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Molecular Ecology (2007)
16
, 1981– 1992 doi: 10.1111/j.1365-294X.2007.03272.x
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd
Blackwell Publishing Ltd
Population differentiation and species cohesion in two
closely related plants adapted to neotropical high-altitude
‘inselbergs’,
Alcantarea imperialis
and
Alcantarea geniculata
(Bromeliaceae)
T. BARBARÁ,
*
G. MARTINELLI,
‡
M. F. FAY,
*
S. J. MAYO
†
and C. LEXER
*
*
Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3DS, UK,
†
Herbarium, Royal Botanic Gardens, Kew,
Richmond, Surrey TW9 3AB, UK,
‡
Instituto de Pesquisas Jardim Botânico do Rio de Janeiro (JBRJ), Rua Pacheco Leão 915,
22460-030 Rio de Janeiro, Brazil
Abstract
Isolated granitic rock outcrops or ‘inselbergs’ may provide a window into the molecular
ecology and genetics of continental radiations under simplified conditions, in analogy to
the use of oceanic islands in studies of species radiations. Patterns of variability and gene
flow in inselberg species have never been thoroughly evaluated in comparison to related
taxa with more continuous distribution ranges, or to other species in the same kingdom in
general. We use nuclear microsatellites to study population differentiation and gene flow
in two diploid, perennial plants adapted to high-altitude neotropical inselbergs,
Alcanta-
rea imperialis
and
Alcantarea geniculata
(Bromeliaceae). Population differentiation is pro-
nounced in both taxa, especially in
A. imperialis
. Gene flow in this species is considerably
lower than expected from the literature on plants in general and Bromeliaceae in particular,
and too low to prevent differentiation due to drift (
N
e
m
< 1), unless selection coefficients/
effect sizes of favourable alleles are great enough to maintain species cohesion. Low gene
flow in
A. imperialis
indicates that the ability of pollinating bats to promote gene exchange
between inselbergs is smaller than previously assumed. Population subdivision in one
inselberg population of
A. imperialis
appears to be associated with the presence of two
colour morphs that differ in the coloration of rosettes and bracts. Our results indicate a high
potential for inselbergs as venues for studies of the molecular ecology and genetics of
continental radiations, such as the one that gave rise to the extraordinary diversity of adaptive
strategies and phenotypes seen in Bromeliaceae.
Keywords
:adaptive radiation, bromeliad, gene flow, inselberg, microsatellites, species cohesion
Received 17 September 2006; revision received 21 November 2006; accepted 11 December 2006
Introduction
Experimental observations on islands have a long tradition
in studies of biogeography and evolution (Darwin 1859;
MacArthur & Wilson 1967). Similar to oceanic islands,
isolated granitic rock outcrops or inselbergs (from German
Insel = island and Berg = mountain) have been suggested
as model systems for ecological and evolutionary studies
(Prance 1996; Porembski & Barthlott 2000). The insular
nature of these ancient monoliths harboring specialized
animal and plant life has raised the expectation that
inselbergs may indeed represent ‘terrestrial habitat islands’,
resembling oceanic islands as models for studying ecological
and evolutionary processes (Porembski & Barthlott 2000).
If this view is correct, then inselbergs may contribute to
much-needed research towards integrating genetics into
the ecological theory of adaptive radiation, as encouraged
by Schluter (2000). In particular, inselberg radiations may
then provide a window on the molecular ecology and
genetics of continental adaptive radiations, complementary
to the much celebrated examples of radiations on islands
(Schluter 2000; Emerson 2002).
Correspondence: Thelma Barbará, Fax: +44(0)20 8332 5310;
E-mail: thelmabarbarakew@yahoo.co.uk and t.barbara@kew.org
1982
T. BARBARÁ
ET AL.
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd
An important aspect is that inselbergs are thought to
provide a simpler setting for evolutionary studies than
other systems of terrestrial habitat islands, such as contin-
ental mountain ranges (Schonswetter
et al
. 2005; Hughes
& Eastwood 2006) or the ‘sky islands’ of western North
America (Knowles 2001; DeChaine & Martin 2005; Smith &
Farrell 2005). Indeed, from an experimental evolutionary
biologist’s point of view, inselbergs may be more similar to
terrestrial lakes, such as those utilized for studies of eco-
type differentiation, speciation, and adaptive radiation in
lake whitefish (Campbell & Bernatchez 2004), cichlids
(Barluenga
et al
. 2006), or sticklebacks (Schluter 2000). To
our knowledge, however, patterns of genetic variability
and gene flow in inselberg species have never been evalu-
ated in comparison to related taxa with more continuous
distribution ranges, or to patterns of gene flow in other
organisms in general. Here, we aim at filling this gap. A
recent review of the role of gene flow and selection in the
maintenance of species cohesion (Morjan & Rieseberg
2004) allows us to put our data in the context of results in
many other species.
Our study is focused on inselberg-dwelling plants in the
Bromeliaceae (bromeliads), a family that represents a par-
ticularly striking and phylogenetically well-characterized
continental adaptive radiation (Brown & Gilmartin 1989;
Givnish
et al
. 1997; Smith & Till 1999; Benzing 2000; Barfuss
et al
. 2005). Bromeliaceae is one of the predominant plant
families found on South American inselbergs (Barthlott
et al
. 1993; Safford & Martinelli 2000). Population genetic
studies exist for several bromeliads in different subfamilies
(Soltis
et al
. 1987; Murawski & Hamrick 1990; Izquierdo &
Pinero 2000; Sarthou
et al
. 2001; Gonzalez-Astorga
et al
.
2004; Sgorbati
et al
. 2004; Cavallari
et al
. 2006), which
allows us to compare genetic attributes of related species
with varying breeding systems. Here, we focus on
Alcanta-
rea imperialis
(Carriere) Harms and
Alcantarea geniculata
(Wawra) J.R. Grant, two inselberg-adapted bromeliads in
the Atlantic Rainforest of Brazil, one of the world’s most
important and vulnerable biodiversity ‘hotspots’ (Myers
et al
. 2000).
Alcantarea imperialis
and
A. geniculata
are perennial,
rupicolous plants (they grow on rocks with very limited
substrate). They are characterized by hermaphrodite flowers
and mixed mating or outcrossing breeding systems with
animal pollination and wind-based seed dispersal
(Martinelli 1994; Safford & Martinelli 2000). Their vegetative
structures form ‘tanks’ that are able to hold many litres of
water (up to 40 L in some cases), thus providing an impor-
tant resource base for associated biota in this harsh high-
altitude environment (Benzing 2000).
Alcantarea imperialis
and
A. geniculata
are part of a larger radiation that includes
numerous other taxa of the closely related genera
Alcanta-
rea
and
Vriesea
, many of which occur on inselbergs (Safford
& Martinelli 2000). However, these two diploid perennial
species are especially suitable for our purpose. Pollinator
observations and hand-pollination experiments in
A. impe-
rialis
indicate that it favours outcrossing over selfing (Mar-
tinelli 1994), and thus this species represents a conservative
model case for assessing the effect of inselberg adaptation
on population connectivity. Less information exists about
the pollination syndrome of the closely related
A. genicu-
lata
(Porsch 1935; Martinelli 1994), but we anticipated that
inspection of Hardy–Weinberg proportions would pro-
vide us with clues about its breeding system at the outset
of the study. The two species are clearly recognizable
based on consistent vegetative features (Martinelli 1994)
and they co-occur on some inselbergs, which makes a joint
analysis sensible.
Here, we address the following questions related to the
molecular ecology and population genetics of
Alcantarea
spp. on neotropical high-altitude inselbergs:
1
How do levels of population differentiation in these
inselberg species compare to other bromeliads, or to
other plants in general?
2
Are the observed levels of differentiation compatible
with the view of inselbergs as ‘terrestrial islands’, and if
yes, what are the relative roles of gene flow and selection in
maintaining species cohesion in the face of fragmentation?
3
To what extent are molecular marker-based breeding sys-
tem parameters congruent with knowledge of the pollina-
tion syndromes of these two closely related congeners?
4
Is there evidence for population subdivision on inselbergs?
We use our data to assess the usefulness of inselbergs
as model cases for studying the molecular ecology and
genetics of a continental plant radiation, and we comment
on implications for conservation in this world biodiversity
‘hotspot’.
Materials and methods
Population sampling
A total of eight populations of
Alcantarea imperialis
and
Alcantarea geniculata
were sampled on high-altitude
granitic inselbergs located in the Atlantic Rainforest of
southeastern Brazil (states of Rio de Janeiro and Minas
Gerais) (Fig. 1). Sampling on high-altitude outcrops in the
tropics is a costly and demanding procedure (few localities
are accessible without a helicopter and rappelling), and
thus our sampling design was chosen to extract a maximum
of information without sampling all populations of each
species. Rather, populations were sampled in such a way
as to represent the species range of each of these endemics
and to provide a broad range of geographical distances
between populations, including both neighbouring and
also more distant population pairs for each species. The
POPULATION DIFFERENTIATION ON INSELBERGS
1983
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd
names, abbreviations, and geographical coordinates of the
sampled populations are as follows:
A. imperialis
: Imperialis
‘Irmã Menor’ or IIM (22
°
24.401
′
S, 43
°
12.143
′
W), Imperialis
‘Macaé-de-Cima’ or IMC (22
°
22.176
′
S, 42
°
29.774
′
W),
Imperialis ‘Juíz-de-Fora’ or IJF (21
°
47.922
′
S, 43
°
22.243
′
W),
and Imperialis ‘Vale das Videiras’ or IVV (22
°
25.870
′
S,
43
°
16.228
′
W);
A. geniculata
: Geniculata ‘Irmã Menor’ or
GIM (22
°
24.401
′
S, 43
°
12.143
′
W), Geniculata ‘Ricardo Clearing’
GRC (22
°
25.044
′
S, 43
°
13.262
′
W), Geniculata ‘Ricardo Rock’
GRR (22
°
25.232
′
S, 43
°
13.183
′
W), and Geniculata ‘Reserva
Privada’ GRP (22
°
24.960
′
S, 43
°
12.844
′
W).
The altitudes of the sampled populations ranged
between 872 m and 1310 m above sea level. Geographical
distances between populations ranged from 7 to 110 km
with an average of 68 km for the Atlantic Rainforest
endemic
A. imperialis
, and from 0.4 to 2.4 km with an aver-
age of 1.4 km for the narrow endemic
A. geniculata
. The
species co-occur on one of the sampled inselbergs, namely
the rock outcrop ‘Irmã Menor’ (populations IIM and GIM).
Population IMC of
A. imperialis
consisted of plants of two
different colour morphs: one with green and one with red
rosettes and bracts. The two colour morphs were found
interdigitated at roughly equal frequency with no obvious
spatial pattern. Inspection of juvenile plants in the popula-
tion suggested that colour morphs segregated like a herit-
able character with simple Mendelian mode of inheritance,
but controlled crosses have not yet been analysed. Sample
sizes for all populations are given in Table 2. For each
plant, leaf material for DNA extraction was collected in
silica gel.
Molecular markers and genotyping assays
Six of the eight microsatellite markers used in this study
were assayed for the first time in
Alcantarea
spp. by cross-
species amplification from the related bromeliad genera
Tillandsia
and
Guzmania
(markers e6, p2p19, e19, e6b, and
CT5; Boneh
et al
. 2003) and from
Pitcairnia
(Pit8; Sarthou
et al
. 2003). Two additional markers, Ai4.10 and Ai4.3, were
isolated de novo from
A. imperialis
and are reported in
more detail elsewhere, in combination with new markers
from another bromeliad species (Palma-Silva
et al
. 2007).
Repeat types and molecular size ranges for all eight markers
in
A. imperialis
and
A. geniculata
are given in Table 1.
For molecular genotyping, total genomic DNA was
extracted from silica gel-dried leaves using a modified
approach based on Doyle & Doyle (1987), and DNA was
quantified using an Eppendorf BioPhotometer. The eight
nuclear microsatellites were polymerase chain reaction
(PCR)-amplified following methods described previously
by Burke
et al
. (2002), making use of a standard touchdown
cycling programme with an annealing temperature (
T
a
) of
48
°
C and either FAM- or JOE-labelled forward primers, or
a three-primer protocol including unlabelled M13-tagged
forward and unlabelled/untagged reverse primers for
each marker, and a third ‘universal’ M13-primer labelled
with one of the fluorescent dyes, FAM or JOE (Applied Bio-
systems). Microsatellite genotypes were resolved on an
ABI PRISM 3100 Genetic Analyser (Applied Biosystems),
making use of the different fluorescent dyes for duplexing.
Molecular sizes in base pairs were determined using the
Fig. 1 Distribution map of Alcantarea
inselberg populations sampled in the
Atlantic Rainforest of Brazil. For populatio
n
abbreviations and details see Materials and
methods.
1984
T. BARBARÁ
ET AL.
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd
GENESCAN-500 ROX size standard (Applied Biosystems),
and result files from the sequencers were analysed using
genescan
and
genotyper
software (Applied Biosystems).
Data analysis
Genetic diversity of the sampled loci and populations.
In order
to characterize the microsatellite loci in the two study
species, the number of alleles (
A
), expected heterozygosity
(
H
E
), observed heterozygosity (
H
O
), and the within- and
total-population inbreeding coefficients
F
IS
and
F
IT
were
calculated for each locus using the computer programs
msa
(Dieringer & Schlotterer 2003) and
fstat
(Goudet
1995). In addition, departures from Hardy–Weinberg
equilibrium (HWE) for each locus within populations of
each species were tested using
fstat
, in order to explore
the possibility that particular loci may deviate from
HWE within populations because of null alleles (= allele
nonamplification). Subsequently, each population was
characterized using the variance in allele size (Var),
H
E
,
and
H
O
calculated by
msa
and allelic richness in
fstat
(Goudet et al. 1995). All genetic diversity parameters were
corrected for sample size in msa and fstat. Departures
from HWE for each population were identified using exact
tests in genepop (Raymond & Rousset 1995).
Tests of basic assumptions underlying indirect estimation
of gene flow
To determine if the sampled Alcantarea inselberg
populations are likely to meet the equilibrium conditions
required for the indirect estimation of gene flow via F-
statistics, the possibility of founder effects due to recent
colonization (genetic ‘bottlenecks’) was tested using the
‘sign test’ and ‘Wilcoxon sign-rank’ test in the bottleneck
program (Piry et al. 1999). Both tests are able to detect
recent reductions in effective population size due to
genetic bottlenecks. The analyses were carried out both for
the ‘infinite allele model’ (IAM) and for the ‘two-phased
mutation model’ (TPM) recommended for microsatellites
in the user manual. The detection of recent bottlenecks
would indicate that Alcantarea inselberg populations have
not yet reached an equilibrium between gene flow and
genetic drift, which may render the indirect estimation of
gene flow via FST difficult (Whitlock 1992). Also, correlations
between genetic and geographical distance (isolation by
distance; Slatkin 1993) were tested using nonparametric
Table 1 Characterization of microsatellite markers in ‘inselberg’ populations of Alcantarea imperialis and Alcantarea geniculata, including
marker source, repeat type, molecular size range in each Alcantarea spp. in base pairs (bp), number of alleles (A), expected (HE) and observed
(HO) heterozygosity, within-population inbreeding coefficient (FIS), and total-population inbreeding coefficient (FIT) in each species
Locus
Repeat
type
A. imperialis A. geniculata
Size range (bp) AH
EHOFIS FIT Size range (bp) AH
EHOFIS FIT
Ai4.10† di 184–188 3 0.446 0.066 −0.007 +0.879 184–191 4 0.554 0.408 +0.175 +0.297
E19‡ di 115–123 4 0.509 0.402 +0.000 +0.264 105–139 6 0.104 0.083 +0.196 +0.202
E6‡ tri 107–128 3 0.510 0.405 +0.002 +0.309 107–128 3 0.275 0.301 −0.215 −0.058
Ai4.3† di 191–195 3 0.496 0.279 +0.002 +0.389 191 1 — — — —
CT5‡ di 157–195 14 0.723 0.448 −0.002 +0.346 159–187 8 0.707 0.570 +0.060 +0.238
E6b‡ tri 128–146 7 0.781 0.475 +0.001 +0.420 128–153 5 0.135 0.128 +0.043 +0.054
P2p19‡ tri 185–214 9 0.812 0.491 +0.001* +0.324 178–208 5 0.590 0.526 +0.062 +0.128
Pit8§ di 280–318 9 0.646 0.328 −0.002 +0.575 280–314 7 0.635 0.481 +0.210*** +0.255
†Microsatellite markers isolated de novo from Alcantarea imperialis in the Jodrell Laboratory at RBG Kew (Palma-Silva et al. 2007). ‡Markers
isolated by Boneh et al. (2003). §Marker isolated by Sarthou et al. (2003). Significant FIS values for particular loci are indicated by asterisks
(*P < 0.05, ***P < 0.005).
Table 2 Characterization of ‘inselberg’ populations of Alcantarea
imperialis and Alcantarea geniculata with eight nuclear micro-
satellite markers, including the number of chromosomes sampled
(N), variance in allele size (Var), allelic richness, as well as
expected (HE) and observed (HO) heterozygosities. Departures
from Hardy–Weinberg equilibrium are indicated by asterisks
(*P < 0.05, ***P < 0.005)
Species Population NVar
Allelic
richness HEHO
A
. imperialis IIM 40 16.4 2.20 0.327 0.304
IMC 112 13.3 2.29 0.395 0.365
IJF 52 28.6 2.68 0.452 0.400*
IVV 44 21.9 2.75 0.421 0.362
Overall: 248 27.96 6.25 0.615 0.362
A
. geniculata GIM 52 26.5 2.66 0.455 0.408
GRC 62 22.9 2.45 0.383 0.333***
GRR 18 17.4 2.13 0.341 0.355
GRP 36 21.5 2.21 0.343 0.330
Overall: 168 23.84 5.25 0.429 0.357
POPULATION DIFFERENTIATION ON INSELBERGS 1985
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd
Mantel tests in fstat with 10 000 randomizations for each
species. Isolation by distance may lead to serious departures
from an island migration model which may complicate the
indirect estimation of gene flow (Whitlock 1992).
Indirect analysis of gene flow via F-statistics and
population phylogeny
Analysis of molecular variance (amova) in arlequin
(Excoffier et al. 2005) was used to obtain F-statistics for
microsatellites at different hierarchical levels. We tested
the hierarchies ‘among species’, ‘among populations
within species’, and ‘within populations’ for the entire data
set. Subsequently, separate amova models were analysed
to test the distribution of genetic variance among and
within populations of each species. In addition, FST
between pairs of populations and between the two colour
morphs present in population IMC was estimated using
msa (Dieringer & Schlotterer 2003). The significance of
each F-statistic was tested through 10 000 permutations at
the appropriate hierarchical level in arlequin or msa. To
depict relationships between populations and species in a
graphical way, a neighbour-joining (NJ) tree was constructed
based on the chord distance of Cavalli-Sforza & Edwards
(1967). One thousand bootstrap replicates of the distance
matrix were obtained in msa, and NJ trees were generated
and analysed in phylip 3.6 (Felsenstein 2004).
Effective population sizes and migration rates
Theta (4Neµ, with Ne = effective population size and
µ = mutation rate) for populations of A. imperialis and A.
geniculata and the effective number of migrants (Nem)
between pairs of populations were estimated following a
coalescent theory and maximum-likelihood-based approach
using migrate 2.0.6 (Beerli & Felsenstein 1999). Pairwise
analysis was used because of unfavourable trade-offs
between analysis run-time and gains in precision when
multiple populations are analysed simultaneously. Local
gene flow between pairs of populations can be estimated
with confidence even if not all populations were sampled,
as long as migration rates from unsampled populations
are expected to be low (Beerli 2004). Pairwise analysis
allowed the estimation of intraspecific gene flow (Nem) into
each inselberg population in each species. In addition,
interspecific Nem for populations of A. imperialis and A.
geniculata co-occurring in sympatry on inselberg Irmã
Menor (populations IIM and GIM) was estimated in both
directions. In each case, genetic divergence between pairs
of populations (FST; Weir & Cockerham 1984) was used to
obtain initial start values for the estimation of theta and
Nem. The computations were carried out under both the
IAM and the stepwise mutation model (SMM), and
effective population sizes were estimated from theta
values by assuming a microsatellite mutation rate of 10−3
per gamete per generation (Zhang & Hewitt 2003).
Bayesian genetic structure analysis
Bayesian analysis in structure version 2 (Pritchard et al.
2000) was used to obtain additional insights into patterns
of gene flow and population subdivision within Alcantarea
inselberg populations. Our aim was to determine the most
likely number of populations (K) for each species, and to
estimate admixture proportions (Q) for individuals of each
population. Preliminary runs revealed that the variances
of likelihood estimates relative to the actual likelihoods
for models with different K were not satisfactory in
A. geniculata, despite excessive run lengths. This probably
was the case because allelic diversities were low in this
narrow endemic, and one locus (Ai4.3) was even fixed for
a single allele in this species (Table 1). Therefore, Bayesian
genetic structure analysis was carried out for the more
variable species A. imperialis only. ‘Burn-in’ lengths of
50 000 and run lengths of 1 000 000 were identified as being
appropriate based on the diagnostic tools available in
structure. The analyses were carried out under the
admixture model for independent allele frequencies, and
all possible models for A. imperialis from K = 1 to K = 8
were evaluated based on the natural logarithm of their
probability and on their variances. Individual and average
admixture proportions (Q) for each sampled population in
each genetic cluster found by structure were recorded for
the model with the highest probability.
Results
Genetic diversity and equilibria in Alcantarea inselberg
populations
All eight microsatellite loci were polymorphic, with up to
14 alleles per locus and gene diversities (HE) of up to 0.812
in Alcantarea imperialis, and up to eight alleles per locus and
HE up to 0.707 in Alcantarea geniculata (Table 1). One locus,
Ai4.3, was fixed for a single allele in the narrow endemic
A. geniculata but was nevertheless polymorphic in A. imperialis
(Table 1). Low or nonsignificant within-population inbreeding
coefficients (FIS) for most loci indicate near-random mating
within populations, whereas high total-population inbreeding
coefficients (FIT) reflect species-level homozygote excess
due to genetic subdivision (‘Wahlund’ principle; Hartl &
Clark 1997) (Table 1). One locus (P2p19) displayed an
overall departure from Hardy–Weinberg equilibrium
(HWE) within populations in A. imperialis in the form of a
heterozygote deficit and one did so in A. geniculata (Pit8),
resulting in significantly positive within-population inbreeding
coefficients (FIS) in these two cases (Table 1). As no locus
displayed consistent departures from HWE across all
1986 T. BARBARÁ ET AL.
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd
populations, it is likely that the observed departures from
HWE reflect occasional departures from random mating
rather than the presence of null alleles.
Genetic diversity evaluated at the population level was
always higher in A. imperialis than in A. geniculata, regard-
less of whether it was estimated via the variance in allele
size (Var), allelic richness, expected (HE), or observed
heterozygosities (HO) (Table 2), which probably reflects
the different geographical distribution ranges of the two
species (endemic vs. narrow endemic) or differences in
pollination syndromes (discussed below). Population IJF in
A. imperialis and population GRC in A. geniculata displayed
significant heterozygote deficits, whereas all other popula-
tions were in HWE (Table 2). Neither the sign test nor the
Wilcoxon sign-rank test for recent population bottlenecks
was significant for any of the populations, regardless of the
mutation model used (not shown). This indicates that
populations of the two species are not recently colonized and
are therefore likely to have reached equilibrium, which is
a prerequisite for the indirect estimation of gene flow.
Mantel tests in each species indicated no significant
correlation between geographical and genetic distance
(not shown), thus suggesting the absence of isolation by
distance in inselberg populations of Alcantarea.
Genetic structure and gene flow between Alcantarea
inselberg populations
An NJ tree based on microsatellite genetic distances (Fig. 2)
separated inselberg populations of A. imperialis and
A. geniculata with high bootstrap support. As indicated by
the branch lengths in Fig. 2, genetic distances between
populations of A. imperialis were sometimes nearly as large
as genetic distances between the two species, reflecting a
high degree of genetic isolation among A. imperialis inselberg
populations. In agreement with these results, amova
attributed a significant proportion of the genetic variance
(28%; P < 0.05) to the ‘among species’ level, and a similarly
high and significant proportion to ‘among populations
within species’ (25%; P < 0.001) (Table 3). Separate amova
models for each species revealed that a higher proportion
of the genetic variance resided ‘among populations’ in
A. imperialis (44%; P < 0.001) than in A. geniculata (11%;
P < 0.001) (Table 3). Individual FST estimates between pairs
of populations ranged from 0.166 to 0.535 for A. imperialis
and from 0.082 to 0.142 for A. geniculata (all P values < 0.005
Table 3 Results of analysis of molecular variance (amova) for three different hierarchical models, a three-level model including both
A
lcantarea species, and separate two-level models for each species. The significance of each FST analogue was tested through 10 000
permutations at the appropriate hierarchical level
Model Partitioning Variation (percentage) F-statistic P
Three levels — both species Among species 28 FCT = 0.280 < 0.05
Among populations within species 25 FSC = 0.351 < 0.001
Within populations 47 FST = 0.533 < 0.001
Two levels — A. imperialis Among populations 44 FST = 0.434 < 0.001
Within populations 56 FIS = 0.099
FIT = 0.490
Two levels — A. geniculata Among populations 11 FST = 0.111 < 0.001
Within populations 89 FIS = 0.094
FIT = 0.195
Fig. 2 Unrooted neighbour-joining tree of populations based on
Cavalli-Sforza & Edward’s (1967) chord distance, including
b
ootstrap support values in percent. A scale for genetic distance is
provided at the bottom of the graph. For population abbreviations
see Materials and methods.
POPULATION DIFFERENTIATION ON INSELBERGS 1987
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd
in both species; not shown). Interspecific pairwise FST
averaged 0.496 (maximum = 0.554) and was lowest between
the sympatric populations IIM and GIM (FST = 0.412)
and between population GIM of A. geniculata and the
neighbouring population IVV of A. imperialis (FST = 0.368;
distance = 7 km). A high and significant proportion of the
genetic variance was observed within populations of each
species (56% and 89% in separate amova models for
A. imperialis and A. geniculata, respectively) (Table 3), as
expected for perennial plants with mixed or outcrossing
breeding systems.
Maximum-likelihood-based estimates of migration rates
(Nem; ‘gene flow’) were low in A. geniculata and extremely
low in A. imperialis (Fig. 3), consistent with the microsatel-
lite genetic distances (Fig. 2) and F-statistics from amova
(Table 3; above). In fact, all Nem estimates for A. imperialis
were < 1 migrant per generation, which has traditionally
been regarded as the minimum required for maintaining
species cohesion (Fig. 3). In A. geniculata, seven out of 12
Nem estimates were < 1. Migration rates between the only
pair of populations of A. imperialis and A. geniculata sam-
pled in sympatry on the same inselberg were Nem = 0.45
and 0.73 migrants per generation, depending on the
direction of the analysis, suggestive of very low levels of
interspecific gene flow in sympatry (Fig. 3).
Bayesian genetic structure analysis in A. imperialis
Bayesian genetic structure analysis confirmed the
pronounced genetic structure in A. imperialis and yielded
Fig. 3 Effective population sizes (Ne) and
effective number of migrants (Nem) for
inselberg populations of Alcantarea imperialis
and Alcantarea geniculata, estimated followin
g
Beerli & Felsenstein (1999) under the SMM
with migrate 2.0.6. (A) Effective population
size for each sampled population of A. imperialis
and migration into that population from
other inselbergs. (B) Effective population siz
e
for each population of A. geniculata and
migration into that population from other
inselbergs. (C) Effective population size and
migration for populations of A. imperialis
and A. geniculata co-occurring on one
‘inselberg’, Irmã Menor. Population
comparisons with Nem < 1 are indicated by
empty arrows, and comparisons for which
intraspecific Nem (A and B) was smaller than
interspecific Nem in sympatry (C) are
indicated by asterisks. For population
abbreviations see Materials and methods.
1988 T. BARBARÁ ET AL.
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd
additional insights into patterns of gene flow and
population subdivision in this species. A model of K = 5
populations was best able to capture the variation in the
data, based on both an abrupt slowing down of the change
in probability of the data (ln prob) for models with a
number of populations K > 5 and a noticeable increase
in the variance with K > 5; the ln prob for the K = 5
population model was −1329.4 with a variance of ln
likelihood of 109.7, whereas for the K = 6 model the
parameter values were −1319.4 and 136.2 for ln prob and
variance, respectively. Based on the small difference in ln
prob and the increase in variance, the simpler K = 5 model
was chosen to represent the data.
The average admixture proportion for each population
of A. imperialis among the five different ‘genetic clusters’
found by the Bayesian analysis is given in Table 4, and the
admixture proportions (Q) of individual plants in each of
the four clusters are depicted graphically in Fig. 4. The
graph reveals that some plants from population IVV
are admixed with alleles from the same ‘genetic cluster’
that makes up population IIM. These two inselberg popu-
lations are separated by only 7.5 km. Also, population
IMC, the population containing plants of two different
colour morphs, showed signs of admixture between two
‘genetic clusters’ not found elsewhere in the data set
(Fig. 4).
Discussion
Population differentiation and species cohesion in
fragmented Alcantarea inselberg populations
The forces responsible for the maintenance of species
cohesion have received great interest in recent years
(Ehrlich & Raven 1969; Morjan & Rieseberg 2004), and this
allows us to put our present results on Alcantarea inselberg
species in the context of gene flow estimates from many
other taxa. The genetic parameters estimated here indicate
that gene flow between inselberg populations of Alcantarea
imperialis and Alcantarea geniculata is much weaker than
normally expected for diploid perennial outcrossing
plants. Estimates of the effective number of migrants (Nem)
indicate that gene flow is < 1 for all pairwise population
comparisons of A. imperialis and for seven out of 12
comparisons in A. geniculata (Fig. 3). Thus gene flow in
A. imperialis would appear to be too low to prevent
differentiation because of genetic drift (Wright 1931),
unless selection coefficients/effect sizes of favourable
alleles are great enough to maintain species cohesion
(Morjan & Rieseberg 2004). This pattern is less pronounced
in the narrow endemic A. geniculata, where each population
is interconnected to at least one other population with
Nem > 1 (Fig. 3). In accordance with low levels of gene
flow (Nem), genetic distances and branch lengths in our
neighbour-joining tree of populations are almost as large
within as between species (Fig. 2). Likewise, the proportion
of genetic variance of amova residing between populations
of each species approaches that of the variance resid-
ing between species (25% vs. 28%, respectively; Table 3).
Strong population subdivision is also visible from high
total inbreeding coefficients FIT (Wahlund effect; Hartl &
Clark 1997) despite low estimates of within-population
inbreeding FIS (Table 1; Table 3) and only modest depart-
ures from HWE for most populations (Table 2).
If we compare genetic differentiation between popula-
tions of A. imperialis (FST = 0.434 in single species amova;
Table 3) with estimates of FST from the literature database
of Morjan & Rieseberg (2004), a surprising result emerges:
FST in A. imperialis falls far outside the interquartile range
Table 4 Average admixture proportion for each sampled
population of Alcantarea imperialis (rows) among each of five
‘genetic clusters’ (columns) inferred by Bayesian analysis in
structure (Pritchard et al. 2000). For population abbreviations
see Materials and methods
Population
Cluster
IIIIII IV V
IIM 0.010 0.011 0.019 0.019 0.94
2
IMC 0.015 0.691 0.280 0.006 0.00
7
IJF 0.961 0.014 0.015 0.006 0.00
5
IVV 0.051 0.008 0.014 0.438 0.48
9
Fig. 4 Bayesian admixture proportions (Q)
of individual plants of A. imperialis for
a K = 5 population model. The K = 5 ‘gen-
etic clusters’ identified by structure are
indicated in different shades of grey. For
population abbreviations see Materials and
methods.
POPULATION DIFFERENTIATION ON INSELBERGS 1989
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd
(= typical expectation) for FST in outcrossing plants or
plants with mixed mating systems (Fig. 5). Rather, it falls
within the interquartile range of plants with selfing mating
systems (Fig. 5). We note that controlled pollination experi-
ments and observations of ovule penetration in A. imperi-
alis suggest that this species, although self-compatible,
favours outcrossing (Martinelli 1994). Ovule penetration
was clearly higher after cross-pollination (range: 52–75%)
compared to self-pollination (range: 0–29%) at 96 h, the
earliest time point at which penetration was observed in
this species (Martinelli 1994). Although mixed pollination
experiments would be desirable, the concordance of most
of our studied populations with HWE (Table 2) confirms
the predominantly outcrossing nature of this species. Also,
our molecular marker data did not reveal a single case of
clonal origin for plants at the adult stage (= all sampled
plants had unique genotypes). Such unusually high popu-
lation divergence in a preferentially outcrossing species
indicates strongly restricted gene flow due to fragmented
distribution on inselbergs. Genetic divergence for A. geni-
culata, on the contrary, was more similar to what would be
expected for a predominantly outcrossing species (FST = 0.111;
Fig. 5), most likely due to greater population connectivity
associated with its much narrower endemic range.
How does FST in A. imperialis compare with other bro-
meliads then? Population divergence in bromeliads with
comparable (mixed or outcrossing) mating systems and
more continuous distributions are typically much lower
than in A. imperialis, for example FST = 0.043 (Tillandsia
ionantha; Soltis et al. 1987), 0.196 (Aechmea tuitensis;
Izquierdo & Pinero 2000), or 0.080–0.160 (Encholirium spp.;
Cavallari et al. 2006), with no obvious difference between
the size of geographical distribution ranges or the type of
molecular marker used. Again, bromeliads with FST values
as high as those in A. imperialis are normally taxa with
increased levels of selfing/cloning: population divergence
was estimated as FST = 0.390 for Tillandsia achyrostachys
(clonal reproduction is documented for this species;
Gonzalez-Astorga 2004), and as FST = 0.356 for Aechmea
magdalenae (tendency to clone documented as well;
Murawski & Hamrick 1990). In Tillandsia recurvata, a spe-
cies expected to self-pollinate based on its floral morpho-
logy, FST was as high as 0.906 (Soltis et al. 1987), and in Puya
raimondii, a notorious inbreeder, the related differentiation
parameter GST was as high as 0.961 (Sgorbati et al. 2004).
This translates into FST = 0.966 following Cockerham &
Weir (1987). Hence, just as in our earlier comparison to the
general plant database, A. imperialis exhibits much greater
population differentiation than expected based on its well-
documented outcrossing mating system. Again, this points
to restricted gene flow due to fragmented distribution on
inselbergs as the most likely cause of high FST. The only
other genetic survey of inselberg bromeliads that we are
aware of, on the mixed outcrosser Pitcairnia geyskesii on
rock outcrops of French Guiana, estimated FST = 0.322
(Sarthou et al. 2001), which is lower but similar to our esti-
mate for A. imperialis. Taken together, their study and ours
indicate high degrees of population isolation in inselberg
species for different subfamilies of Bromeliaceae.
Correspondence between molecular marker data and
pollination syndromes of Alcantarea spp.
Previous work on Alcantarea spp. (Martinelli 1994; Safford
& Martinelli 2000) allows us to place our molecular marker
data in the context of the pollination syndromes of these
species. Extensive fieldwork on A. imperialis (Martinelli
1994) indicates that flowers are visited by a wide range of
animals including hummingbirds (Clytolaema rubricauda,
Leucochloris albicollis, Heliothryx aurita, and Melanothrochilus
fuscus), hawkmoths (Sphingidae), and bats (Anoura
caudifera and Artibeus lituratus). However, hummingbirds
visit flowers only during the beginning of anthesis and
after senescence (the flowers are open during the night),
and only bats appear to be able to promote pollination
(Martinelli 1994). With this in mind, the extremely low
levels of gene flow observed in A. imperialis are surprising
— bats are thought to be efficient pollinators and should
thus be able to promote gene exchange between plant
populations situated on inselbergs (Sazima et al. 1989;
Sazima et al. 1999). Our data indicate that the ability of bats
to promote gene flow between inselberg populations may
be smaller than previously assumed.
Fig. 5 Comparison of FST in Alcantarea imperialis and Alcantarea
g
eniculata with estimates from the plant literature. FST in A.
imperialis (imp) and A. geniculata (gen) is compared to 135 nuclear
marker-based estimates of population divergence (FST or GST) i
n
plants with outcrossing, mixed, or selfing breeding systems from th
e
literature database of Morjan & Rieseberg (2004). The boxplots allow
comparison of FST in Alcantarea spp. with the interquartile ranges
(boxes) for plants with different breeding systems.
1990 T. BARBARÁ ET AL.
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd
Suggested pollinators of A. geniculata are bees and sph-
ingid moths (Knuth 1904; Porsch 1935), but these sugges-
tions may be based on superficial observation of visitors to
flowers or simply on speculative comments based on floral
morphology of cultivated specimens (Martinelli 1994).
Nevertheless, owing to its narrowly endemic range, the
geographical distances between outcrops on which these
populations occur tend to be very small (often < 1 km).
These distances may easily be covered by any of these
potential pollinators, and dispersal of the small seeds by
wind should easily be facilitated as well. Future studies of
Alcantarea spp. should include molecular-marker-based
estimation of breeding system parameters from progeny
arrays (Ritland 2002), to allow a more accurate assessment
of breeding systems under natural conditions.
Population subdivision on inselbergs
Our Bayesian analysis following Pritchard et al. (2000)
provides some first insights into fine-scale genetic
structure in A. imperialis inselberg populations. First, fine-
scale patterns of gene flow between the geographically
neighbouring populations IIM and IVV become apparent
in Fig. 4, with population IVV exhibiting a proportion of
plants belonging to the same ‘genetic cluster’ as all plants
of its geographical neighbour IIM (cluster 1; Fig. 4; Table 4).
Second, population subdivision becomes apparent for
IMC, the population with two different colour morphs
present (plants with red and plants with green rosettes and
bracts; see Materials and methods). The Bayesian analysis
identified two genetic clusters exclusive to this population
(clusters 2 and 3; Fig. 4; Table 4). Genetic divergence
between the two sympatric morphs was low (FST = 0.099)
but indeed significant at the 0.001 level when compared to
10 000 random permutations of alleles between morphs.
It is thus possible that genetic subdivision in population
IMC is indeed associated with colour morphs, although
the origin of morphs (secondary contact vs. origin in sym-
patry), their genetic basis (monogenic or oligogenic), and
the actual mechanism maintaining isolation (ecological or
pollinator-mediated), must remain speculative at present.
It is certain that colour morphs arise easily and repeatedly
in Bromeliaceae, as evidenced by the multitude of spon-
taneous colour variants found in breeders’ collections and
among horticultural varieties.
Inselbergs as venues for molecular ecology studies
Patterns of diversity and gene flow in A. imperialis indicate
that fragmented distribution on inselbergs can lead to
unusually high levels of population differentiation, thus
indicating restricted gene flow and decreased population
connectivity even in species with predominantly outcrossing
mating systems. This supports the view that these granitic
rock outcrops, widely distributed throughout tropical and
temperate regions of the world (Porembski & Barthlott
2000), may indeed be comparable to oceanic islands in their
effects on patterns of genetic variability and gene flow.
Consequently, inselbergs are indeed promising venues
for ecological and evolutionary studies as suggested by
ecologists (Porembski & Barthlott 2000), similar to the
use of oceanic islands in biogeography and evolutionary
biology (MacArthur & Wilson 1967).
When compared to other types of ‘terrestrial archipe-
lagos’, inselbergs may be most similar in nature to the ‘sky
island’ populations of the North American cordillera sys-
tem. Phylogeographical studies based on mitochondrial
DNA in animals and plastid DNA polymorphisms in
plants indicate that ‘sky island’ populations in the western
USA underwent extensive genetic divergence in response
to fragmentation associated with recent palaeoclimatic
cycles (Knowles 2001; DeChaine & Martin 2005; Smith &
Farrell 2005). Although tropical inselberg populations may
have been affected by range expansions and contractions
of similar magnitude, they differ from North American
‘sky islands’ in many ways. Perhaps most importantly,
gradients in humidity, temperature, and irradiation
between tropical inselbergs and their surrounding rain-
forest will be much steeper for a herbaceous plant. Also,
changes in soil substrates will be more severe for tropical
inselbergs, with tremendous differences in nutrient avail-
ability between outcrops and their surrounding forest
(Porembski & Barthlott 2000). It is this clear ecological
differentiation between outcrops and their surrounding
forest — an inhospitable matrix that prevents gene flow —
that has encouraged comparisons with oceanic islands.
The inselbergs of the South American Atlantic Rainfor-
est offer a huge variety of abiotic environmental conditions
(Safford & Martinelli 2000), ranging from high-altitude
rock outcrops (studied here) to coastal lowland inselbergs
subject to completely different temperature regimes and
high salinity from ocean spray — the preferred habitats
of closely related species such as Alcantarea glaziouana
or Alcantarea regina (Martinelli 1994). Such island-like dis-
tributions of populations across different environments
are typically regarded as conducive to ecological speci-
ation and adaptive radiation (MacArthur & Wilson 1967;
Schluter 2000). We also note that low levels of interspecific
gene flow (Nem) appear to be possible on inselbergs on
which A. imperialis and A. geniculata co-occur (Fig. 3), in
agreement with the view that hybridization may form an
integral part of species radiations (Seehausen 2004).
Implications for conservation
The Brazilian Atlantic Rainforest is one of the world’s most
critically endangered biodiversity hotspots (Myers et al.
2000), the primary conservation problem from a genetic
POPULATION DIFFERENTIATION ON INSELBERGS 1991
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd
standpoint being rapid landscape and habitat fragmentation
due to logging and urban development. Although neither
A. imperialis nor A. geniculata occur in the rainforest matrix
in which their habitats are embedded, their pollinators
may nevertheless depend directly on the forest for shelter,
breeding, and alternative sources of food. Preservation of
surrounding forest may therefore be more crucial to the
survival of these inselberg species than their naturally
fragmented distribution patterns suggest.
High levels of population differentiation in A. imperialis
(43% of genetic variance residing between populations;
Table 3) indicate the need to consider a sufficient number
of inselbergs when devising in situ or ex situ conservation
plans in this ecologically and horticulturally important
species. For A. geniculata, the need to preserve many locations
is less clear from the partitioning of the genetic variance (11%
of variance between populations; Table 3), but its extremely
narrow range and lower diversity (Table 2) call for close
monitoring and preventive action. Calamities such as natural
or man-induced fires in some of its locations would easily
make this species vulnerable to stochastic factors typical
for small populations (Frankham et al. 2002). On a positive
note, one population of A. geniculata studied here, popu-
lation GRP, is already under protection. This population
is located in a Private Nature Reserve (RPPN ‘Pedra do
Amarylis’). Genetic diversity in this population is within
the normal range of this species (Table 2), and its effective
population size (Ne) is the largest observed for A. geniculata
to date (Fig. 3). Thus, community-based in situ conserva-
tion of this narrow endemic is already proving effective.
Acknowledgements
We are thankful to Pedro Cavalcanti (‘Jardims de Altitude’ eco-
tourism), Ricardo Alvarez, and Clarisse Palma-Silva for support
during field collections, the Brazilian IBAMA for processing of
collection/export permits, Jeffrey Joseph for help in the labora-
tory, Myriam Heuertz for helpful discussions on data analysis,
Carrie Morjan & Loren Rieseberg for sharing FST literature data,
and Alex Widmer and Clarisse Palma-Silva for helpful comments
on the manuscript. Fieldwork was supported by the Kew Over-
seas Fieldwork Committee and British Airways. Thelma Barbará’s
PhD thesis work was supported in part by a ‘Prance Fellowship in
Neotropical Botany’ Award.
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This work forms part of Thelma Barbará’s PhD thesis work on the
molecular population genetics of Alcantarea spp. adapted to
inselbergs in the Atlantic Rainforest of Brazil. Gustavo Martinelli
has long-standing interests in the ecology, systematics, and repro-
ductive biology of Bromeliaceae in southeastern Brazil. Simon
Mayo’s and Mike Fay’s work is focused primarily on the evolution
and systematics of tropical and temperate monocot families.
Christian Lexer’s main interest is on the genetics of speciation
in selected plant groups.
