma 6 af*g j
Fagus12 La Palma 12 1 abdeg hij l
Fagus13 La Palma 13 abceg hj kl
Fagus14 La Palma 9 1 ae hj k
Fagus15 El Hierro 10 abeg hij
F. jubata Madeira 27 (3)
Fjuba1 Central peaks 3 hij
Fjuba2 Central peaks 5 hj
Fjuba3 Central peaks 5 hj
Fjuba4 Central peaks 4 hj
Fjuba5 Central peaks 4 hj m
Fjuba6 Curral das Freiras 5 3 h
Fjuba7 Bica da Cana 3 hj o
F. francoi Azores 33 (1)
Ffran1 S ˜ao Miguel 5 abce
Ffran2 S ˜ao Miguel 5 e m n
Ffran3 Terceira 4 be n
Ffran4 Faial 4 bcdefg n
Ffran5 Pico 5 defg
Ffran6 S ˜ao Jorge 5 befg n
Ffran7 Flores 5 m n
F. petraea Azores 27 (1)
Fpetr1 Santa Mar´ıa 4
Fpetr2 Graciosa 3 k n
Fpetr3 Graciosa 3 kl n
Fpetr4 Faial 4 k n
Fpetr5 Pico 5 k o n
Fpetr6 S ˜ao Jorge 4 kl o n
Fpetr7 Flores 4 n
Interspecific Genetic Structure and Phylogenetic Analyses
The analyses performed were based on the assump-
tions that (i) despite the fact that AFLP markers behave
as dominant markers, they could be used in simulated
genotype-based analyses of diploid species such as the
Macaronesian red fescues; (ii) comigrating fragments are
considered homologous loci; (iii) genetic distances be-
tween heterozygous and homozygous individuals for
specific loci are compensated across all surveyed loci;
and (iv) even if loci might not all reconstruct the same
coalescent history, the predominant sharing of homol-
ogous fragments indicate a common ancestry. Despite
the problems associated with some of these assumptions,
and given the high number of markers randomly gener-
ated from the whole genome, AFLPs has demonstrated to
be a suitable technique to recover phylogeographic sig-
nal among closely related taxa and populations (Despres
et al., 2003; Koopman, 2005; Pimentel et al., 2007; Althoff
et al., 2007).
Bayesian analyses were conducted at the interspecific
level, trying to estimate the genetic structure of taxa as a
preliminary step for further testing of evolutionary spe-
ciation and colonization hypotheses. First, genetic struc-
ture was quantified at the species level according to
the unbiased Bayesian-derived estimate Gβ
st, related to
Wright’s Fst coefficient, based on the fixed-effect models
proposed by Nei and Chesser (1983) using the program
Hickory v.1.0.4 (Holsinger et al., 2002). Hickory’s default
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736 SYSTEMATIC BIOLOGY VOL. 57
values (burn-in set to 50,000, sampling set to 250,000,
and thin set to 50) were used to specify the prior distri-
butions. AFLP data were analysed assuming two mod-
els: (1) free model and (2) θβ=0 model, where θβis the
among-populations fixation index. The deviance infor-
mation criterion (DIC) was used to choose the model that
best fitted to the data, based on their lower DIC values
(Holsinger and Wallace 2004). A lower DIC value of the
free model than the θβ=0 model would be indicative of
the existence of genetic structure in the data.
Second, to infer the spatial pattern of genetic diver-
gence of the Macaronesian red Festuca, Bayesian model-
based analysis was performed with STRUCTURE v.2.2
(Pritchard et al., 2000; Pritchard and Wen, 2004; Falush
et al., 2007). The F model with admixed ancestry was
used to estimate posterior probabilities of any predefined
number (K) of groups (hereafter Bayesian groups), and
individual percentages of membership were assigned to
them according to their AFLP multilocus profiles (Falush
et al., 2003, 2007). As a first strategy, a burn-in and a
Monte Carlo Markov chain (MCMC) of 5000 and 20,000
iterations, respectively, were used in all these searches.
Interspecific groupings were analyzed for different K
groups of 50 runs each, analyzing simultaneously the
studied populations of the Macaronesian red Festuca.
A second strategy, involving 5 ×105burn-in, 3 ×106
MCMC iterations, and 5 repetitions for K =1–7 did not
show any convergence into a unique clustering mode,
so the first strategy was chosen to obtain reliable es-
timates for computing K (rate of change in the log
probability of data between successive K values; Evanno
et al., 2005). The choice of the best K values was based
on the following criteria: (i) selection of the higher K;
and (ii) the higher probability value (lnP(D); Pritchard
and Wen, 2004) and the stability of clustering schemes
(measured by a similarity coefficient [SC]) between dif-
ferent runs (Rosenberg et al., 2002). SC of >0.85 was
taken as a measure of high stability. This coefficient was
computed with the Matlab software (The Mathworks,
1994). We also considered the inference of a common in-
dividual membership (the αparameter) for the ancestral
groups to compare it to αfrom non-structured groups
(∼1/K; Pritchard and Wen, 2004). Small αvalues im-
ply that most individuals derive essentially from one or
another ancestral Bayesian group, whereas α>1 val-
ues imply that most individuals are admixed (Pritchard
and Wen, 2004).
Third, unrooted evolutionary relationships among the
studied Macaronesian red Festuca samples were recon-
structed based on a Bayesian approach to model AFLP
marker evolution by nucleotide substitution and MCMC
simulations (Luo et al., 2007). The genetic model assumes
that a band might be lost due to mutations in the adap-
tor +restriction enzyme recognition sites or by a gain of
a restriction site in the intermediate region. It was also
assumed that all sites evolved independently with the
same rate according to a Jukes-Cantor model. A total of
1.3 ×106generations were simulated using simultane-
ously five parallel chains. Thirteen thousand trees were
sampled discarding the first 800 trees per parallel chain
to ensure convergence, totaling 9000 final trees for anal-
ysis. Preburn cycles were set to 10,000, tuning interval to
500, alphaA-tune =10, alphaS-tune =0.625, lambdaL-
tune =0.05, and lambdaG-tune =0.05. All simulations
were done with the software aflp v.1.01, kindly provided
by R. Luo. The posterior distribution of trees was sum-
marized through the posterior probabilities of common
clades to all trees obtained from the aflp v.1.0.1 output.
These probabilities were used to generate a priority con-
sensus tree (PCT) in which compatible common clades
were successively added to the tree according to their
decreasing order of probability. Clades with probabili-
ties less than 0.15 were treated as polytomies. The final
Newick string of the PCT was edited with the Dendro-
scope tree viewer (Huson et al., 2007)
Species Colonization and Speciation Hypotheses
Due to the lack of any reliable sister group root that
could provide a clearer direction of colonization and dis-
persal routes of the Macaronesian red fescues, and in
order to infer the probability of shared history among
species, the correlation structure of standardized resid-
uals between species pairs were obtained according to
Nicholson et al. (2002). Briefly, for each of the Bayesian
groups defined according to clustering and phylogenetic
analyses (i.e., F. agustinii, F. jubata, F. francoi, F. petraea) and
each of the studied loci, the residuals were calculated by
comparing estimated allele frequencies with those of the
hypothetical ancestral population. Allele frequencies of
the ancestor were obtained from the highest probabil-
ity run out of 50 runs (best value for K =4) using the
no-admixture model with correlated allele frequencies.
The testing of the colonization and speciation history
of the Macaronesian red fescues was done under the null
hypothesis of pairwise independent colonization events
from continental ancestors, each of them rendering a
distinct species. This hypothesis was based on the un-
resolved basal placements of the Canarian, Madeiran,
and Azorean red Festuca lineages (Festuca sect. Aulaxyper
clade) obtained in the Loliinae phylogenies of Catal´an
(2006) and Inda et al. (2008). In this case, the absence
of correlated residuals would suggest independent evo-
lution between the compared species, whereas a nega-
tive or a positive correlation would suggest divergent or
shared evolution, respectively. Shared evolution is com-
patible with a single origin from a continental source
followed by interarchipelago dispersals and subsequent
speciations.
Infraspecific Genetic Structure and Phylogenetic Analyses
Similar approaches to those previously assayed at
the species level were undertaken at infraspecific level,
to determine the genetic divergence and phylogenetic
patterns of populations within species. Establishment
of specific colonization hypothesis was preceded by
(i) the detection of genetic homogeneous groups by
mean of Bayesian STRUCTURE analysis, and (ii) the
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EREZ ET AL.—PHYLOGEOG RAPHY OF THE MACAR ONES IAN RED FES CUES 737
reconstruction of rooted phylogenetic relationships
among them using the infraspecific topologies of the
PCT.
In order to establish infraspecific groups, STRUC-
TURE analysis for different K values were tested for each
species, ranging from 1 to the number of sampled geo-
graphical populations plus two. At least 10 independent
runs were computed for each K to adjust better the num-
ber of hypothetical groups within each species. A burn-in
and an MCMC of 105and 106iterations were performed,
respectively. Substructuring of F. agustinii K=2 groups
was based on 25 repetitions for each K with a burn-in
and MCMC of 5 ×103and 104iterations, respectively.
The choice of the best K values was based on previous
interspecific criteria.
Infraspecific dispersal routes were generated accord-
ing to the genetic composition of STRUCTURE Bayesian
groups. It was assumed that populations within a
Bayesian group were highly related to each other and
consequently, infragroup population connections were
favored over intergroup population connections. The
root and order of colonization of the dispersal route with
the highest correlation values (see next section) were
established superimposing K =2 Bayesian groups for
F. agustinii and K =3 Bayesian groups for F. francoi and
F. petraea on the PCT. All individuals in a PCT clade con-
taining a Bayesian group were then treated as a single
genetic unit. In addition, given that it was of particular
interest to evaluate the contribution of the easternmost
Canarian F. agustinii individuals as a potential starting
point of colonization, the eastern Bayesian group was
further subdivided in the PCT into a Tenerife genetic unit
and Gran Canaria genetic unit (see Results and Table 2).
External nodes (i.e., those related to a Bayesian genetic
unit) were located in the geographic vicinity of its indi-
viduals, whereas the geographic locations of the internal
nodes were allowed to vary among different islands ac-
cording to alternative dispersal hypotheses (see Table 3).
Infraspecific IBD and Dispersal Analysis
The strength of alternative colonization hypotheses for
each of the four species was tested through simple and
partial correlations between a matrix of genetic distance
values and matrices of geographic or dispersal distance
values among populations (Dietz, 1983; Smouse et al.,
1986). Geographic distances are defined as the shortest
linear distance between any given population pair where
dispersion could have occurred in any possible direc-
tion (isolation by distance [IBD] model), whereas dis-
persal models include distances calculated as the sum
of the linear distances connecting all the nodes between
two populations for a given model (e.g., two or more
steps). In some cases, an intermediate virtual node was
created to connect geographically adjacent populations
with other groups or populations. Population pairwise
Gβ
st estimates were calculated based on the free model
of Hickory v.1.04 according to previously indicated
interspecific parameters. Correlation analyses were com-
puted with the program Phylogeographer v.1.1 (Buckler,
1999; Buckler et al., 2006). Significance was assessed by
means of 10,000 permutations.
For each species, different dispersal routes were tested
(see Figs. 3 to 5; Table 3), trying to identify the one that
best explained the colonization patterns within each
archipelago since the founding ancestor had speciated.
We were interested to investigate whether the best col-
onization model followed an intuitive east-to-west dis-
persal pattern, concordant with the geological ages of the
islands in each archipelago, or whether the optimal
models did not respond to this general pattern. Festuca
jubata was excluded from the analysis because in all
tested models the correlation coefficients between
genetic and dispersal distances (r) were not significantly
different from zero.
RESULT S
Genetic Diversity and Structure of the Macaronesian
Red Fescues
One hundred and eighty-one reliable polymorphic
bands were generated across the four studied species
using the two selective primers E-ACC/M-CAA and
E-AAG/M-CAG. All individuals showed unique AFLP
multilocus patterns. Unique and shared bands were ob-
served at both species and population levels (Table 1).
At the species level, F. agustinii showed the higher num-
ber of unique bands (7), followed by F. jubata (3) and
F. francoi (1) and F. petraea (1). However, none of those
bands served to characterize species as they tended to
be in low frequencies. The number of exclusive shared
bands between two species ranged from 1 (F. francoi–
F. petraea)to12(F. agustinii–F. francoi), with a mean
value of 3.6. Considering the whole set of polymorphic
bands (181), the proportions of unique (range 0.56% to
3.87%) and shared (range 0.56% to 6.6%; mean 1.99%)
bands were extremely low. At the population level,
the Madeiran Fjuba6 population was the most diverse
with three unique bands, whereas a single unique band
was observed in the Canarian populations Fagus12 and
Fagus14 (Table 1).
Free-model Bayesian analyses consistently gave lower
DIC scores than θβ=0 models, clearly supporting the
existence of genetic structure among the studied Festuca;
consequently, only free-model estimates of Gβ
st are dis-
cussed. According to Gβ
st, 31.0% of the overall variation
within the Macaronesian red Festuca was attributable
to differences among species; 26% of the total variation
within Festuca was partitioned among archipelagos. Gβ
st
analysis revealed that 22.2% of the overall genetic varia-
tion contained in what was previously considered to be
F. jubata sensu lato (F. jubata +F. francoi) was due to differ-
ences between the two species (see Discussion), whereas
15% was due to differences between the Azorean (F. fran-
coi,F. petraea) species.
Interspecific Genetic Structure and Phylogenetic Analysis
The Bayesian priority consensus tree (PCT) recovered
a high genetic structure among the Macaronesian red
fescues (Fig. 2a, b). Almost all conspecific individuals
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738 SYSTEMATIC BIOLOGY VOL. 57
TABLE 2. Results from Bayesian-model clustering analysis of the Macaronesian red fescues conducted with STRUCTURE and Matlab software.
K indicates the number of predefined Bayesian groups. K is the second-order rate of change of the likelihood distribution (Evanno et al., 2005).
Max lnP(X/K) and ln P(X/K) indicate the highest and the mean probability run values, respectively, for each K. SC is the mean similarity
coefficient between all pairs of runs (Rosenberg et al., 2002). αis the admixture proportion of an individual and N(qK) represents the number of
individuals and the mean proportion (in brackets) of membership of all individuals associated with each inferred Bayesian group in the highest
probability run. An individual was assigned to an inferred group according to its highest proportion of membership. Ø represents an empty
group.
KK Max lnP(X/K) lnP(X/K) SC αN(qK)
All Festuca (F. agustinii +F. Francoi +F. petraea +F. jubata)
1—−24,527.4 −24,568.1 — —
2 103.4 −20,963.5 −21,007.2 0.99 0.03 89(0.99); 126(0.99)
3 1.8 −19,563.1 −19,805.8 0.59 0.03 126(0.99); 60(0.98); 29(0.92)
4 0.1 −18,880.1 −19,205.4 0.38 0.03 126(0.99); 33(0.98); 27(0.97); 29(0.94)
5–7 0.3–1.8 0.18–0.32
F. francoi+F. petraea +F. jubata
1—−9,636.1 −9,662.4 — —
2 80.4 −8,260.3 −8,274.9 0.99 0.05 63(0.97); 26(0.96)
3 8.4 −7,581,2 −7,615.6 0.79 0.04 33(0.99); 27(0.97); 29(0.94)
4 0.3 −7,023.8 −7,390.9 0.90 0.03 5(0.99); 24(0.99); 33(0.99); 27(0.98)
5–6 7.0 0.45–0.48
F. agustinii
1—−11,184,6 −11,199.5 — —
2 195.7 −10,616.0 −10,618.5 0.99 0.07 66(0.97); 60(0.94)
3 2.2 −10,341.1 −10,347.3 0.99 0.09 48(0.89); 30(0.87); 48(0.86)
4 0.9 −10,026.2 −10,064.7 0.48 0.06 12(0.92); 32(0.92); 31(0.86); 51(0.86)
5 1.2 −9,772.9 −9,824,4 0.41 0.05 12(0.93); 32(0.93); 30(0.90); 24(0.87); 28(0.80)
6–17 0.1–8.6 0.00–0.37
Eastern group
2—−5,301.8 −5,352.2 0.59 0.23 38(0.88); 28(0.86)
3—−5,087.6 −5,123.1 0.73 0.09 22(0.88); 32(0.86); 12(0.82)
Western group
2—−4,360.6 −4,372.8 0.66 0.04 45(0.99); 12(0.98)
3—−4,099.1 −4,159.3 0.57 0.04 12(0.97); 38(0.97); 7(0.93)
F. francoi
1—−3,054.3 −3,065.1 — —
2 18.1 −2,856.6 −2,862.9 0.64 0.06 10(0.98); 23(0.98)
3 123.7 −2,556.3 −2,559.2 1.00 0.05 18(0.97); 5(0.96); 10(0.93)
4 4.2 −2,418.3 −2,440.1 0.68 0.05 5(0.95); 13(0.94); 5(0.93); 10(0.93)
5 13.7 −2,325.8 −2,480.7 0.65 0.08 5(0.94); 5(0.91); 10(0.9); 9(0.81); 4(0.78)
6 3.3 −2,918.4 −4,251.1 0.64 0.04 Ø; Ø; 5(0.96); 5(0.94); 10(0.90); 13(0.89)
7–9 0.81 0.53–0.68
F. petraea
1—−1,963.6 −1,972.7 — —
2 17.4 −1,695.1 −1,701.4 0.84 0.12 13(0.95); 14(0.91)
3 1.3 −1,555.6 −1,609.2 0.76 0.05 4(0.99); 8(0.96); 15(0.91)
4 92.4 −1,423.1 −1,427.8 0.99 0.04 4(0.99); 4(0.99); 12(0.97); 7(0.85)
5 5.3 −1,461.3 −1,468.2 0.99 0.04 Ø; 4(0.99); 4(0.99); 12(0.97); 7(0.82)
6 0.5 −1,480.5 −1,482.8 0.99 0.03 Ø; Ø; 4(0.99); 4(0.98); 12(0.96); 7(0.80)
7–9 0.2–0.8 0.66–0.92
F. jubata
1—−2,381.4 −2,396.7 — —
2 290.6 −1,881.0 −1,886.5 1.0 0.03 5(1.00); 24(1.00);
3 1.2 −1,817.0 −3,365.3 0.66 0.04 5(0.99); 4(0.91); 20(0.89);
4 0.4 −1,709.0 −3,535.9 0.48 0.05 5(0.99); 11(0.92); 4(0.89); 9(0.83)
5 0.5 −1,659.9 −2,852.6 0.56 0.05 5(0.98); 4(0.90); 9(0.89); 2(0.88); 9(0.88)
6 0.7 −1,922.3 −3,062.6 0.75 0.03 Ø; Ø; Ø; Ø; 5(0.97); 24(0.97)
7–9 0.5–0.6 0.82–0.92
were monophyletic; only F. francoi showed paraphyly
with F. petraea apparently derived from within it. In gen-
eral, species and geographic branches had weaker sup-
port (Azores: 0.24 posterior probability support [PS], F.
petraea: 0.60 PS, F. agustinii 0.42 PS, and F. jubata: 0.33 PS)
than terminal branches (Fig. 2).
The same trend of genetic divergence among the Mac-
aronesian red Festuca was observed in the Bayesian
analyses performed with STRUCTURE (Fig. 2, Table 2).
Higher values of K and SC and lower values of α
indicated that K =2 appropriately represents the num-
ber of optimal Bayesian groups for the four Macarone-
sian red fescues. For K >2, increasingly higher values of
lnP(D) were observed, indicating the existence of addi-
tional structure; however, lower K and SC <0.59 values
also suggested the existence of a complex structure with
many different groups that generated multiple solutions
for the same K (Table 2). The most robust result (K =2)
clearly separated the Canarian F. agustinii from the re-
maining species (Fig. 2c1), suggesting that this species is
the most divergent one, although sample sizes and ge-
netic diversity could have influenced the splitting order
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TABLE 3. Dispersion models analyzed in three Macaronesian red fescues (F. agustinii,F. francoi, F. petraea). The numbers representabbreviations
of population codes given in Table 1. All connective paths are read from left to right, beginning with the easternmost population(s) of each
archipelago. Populations in parentheses were joined by a proximal geographic hypothetical node. Double slashes represent a principal bi- or
trifurcation. Single slashes represent a secondary bi- or trifurcation. The best five dispersion models of each species have been represented in
Figures 3 to 5.
Model Connective path
F. agustinii
Model 1 (1,2)//(1,2)-(8,9)//(1,2)-3/3-(4,6,7)-5/3-(10,11)-(12,13,14)-15
Model 2 (1,2)//(1,2)-9//(1,2)-3/3-(4,6,7)-5/3-(10,11)-(12,13,14)-15-8
Model 3 (1,2)-3//3-(4,6,7)-5-(8,9)//3-(10,11)-(12,13,14)-15
Model 4 (1,2)-3//3-(4,6,7)-5-9/3-(10,11)-(12,13,14)-15-8
Model 5 (1,2)-3//3-(4,6,7)-5-9//3-(10,11)-(12,13,14)/(12,13,14)-15/(12,13,14)-8
Model 6 (1,2)-(4,6,7)//(4,6,7)-5-(8,9)//(4,6,7)-3-(10,11)-(12,13,14)-15
Model 7 (1,2)-(4,6,7)//(4,6,7)-5-9//(4,6,7)-3-(10,11)-(12,13,14)/(12,13,14)-15/(12,13,14)-8
Model 8 (1,2)-5//5-(8,9)//5-(4,6,7)-3-(10,11)-(12,13,14)-15
Model 9 (1,2)-5//5-9//5-(4,6,7)-3-(10,11)-(12,13,14)/(12,13,14)-15/(12,13,14)-8
Model 10 (1,2)//(1,2)-9//(1,2)-3-(4,6,7)-5//(1,2)-(10,11)-(12,13,14)-15-8
Model 11 (1,2)//(1,2)-3-(4,6,7)-5-9//(1,2)-(10,11)-(12,13,14)-15-8
Model 12 (1,2)-3//3-(10,11)//3-(4,6,7)/(4,6,7)-(12,13,14)-15/(4,6,7)-5-(8,9)
Model 13 (1,2)-(4,6,7)//(4,6,7)-(8,9)//(4,6,7)-(12,13,14)-15//(4,6,7)-3-(10,11)
Model 14 (1,2)-(8,9)-5-(4,6,7)-3-(10,11)-(12,13,14)-15
Model 15 (1,2)-3-(4,6,7)-5-9-8-(10,11)-(12,13,14)-15
Model 16 (1,2)-5//5-(4,6,7)-3//5-9-8-(10,11)-(12,13,14)-15
Model 17 (1,2)//(1,2)-3-(4,6,7)-5//(1,2)-9-(10,11)-(12,13,14)/(12,13,14)-15/(12,13,14)-8
Model 18 (1,2)-3//3-(4,6,7)-5-9-8-(12,13,14)-15//3-(10,11)
Model 19 (1,2)-(4,6,7)//(4,6,7)-3-(10,11)//(4,6,7)-5-9-8-(12,13,14)-15
Model 20 (1,2)-(10,11)//(10,11)-(12,13,14)-15-8//(10,11)-3-(4,6,7)-5-9
Model 21 (1,2)//(1,2)-9//(1,2)-(10,11)/(10,11)-(12,13,14)-15-8/(10,11)-3-(4,6,7)-5
F. francoi
Model 1 (1,2)-3//3-5//3-6-4//3-7
Model 2 (1,2)//(1,2)-7//(1,2)-3/3-5/3-6-4
Model 3 (1,2)-3//3-6-5//3-4//3-7
Model 4 (1,2)//(1,2)-7//(1,2)-3-6-5-4
Model 5 (1,2)-3//3-6-5-4//3-7
Model 6 (1,2)-3//3-6-5-7//3-4
Model 7 (1,2)-3-6-5//5-4//5-7
Model 8 (1,2)-4//4-7//4-6/6-5/6-3
Model 9 (1,2)-5//5-7//5-4//5-6-3
Model 10 (1,2)-4//4-7//4-5//4-6-3
Model 11 (1,2)//(1,2)-4//(1,2)-3/3-6-5/3-7
Model 12 (1,2)//(1,2)-6-5-7//(1,2)-3-4
Model 13 (1,2)//(1,2)-4//(1,2)-3-6-5-7
Model 14 (1,2)-7-3//3-5//3-6-4
Model 15 (1,2)-7//7-3//7-(4,5,6)
F. petraea
Model 1 1-4//4-2-3-6-5//4-7
Model 2 1-(2,3)//(2,3)-4//(2,3)-6-5//(2,3)-7
Model 3 1-(2,3)//(2,3)-4-7//(2,3)-6-5
Model 4 1-(2,4)-(3,5,6)-7
Model 5 1-(2,4)-7-3-6-5
Model 6 1//1-7//1-4-2-3-6-5
Model 7 1//1-7//1-(2,3)/(2,3)-6-5/(2,3)-4
Model 8 1//1-7//1-2-4//1-3-6-5
Model 9 1//1-3-6-5//1-2/2-4/2-7
Model 10 1//1-3-6-5//1-2-4-7
Model 11 1//1-3-6-5//1-4/4-2/4-7
Model 12 1//1-2-4//1-3/3-6-5/3-7
Model 13 1//1-7-(3,5,6)//1-(2,4)
(Rosenberg et al., 2002). Nevertheless, this divergence
should not be interpreted as indicative of ancestry, since
STRUCTURE does not take into account any relationship
of Bayesian groups to an evolutionary root.
Further substructuring was analyzed for the Bayesian
group comprising F. petraea +F. francoi +F. jubata. Here,
K supported the existence of two groups (K =2),
formed by F. jubata and by F. petraea +F. francoi, although
K=3 could also represent the actual number of groups,
because this grouping also showed a relatively high SC =
0.79 and a higher probability than K =2 (Table 2, Fig. 2c2).
Festuca jubata was the most divergent species followed by
the separation of F. francoi and F. petraea in the STRUC-
TURE analyses (Fig. 2c2) and in the PCT (Fig. 2a).
Hypothesis Testing of Species’ Origins
The no-admixture model of STRUCTURE for K =
4 was further assessed to infer the possibility of
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740 SYSTEMATIC BIOLOGY VOL. 57
FIGURE 2. Supraspecific Bayesian phylogenetic and structure analyses of the four Macaronesian red fescues (Festuca sect. Aulaxyper): F. agus-
tinii (purple); F. jubata (dark green); F. francoi (blue); F. petraea (red). (a) Priority consensus tree based on Bayesian MCMC strategy. Population
codes correspond to those indicated in Table 1. Filled triangles represent collapsed whole populations; dotted triangles represent partial pop-
ulations. Values on branches indicate posterior probabilities of clades. (b) PCT constructed as for Figure 2 but with all conspecific individuals
represented by triangles. (c) Diagrams showing the proportion of membership of each individual to the inferred Bayesian groups: (c1) K =2 for
the total number of individuals; (c2) K =2–3 with F. agustinii individuals excluded from the analysis (see comments in text). Gross vertical black
bars separate populations, which are formed by thin vertical colored individual bars.
independent evolution of each of the four Macarone-
sian red fescues. The no-admixture model estimates the
allelic frequencies of the putative ancestor of the ana-
lyzed species and those frequencies are subsequently
used for the correlation analysis of the species’ standard-
ized residuals. This approach was used to investigate
the null hypothesis of independent evolution of allele
frequencies of each pair of the four Macaronesian red
fescues.Exclusion of Fjuba6 from the analysis was based
on previous results obtained with the admixture model
analysis (Fig. 2c2) that showed that this population gen-
erated a non-discrete condition to F. jubata.
According to the correlation structure of standardized
residuals among species, three sets of correlations are
observed: (i) positive correlation between F. francoi and
F. petraea (r=0.264, P<0.01); (ii) pairwise negative cor-
relations between F. agustinii and F. francoi (r=−0.381,
P<0.01), F. agustinii and F. petraea (r=−0.24, P<0.01),
and F. jubata and F. francoi (r=−0.196, P<0.01); and
(iii) no-correlation between F. jubata and F. agustinii (r=
−0.01, P=0.84) and F. jubata and F. petraea (r=−0.055,
P=0.47). Shared evolutionary history is only suggested
for F. petraea and F. francoi. The remaining comparisons
indicate a clear divergent evolutionary history between
F. agustinii and the two Azorean species, despite the rela-
tively high number of shared fragments between F. agus-
tinii and F. francoi (Table 1), and an independent evo-
lutionary pattern between F. jubata and the remaining
taxa. These results are compatible with a single coloniza-
tion event to Azores and two independent colonization
events to Madeira and the Canary Islands, respectively.
Spatial Structure and Phylogeographic Patterns
of Populations
The PCT (Fig. 2a) detected a high genetic structure
among 25 out of the 36 analyzed populations (69.4% of
the total). This included all populations of F. francoi, and
F. petraea,5ofF. jubata, and 6 of F. agustinii that showed all
their individuals clustered monophyletically with mod-
erate to low posterior probabilities.
At the species level, higher values of SC, lnP(D), and
K were observed for K <5 (Table 2). Also, some empty
groups were obtained for K =5orK=6 for F. petraea,F.
francoi, and F. jubata. Consequently, only Bayesian groups
of up to K =4 were taken into account for phylogeo-
graphical analysis. K values supported 2 groups for F.
agustinii and F. jubata, 3 for F. francoi, and 4 for F. petraea
(Table 2).
Festuca agustinii.AtK=2, two Bayesian groups and
one putatively admixed Fagus10 population (Fig. 3a)
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FIGURE 3. (a) Model-based Bayesian analysis of Festuca agustinii. For each K the vertical black bars of the histogram separate different
populations and their colors represent the proportion of individual membership to each inferred Bayesian group. Each Bayesian analysis is
accompanied by a superposition of geographical maps (each for a different K) where populations are located. Dots represent populations and
their color is related to the Bayesian group inferred at the respective K. In italics, the geological age of each island in Ma (million years ago).
(b) Models or dispersion routes of Festuca agustinii in the Canary Islands. Connective lines represent dispersion paths. Ovals frame populations
connected to a proximal ancestor geographic node. Only the five dispersal models with higher correlation with genetic distance are shown (see
Table 4). (c) Superposition of the Bayesian structure groups over the F. agustinii partial priority consensus tree. rvalues indicate the correlation
coefficient of dispersal model 1 matrix with Gβ
st genetic distance matrix.
were inferred, respectively, in this species. In this sense,
Fagus10 emerges as a possible genetic bridge between
the two Bayesian groups. One group was formed by the
eastern Gran Canaria, Tenerife, and Fagus9 (La Gomera)
populations and another group by the western La Palma,
El Hierro, and Fagus8 (La Gomera) populations. The
western group was resolved as monophyletic in the PCT
(Fig. 2a). Within this group, it was also observed a mono-
phyletic group formed by Fagus15 (El Hierro) and Fa-
gus8 (La Gomera) populations. Substructuring revealed
that the eastern group was further subdivided into a
southern Gran Canaria +Gomera group and a Tenerife
group (K =2; Fig. 3a) and that the central populations of
Tenerife are differentiated from the northern and south-
ern ones (K=3; Fig. 3a), with Tenerife Fagus6 showing
more genetic similarity to Gran Canaria than to other
Tenerife populations.
Festuca jubata.AtK=2, individuals of the two inferred
Bayesian groups showed full percentage of membership
to their respective groups. One group included popula-
tion Fjuba6, which diverged first from the clade of the
remaining F. jubata populations in the PCT (Fig. 2a). The
later group was further subdivided at K =3andK=4
but without resulting in a geographic arrangement. The
large geographic distance (12 km) that separates popu-
lation Fjuba7 from populations Fjuba1 to Fjuba5 (Fig. 1)
was not paralleled by any genetic divergence between
those groups of conspecific individuals.
Festuca francoi.AtK=2, an eastern group formed
by S˜ao Miguel populations (Ffran1, Ffran2) was sepa-
rated from the remaining ones, located in the central and
western subarchipelagos (Fig. 4a). The inferred Bayesian
groups were represented by individuals with high pro-
portions of membership to their respective clusters. At
K=3, the central-western group was subdivided into the
western (Ffran7, Flores) and central groups. The K =4
model separated Ffran5 (Pico) from the rest of the central
subgroup. The PCT did not show any basal resolution to
define whether the eastern and western subarchipelago
groups are sister or paraphyletic clades. (Fig. 2a).
Festuca petraea.AtK=2, the Bayesian groups did not
show geographical structure as in one of those groups
there was a close connection of populations Fpetr1 (Santa
Maria), Fpetr4 (Faial), and Fpetr7 (Flores), located in the
eastern, central, and western subarchipelagos, respec-
tively (Fig. 5a). That group was further resolved as a
cline at K =3 and K =4, suggesting a progressive ge-
netic differentiation of populations in a southeastern-
northwestern direction. The second group was formed
exclusively by central populations (Fpetr3, Graciosa;
Fpetr5, Pico; Fpetr6, S˜ao Jorge) and was not further sub-
divided at K =3 to 4. Fpetr2 (Graciosa) showed an
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742 SYSTEMATIC BIOLOGY VOL. 57
FIGURE 4. (a) Model-based Bayesian analysis of Festuca francoi. For each K the vertical black bars of the histogram separate different popula-
tions and their colors represent the proportion of individual membership to each inferred Bayesian group.Each Bayesian analysis is accompanied
by a superposition of geographical maps (each for a different K) where populations are located. Dots represent populations and their color is
related to the Bayesian group inferred at the respective K. In italics, the geological age of each island in Ma (million years ago). (b) Models or
dispersion routes of Festuca francoi in the Azores archipelago. Connective lines represent dispersion paths. Ovals frame populations connected
to a proximal ancestor geographic node. Only the five dispersal models with higher correlation with genetic distance are shown (see Table 4).
(c) Superposition of the Bayesian structure groups over the F. francoi partial priority consensus tree. (c1–c3) Three different placements of the
root (with asterisk) compatible with model 1 dispersal route. rvalues indicates the correlation coefficient of dispersal model 1 matrix with Gβ
st
genetic distance matrix.
admixed constitution, probably connecting Fpetr4 with
other members of the central subarchipelago group. The
PCT indicated that the first group was paraphyletic with
respect to the central subarchipelago group (Fig. 2a).
Hypothesis Testing of Populations’ IBD
and Dispersal Models
Dispersal models were tested at the population level
for the Macaronesian red fescues. Different hypothetical
dispersal routes of populations, deduced from the diver-
gent Bayesian groups obtained from STRUCTURE and
their potential geographical connections, were assayed
for F. agustinii (21), F. francoi (15), and F. petraea (13) (Ta-
ble 3). Populations of F. jubata were excluded from this
analysis, given that none of the dispersal routes tested
resulted in significant correlations.
The five routes that showed the highest simple (r) and
multiple (R2) correlation coefficients between genetic
and dispersal distances (dispersal models) as well as be-
tween genetic and geographic distances (IBD model) are
shown in Figures 3 to 5 and Table 4. Model 1 showed a
higher correlation value than the alternative models in
all cases (Table 4). This dispersal model also had a higher
correlation value than the IBD model. Partial correlations
of model 1|IBD model indicated that the dispersal dis-
tance model 1 explained a significant part of the variation
that was not explained by the IBD model, whereas par-
tial correlations of IBD model |model 1 were nonsignif-
icant, indicating that all the variation explained by the
IBD model was already explained by the dispersal dis-
tance model 1. Also, the differences observed between
the simple coefficient of determination and the coeffi-
cient of multiple determination indicated the relative
merit of adding another source of variation once the first
source was fitted. Model 1 and the multiple coefficient
of determination were similar to each other, whereas the
IBD model was lower than the multiple coefficient, im-
plying that substantial information was gained adding
model 1 once the IBD model was fitted.
In order to clarify the most likely direction of the col-
onization in model 1, Bayesian genetic units derived
from the PCT were considered. Only one configuration
is compatible with model 1 dispersal route for F. agus-
tinii, whereas two are compatible for F. petraea. The PCT
topology of F. francoi (plus F. petraea) clades (Fig. 2a) gen-
erated three alternative dispersal model 1 configurations,
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FIGURE 5. (a) Model-based Bayesian analysis of Festuca petraea. For each K the vertical black bars of the histogram separate different
populations and their colors represent the proportion of individual membership to each inferred Bayesian group. Each Bayesian analysis is
accompanied by a superposition of geographical maps (each for a different K) where populations are located. Dots represent populations and
their color is related to the Bayesian group inferred at the respective K. In italics, the geological age of each island in Ma (million years ago).
(b) Models or dispersion routes of Festuca petraea in the Azores archipelago. Connective lines represent dispersion paths. Ovals frame populations
connected to a proximal ancestor geographic node. Only the five dispersal models with higher correlation with genetic distance are shown (see
Table 4). (c) Superposition of the Bayesian structure groups over the F. petraea partial priority consensus tree. (c1, c2) Two different placements of
the root (with asterisk) compatible with model 1 dispersal route. rvalues indicates the correlation coefficient of dispersal model 1 matrix with
Gβ
st genetic distance matrix.
which agreed with equal number of different geographic
locations of the root node in each of the Bayesian genetic
units.
Model 1 of F. agustinii (Fig. 3b, c) suggests that the col-
onization of the Canary Islands could have started from
northern Tenerife following two bidirectional disper-
sal colonizations, one to the easternmost Gran Canaria
island and central La Gomera island and the other to the
western islands of La Palma, and El Hierro, plus a fur-
ther parallel colonization of southern Tenerife. Sequen-
tial analysis of K =2toK=3 Bayesian groups suggests
that colonization of Gran Canaria and La Gomera must
have been of relatively recent origin, as populations
Fagus1 and Fagus2 (Gran Canaria) and populations Fa-
gus8 and Fagus9 (La Gomera) are not further differen-
tiated from populations Fagus4 or Fagus6 from Central
Tenerife. Other potential configurations in model 1 are
not supported by any of the best five correlation coeffi-
cient models (e.g., placing the root in Gran Canaria or
La Palma genetic units would imply two independent
colonizations to each of the two other genetic units, re-
spectively). A further retrocolonization event suggested
by STRUCTURE analysis and PCT, from the younger is-
land of El Hierro to the older island of La Gomera (Fig.
3a), is supported by models 2, 4, and 5 (Fig. 3b).
F. francoi model 1 (Fig. 4b, c1, c2, c3) supports
a stepping-stone colonization, involving the central
subarchipelago genetic unit as an intermediate step be-
tween the eastern and western subarchipelagos. Nev-
ertheless, the root could not be placed unambiguously
into a unique location, considering that the three genetic
units were connected by one basal polytomy (Fig. 2a).
This generated three different alternative hypotheses to
explain model 1. Sequential analysis of K =2toK=3
Bayesian groups (Fig. 4a) indicates that Ffran 7 popula-
tion (Flores) could have diverged after the main split be-
tween eastern and the western +central subarchipelago
populations. This rules out model 1c1 and 1c3 scenar-
ios, adding support for the stepping-stone east-to-west
colonization route of model 1c2 (Fig. 4c).
For F. petraea, model 1 (Fig. 5b, c1, c2, c3) suggests
the same scenario as for F. francoi, involving a central
subarchipelago intermediate colonization step. Two dif-
ferent model 1 configurations could explain, however,
this colonization pattern (Fig. 5c1, c2). Scenario model
1c1 suggests that the colonization trend of F. petraea
could have started in the central subarchipelago islands
of Faial or Graciosa and that the eastern and western
subarchipelagos would be colonized independently. Un-
der such an assumption, the Fpetr3 +Fpetr5 +Fpetr6
Bayesian group would be of ancient origin, given its ex-
treme divergence from the rest of populations in the K =
2 STRUCTURE analysis (Fig. 5a), although another alter-
native hypothesis would imply a recent divergence event
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744 SYSTEMATIC BIOLOGY VOL. 57
TABLE 4. Simple and partial correlation analyses between genetic Gβ
st distances and geographic or dispersal distances for three Macaronesian
red fescues (F. agustinii, F. francoi, F. petraea). Dispersion models correspond to those described in Figures 3 to 5 and Table 3; they correspond to the
dispersal models that showed the highest correlation with genetic distance. r=simple correlation coefficient between genetic and geographic
or dispersion model distances. P=probability for a random rhigher than observed rafter 10,000 permutations. rM|G=partial correlation
coefficient between genetic and dispersal model distances once geographic distance was fixed. r2=coefficient of determination. R2=coefficient
of multiple determination.
Model rPr2rM|GPrG|MPR2
F.agustinii
Geographic 0.3399 0.0062 0.1156
Model 1 0.6170 <0.001 0.3807 0.5493 <0.001 0.0531 0.4111 0.3824
Model 2 0.5717 <0.001 0.3268 0.4975 0.0051 0.1062 0.3015 0.3344
Model 3 0.5705 <0.001 0.3255 0.4978 <0.001 −0.1171 0.7997 0.3347
Model 4 0.5625 <0.001 0.3164 0.4957 <0.001 −0.1554 0.8723 0.3329
Model 5 0.5526 <0.001 0.3054 0.4658 <0.001 −0.0545 0.6670 0.3074
F. francoi
Geographic 0.6989 0.0145 0.4884
Model 1 0.8446 0.0010 0.7134 0.7030 0.0080 −0.3118 0.8593 0.7412
Model 2 0.8254 0.0013 0.6812 0.6518 0.0140 0.2775 0.1899 0.7058
Model 3 0.8172 0.0014 0.6678 0.6141 0.0273 −0.2019 0.7481 0.6814
Model 4 0.7925 0.0019 0.6280 0.5713 0.0238 0.2712 0.1822 0.6554
Model 5 0.7875 0.0044 0.6201 0.5317 0.0097 −0.1847 0.8090 0.6331
F. petraea
Geographic 0.6900 0.0167 0.4761
Model 1 0.7929 <0.001 0.6287 0.6184 0.0028 −0.3587 0.9364 0.6765
Model 2 0.7598 0.0054 0.5773 0.5156 0.0448 −0.3002 0.8201 0.6154
Model 3 0.7520 <0.001 0.5656 0.5023 0.0272 −0.3137 0.8689 0.6083
Model 4 0.5711 0.0329 0.3261 0.2434 0.2937 0.5183 0.0836 0.5072
Model 5 0.5388 0.0599 0.2903 0.2025 0.3446 0.5405 0.0708 0.4976
that would require extreme genetic changes during col-
onization. Scenario model 1c2 is compatible with a pu-
tative origin of F. petraea in Flores, which agrees with the
close relationship between this species and the Flores F.
francoi Ffran7 population in the PCT (Fig. 2a). In this case,
the colonization would follow a west-to-east direction,
opposite to that of F. francoi.
DISCUSSION
Origin and Diversification of the Macaronesian Red Fescues
In contrast to other endemic angiosperm groups that
show high ecological adaptation and pronounced spe-
ciation in Macaronesia (e.g., Argyranthemum: Francisco-
Ortega et al., 1996; Echium:B¨ohle et al., 1996; Sonchus:
Kim et al., 1996; Tolpis: Moore et al., 2002, Archibald et al.,
2006; and the Macaronesian Crassulaceae clade [i.e., Aeo-
nium, Aichryson, and Monanthes]: Mort et al., 2002; Fair-
field et al., 2004), there are only four endemic species
of red fescues. However, these species are distributed
in all Macaronesian archipelagos, with the exception of
the Cape Verde, making them an ideal model to test dis-
persal models of plants with similar diversity rates in
this large oceanic region of more than 450,000 km2. Fur-
thermore, the recent Pliocene origin of the Macaronesian
red fescues has allowed us to test if alternative multiple
colonization or in situ speciation episodes could have oc-
curred during the short span of 2.5 ±0.9 Ma time that
has elapsed since they diverged from their common con-
tinental Festuca sect. Aulaxyper ancestor (Catal´an, 2006;
Inda et al., 2008).
The close, but unresolved, evolutionary relationships
recovered for three (F. agustinii,F. jubata, the Azorean
group) of the four lineages of Macaronesian red fescues
in previous studies of subtribe Loliinae based on plas-
tid and nuclear sequence data (Inda et al., 2008) are
not inconsistent with at least three independent long-
distance dispersal events from the continent to each sep-
arate archipelago (i.e., the Canaries, Madeira, and the
Azores). Moreover, the strong sister group relationship
recovered for the two Azorean species indicated that they
probably evolved from a common ancestor that colo-
nized the Azores during the Pleistocene (ca. 1.1 ±0.6 Ma;
cf. Inda et al., 2008). These hypotheses have been partially
confirmed and further illuminated by the present study.
The relatively high number of shared AFLP fragments
among populations of different island species (Table 1)
and the low number of unique, non-fixed fragments in
species and populations suggest a recent origin of these
species. The STRUCTURE and PCT analyses illustrate
the greater divergence of the Canarian F. agustinii with
respect to the remaining species and the separation of the
Madeiran F. jubata from the Azorean group of the closely
related F. francoi and F. petraea (Fig. 2a, b, c1, c2). The
possession of the highest number of private fragments
further highlights the genetic distinctness of F. agustinii
from the others (Table 1). All these data support a more
ancient isolation of F. agustinii in the Canary Islands and
of F. jubata in Madeira and a more recent isolation of
F. francoi and F. petraea in the Azores but do not allow
inferences to be made about the mono- or polyphyletic
origin of the group. Some drawbacks that could preclude
the resolution of the origins of the Macaronesian red fes-
cues relate to the present restricted distribution of the
close relative F. rivularis in SW Europe and the possi-
ble extinction of other close ancestral diploid lineages in
this region and in NW Africa after the colonization of
the oceanic islands. This has also been hypothesized for
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other angiosperms (Tolpis: Moore et al., 2002; Androcym-
bium: Caujap´e-Castells, 2004).
The analysis of correlation of standardized residu-
als based on hypervariable AFLP data has allowed
us to infer a possible evolutionary scenario related to
the origin and divergence of these Atlantic oceanic
grasses, although it should be considered a preliminary
hypothesis, given that the use of correlations among
residuals to detect deviations from the model of inde-
pendent evolution (such as shared history or migration)
has not been extensively evaluated (G. Nicholson, per-
sonal communication). Our results suggest three colo-
nization events: one to the Canary Islands probably from
northwest Africa, resulting in F. agustinii, a second one
to Madeira, probably from southwest Europe, resulting
in F. jubata, and a third one to Azores, probably from
western Europe, resulting in the ancestor of F. francoi
and F. petraea.
The priority consensus tree (PCT) suggests a possible
scenario where F. petraea could have diverged from F.
francoi, as indicated by the PCT node that locates Ffran 7
(Flores) as a sister population to F. petraea. Assumed evi-
dence for the existence of breeding barriers between the
two Azorean species (St-Yves, 1922; Dias, 2001) has been
corroborated by our genetic data: (a) Bayesian results
that clearly discriminated individuals of each species
(Fig. 2); and (b) Bayesian Gβ
st that give an estimate of
15%, indicative of a pronounced genetic differentiation
(Hartl and Clark, 1997). Although multiple colonization
events cannot be totally ruled out because westerly wind
currents may facilitate the transport of diaspores to the
Azores, the archipelago is an extremely difficult target
for such events given that it has a small land area rel-
ative to the Atlantic Ocean and is isolated by at least
800 km from any source of colonization (Fig. 1). Adap-
tive speciation following a single colonization event is
therefore consistent with the data and the most proba-
ble scenario to explain the existence of divergent sym-
patric gene pools in the archipelago (see Moore et al.,
2002). The possibility of adaptive speciation is suggested
by differences in morphology and ecological habitats.
Thus, F. petraea and F. francoi are associated with coastal
and with humid highland environments, respectively,
with few contact zones except in the coastal cliffs of the
westernmost islands of Flores and Corvo (Dias, 2001).
Ecological speciation could have been prompted by ge-
ographic, stochastic, and genetic factors (Jorgensen and
Olensen, 2001). First, the remoteness of the Azores could
have limited the number of colonizing events, restrict-
ing competition and predation from well-established or-
ganisms (Sj¨ogren, 1973). Second, the Azores offer a rich
variety of ecological habitats for colonizers, including
coastal, humid, forest, and prairie environments (Dias,
2001). Finally, genetic drift processes associated with the
colonization of oceanic islands tend to favor complexes
of epistatic genes already present in the ancestral pop-
ulation conferring adaptation to particular environmen-
tal settings (Crow and Kimura, 1970). In situ speciation
probably occurred very recently, in the Pleistocene (cf.
Inda et al., 2008), exemplifying a rapid radiation event of
Macaronesian plants. This example might also serve as a
useful model to understand the evolutionary processes
that fostered a rapid speciation through ecological adap-
tation to coastal environments of mountain ancestors in
the closely related continental polyploid complexes of
Festuca sect. Aulaxyper (Catal´an, 2006; Inda et al., 2008).
Colonization and Dispersal Routes of F. agustinii,
F. francoi, and F. petraea
Dispersal model tests were a major tool in select-
ing optimal colonization routes of the Macaronesian red
fescues. To deduce the ancestry of a Bayesian group, se-
quential analysis of increasing K models from STRUC-
TURE and the topology of the PCT was considered in
each species.
In the Canarian F. agustinii, Bayesian methods sug-
gest that the younger westernmost island populations
were derived from those located in the older eastern-
most islands, likely from Tenerife island (Fig. 3c2). This
hypothesis assumes that geological older islands were
colonized before the relatively younger islands of La
Palma and El Hierro but without a defined east-to-west
stepping stone colonization route. The detected retrocol-
onization event suggested by STRUCTURE analysis and
PCT from the younger island of El Hierro to the older
island of La Gomera indicates that island dispersal is not
dependent on linear geographic or chronological rela-
tionship as IBD and genetic distance matrix correlations
had always lower values than dispersal model and ge-
netic distance matrix correlations. It also indicates that
a single island could have been colonized several times
from relative different genetic pools, possibly of different
evolutionary origins. The La Palma populations Fagus10
and to a lesser extent individuals of Fagus11 exhibited
a decreasing proportion of membership to the eastern
Bayesian group. This situation could be interpreted in
two different ways in the context of a Bayesian analysis
(Rosenberg et al., 2002): (i) they could represent grada-
tions of allele frequencies between eastern and western
populations and thus might be interpreted as genetic
bridges between the two Bayesian groups; or (ii) they
could also represent an admixture zone between both
groups, in which case these populations might have re-
sulted from a relatively recent colonization from Tenerife
that hybridized with already established populations on
La Palma. Dispersal models and the PCT indicate that
the genetic relationships between the optimal Bayesian
groups is best explained if La Palma was colonized only
once from northern Tenerife. Models 1 and 4 also suggest
two southward dispersal routes along the longitudinal
axes of Tenerife and La Palma–El Hierro, respectively.
The PCT and STRUCTURE analyses suggest that the
initial colonization for the Azorean endemic F. francoi
could have occurred in the eastern subarchipelago of the
Azores, because this region harbors the first diverging
group from the common ancestor (Fig. 4a). According
to the splitting order of divergence of the PCT Bayesian
genetic units and the dispersal model 1 (Fig. 4b, c2), an
initial westward dispersal occurred from S˜ao Miguel to
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746 SYSTEMATIC BIOLOGY VOL. 57
the central subarchipelago islands; a second westwards
dispersal is inferred from Terceira to Flores. The STRUC-
TURE analysis supported the existence of such groups
(Fig. 4a, K =2), indicating a close relationship between
the putatively younger western archipelago and the cen-
tral subarchipelago group. Model 1 further suggests two
southwestern parallel dispersal events to the younger is-
lands of the central subarchipelago. This would be com-
patible with the volcanic events associated to the recent
ages of those islands, indicating fast secondary dispersals
from the main island of Terceira to the newly emerged
islands of S˜ao Jorge, Faial, and Pico.
In the Azorean F. petraea, genetic divergence from a
common ancestor might have occurred in two scenarios.
Scenario 1 (Fig. 5c1) suggests a bidirectional cen-
tripetal colonization from the central subarchipelago
Faial or Graciosa islands. Starting from these central sub-
archipelago islands a hypothetical ancestral colonization
event reached the proximal Sa˜o Jorge and Pico islands.
STRUCTURE genetic analysis (Fig 5a) indicates that the
Graciosa Fpetr2 population represents a possible genetic
bridge between the two Bayesian groups. Posterior dis-
persions started from Graciosa or Faial to the western-
most Flores and the easternmost Santa Mar´ıa islands.
The second scenario (scenario 2), involving Flores as the
starting point of colonization (Fig. 5c2), is more compat-
ible with the interspecific relationship found between an
older paraphyletic F. francoi and a more recently derived
F. petraea.Festuca petraea appears as the sister clade of
Flores Ffran 7 population in the PCT (Fig. 2a). Thus, sce-
nario 2 is supported by dispersal analysis and by infra
and interspecific genetic relationships, whereas STRUC-
TURE analysis is inconclusive to discriminate between
the two scenarios. Model 1 dispersal analysis suggests
that within the central subarchipelago of the Azores, a
possible recolonization could have resulted in the con-
nection of a younger island Faial Fpetr4 ancestor with an
older Graciosa Fpetr2 population descendant. Although
this suggests dispersal routes with an inverse direction
to the geological age of the islands, it should be noted
that the Azorean populations of F. petraea exist within a
dynamic volcanic environment. Graciosa has been the
source of eruptions with significant emission of lava
and pyroclastic flows (Global Vulcanism Program [GVP],
2006), the latter with high destructive power. Conse-
quently, it is possible that extinction of past populations
of Graciosa could have been followed by a recolonization
from younger island populations.
Long-Distance Oceanic Colonization and East-to-West
Dispersal Hypotheses Related to the Endemic
Macaronesian Flora
Our population-based phylogeographical study of the
Macaronesian red fescues provides insights into the col-
onization and speciation processes followed by these re-
cently evolved plants in the three Atlantic archipelagos.
They could serve as models to understand similar distri-
bution patterns observed in other endemic Macaronesian
angiosperms and to assess various dispersal and ecolog-
ical theories of speciation in oceanic islands. Our data in-
dicate that multiple colonizations likely happened from
mainland continental ancestors. Within species, three
different colonization patterns were observed: a predom-
inant east-to-west colonization pattern, concordant with
the geological ages of the islands, was selected as the op-
timal model for the Azorean F. francoi; for the Canarian F.
agustinii a bidirectional centripetal colonization pattern
was inferred, whereas for F. petraea a counterintuitive di-
rection of colonization (west-to-east) is suggested (Figs.
3 to 5; Tables 3 and 4). Whereas the geological eastern-to-
western dispersal pattern recovered for F. francoi agrees
with a likely arrival of continental founders to the more
ancient and geographically close island of S¨ao Miguel,
followed by the subsequent dispersal of the new species
to the west, the origin and initial dispersal of F. petraea
could have occurred in any of the three subarchipela-
gos where F. francoi was already present. The Bayesian
approaches and dispersal model 1 support an earlier ori-
gin in the central subarchipelago, though a western sub-
archipelago origin (PCT; Fig. 2a) could be also possible.
These general models also varied considerably in sec-
ondary dispersals and back-colonizations, reflecting the
different volcanic activities and interislands dispersal
possibilities of the two archipelagos. Different disper-
sal scenarios have been proposed for the stepping-stone
interisland population colonizations within some Mac-
aronesian archipelagos. Hess et al. (2000) suggested inde-
pendent bird-mediated dispersal patterns for the fleshy
and lipid-rich fruits of Olea europaea in the Madeiran and
the Canary Islands. The intricate dispersal routes ob-
served within F. agustinii in the Canary Islands and of
F. petraea in the Azores do not seem to be connected with
migratory bird routes. Rather, they reflect a more an-
cient volcanism-associated island formation pattern that
has been also observed in other Macaronesian endemics
(e.g., Deschampsia foliosa: M. Sequeira and P. Catal´an, un-
published data). The older and more stable Canary Isles
have experienced a low number of secondary dispersals,
probably due to the east-to-west geographically scaled
ages of the islands, which favored the saltatory route
of F. agustinii populations from Gran Canaria to Tener-
ife to La Gomera to La Palma to El Hierro (models 1
and 4). Nonetheless, the geographical proximity of the
western island also allowed for potential secondary in-
vasions from Tenerife to La Palma (models 1 to 5) and
a potential back-colonization from La Palma and El Hi-
erro to La Gomera (model 5 and models 2 and 4, re-
spectively). In the young and highly dynamic Azores
isles, secondary dispersal events have been more pro-
nounced and have occurred in different ways. They have
been manifested in both the secondary colonizations of
younger islands from older islands (F. francoi popula-
tions; model 1) and in the back-colonization of older is-
lands subjected to volcanism-mediated extinctions from
younger islands (F. petraea populations, model 1) within
the central subarchipelago. Also, the long geographical
distances that separate the eastern, central, and western
Azores subarchipelagos seem to have acted as barriers
preventing any recent gene flow among them.
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In agreement with Carine et al. (2004), our results
show that the multiple congeneric colonizations of the
Macaronesian red fescues’ ancestors occurred because
the three different archipelagos were colonized indepen-
dently. We predict that this fact might have operated in
most of the species-poor Macaronesian endemic group
plants, irrespectively of the colonization time, but per-
haps not of their dispersal abilities. Finally, our data con-
cur with the window-of-opportunity hypothesis (Carine,
2005) at the population level, as suggested by the sec-
ondary back-colonization of the older island of Graciosa
by younger F. petraea lineages after local extinction of
the initial founders. Despite the advances made, there
is still the paradox of why, if the Macaronesian islands
are so easy to colonize, there is clearly isolation, as evi-
denced by the high genetic divergence observed among
the Macaronesian red fescues and by the lack of genetic
exchange between continent and islands and back again
(e.g., Androcymbium: Caujap´e-Castells, 2004).
ACKNOWLEDG MENTS
We thank M. Carine, P. Comes, associate editor R. Mason-Gamer,
and reviewers F. Blattner, K. Holsinger, and M. Mort for their valuable
comments and critical review of an earlier version of the manuscript;
R. Luo for his advise on Bayesian analysis of genotypes relation-
ships; G. Nicholson for helpful discussion on correlation models of
residuals; E. P´erez-Collazos for technical advise on AFLP protocols;
and J. Caujap´e for providing us with some F. agustinii samples from
Gran Canaria. This work has been supported by two Spanish Min-
istry of Education and Science (REN2003-02818/GLO and CGL2006-
00319/BOS) and one Portuguese Ministry of Science and Technology
(POCTI/BME/39640/2001) research grant projects. A. D´ıaz-P´erez was
supported by a Central University of Venezuela CDCH PhD fellowship.
Bayesian analyses were conducted at the cluster computer system of
the BIFI Research Institute (University of Zaragoza, Spain).
REFERENCES
Althoff, D. M., M. A. Gitzendanner,and K. A. Segraves. 2007. The utility
of amplified fragment length polymorphisms in phylogenetics: A
comparison of homology within and between genomes. Syst. Biol.
56:477–484.
Archibald, J. K., D. Crawford, A. Santos-Guerra, and M. E. Mort. 2006.
The utility of automated analysis of inter-simple sequence repeat loci
(ISSR) for resolving relationships in the Canary Island species Tolpis
(Asteraceae). Am. J. Bot. 93:1154–1162.
Bassam, B., G. Caetano-Anolles, and P. Gresshoff. 1991. Fast and sensi-
tive silver staining of DNA in polyacrylamide gels. Anal. Biochem.
196:80–83.
Batista, F., and P. Sosa. 2002. Allozyme diversity in natural populations
of Viola palmensis Webb & Berth. (Violaceae) from La Palma (Canary
Islands): Implications for conservation genetic. Ann. Bot. 90:725–733.
Bensch, S., and M. ˚
Akesson. 2005. Ten years of AFLP in ecology and
evolution: Why so few animals? Mol. Ecol. 14:2899–2914.
B¨ohle, U. R., H. H. Hilger, and W. Martin. 1996. Island colonization and
evolution of the insular woody habit in Echium L. (Boraginaceae).
Proc. Natl. Acad. Sci. USA 93:11740–11745.
Bohs, L., and R. Olmstead. 2001. A reassessment of Normania and
Triguera (Solanaceae). Plant Syst. Evol. 228:33–48.
Bolos, O. 1996. Acerca de la flora macaron´esica. An. Jard. Bot. Madr.
54:457–461.
Bonin, A. E. Bellemain, P. B. Eidesen, F. Pompanon, C. Brochmann,
and P. Taberlet. 2004. How to track and assess genotyping errors in
population genetics studies. Mol. Ecol. 13:3261–3273.
Bonin, A., D. Ehrich, and S. Manel 2007. Statistical analysis of ampli-
fied fragment length polymorphism data: A toolbox for molecular
ecologists and evolutionists. Mol. Ecol. 16:3737–3758.
Buckler, E. S., IV. 1999. Phylogeographer: A tool for developing and
testing phylogeographic hypotheses, 0.3 ed. Availableat http://stat-
gen.ncsu.edu/buckler.
Buckler, E. S., IV, M. M. Goodman, T. P. Holtsford, J. F. Doebley, and G.
S´anchez. 2006. Phylogeography of the wild subspecies of Zea mays.
Maydica 51:123–134.
Carracedo, J. 1999. Growth, structure, instability and collapse of Ca-
narian volcanoes and comparisons with Hawaiian volcanoes. J. Vol-
canol. Geotherm. Res. 94:1–19.
Carine, M. A. 2005. Spatio-temporal relationships of the Macaronesian
endemic flora: A relictual series or windows of opportunity? Taxon
54:895–903.
Carine, M. A., S. Russell, A. Santos-Guerra, and J. Francisco-Ortega.
2004. Relationships of the Macaronesian and Mediterranean floras:
Molecular evidence for multiple colonizations into Macaronesia and
back-colonization of the continent in Convolvulus (Convolvulaceae).
Am. J. Bot. 91:1070–1085.
Carlquist, S. 1965. Island life. Natural History Press, Garden City, New
York.
Catal´an, P. 2006. Phylogeny and evolution of Festuca L. and related
genera of subtribe Loliinae (Poeae, Poaceae). Pages 255–303 in Plant
genome: Biodiversity and evolution (A. Sharma and A. Sharma,
eds.). Science Publishers, Enfield, New Hampshire.
Caujap´e-Castells, J. 2004. Boomerangs of biodiversity? The interchange
of biodiversity between mainland North Africa and the Canary Is-
lands as inferred from cpDNA RFLPs in genus Androcymbium. Bot.
Macaronesica 25:53–69.
Comes, H. P. 2004. The Mediterranean region—A hotspot for plant
biogeographic research. New Phytol. 164:11–14.
Crow, J., and M. Kimura.1970. An introduction to population genetics
theory. Harper & Row, New York.
Darwin, C. 1859. On the origin of species by means of natural selection.
J. Murray, London.
Despres, L., L. Gielly, B. Redoubet, and P. Taberlet. 2003. Using AFLP to
resolve phylogenetic relationships in a morphologically diversified
plant species complex when nuclear and chloroplast sequences fail
to reveal variability. Mol. Phylogenet. Evol. 27:185–196.
Dias, E. 2001. Ecologia e classifica¸c˜ao da vegeta¸c˜ao natural dos A¸cores.
Cadernos de Botˆanica, No 3. Herb ´ario da Universidade dos A¸cores.
Angra do Hero´ısmo.
Dietz, J. 1983. Permutation tests for association between two distance
matrices. Syst. Zool. 32:21–26.
Doyle, J. L., and J. J. Doyle. 1987. A rapid DNA isolation procedure
for small quantities of fresh leaf tissue. Phytochem. Bull. 19:11–
15.
Emerson, B. C. 2002. Evolution on oceanic islands; molecular phyloge-
netic approaches to understanding pattern and process. Mol. Ecol.
11:951–966.
Emerson, B. C. 2003. Genes, geology and biodiversity: Faunal and floral
diversity on the island of Gran Canaria. Anim. Biodivers. Conserv.
26:9–20.
Evanno, G., S. Regnaut, and J. Goudet. 2005. Detecting the number of
clusters of individuals using the software STRUCTURE: A simula-
tion study. Mol. Ecol. 14:2611–2620.
Fairfield, K. N., M. E. Mort, and A. Santos-Guerra. 2004. Phylogenetics
and evolution of the Macaronesian endemic Aichryson (Crassulaceae)
inferred from nuclear and chloroplast DNA sequence data. Plant
Syst. Evol. 248:71–83.
Falush, D., M. Stephens, and J. Pritchard. 2003. Inference of population
structure using multilocus genotype data: Linked loci and correlated
allele frequencies. Genetics 164:1567–1587.
Falush, D., M. Stephens, and J. K. Pritchard. 2007. Inference of popu-
lation structure using multilocus genotype data: Dominant markers
and null alleles. Mol. Ecol. Notes 7:574–578.
Francisco-Ortega J., J. Fuertes-Aguilar, S. C. Kim, A. Santos-Guerra, D.
J. Crawford, and R. K Jansen. 2002. Phylogeny of the Macaronesian
endemic Crambe section Dendrocrambe (Brassicaceae) based on inter-
nal transcribed spacer sequences of nuclear ribosomal DNA. Am. J.
Bot. 89:1984–1990.
Francisco-Ortega, J., R. Jansen, and A. Santos-Guerra. 1996. Chloroplast
DNA evidence of colonization, adaptive radiation, and hybridization
in the evolution of the Macaronesian flora. Proc. Natl. Acad. Sci. USA
93:4085–4090.
by guest on December 27, 2015http://sysbio.oxfordjournals.org/Downloaded from
748 SYSTEMATIC BIOLOGY VOL. 57
Francisco-Ortega J., S. J. Park, A. Santos-Guerra, A. Benabid, and R. K.
Jansen. 1997a. Origin and evolution of the endemic Macaronesian
Inuleae (Asteraceae): Evidence from the internal transcriber spacers
of nuclear ribosomal DNA. Biol. J. Linnean Soc. 72:77–97.
Francisco-Ortega J., A. Santos-Guerra, A. Hines, and R. K. Jansen 1997b.
Molecular evidence for a Mediterreanean origin of the Macarone-
sian endemic genus Argyranthemum (Asteraceae). Am. J Bot. 84:1595–
1613.
Francisco-Ortega, J., A. Santos-Guerra, S. Kim, and D. Crawford. 2000.
Plant genetic diversity in the Canary Islands: A conservation per-
spective. Am. J. Bot. 87:909–919.
Frankham, R. 1997. Do island populations have less genetic variation
than mainland populations? Heredity 78:311–327.
Fuertes-Aguilar J., M. F.Ray, J. Francisco-Ortega, A. Santos-Guerra, and
R. K Jansen. 2002. Molecular evidence from chloroplast and nuclear
markers for multiple colonizations of Lavatera (Malvaceae) in the
Canary Islands. Syst. Bot. 27:74–83.
Garc´ıa-Talavera, F. 1999. La Macaronesia: Consideraciones geol ´ogicas,
biogeogr´aficas y paleoecol ´ogicas. Pages 39–63 in Ecolog´ıa y cultura
en Canarias (J. Fern´andez-Palacios, J. Bacallado, and J. Belmonte,
eds.). Museo de la Ciencia, Cabildo Insular de Tenerife, Junta Santa
Cruz de Tenerife, Isla Canaria, Espa ˜na.
Geldmacher, J., P. van den Bogaard, K. Hoernle, and H. Schmincke.
2000. The 40Ar/39 Ar age dating of the Madeira Archipelago and
hotspot track (eastern North Atlantic). Geochem. Geophys. Geosyst.
1, Paper number 1999GC000018.
Gillespie, R., and G. Roderick. 2002. Arthropods on islands: Coloniza-
tion, speciation, and conservation. Annu. Rev. Entomol. 47:595–632.
Global Volcanism Program (GVP). 2006. Graciosa. Smithso-
nian National Museum of Natural History. Available at
http://www.volcano.si.edu.
G¨ubbitz, T., R. Thorpe, and A. Malhotra. 2005. The dynamics of genet-
ics and morphological variation on volcanic islands. Proc. R. Soc. B
272:751–757.
Hartl, D., and A. Clark. 1997. Principles of population genetics. Sinauer
Associates, Sunderland, Massachusetts.
Helfgott, D. M., J. Francisco-Ortega, A. Santos-Guerra, R. K. Jansen,
and B. B. Simpson. 2000. Biogeography and breeding system evolu-
tion of the woody Bencomia alliance (Rosaceae) in Macaronesia based
on ITS sequence data. Syst. Bot. 25:82–97.
Hess, J., J. Kadereit, and P. Vargas. 2000. The colonization history of
Olea europaea L. in Macaronesia based on internal transcribed spacer 1
(ITS-1) sequences, randomly amplified polymorphic DNAs (RAPD),
and intersimple sequence repeats (ISSR). Mol. Ecol. 9:857–868.
Holsinger, K., P. Lewis, and D. Dey. 2002. A Bayesian approach to
inferring population structure from dominant markers. Mol. Ecol.
11:1157–1164.
Holsinger, K., and L. Wallace. 2004. Bayesian approaches for the anal-
ysis of population genetic structure: An example from Platanthera
leucophaea (Orchidaceae). Mol. Ecol. 13:887–894.
Humphries, C. J. 1979. Endemism and evolution in Macaronesia. Pages
171–200 in Plant and islands (D. Bramwell, ed.). Academic Press,
London, UK.
Huson, D. H., D. Richter, C. Rausch, T. Dezulian, M. Franz, and R.
Rupp. 2007. Dendroscope: An interactive viewer for large phyloge-
netic trees. BMC Bioinformatics 8:460.
Inda, L. A., J. G. Segarra-Moragues, J. M ¨uller, P. M. Peterson, and P.
Catal´an. 2008. Dated historical biogeography of the temperate Loli-
inae (Poaceae, Pooideae) grasses in the northern and southern hemi-
spheres. Mol. Phylogen. Evol. 46:932–957.
Jorgensen, T., and J. Olesen. 2001. Adaptive radiation of island plants:
Evidence from Aeonium (Crassulaceae) of the Canary Islands. Per-
spect. Plant Ecol. Evol. Syst. 4:29–42.
Jorgensen, T. H., and J. Frydenberg. 1999. Diversification in insular
plants: Inferring the phylogenetic relationships in Aeonium (Crassu-
laceae) using ITS sequences of nuclear ribosomal DNA. Nord. J. Bot.
19:613–621.
Kim, S., D. Crawford, J. Francisco-Ortega, and A. Santos-Guerra. 1996.
A common origin for woody Sonchus and five related genera in the
Macaronesian islands: Molecular evidence for extensive radiation.
Proc. Natl. Acad. Sci. USA 93:7743–7748.
Kim, S., D. Crawford, J. Francisco-Ortega, and A. Santos-Guerra. 1999.
Adaptive radiation and genetic differentiation in the woody Sonchus
alliance (Asteraceae: Sonchinae) in the Canary islands. Plant Syst.
Evol. 215:101–118.
Kim, S., C. Lee, and A. Santos-Guerra. 2005. Genetic analysis and
conservation of the endangered Canary Island woody sow-thistle,
Sonchus gandogeri (Asteraceae). J. Plant Res. 118:147–153.
Koopman, W. 2005. Phylogenetic signal in AFLP data sets. Syst. Biol.
54:197–217.
Koopman, W., M. Zevenbergen, and R. van den Berg. 2001. Species
relationships in Lactuca S. L. (Lactuceae, Asteraceae) inferred from
AFLP fingerprints. Am. J. Bot. 10:1881–1887.
Luo, R., A. Hipp, and B. Larget. 2007. A Bayesian model
of AFLP marker evolution and phylogenetic inference.
Stat. Appl. Genet. Mol. Biol. 6(1), article 11. Available at
http://www.bepress.com/sagmb/vol6/iss1/art11.
MacArthur, R. H., and E. O. Wilson. 1967. The theory of island biogeog-
raphy. Priceton University Press, Princeton, New Jersey.
Masson, D., A. Watts, M. Gee, R. Urgeles, N. Mitchell, T. Le Bas, and
M. Canals. 2002. Slope failures on the flanks of the western Canary
Islands. Earth Sci. Rev. 57:1–35.
Mayr, E. 1942. Systematics and the origin of the species. Columbia
University Press, New York.
Molero, J., T. Garnatge, A. Rovira, N. Garc´ıa-Jacas, and A. Susanna.
2002. Karyological evolution and molecular phylogeny in Macarone-
sian dendroid spurges (Euphorbia subsect. Pachycladae). Plant Syst.
Evol. 231:109–132.
Moore, M., J. Francisco-Ortega, A. Santos-Guerra, and R. Jansen. 2002.
Chloroplast DNA evidence for the roles of island colonization and
extinction in Tolpis (Asteraceae: Lactuceae). Am. J. Bot. 89:518–526.
Mort, M. E., D. E. Soltis, P. S. Soltis, J. Francisco-Ortega, A. Santos-
Guerra. 2002. Phylogenetics and evolution of the Macaronesian clade
of Crassulaceae inferred from nuclear and chloroplast sequence data.
Syst. Bot. 27:271–288.
Nei, M., and R. Chesser. 1983. Estimation of fixation indices and gene
diversities. Ann. Hum. Genet. 47:253–259.
Nicholson, G., A. Smith, F. J´onsson, O. G ´usstafsson, K. Stef ´ansson, and
P. Donnelly. 2002. Assessing population differentiation and isolation
from single-nucleotide polymorphism data. J. R. Stat. Soc. Ser.B-Stat.
Methodol. 64:695–715.
Ogden, R., and S. Thorpe. 2002. The usefulness of amplified fragment
length polymorphism markers for taxon discrimination across grad-
uated fine evolutionary levels in Caribbean Anolis lizards. Mol. Ecol.
11:437–445.
Oliva, F., J. Caujap´e-Castells, J. Naranjo, J. Navarro, J. Acebes, and D.
Bramwell. 2004. Variaci´on gen ´etica de los Lotus L. (Fabaceae: Loteae)
de pinar en Gran Canaria. Bot. Macaronesica 25:31–52.
Panero, J., J. Francisco-Ortega, R. Jansen, and A. Santos-Guerra. 1999.
Molecular evidence for multiple origins of woodiness and a New
World biogeographic connection of the Macaronesian Island en-
demic Pericallis (Asteraceae: Senecioneae). Proc. Natl. Acad. Sci. USA
96:13886–13891.
Paulay, G., and C. Meyer. 2002. Diversification in the Tropical Pacific:
Comparisons between marine and terrestrial systems and the im-
portance of founder speciation. Integr. Comp. Biol. 42:922–934.
Percy, D., and Q. Cronk. 2002. Different fates of island brooms: Con-
trasting evolution in Adenocarpus,Genista, and Teline (Genisteae,
Fabaceae) in the Canary Islands and Madeira. Am. J. Bot. 89:854–
864.
Pimentel, M., E. Sahuquillo, and P. Catal ´an.. 2007.Genetic diversity and
spatial correlation patterns unravel the biogeographical history of
the European sweet vernal grasses (Anthoxanthum L., Poaceae). Mol.
Phylogen. Evol. 44:667–684.
Pi˜neiro, R., J. F. Aguilar JF, D. D. Munt, and G. N. Feliner. 2007.
Ecology matters: Atlantic-Mediterranean disjunction in the sand-
dune shrub Armeria pungens (Plumbaginaceae). Mol. Ecol. 16:2155–
2171.
Pompanon, F.,A.Bonin, E. Bellemain, and P. Taberlet. 2005. Genotyping
errors: Causes, consequences and solutions. Nat. Rev. Genet. 6:847–
859.
Pritchard, J., M. Stephens, and P. Donelly. 2000. Inference of popu-
lation structure using multilocus genotype data. Genetics 155:945–
959.
Pritchard, J., and W. Wen. 2004. Documentation for structure software.
Version 2. Chicago. Available at http://pritch.bsd.uchicago.edu.
by guest on December 27, 2015http://sysbio.oxfordjournals.org/Downloaded from
2008 D´
IAZ-P ´
EREZ ET AL.—PHYLOGEOG RAPHY OF THE MACAR ONES IAN RED FES CUES 749
Prohens, J., G. Anderson, F. Herraiz, G. Bernardello, A. Santos-Guerra,
D. Crawford, and F. Nuez. 2007. Genetic diversity and conservation
of the endangered Canary Island endemics: Solanum vespertilio and
S. lidii (Solanaceae). Genet. Resour. Crop. Evol. 54:451–464.
Rosenberg, N., J. Pritchard, J. Weber, H. Cann, K. Kidd, L. Zhivotovsky,
and M. Feldman. 2002. Genetic structure of human populations. Sci-
ence 298:2381–2385.
Saint-Yves, A. 1922. Les Festuca (subgenus Eu-Festuca) de l’Afrique du
Nord et des les Isles Atlantiques. Candollea 1:1–63.
S´anchez, J., J. Reyes-Betancort, S. Scholz, and J. Caujap´e-Castells. 2004.
Patrones de variaci´on gen ´etica poblacional en el endemismo canario
Matthiola bolleana Webb Ex Christ. Bot. Macaronesica 25:3–13.
Santos-Guerra, A. 1999. Origen y evoluci´on de la flora canaria. Pages
107–131 in Ecolog´ıa y cultura en Canarias (J. M. Fern ´andez, J. J. Ba-
callado, and J. Belmonte, eds.). Organismo Aut´onomo, Complejo In-
sular de Museos y Centros, Tenerife, Spain.
Silvertown, J. 2004. The ghost of competition past in the phylogeny of
island endemic plants. J. Ecol. 92:168–173.
Sj¨ogren, E. 1973. Recent changes in the vascular flora and vegetation of
the Azores Islands. Mem´orias da Sociedade Broteriana 22:1–453.
Smouse, P., J. Long, and R. Sokal. 1986. Multiple regression and corre-
lation extensions of the Mantel test of matrix correspondence. Syst.
Zool. 35:627–632.
The Mathworks, Inc. 1994. MATLAB
Rfor Windows. Version 4.2.
Massachusetts.
Valad˜ao, P., J. Gaspar, G. Queiroz, and T. Ferreira. 2002. Landslides
density map of S. Miguel Island, Azores archipelago. Nat. Hazards
Earth Syst. Sci. 2:51–56.
Vargas, P., C. Morton, and S. Jury. 1999. Biogeographic patterns
in Mediterranean and Macaronesian species of Saxifraga (Saxifra-
gaceae) inferred form phylogenetic analyses of ITS sequences. Am.
J. Bot. 86:724–734.
Whittaker, R. J. 1998. Island biogeography: Ecology, evolution and con-
servartion. Oxford University Press, New York.
First submitted 18 December 2007; reviews returned 14 March 2008;
final acceptance 6 May 2008
Associate Editor: Roberta Mason-Gamer
by guest on December 27, 2015http://sysbio.oxfordjournals.org/Downloaded from
