Download pdf - 2014 - Population genetics of the endemic and endangered Vriesea minarum (Bromeliaceae) in the Iron Quadrangle, Espinhaço Range, Brazil

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American Journal of Botany 101 ( 7 ): 1167 – 1175 , 2014 .

American Journal of Botany 101 ( 7 ): 1167 – 1175 , 2014 ; http://www.amjbot.org/ © 2014 Botanical Society of America

The Iron Quadrangle (IQ) region in southeastern Brazil is one of the most relevant centers of Bromeliaceae species richness, with a high number of endemic and endangered taxa ( Versieux and Wendt, 2007 ; Versieux et al., 2008 , 2010b ). The region is renowned worldwide for its mineral deposits and lies between two global biodiversity hotspots: the Atlantic Forest and the Brazilian Cerrado ( Mittermeier et al., 2004 ). The peaks of the IQ mountains, which may reach up to 1850 m a.s.l., are topped with quartzite and, predominantly, ironstone outcrops ( Jacobi et al., 2007 ; Jacobi and Carmo, 2012 ). Ironstone out-crops are constituted basically of hematite (Fe 2 O 3 ) and goethite (FeOOH) ( Spier et al., 2007 ; Shuster et al., 2012 ) and are rap-idly losing area to iron ore opencast mining. Their removal is threatening the survival of several plant species adapted to this peculiar environment ( Jacobi et al., 2007 ; Versieux and Wendt, 2007 ; Jacobi and Carmo, 2008 , 2012 ).

Bromeliaceae plants have an ecological importance in rocky outcrops, where many other plant species have diffi culty grow-ing, and several species have been threatened by habitat loss from human activities ( Porembski et al., 1998 ; Benzing, 2000 ; Versieux et al., 2010a ; Marques et al., 2012 ). Vriesea minarum L.B.Sm. is a rupicolous bromeliad endemic to the IQ that is threatened and currently listed as endangered in the Brazilian plant species red list ( CNCFlora, 2013 ). It is offi cially protected only within the Rola-Moça State Park (PESRM), and a few re-cords place it in small private reserves ( Versieux, 2011 ).

Understanding the historical-evolutionary processes respon-sible for the distribution patterns of genetic variation in plant populations, such as those occurring in the IQ, is still a largely unexplored research fi eld. Although new research has been conducted to understand microevolutionary patterns of brome-liads from Atlantic Forest inselbergs (e.g., Barbará et al., 2007a , 2009 ; Palma-Silva et al., 2011 ), bromeliads from the Espinhaço Range remain less studied (e.g., Cavallari et al., 2006 ). The Espinhaço Range has one of the richest fl oras in the world with hundreds of endemic species ( Giulietti et al., 1997 ). Unveiling the distribution patterns of genetic diversity for endemic plants may help conserve the biodiversity in this area, as shown for similar environments in other parts of the world ( Butcher et al., 2009 ). Accurate identifi cation of populations or portions of a species range that contain the greatest allelic variation, and hence evolutionary potential, can assist in the designation of priority protected areas and help capture genetic variation to be

1 Manuscript received 2 November 2013; revision accepted 27 May 2014. The authors thank CAPES for a scholarship to P.L., FAPESB for fi nancial

support (PNX0014/2009), CNPq for a fellowship to C.V.D.B. (PQ1D), C.M.J. (PQ2), and L.M.V. (PQ2). The authors thank two anonymous reviewers and Editor-in-Chief Judy Jernstedt for much appreciated comments, Maria Cristina López-Roberts for training in molecular laboratory techniques, and Dra. Vânia C. Azevedo, Dr. Sérgio Maia Queiroz Lima, and Dr. Fábio Vieira for comments on an earlier version of this manuscript.

5 Author for correspondence (e-mail: [email protected])

doi:10.3732/ajb.1300388

POPULATION GENETICS OF THE ENDEMIC AND ENDANGERED VRIESEA MINARUM (BROMELIACEAE) IN THE IRON

QUADRANGLE, ESPINHAÇO RANGE, BRAZIL 1

PÂMELA LAVOR 2,5 , CÁSSIO VAN DEN BERG 3 , CLAUDIA M. JACOBI 4 , FLÁVIO F. CARMO 4 , AND LEONARDO M. VERSIEUX 2

2 Programa de Pós-Graduação em Sistemática e Evolução, Laboratório de Botânica Sistemática, Departamento de Botânica e Zoologia, Universidade Federal do Rio Grande do Norte, Lagoa Nova, Natal, RN 59072-970, Brazil; 3 Departamento de Ciências

Biológicas, Universidade Estadual de Feira de Santana, Av. Transnordestina. s.n., Feira de Santana, Bahia, BA 44036-900, Brazil; and 4 Departamento de Biologia Geral, Universidade Federal de Minas Gerais, Belo Horizonte, MG 31270-901, Brazil

• Premise of the study: Knowledge about genetic variability in plant populations is one of the main branches of conservation genetics, linking genetic data to conservation strategies. Vriesea minarum is a bromeliad endemic to the Iron Quadrangle region (southeastern Brazil), occurring on mountaintop rock outcrops. It is listed as endangered due to habitat loss, particularly from iron ore mining. Thus, determining the structure and genetic diversity of V. minarum populations could help develop strategies to conserve the species.

• Methods: We studied the genetic structure of 12 populations of V. minarum using 10 microsatellite loci transferred from other species of Bromeliaceae. Statistical analyses to compare and describe the genetic diversity of each population were performed, and genetic structure within and among populations, isolation by distance, and Bayesian structure were also analyzed.

• Key results: Our results show high inbreeding ( G IS = 0.376) and low population structure ( F ST = 0.088), possibly related to high gene fl ow due to great pollinator effi ciency and/or effi cient seed dispersal, thus leading to high connectivity among populations of these fragmented rock outcrops. Two clusters were observed, corresponding to the basins of rivers São Francisco and Doce.

• Conclusions: Gene fl ow among populations is high but, given the rate of habitat loss to mining, most populations are vulnerable and will become increasingly isolated if no action is taken to preserve them. Thus, conservation of this species depends on in situ and ex situ actions, such as controlling overexploitation and creating a germoplasm bank.

Key words: Brazil; Bromeliaceae; bromeliad; conservation; genetic variability; Iron Quadrangle; microsatellite; Minas Gerais; Tillandsioideae; Vriesea minarum .

1168 AMERICAN JOURNAL OF BOTANY [Vol. 101

Range in Minas Gerais state), we hypothesized that the coloni-zation of the IQ by V. minarum ancestors occurred from the south or southeastern border of the distribution. This fi rst hypothesis south/southeast to west and north, assumes that migration oc-curred from Rio de Janeiro toward the west and north, via a step-ping-stone model (see map in Versieux, 2011 ). If this is correct, we would expect south/southeastern populations to be older and with higher genetic variability than populations from the north-ern border, which are less diverse and possibly younger. A second hypothesis is that V. minarum had an older and broader distribu-tion within the IQ in the past, and due to recent effects (habitat loss mainly concentrated during the two last centuries), its popu-lations are now more fragmented and isolated on mountaintops.

Taxonomically, some populations found along the core distri-bution area of V. minarum were once considered as part of a distinct taxon: V. ouroensis W. Weber. The latter species was placed as a synonym of V. minarum based on a morphological review of the yellow-fl owered Vriesea species of the Espinhaço Range ( Versieux, 2011 ). Molecular data may confi rm whether the populations from the Ouro Preto/Ouro Branco area (treated earlier as V. ouroensis ) form a distinct cluster. Additionally, in-vestigating the genetic structure of V. minarum may shed light on the genetic patterns of similarly isolated species, also nar-rowly endemic to a few mountaintops.

used in reintroduction and ex situ preservation programs ( Moritz, 1994 ; Holtsford and Hancock, 1998 ; Frankham et al., 2002 ; Moreira et al., 2010 ).

Besides the potential conservation implications of this work, our study is also motivated by taxonomic and biogeographic questions, which may help us understand the origin of the many endemic species of Vriesea in the Espinhaço Range, using V. minarum as an example. Phylogenetic knowledge of the large genus Vriesea is still fragmentary and far from satisfactory. Re-cent phylogenetic hypotheses suggest that the species of Vriesea in the Espinhaço Range are derived from Atlantic Forest ances-tors ( Versieux et al., 2012 ). Most Vriesea species are restricted to the Atlantic Forest domain. The genus Vriesea prefers meso-phytic habitats and, along the Espinhaço Range, species grow as epiphytes preferably on mountaintops where mist and fog con-stantly form ( Versieux and Wendt, 2006 ). According to Versieux and Wendt (2007) , the southern Espinhaço Range shares brome-liad taxa with the forested areas of the core Atlantic Forest, near the coast of southeastern of Brazil. This similarity would be a consequence of their proximity, allowing dispersal between these two ecoregions ( Versieux and Wendt, 2007 ).

As V. minarum belongs to a phenetically delimited group of rupicolous yellow-fl owered species that occurs in southeastern Brazil (from Rio de Janeiro state to central and north Espinhaço

Fig. 1. Map of iron formations in the Iron Quadrangle in southeastern Brazil, highlighting location of the Vriesea minarum populations sampled (modifi ed from Garcia et al., 2009 ).

LAVOR ET AL .— POPULATION GENETICS OF VRIESEA MINARUM 1169July 2014]

individuals were sampled ( Table 2 ). Only in Itatiaiuçu, the 5 m distance could not be strictly followed due to the low number of individuals remaining in this sampling site (six individuals). Leaf samples measuring approximately 3 × 3 cm 2 were cut from the middle portion of healthy leaves using sterilized scissors and immediately stored in plastic bags containing silica gel for dehydration. The genomic DNA was extracted from a total of 206 individuals following the protocol of Doyle and Doyle (1990) .

Microsatellite analysis — Ten microsatellite loci previously described and tested in V. minarum during a preliminary screening ( Lavor et al., 2013 ) were used: from Vriesea gigantea Gaud. ( Palma-Silva et al., 2007 ) loci VgB10, VgC01, VgF02, VgG03, and VgG5; from Tillandsia fasciculata Sw. and Guz-mania monostachya Rusby ( Boneh et al., 2003 ) loci e6, p2p19, and e19; and from Fosterella rusbyi (Mez) L.B.Sm. ( Wöhrmann et al., 2012 ) loci ngFos_6 and ngFos_22. The amplifi cations were done using GeneAmp PCR system 9700 (Applied Biosystems) and Swift Maxpro (Esco) thermocyclers following the protocols described by Lavor et al. (2013) . The microsatellite alleles were resolved on an ABI 3130XL Genetic Analyzer (Applied Biosystems), and they were precisely sized using the GeneMapper 4.0 (Applied Biosystems) software. The microsatellite data were tested for genotyping errors resulting from stutter-ing, short allele dominance, and null alleles using software Microchecker 2.2.3 (Brookfi eld 1 estimator; van Oosterhout et al., 2004 ).

Statistical analysis — Genetic diversity— Levels of genetic diversity within populations and loci were described by calculating the following parameters: A, number of alleles; Rs, allelic richness; Gd, gene diversity; Apr, number of private alleles; H O , observed heterozygosity; and H E , expected heterozygosity. All genetic diversity parameters were estimated with the software GenAlEx 6.5 ( Peakall and Smouse, 2012 ), PopGene 1.31 software ( Yeh et al., 1999 ) and Fstat version 2.9.3 ( Goudet, 1995 ).

To test for statistical deviations from the Hardy–Weinberg equilibrium, the software Genepop, online version 4.3 ( Raymond and Rosset, 1995 ) was used. The software Fstat version 2.9.3 ( Goudet, 1995 ) was used to estimate the in-breeding coeffi cient in populations ( F IS ) ( Weir and Cockerham, 1984 ), and the software GenAlEx 6.5 ( Peakall and Smouse, 2012 ) was used to estimate the inbreeding coeffi cient ( G IS ) ( Nei and Chesser, 1983 ).

Genetic structure within and among populations — Measures of genetic differentiation F ST ( Wright, 1965 ), G ST , G ″ ST and Jost’s D ( Meirmans and Hedrick, 2011 ) were performed with GenAlEx 6.5 ( Peakall and Smouse, 2012 ).

The aims of the present work were to (1) assess the intra- and interpopulation genetic diversity and structure of V. minarum populations using microsatellite markers; (2) quantify the de-gree of genetic differentiation among populations (including those from Ouro Preto/Ouro Branco regions, once treated as a distinct taxon); (3) provide molecular data to discuss biogeo-graphic patterns within the IQ and (4) propose conservation ac-tions for the species.

MATERIALS AND METHODS

Study species — Vriesea minarum is a rupicolous bromeliad endemic to the Iron Quadrangle region, a mountain area of about 7200 km 2 located at the southern portion of the Espinhaço Range, in southeastern of Brazil ( Versieux, 2011 ) ( Fig. 1 ). Vriesea minarum reproduces by clonal growth through the production of shoots at the base of the mother plant or on the axil of old leaves, as well as by sexual reproduction by seeds ( Fig. 2 ) ( Versieux, 2011 ).

Population sampling and DNA extraction — Leaf samples were collected between January and February 2012 in 10 locations, totaling 12 populations across the IQ, Minas Gerais state ( Fig. 1 ). The regional climate is temperate highland tropical with dry winters—Cwb according to Köppen classifi cation—and most of the rain falls from November to January ( Nimer and Brandão, 1989 ). Populations were previously known from herbarium records ( Versieux, 2011 ) or were searched for in feasible habitats, particularly on mountaintops (1300–1600 m a.s.l.) where iron ore deposits form outcrops or on quartzite outcrops, where it can also occasionally grow. The minimum distance between populations was 1.7 km (Rola-Moça 1 to Rola-Moça 2), and the maximum was 92.5 km (Itatiaiuçu to Lavras Novas; Fig. 1 ).

All individuals were georeferenced with a global positioning system (GPS), but because the species is offi cially threatened we do not provide the exact location for each population sampled. Vouchers for all populations surveyed are deposited in the Herbarium of the Federal University of Rio Grande do Norte (UFRN) and Federal University of Rio de Janeiro (RFA) (Appendix 1).

Leaves were collected at a minimum distance of 5 m between individuals to avoid sampling clones or close relatives. In most of the populations at least 16

Fig. 2. Specimens of Vriesea minarum in their habitat. (A) Blooming individual from Lavras Novas, municipality of Ouro Preto, MG in Brazil. (B) Fruiting individual with Tamanduá mine in the background, municipality of Nova Lima, MG (Images: P. Lavor).


TAB

LE 1

. M

easu

res

of g

enet

ic d

iver

sity

for

10

mic

rosa

telli

te lo

ci in

12

popu

latio

ns o

f V

ries

ea m

inar

um .

Loc

us A

R

sG

d H

O H

E F

ST

G ST

G

″ ST

Des

t G

IS

e66

1.63

90.

295

0.27

00.

325

0.10

7*0.

074*

0.11

2*0.

035*

0.13

1 ns

e19

111.

482

0.23

20.

146

0.23

10.

065

0.02

10.

029

0.00

70.

307

ngFo

s_6

31.

499

0.23

20.

231

0.25

70.

102

0.06

80.

095

0.02

40.

059 n

s ng

Fos_

2213

2.85

70.

768

0.54

00.

762

0.07

20.

014

0.05

80.

044

0.37

6p2

p19

122.

838

0.75

00.

550

0.76

40.

096*

*0.

049*

*0.

196*

*0.

151*

*0.

308

VgB

1022

3.60

20.

904

0.66

30.

930

0.07

7**

0.03

4**

0.36

8**

0.34

3**

0.31

4V

gC01

223.

554

0.88

50.

602

0.92

00.

093*

*0.

046*

*0.

405*

*0.

373*

*0.

383

VgF

0217

3.59

00.

945

0.39

70.

928

0.08

60.

004

0.06

10.

057

0.69

6V

gG03

81.

883

0.44

10.

294

0.42

10.

081

0.03

60.

068

0.03

00.

337

VgG

0517

2.80

00.

737

0.55

00.

742

0.10

2**

0.05

0**

0.18

5**

0.13

9**

0.34

5M

ean

132.

678

0.61

80.

424

0.62

80.

088*

*0.

036*

*0.

098*

*0.

062*

*0.

376*

*

Not

es: A

= n

umbe

r of

alle

les;

Rs

= a

llelic

ric

hnes

s; G

d =

gen

e di

vers

ity; H

O =

obs

erve

d he

tero

zygo

sity

; H E

= e

xpec

ted

hete

rozy

gosi

ty; F

ST =

gen

etic

dif

fere

ntia

tion

amon

g po

pula

tions

; G ST

=

anal

og o

f F

ST , a

djus

ted

for

bias

; G ″ S

T =

Hed

rick

’s s

tand

ardi

zed

G ST

; Des

t = J

ost’s

est

imat

e of

dif

fere

ntia

tion;

G IS

= in

bree

ding

coe

ffi c

ient

; ns

= n

ot s

igni

fi can

t ( P

> 0

.05)

dep

artu

re f

rom

Har

dy–

Wei

nber

g eq

uilib

rium

. * P

< 0

.05,

** P

< 0

.01,

***

P <

0.0

01.

Signifi cance was tested by resampling with 1000 permutations. F ST was chosen because R ST requires a stepwise mutation model, which is rarely observed for microsatellite loci (revised in Meirmans and Hedrick, 2011 ).

Partitioning of genetic diversity among and within populations was exam-ined by analysis of molecular variance (AMOVA) performed with the software Arlequin ver 3.5.1.3 ( Excoffi er and Lischer, 2010 ), and the signifi cance was estimated using R statistics and tested using 10 000 permutations.

Bayesian structure analysis — Population structure was searched for with the Bayesian clustering method implemented in the program STRUCTURE 2.3.4 ( Pritchard et al., 2000 ). To determine the most likely number of clusters ( K ), we conditioned our data to various values of K ranging from 1 to 12. The analyses were carried out under the admixture model (Q), assuming dependent allele frequencies and using a burn-in period of 50 000 replicates and 500 000 Markov chain Monte Carlo replicates, with 10 iterations per K to confi rm stabilization of the summary statistics ( Pritchard et al., 2000 ). To determine the most likely number of clusters ( K ), the method of Evanno et al. (2005) was employed, based upon the ad hoc measure Δ K , from the online software STRUCTURE HAR-VESTER v0.6.93 ( Earl and von Holdt, 2012 ).

Isolation by distance — A Mantel test ( Mantel, 1967 ) was conducted in the GenAlEx 6.5 software ( Peakall and Smouse, 2012 ) using the straight line dis-tance among populations measured in GoogleEarth based on our GPS collec-tion points associated to the Nei’s unbiased genetic distances ( Nei, 1978 ). The statistical signifi cance was tested using 10 000 permutations.

RESULTS

Variation across loci — The 10 polymorphic loci had an aver-age number of alleles ( A ) of 13, ranging from 3 (ngFos_6) to 22 (VgB10 and VgC01) alleles per locus ( Table 1 ). The mean al-lelic richness (Rs) was 2.678 and mean gene diversity (Gd) per locus was 0.618. The observed ( H O ) and expected ( H E ) hetero-zygosities for all loci ranged from 0.146 to 0.663 and 0.231 to 0.930, respectively ( Table 1 ). Only two loci (e6 and ngFos_6) were in Hardy–Weinberg equilibrium. The mean inbreeding coeffi cient on average, was high ( G IS = 0.376; P < 0.01). Ge-netic differentiation among the 12 populations was low ( F ST = 0.088, G ST = 0.036, G ″ ST = 0.098 and Jost’s D = 0.062; Table 1 ), although all were signifi cant ( P < 0.01).

Patterns of genetic variability across populations — Private alleles were distributed in all populations, ranging from 1 (MSer) to 4 (MTam). Observed and expected heterozygosities per population varied from 0.297 to 0.475 and 0.529 to 0.620, respectively ( Table 2 ). All populations departed signifi cantly from Hardy–Weinberg equilibrium ( P < 0.01). The MTam pop-ulation had the greatest allelic richness (2.685), while SCal and SGan2 showed the most gene diversity (0.677). The highest in-breeding coeffi cient index ( F IS ) was 0.572 in the SIta popula-tion ( Table 2 ).

Patterns of population divergence and gene fl ow — The Mantel test indicated low correlation between genetic (Nei’s unbiased distance) and geographical distance ( r 2 = 0.170; P < 0.05), suggesting poor, or absent, isolation by distance among populations ( Fig. 3 ). The partition of variances in the AMOVA is 8.85% between populations and 81.15 within, a value very similar to the F ST ( Table 3 ). Genomic admixture analysis with the program STRUCTURE identifi ed K = 2 as the most likely num-ber of genetic clusters. One cluster grouped populations from Serra da Piedade, Pedra Rachada, Serra do Rola-Moça, Serra do Itatiaiuçu, Morro do Tamanduá, and Serra da Calçada. The second grouped populations from Serra do Gandarela (1 and 2),


was formed by the populations from Ouro Preto (Lavras Novas) and Ouro Branco, supporting the view that V. ouroensis is in fact a synonym of V. minarum ( Versieux, 2011 ).

The two observed clusters refl ect mountain range positioning within distinct river basins, suggesting that the forested valleys of the Espinhaço range or also the mountain ridges that separate basins are barriers for migration and pollinators. While the pop-ulations Serra do Gandarela, Lavras Novas, and Serra de Ouro Branco belong to the Doce river basin, the populations of Serra da Piedade, Pedra Rachada, Serra do Rola-Moça, Serra do Itatiaiuçu, Morro do Tamanduá, Serra da Calçada, and Marinho da Serra are nested within the São Francisco river basin (see Geopark Quadrilátero Ferrífero, 2014 ). The only population that did not refl ect river basin positioning in the cluster analysis was Marinho da Serra, which is located in the São Francisco river basin, but appeared as part of the Doce river cluster. The proximity of Marinho da Serra population to the watershed of the São Francisco and Doce rivers may account for this re-sult. Factors responsible for such division need to be further

Lavras Novas, Serra do Ouro Branco, and Marinho da Serra ( Fig. 4 ).

DISCUSSION

Patterns of genetic diversity in Vriesea minarum — The values of observed and expected heterozygosity in loci are sim-ilar to other species of Brazilian Bromeliaceae, such as in the inselberg species of Alcantarea imperialis (Carrière) J.R. Grant and A. geniculata (Wawra) J.R. Grant ( Barbará et al., 2007a ); in V. gigantea ( Palma-Silva et al., 2009 ), and in Bromelia antia-cantha Bertoloni ( Zanella et al., 2011 ), all of them from the Atlantic forest. In all these studies, the observed heterozygosity was lower than expected, due to a defi cit of heterozygotes. The same pattern was observed in V. minarum ( Table 4 ). The ex-cess of homozygotes could be attributed to selfi ng or biparental inbreeding since V. minarum exhibits a mixed reproductive strategy (outcrossing + self-compatibility, P. Lavor, unpub-lished manuscript) or might just be a general pattern in the fam-ily, considering that all species studied so far showed similar results.

Some of the genetic diversity parameters of V. minarum were lower than those found in other Bromeliaceae studies, such as the mean allelic richness, number of alleles, and private alleles (see Barbará et al., 2007a , 2009 ; Palma-Silva et al., 2009 , 2011 ). These values may have resulted from the transferability of all microsatellite loci from other species in our study ( Barbará et al., 2007b ) or due to the narrow distributional range of V. minarum , compared with some other species (see Table 4 ).

Patterns of population genetic structure in Vriesea mina-rum — The low population genetic structure (mean F ST = 0.088) of V. minarum compared with other studies of Bromeliaceae population genetics using microsatellites ( Table 4 ) and AMOVA results showing that 8.85% of genetic variance resided “among populations” ( Table 3 ) indicates high gene fl ow among popula-tions, which agrees with the close distribution of the popula-tions of V. minarum , separated by a mean distance of 39 km ( Table 4 ). These values could be explained by either seed or pollen dispersal effi ciency, or both. No well-supported cluster

TABLE 2. Sampling locality in the state of Minas Gerais, approximate elevation, number of samples in each population, and measures of genetic diversity of the 12 populations studied in all loci of Vriesea minarum .

Locality, Municipality PopulationElevation (m a.s.l.) N A Apr Rs Gd H O H E F IS

Pedra-Rachada, Mun. Sabará PRac 1300 20 6 4 2.520 0.600 0.469 0.578*** 0.225Santuário Estadual Nossa Senhora de

Piedade, Mun. CaetéSPie 1600 20 6 3 2.532 0.615 0.448 0.560*** 0.231

Serra do Ouro Branco, Mun. Ouro Branco SOBr 1400 20 6 3 2.578 0.633 0.423 0.588*** 0.332Parque Estadual da Serra do Rola-Moça

1, Mun. Nova LimaSRm1 1500 19 6 2 2.518 0.603 0.354 0.565*** 0.415

Serra da Calçada, Mun. Brumadinho SCal 1500 20 7 3 2.618 0.677 0.444 0.608*** 0.299Marinho da Serra, Mun. Moeda MSer 1500 16 5 1 2.444 0.598 0.397 0.529** 0.288Pedra Grande, Serra do Itatiaiuçu, Mun. Igarapé SIta 1200 6 3 1 2.573 0.693 0.297 0.533** 0.572Morro do Tamanduá, Mun. Nova Lima MTam 1500 20 6 4 2.685 0.639 0.386 0.599*** 0.396Lavras Novas, Mun. Ouro Preto LNov 1400 16 5 2 2.586 0.613 0.336 0.562*** 0.452Serra do Gandarela 1, Mun. Rio Acima SGan1 1600 19 5 3 2.518 0.612 0.400 0.567*** 0.346Serra do Gandarela 2, Mun. Rio Acima SGan2 1600 20 6 4 2.666 0.677 0.475 0.620*** 0.267Parque Estadual da Serra do Rola-Moça 2, Mun. Nova Lima SRm2 1500 10 5 1 2.548 0.671 0.444 0.565*** 0.270

Notes : N = sample size; A = number of alleles; Apr = number of private alleles; Rs = allelic richness; Gd = gene diversity; H O = observed heterozygosity; H E = expected heterozygosity; F IS = inbreeding coeffi cient in populations. Departures from Hardy–Weinberg equilibrium are indicated by asterisks: **P < 0.01 , ***P < 0.001.

Fig. 3. Relationship between genetic divergence, based on Nei’s (1978) unbiased distance for nuclear microsatellites, and geographical dis-tances (km) among 12 populations of Vriesea minarum (Mantel test cor-relation: r 2 = 0.170; P < 0.05).


the night (P. Lavor, unpublished manuscript). Also, this taxon has been traditionally placed in section Xiphion , which is char-acterized by dull-colored fl owers and bracts and bat pollination ( Smith and Downs, 1977 ). Bats are considered good pollen car-riers, enabling pollen dispersal among separate patches ( Martins and Gribel, 2007 ). They are deemed important in maintaining population connectivity in V. gigantea ( Palma-Silva et al., 2009 ) and several other tropical plant species populations with low F ST values (see review by Fleming et al., 2009 ).

The lack of structure could also be explained from a histori-cal biogeographical perspective, considering a possible sce-nario of a recent fragmentation among populations of V. minarum , concomitant with the rapid loss of many ironstone outcrops in the past decades throughout the IQ ( Jacobi and Carmo, 2012 ). In this case, some currently disconnected popu-lations may have been connected by extinct outcrops, support-ing our second biogeographical hypothesis. Similar results have been reported for other threatened plant species that have undergone recent habitat loss and fragmentation ( Ding et al., 2008 ).

Conservation — The fl oristic diversity of the IQ outcrops, in particular ironstones, was overlooked until recently, and their protection is challenging because of the escalating habitat loss by iron ore mining ( Jacobi et al., 2007 , 2008 , 2011 ). As noted by Versieux (2011) , V. minarum has been recorded in only one state reserve: the Rola-Moça State Park. Even within this protected area, poaching and other threats are problems for the conservation of the species ( Biodiversitas, 2007 ). The type locality (Serra da Piedade, Caeté) is a private protected area (Catholic Sanctuary), but the protection is inadequate ( Versieux, 2011 ). The other populations of V. minarum (e.g., Serra do Itatiauçu, Serra do Gandarela, Serra da Pedra Rachada, and Serra da Moeda), are not under legal protection and are therefore exposed to many risks. In 2011, a large area of Marinho da Serra was burned, decimating the population that we sam-pled, and in the same year, another fi re also affected the popula-tions of Serra do Rola-Moça State Park, leaving only a single population intact.

The most devastating impact is that of iron ore mining, ac-cording to Jacobi and Carmo (2012) , which has consumed about 40% of the total area of the ironstone outcrops in the last four decades. The habitat loss is happening so fast that one pop-ulation (SIta) sampled in this study was totally lost within 1 yr.

investigated, focusing on seed and pollinator mobility within each river basin.

Lack of population structuring using inter-simple sequence repeat (ISSR) markers was reported by Ribeiro et al. (2013) in Vriesea cacuminis L.B. Sm., another rare rupicolous species from Minas Gerais. This outcrossing species is morphologi-cally related to V. minarum ( Versieux, 2011 ), and its low popu-lation genetic structuring was related to clone longevity and connection among populations by efficient pollinators. The authors of this study interpreted this last factor as the main cause of the low structure and high diversity indices observed ( Ribeiro et al., 2013 ).

A recent study with V. gigantea , an epiphyte from the Atlan-tic Forest understory, showed that seed dispersal occurs only within a short distance ( Paggi et al., 2010 ), although seeds are plumose and dispersed by wind like all Tillandsioideae subfam-ily members ( Madison, 1977 ; Benzing, 1990 ). On the basis of our results, we believe that seed dispersal in V. minarum is more effective and not directly comparable to the results of Paggi et al. (2010) . Vriesea minarum is restricted to rocky sys-tems of open environments, generally above 1000 m a.s.l, where the high incidence of winds can enhance their dispersal. Besides, the usually low stature of the rupicolous vegetation offers fewer obstacles to dispersal than a forested area. Addi-tionally, fruit ripening occurs from May to October ( Versieux, 2011 ), corresponding to the dry season, thus avoiding seed drenching and clustering within the capsule or close to the dis-persing plant.

In addition, the lack of genetic structure can also be explained by pollinator behavior and by historical colonization processes. Although data on the reproductive biology of V. minarum are still preliminary, ongoing fi eld observations suggest humming-bird pollination due to the early morning anthesis and tubular, odorless yellow corolla. Bat pollination is not excluded, since fl owers last 2 days, do not close at night, have stigmatic recep-tivity only in the late afternoon, and nectar is produced during

Fig. 4. Results of Bayesian analysis of 206 individuals from 12 populations of Vriesea minarum , occurring in the Iron Quadrangle, southeastern Bra-zil, based on 10 microsatellite loci, using STRUCTURE software. Graphic representation of the different genetic pools for K = 2. Populations are separated by vertical bars (for population abbreviations, see Table 2 ).

TABLE 3. AMOVA of 12 populations of Vriesea minarum .

Source of variation df Sum of squares Variation %

Among populations 11 46323.388 94.79375 8.85*Within populations 400 390472.643 976.18161 91.15*

Note: P values: * P < 0.05.


BENZING , D. H. 1990 . Vascular epiphytes: General biology and related biota, Cambridge University Press, Cambridge, UK.

BENZING , D. H. 2000 . Introduction . In D. H. Benzing [ed.], Bromeliaceae: Profi le of an adaptive radiation. Cambridge University Press, Cambridge, UK.

BIODIVERSITAS . 2007 . Plano de manejo do Parque Estadual da Serra do Rola-Moça. Website http://www.biodiversitas.org.br/planosdemanejo/pesrm/uc34e1.htm [accessed 20 November 2012].

BOISSELIER-DUBAYLE , M. C. , R. LEBLOIS , S. SAMADI , J. LAMBOURDIERE , AND C. SARTHOU . 2010 . Genetic structure of the xerophilous bromeliad Pitcairnia geyskesii on inselbergs in French Guiana—A test of the forest refuge hypothesis. Ecography 33 : 175 – 184 .

BONEH , L. , P. KUPERUS , AND P. H. VAN TIENDEREN . 2003 . Microsatellites in the bromeliads Tillandsia fasciculata and Guzmania monostachya. Molecular Ecology 3 : 302 – 303 .

BUTCHER , P. , S. MCNEE , AND S. KRAUSS . 2009 . Genetic impacts of habitat loss on the rare ironstone endemic Tetratheca paynterae subsp. payn-terae. Conservation Genetics 10 : 1735 – 1746 .

CAVALLARI , M. M. , R. C. FORZZA , E. A. VEASEY , M. I. ZUCCHI , AND G. C. X. OLIVEIRA . 2006 . Genetic variation in three endangered species of Encholirium (Bromeliaceae) from Cadeia do Espinhaço, Brazil, detected using RAPD markers. Biodiversity and Conservation 15 : 4357 – 4373 .

CNCFLORA . 2013 . Lista Vermelha de Espécies. Website http://cncfl ora.jbrj.gov.br/portal/pt-br/redlisting [acessed 1 March 2013].

DING , G. , D. Z. ZHANG , X. Y. DING , Q. ZHOU , AND W. C. ZHANG . 2008 . Genetic variation and conservation of the endangered Chinese en-demic herb Dendrobium offi cinale based on SRAP analysis. Plant Systematics and Evolution 276 : 149 – 156 .

DOYLE , J. J. , AND J. L. DOYLE . 1990 . Isolation of plant DNA from fresh tissue. Focus (San Francisco, Calif.) 12 : 13 – 15 .

EARL , D. A. , AND B. M. VON HOLDT . 2012 . STRUCTURE HARVESTER : A website and program for visualizing STRUCTURE output and imple-menting the Evanno method. Conservation Genetics Resources 4 : 359 – 361 .

EVANNO , G. , S. REGNAUT , AND J. GOUDET . 2005 . Detecting the number of clusters of individuals using the software STRUCTURE : A simulation study. Molecular Ecology 14 : 2611 – 2620 .

EXCOFFIER , L. , AND H. E. L. LISCHER . 2010 . Arlequin suite ver 3.5: A new series of programs to perform population genetics analyses under Linux and Windows. Molecular Ecology Resources 10 : 564 – 567 .

FLEMING , T. H. , C. GEISELMAN , AND W. J. KRESS . 2009 . The evolution of bat pollination: A phylogenetic perspective. Annals of Botany 104 : 1017 – 1043 .

FRANKHAM , R. , J. D. BALLOU , AND D. A. BRISCOE . 2002 . Introduction to conservation genetics. Cambridge University Press, Cambridge, UK.

GARCIA , L. C. , F. V. BARROS , AND J. P. LEMOS-FILHO . 2009 . Fructifi cation phenology as an important tool in the recovery of iron mining areas in Minas Gerais, Brazil. Brazilian Journal of Biology 69 : 887 – 893 .

Other populations at imminent risk of disappearing due to mining are Pedra Rachada, Morro do Tamanduá, and Serra do Gandarela 1 and 2. The populations of Lavras Novas, Serra do Ouro Branco, and Serra do Gandarela 1 had the highest F IS values, suggesting that endogamy is occurring.

As conservation approaches, fi rst, laws addressing mineral extraction should be created and enforced, to prevent loss of the entire habitat ( Ding et al., 2008 ) since, independently of population genetic diversity, other organisms (such as polli-nators and dispersers) depend on the fl ora of ironstone out-crops ( Porembski et al., 1998 ; Benzing, 2000 ; Versieux et al., 2010a ; Marques et al., 2012 ). Second, a germplasm bank should be created with seeds and individuals from different popula-tions, especially those of Pedra Rachada, Morro do Tamanduá, and Serra do Gandarela 2, which had the largest numbers of private alleles and are next to areas where mining is currently expanding.

Given the little structure observed, we cannot point to any population that should be prioritized for conservation, but our suggestion is to have large, well-established, conserved populations along each of the IQ borders, ideally preserving the maximum number of populations from each of the two clusters found in our analysis. The presence of private al-leles may be a consequence of the large total number of al-leles found in each locus, the large expected heterozygosities, and the small sample sizes at each of the sampling sites. In situ and ex situ conservation efforts must be pursued to pre-vent bottleneck effects, mainly due to the loss of whole pop-ulations, leaving few individuals, that can lead to species extinction.

LITERATURE CITED

BARBARÁ , T. , G. MARTINELLI , M. F. FAY , S. J. MAYO , AND C. LEXER . 2007a . Population differentiation and species cohesion in two closely related plants adapted to neotropical high-altitude ‘inselbergs’, Alcantarea imperialis and Alcantarea geniculata (Bromeliaceae). Molecular Ecology 16 : 1981 – 1992 .

BARBARÁ , T. , G. MARTINELLI , C. PALMA-SILVA , M. F. FAY , S. J. MAYO , AND C. LEXER . 2009 . Genetic relationships and variation in reproductive strategies in four closely related bromeliads adapted to neotropical ‘inselbergs’: Alcantarea glaziouana, A. regina, A. geniculata and A. imperialis (Bromeliaceae). Annals of Botany 103 : 65 – 77 .

BARBARÁ , T. , C. PALMA-SILVA , G. M. PAGGI , F. BERED , M. F. FAY , AND C. LEXER . 2007b . Cross-species transfer of nuclear microsatellite mark-ers: Potential and limitations. Molecular Ecology 16 : 3759 – 3767 .

TABLE 4. Mating system, population structure, geographical distribution, and mean distance among populations of some Bromeliaceae species studied with microsatellite loci.

SpeciesMating system Mean F ST Mean F IS Geographical distribution

Mean distance (min–max) among populations (km)

No. of populations Reference

Alcantarea geniculata Out 0.111 0.094 Rio de Janeiro, Brazil 1.04 (0.4–2.4) 4 Barbará et al., 2007a Alcantarea imperialis Out 0.434 0.099 Rio de Janeiro, Brazil 68 (7–100) 4 Barbará et al., 2007a Alcantarea glaziouana Out 0.217 0.156 Rio de Janeiro, Brazil 25.2 (3.5–47) 5 Barbará et al., 2009 Alcantarea regina Out 0.195 −0.051 Rio de Janeiro, Brazil 1.5 2 Barbará et al., 2009 Vriesea gigantea Mix 0.211 0.273 South and Southeast Brazil 817 (34–1600) 13 Palma-Silva et al., 2009 Pitcairnia geyskesii ND 0.156 0.125 French Guyana and Suriname 146.7 (1.2–292.2) 14 Boisselier-Dubayle et al., 2010 Bromelia antiacantha Out 0.224 0.431 Southeastern Brazil 340.5 (2–679) 8 Zanella et al., 2011 Pitcairnia albifl os Out 0.336 0.109 Rio de Janeiro, Brazil 16 (2–30) 5 Palma-Silva et al., 2011 Pitcairnia staminea Aut 0.260 0.240 Rio de Janeiro, Brazil 16 (2–30) 5 Palma-Silva et al., 2011 Vriesea minarum Mix 0.088 0.341 Minas Gerais, Brazil 47.1 (1.70–92.5) 12 P. Lavor, this manuscript

Notes: Aut = autogamous; Mix = mixed; ND = not determined; Out = outcrossing (adapted from Zanella et al., 2012 ).


PALMA-SILVA , C. , M. M. CAVALLARI , T. BARBARÁ , C. LEXER , M. A. GIMENES , F. BERED , AND M. H. BODANESE-ZANETTINI . 2007 . A set of polymor-phic microsatellite loci for Vriesea gigantea and Alcantarea imperia-lis (Bromeliaceae) and cross-amplifi cation in other bromeliad species. Molecular Ecology Notes 7 : 654 – 657 .

PALMA-SILVA , C. , C. LEXER , G. M. PAGGI , T. BARBARÁ , F. BERED , AND M. H. BODANESE-ZANETTINI . 2009 . Range-wide patterns of nuclear and chloroplast DNA diversity in Vriesea gigantea (Bromeliaceae), a neo-tropical forest species. Heredity 103 : 503 – 512 .

PALMA-SILVA , C. , T. WENDT , F. PINHEIRO , T. BARBARÁ , M. F. FAY , S. COZZOLINO , AND C. LEXER . 2011 . Sympatric bromeliad species ( Pit-cairnia spp.) facilitate tests of mechanisms involved in species cohe-sion and reproductive isolation in Neotropical inselbergs. Molecular Ecology 20 : 3185 – 3201 .

PEAKALL , R. , AND P. F. SMOUSE . 2012 . GenAlEx 6.5: Genetic analysis in Excel. Population genetic software for teaching and research—An up-date. Bioinformatics 28 : 2537 – 2539 .

POREMBSKI , S. , G. MARTINELLI , R. OHLEMULLER , AND W. BARTHLOTT . 1998 . Diversity and ecology of saxicolous vegetation mats on inselbergs in the Brazilian Atlantic rainforest. Diversity & Distributions 4 : 107 – 119 .

PRITCHARD , J. K. , M. STEPHENS , AND P. DONNELLY . 2000 . Inference of population structure using multilocus genotype data. Genetics 155 : 945 – 959 .

RAYMOND , M. , AND F. ROSSET . 1995 . GENEPOP: Population genetics soft-ware for exact test and ecumenism. Journal of Heredity 86 : 248 – 249 .

RIBEIRO , P. C. C. , L. C. PINHEIRO , R. DOMINGUES , R. C. FORZZA , M. A. MACHADO , AND L. F. VICCINI . 2013 . Genetic diversity of Vriesea ca-cuminis (Bromeliaceae): An endangered and endemic Brazilian spe-cies. Genetics and Molecular Research 12 : 1934 – 1943 .

SHUSTER , D. L. , K. A. FARLEY , P. M. VASCONCELOS , G. BALCO , H. S. MONTEIRO , K. WALTENBERG , AND J. O. STONE . 2012 . Cosmogenic He 3 in hematite and goethite from Brazilian “canga” duricrust demon-strates the extreme stability of these surfaces. Earth and Planetary Science Letters 329-–330 : 41 – 50 .

SMITH , L. B. , AND R. J. DOWNS . 1977 . Tillandsioideae (Bromeliaceae). Flora neotropica monograph 14, vol. 2. Hafner Press, New York, New York, USA.

SPIER , C. A. , S. M. B. OLIVEIRA , A. N. SIAL , AND F. J. RIOS . 2007 . Geochemistry and genesis of the banded iron formations of the Cauê Formation, Quadrilátero Ferrífero, Minas Gerais, Brazil. Precambrian Research 152 : 170 – 206 .

VAN OOSTERHOUT , C. , W. F. HUTCHINSON , D. P. M. WILLS , AND P. SHIPLEY . 2004 . MICRO-CHECKER: Software for identifying and correcting genotyping errors in microsatellite data. Molecular Ecology Notes 4 : 535 – 538 .

VERSIEUX , L. M. 2011 . Brazilian plants urgently needing conservation: The case of Vriesea minarum (Bromeliaceae). Phytotaxa 28 : 35 – 49 .

VERSIEUX , L. M. , T. BARBARÁ , M. G. L. WANDERLEY , A. CALVENTE , M. F. FAY , AND C. LEXER . 2012 . Molecular phylogenetics of the Brazilian giant bromeliads ( Alcantarea , Bromeliaceae): implications for mor-phological evolution and biogeography. Molecular Phylogenetics and Evolution 64 : 177 – 189 .

VERSIEUX , L. M. , P. M. ELBL , M. G. L. WANDERLEY , AND N. L. D. MENEZES . 2010a . Alcantarea (Bromeliaceae) leaf anatomical characteriza-tion and its systematic implications. Nordic Journal of Botany 28 : 385 – 397 .

VERSIEUX , L. M. , R. B. LOUZADA , P. L. VIANA , N. MOTA , AND M. G. L. WANDERLEY . 2010b . An illustrated checklist of Bromeliaceae from Parque Estadual do Rio Preto, Minas Gerais, Brazil, with notes on phytogeography and one new species of Cryptanthus. Phytotaxa 10 : 1 – 6 .

VERSIEUX , L. M. , AND T. WENDT . 2006 . Checklist of Bromeliaceae of Minas Gerais, Brazil, with notes on taxonomy and endemism. Selbyana 27 : 107 – 146 .

VERSIEUX , L. M. , AND T. WENDT . 2007 . Bromeliaceae diversity and con-servation in Minas Gerais state. Brazil. Biodiversity and Conservation 16 : 2989 – 3009 .

GEOPARK QUADRILÁTERO FERRÍFERO . 2014 . Mapas: Bacias hidrográfi cas no quadrilátero ferrífero. Website http://www.geoparkquadrilatero.org/?pg=conteudo&id=186&L=PTBR&_Mapas [accessed 04/04/2014].

GIULIETTI , A. M. , J. H. PIRANI , AND R. M. HARLEY . 1997 . Espinhaço Range region, eastern Brazil. In S. D. Davis, V. H. Heywood, O. Herrera-MacBryde, J. Villa-Lobos, and A. C. Hamilton [eds.], Centres of plant diversity: A guide and strategy for their conservation, vol. 3, 397–404. Information Press, Oxford, UK.

GOUDET , J. 1995 . FSTAT (vers. 1.2): A computer program to calculate F -statistics. Journal of Heredity 86 : 485 – 486 .

HOLTSFORD , T. P. , AND J. F. HANCOCK . 1998 . Evolution, population genet-ics and germplasm preservation. HortScience 33 : 3 – 5 .

JACOBI , C. M. , AND F. F. CARMO . 2008 . The contribution of ironstone out-crops to plant diversity in the Iron Quadrangle, a threatened Brazilian landscape. Ambio 37 : 324 – 326 .

JACOBI , C. M. , AND F. F. CARMO [organizers]. 2012 . Diversidade fl orística nas Cangas do Quadrilátero Ferrífero. IDM Ltda, Belo Horizonte, Minas Gerais, Brazil.

JACOBI , C. M. , F. F. CARMO , AND I. C. CAMPOS . 2011 . Soaring extinction threats to endemic plants in Brazilian metal-rich regions. Ambio 40 : 540 – 543 .

JACOBI , C. M. , F. F. CARMO , AND R. C. VINCENT . 2008 . Vegetação sobre canga e seu potencial para reabilitação ambiental no Quadrilátero Ferrífero, MG. Revista Árvore 32 : 345 – 353 .

JACOBI , C. M. , F. F. CARMO , R. C. VINCENT , AND J. R. STEHMANN . 2007 . Plant communities on ironstone outcrops—A diverse and endangered Brazilian ecosystem. Biodiversity and Conservation 16 : 2185 – 2200 .

LAVOR , P. , C. VAN DEN BERG , AND L. M. VERSIEUX . 2013 . Transferability of 10 nuclear microsatellite primers to Vriesea minarum (Bromeliaceae), a narrowly endemic and threatened species from Brazil. Brazilian Journal of Botany 36 : 165 – 168 .

MADISON , M. 1977 . Vascular epiphytes: Their systematic occurrence and salient features. Selbyana 2 : 1 – 13 .

MANTEL , N. 1967 . The detection of disease clustering and a generalized regression approach. Cancer Research 27 : 209 – 220 .

MARQUES , A. R. , J. P. LEMOS FILHO , AND R. C. MOTA . 2012 . Diversity and conservation status of bromeliads from Serra da Piedade, Minas Gerais, Brazil. Rodriguésia 63 : 243 – 255 .

MARTINS , R. L. , AND R. GRIBEL . 2007 . Polinização de Caryocar villosum (Aubl.) Pers. (Caryocaraceae) uma árvore emergente da Amazônia Central. Revista Brasileira de Botânica 30 : 37 – 45 .

MEIRMANS , P. G. , AND P. W. HEDRICK . 2011 . Assessing population struc-ture: F ST and related measures. Molecular Ecology Resources 11 : 5 – 18 .

MITTERMEIER , R. A. , P. R. GIL , M. HOFFMAN , J. PILGRIM , T. BROOKS , C. G. MITTERMEIER , J. LAMOREUX , AND G. A. B. FONSECA . 2004 . Hotspots revisited: Earth’s biologically richest and most endangered terres-trial ecoregions. CEMEX & Agrupación Sierra Madre, Mexico City, Mexico.

MOREIRA , R. G. , R. A. MCCAULEY , A. C. CORTÉS-PALOMEC , G. W. FERNANDES , AND K. OYAMA . 2010 . Spatial genetic structure of Coccoloba cereif-era (Polygonaceae), a critically endangered microendemic species of Brazilian rupestrian fi elds. Conservation Genetics 11 : 1247 – 1255 .

MORITZ , C. 1994 . Defi ning “evolutionarily signifi cant units” for conserva-tion. Trends in Ecology & Evolution 9 : 373 – 375 .

NEI , M. 1978 . Estimation of average heterozygosity and genetic distance from a small number of individuals. Genetics 89 : 583 – 590 .

NEI , M. , AND R. K. CHESSER . 1983 . Estimation of fi xation indexes and gene diversities. Annals of Human Genetics 47 : 253 – 259 .

NIMER , E. , AND A. M. P. M. BRANDÃO . 1989 . Balanço hídrico e clima da região dos Cerrados. Instituto Brasileiro de Geografi a e Estatística (IBGE), Rio de Janeiro, Brazil.

PAGGI , G. M. , J. A. T. SAMPAIO , M. BRUXEL , C. M. ZANELLA , M. GOETZE , M. V. BÜTTOW , C. PALMA-SILVA , AND F. BERED . 2010 . Seed dispersal and population structure in Vriesea gigantea , a bromeliad from the Brazilian Atlantic rainforest. Botanical Journal of the Linnean Society 164 : 317 – 325 .


VERSIEUX , L. M. , T. WENDT , R. B. LOUZADA , AND M. G. L. WANDERLEY . 2008 . Bromeliaceae da Cadeia do Espinhaço. Megadiversidade 4 : 98 – 110 .

WEIR , B. , AND C. COCKERHAM . 1984 . Estimating F statistics for the analy-sis of population structure. Evolution 38 : 1358 – 1370 .

WÖHRMANN , T. , N. WAGNER , F. KRAPP , B. HUETTEL , AND K. WEISING . 2012 . Development of microsatellite markers in Fosterella rusbyi (Bro-meliaceae) using 454 pyrosequencing. American Journal of Botany 99 : e160 – e163 .

WRIGHT , S. 1965 . The interpretation of population structure by F -statistics with special regard to systems of mating. Evolution 19 : 395 – 420 .

YEH , F. C. , R. YANG , AND T. BOYLE . 1999 . POPGENE 1.32: Population ge-netic analysis [online]. Website http://www.ualberta.ca/~fyeh/popgene_download.html [accessed 05 September 2012].

ZANELLA , C. M. , M. BRUXEL , G. M. PAGGI , M. GOETZE , M. V. BUTTOW , F. W. CIDADE , AND F. BERED . 2011 . Genetic structure and phenotypic varia-tion in wild populations of the medicinal tetraploid species Bromelia an-tiacantha (Bromeliaceae). American Journal of Botany 98 : 1511 – 1519 .

ZANELLA , C. M. , A. JANKE , C. PALMA-SILVA , E. KALTCHUK-SANTOS , F. G. PINHEIRO , G. M. PAGGI , L. E. S. SOARES , ET AL . 2012 . Genetics, evo-lution and conservation of Bromeliaceae. Genetics and Molecular Biology 35 : 1020 – 1026 .

APPENDIX 1. Voucher information for populations of Vriesea minarum sampled in the present work, deposited in herbaria collections (Federal University of Rio Grande do Norte—UFRN, and Federal University of Rio de Janeiro—RFA).

Brazil, Minas Gerais, Mun. Sabará, Pedra Rachada, 09 May 2004, L. M. Versieux et al., 179 (RFA). Mun. Caeté, Serra da Piedade, 04 Jan 2012, P. Lavor & L. M. Versieux 1 (UFRN). Mun. Ouro Branco, Serra do Ouro Branco, 05 Jan 2012, L. M. Versieux et al. 517 (UFRN). Mun. Nova Lima, Morro do Tamanduá, 08 Feb 2012, P. Lavor & F.

F. Carmo 2 (UFRN). Mun. Ouro Preto, Lavras Novas, 12 Feb 2012, P. Lavor & F. F. Carmo 3, 4 (UFRN). Mun. Santa Bárbara, Serra do Gandarela, 28 Feb 2012, P. Lavor & F. F. Carmo 5, 6 (UFRN). Mun. Nova Lima, Parque Estadual do Rola-Moça, 15 Mar 2012, P. Lavor 7 (UFRN).