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e470
American Journal of Botany: e470–e473, 2012; http://www.amjbot.org/ © 2012 Botanical Society of America
American Journal of Botany: e470–e473. 2012.
The genus Dyckia Schult. f. (Bromeliaceae) currently com-
prises 147 described species of xerophytic, terrestrial, or epi-
lithic rosette plants with showy yellow, red, or orange fl owers
( Smith and Downs, 1974 ). The genus is distributed across east-
ern South America, with a center of diversity in the cerrado
biome of Brazil and adjacent countries. Species of Dyckia and
of its closest relative Encholirium Mart. ex Schult. f. typically
inhabit azonal, arid, or rupicolous habitats that are character-
ized by poor soil, little water supply, high temperatures, and
strong sun exposure. Pollination is mainly by hummingbirds
and insects. Fruits are capsules that release winged, wind-dis-
persed seeds upon maturity ( Smith and Downs, 1974 ).
Little is known about infrageneric relationships within Dy-
ckia , the genetic structure and variation within its species, and
the mechanisms of speciation. This paucity of information is
in part due to the fact that many Dyckia species are rare and
narrow endemics, which are barely represented in herbaria
and living collections. Some species are even known from
their type locality only. Another problem is the high degree of
intraspecifi c morphological plasticity, which makes species
delimitation in Dyckia notoriously diffi cult. We have initiated
a genus-wide molecular phylogenetic study of Dyckia , based
on plastid and nuclear DNA sequences. Our preliminary results
indicate very low levels of plastid sequence divergence, sug-
gesting a young age of the genus (Krapp, unpublished data).
Whereas chloroplast haplotypes are often shared between species,
haplotype networks based on plastid DNA show a pronounced
geographical pattern across the distributional range of the genus.
Chloroplast simple sequence repeats (cpSSRs), also called
chloroplast microsatellites, are among the most sensitive tools
for evaluating plastid DNA variation ( Ebert and Peakall, 2009 ).
To achieve a better-resolved genus-wide plastid phylogeogra-
phy of Dyckia , we developed a set of 12 polymorphic cpSSR
markers based on de novo 454 sequencing.
METHODS AND RESULTS
Total genomic DNA was isolated from one individual plant of Dyckia
marnier-lapostollei L. B. Sm. var. estevesii Rauh from Goiania, central Brazil
(see Appendix 1), using a modifi ed cetyltrimethylammonium bromide
(CTAB) procedure ( Tel-Zur et al., 1999 ). This species was chosen because its
plastid haplotype takes a central position in a statistical parsimony network,
suggesting an ancestral state within the genus (Krapp, unpublished results).
Fragmentation of a 5- μ g DNA aliquot by nebulization, preparation of bar-
coded libraries, and shotgun sequencing on a Roche 454 GS-FLX with the
Titanium Sequencing Kit XLR70 and the Titanium PicoTiterPlate Kit (Roche
Diagnostics, Penzberg, Germany) were performed as described previously
( Wöhrmann et al., 2012a ). Altogether, 59 624 reads were obtained from three
independent runs. The proportion of a single 454 sequencing lane devoted to
D. marnier-lapostollei was 4.2%, 2.1%, and 4.1% in the fi rst, second, and
third run, respectively. Sequences of plastid origin were identifi ed using the
BLAST function of the software package Geneious ( Drummond et al., 2010 ).
The fully sequenced plastome of Typha latifolia L. ( Guisinger et al., 2010 )
1 Manuscript received 23 March 2012; revision accepted 19 June 2012.
The authors thank J. Peters, N. Schütz, and the Botanical Gardens of
Heidelberg, Bonn, and Vienna for providing plant material, as well as R. B.
Louzada, G. Cruz, and A. M. Wanderley for help during fi eld work. F.K. and
D.S.B.P. are supported by fellowship grants of the Otto-Braun-Fonds
(Melsungen) and the Fundação de Amparo à Ciência e Tecnologia do Estado
de Pernambuco (FACEPE), respectively. This work was supported by
PNADB/CAPES and DAAD/CAPES in the frame of a PROBRAL project.
5 Author for correspondence: weising@uni-kassel.de
doi:10.3732/ajb.1200153
AJB PRIMER NOTES & PROTOCOLS IN THE PLANT SCIENCES
A SET OF PLASTID MICROSATELLITE LOCI FOR THE
GENUS DYCKIA (BROMELIACEAE) DERIVED FROM 454
PYROSEQUENCING
1
F LORIAN K RAPP 2 , T INA W ÖHRMANN 2 , D IEGO SOTERO DE BARROS P INANGÉ 3 , A NA MARIA
B ENKO-ISEPPON 3 , B RUNO H UETTEL 4 , AND KURT W EISING 2,5
2 Plant Molecular Systematics, Department of Sciences, University of Kassel, D-34132 Kassel, Germany;
3 Genetics Department,
CCB, Universidade Federal de Pernambuco (UFPE), Av. Prof. Moraes Rego 1235, 50670-420, Recife, Pernambuco, Brazil; and
4 Max Planck Institute for Plant Breeding Research, Carl-von-Linné-Weg 10, 50829 Cologne, Germany
• Premise of the study: Phylogeographical analyses of Dyckia (Bromeliaceae) suffer from low levels of sequence variation.
Plastid microsatellite markers were developed to achieve a better-resolved genus-wide plastid genealogy of Dyckia .
• Methods and Results: Approximately 84% of the D. marnier-lapostollei plastome was sequenced using 454 technology. Flank-
ing primer pairs were designed for 34 out of 36 chloroplast simple sequence repeats (cpSSRs) detected, and 12 loci were further
characterized by genotyping Dyckia samples at the level of populations and species. Three, fi ve, and six cpSSRs were polymor-
phic among four individuals of D. limae , 12 individuals of D. dissitifl ora , and 12 of D. pernambucana , respectively, with two
to three alleles per locus and species. All loci were polymorphic among 19 different Dyckia species, with three to 10 alleles per
locus. Ten primer pairs cross-amplifi ed with bromeliad genera from fi ve subfamilies.
• Conclusions: The set of 12 cpSSR markers provides a toolbox to analyze phylogeographical patterns of Dyckia species.
Key words: Bromeliaceae; cpSSR; Dyckia ; plastome; population genetics.
e471
December 2012] AJB PRIMER NOTES & PROTOCOLS— DYCKIA CPSSRS
clamp of up to three nucleotides at the 3 ′ end. For three loci (DSSR-L01,
DSSR-L04, and DSSR-L06; Table 1 ) , consensus primers were derived from
alignments of the D. marnier-lapostollei sequence with sequence data previ-
ously generated by Sanger sequencing of the same loci in other Dyckia spe-
cies (Krapp, unpublished data).
Primer functionality was initially tested on a single accession each of
D. marnier-lapostollei , D. dissitifl ora Schult. f., and D. pernambucana
L. B. Sm. PCRs were carried out in 10- μ L volumes using a Biometra T1-cycler
or a Biometra T-Gradient cycler (Biometra GmbH, Göttingen, Germany), using
was taken as a reference. A total of 3826 plastid reads were assembled into 77
contigs and 12 singletons, which together represent 113 449 bases of the D.
marnier-lapostollei plastome (counting the two inverted repeats only once).
This corresponds to an overall coverage of ~84%, when compared to the
T. latifolia plastome. A total of 36 mononucleotide repeats with ≥ 10 bases were
detected (181 with ≥ 7 bases) using the FIND function of PhyDE ( Müller
et al., 2010 ). Besides two short dinucleotide repeats, each with an (AT)
5
motif, no other types of SSRs were observed. Flanking primer pairs were
designed by eye for 34 loci, with a default length of 20 nucleotides and a GC
T ABLE 1. Characteristics of 12 chloroplast microsatellite primer pairs developed in Dyckia marnier-lapostollei var. estevesii .
Locus Primer sequences (5 ′ –3 ′ ) Position Repeat motif Size (bp) GenBank accession no.
a
DSSR-L01 F: GTCAATTTTCAAGTTCAGCC atp B- rbc L (T)
13 C(A) 10 75 JQ743912
R: TCACGATTTCATCTACTTGC intergenic
DSSR-L04 F: AAAGGATGAGATCAATTCGG ndh A (T)
9 * 94 JQ743913
R: AAGATACATCGGAAAGTCCC intronic
DSSR-L06 F: ATTGATTGAATAAACCGGGG trn K-UUU- rps 16 (T)
13 77 JQ743914
R: TAAATAAGAAATTGGAATGG intergenic
DSSR-N01 F: GTTCCCAGTAAGAACCAACC rpo C1 (T)
14 102 JQ743915
R: CTCAATAATTTCACATTTCC intronic
DSSR-N04 F: GAAATCAATGTGTCGATTCC clp P (T)
11 87 JQ743916
R: TTTNAATAGAAAGAAGACCC intronic
DSSR-N05 F: TGAGATGAGTTTTGGCTCCC clp P (A)
12 85 JQ743917
R: AACAATACATCAATGATAGG intronic
DSSR-N07 F: ATTATATACATCTGAAACCC trn P-UGG- psa J (A)
13 74 JQ743918
R: CTTCCTCCTGAGCATTACGG intergenic
DSSR-N10 F: TNAATCAATATGGCGAAGGC clp P (T)
10 79 JQ743919
R: ATTCCCTCACGCTTGGCGCC intronic
DSSR-N11 F: ATAGATAAAATTATCGGGCC ndh G- ndh I (A)
18 100 JQ743920
R: AAATTTAAACTACATATTCC intergenic
DSSR-N15 F: CTTCCATTTATCCATATCCC rpl 16 (T)
11 64 JQ743921
R: AAAATAAATCTGATTATGGG intronic
DSSR-N16 F: TTATACCAAATGATCAATCG rpl 16- rps 3 (T)
13 90 JQ743922
R: ACTCTTTCATTCTTTTTCCG intergenic
DSSR-N18 F: AAATAGGTAATCTATTCCCC psb K- psb I (A)
15 63 JQ743923
R: GTAAGCATTACACAATCTCC intergenic
a GenBank accession numbers of the sequences on which the primers are based.
* The SSR motif at DSSR-L04 had only nine T residues in D. marnier-lapostollei , but had up to 14 T residues in other Dyckia species for which sequence
data were available for primer design.
T ABLE 2. Observed allele sizes at 12 chloroplast microsatellite loci in three populations of D. dissitifl ora and D. pernambucana and one population of
D. limae , allele numbers and size range in 19 different Dyckia species (one individual each), and cross-amplifi cation in eight additional genera of
Bromeliaceae (see Appendix 1 ).
Allele sizes
D. dissitifl ora D. pernambucana D. limae 19 Dyckia species
Cross-amplifi cation in other bromeliad
genera
†
Locus
Cachoeira*
( N = 4)
Lajes*
( N = 4)
Morrão*
( N = 4)
Aldeia*
( N = 4)
Brejo*
( N = 4)
Papagaio*
( N = 4)
Jerusalém*
( N = 4) No. of alleles
Size range
(bp) En De Fo Pi Pu An He Ti
DSSR-L01 75 75, 76 75, 76 77 75, 76 77 76, 77 9 72–80 + + + + + + + —
DSSR-L04 98 98 98 99 97 98 99 6 94–99 + + + + + + + +
DSSR-L06 79 79, 80 79, 80 78 78 78 78 8 73–82 + + + + + + + +
DSSR-N01 102 102 102 102 102 102 102 8 98–109 + + + + + + + +
DSSR-N04 91 91 91 91 92 91 91 8 87–98 + + — — — — — —
DSSR-N05 86 86 86 85, 86 85, 86 86 85, 86 4 84–87 + + + + + + + +
DSSR-N07 74 74 74 74 74 74 74 5 71–75 + + — + + + + +
DSSR-N10 81 79 79, 81 79 79 79 79 3 79–81 + + + + + + + +
DSSR-N11 99 96 100 99 97 98 98 98 10 94–104 + + + — — — + —
DSSR-N15 65 65 65 65 65 65 65 3 64–66 + + + + + + + +
DSSR-N16 90 90 90 90 90 90 90 5 87–91 + + + + + + + +
DSSR-N18 68 72, 73 68, 72 66 66 67 62, 66 9 62–73 + + + + + + + +
Note : + = amplifi cation; — = no amplifi cation; An = Ananas (Bromelioideae); De = Deuterocohnia ; En = Encholirium ; Fo = Fosterella ; Pi = Pitcairnia
(all Pitcairnioideae); He = Hechtia (Hechtioideae); Pu = Puya (Puyoideae); Ti = Tillandsia (Tillandsoideae).
* Locality information for the populations is provided in Appendix 1 .
† Single PCR product in the expected size range.
e472 AMERICAN JOURNAL OF BOTANY [Vol. 0
the indirect fl uorescence labeling procedure described by Schuelke (2000) .
Each assay contained approximately 1 ng of template DNA, 1 × Mango- Ta q
reaction buffer (Bioline, Taunton, Massachusetts, USA), 1.5 mM MgCl
2 , 0.2 mM
of each dNTP, 0.04 μ M forward primer carrying a 5 ′ -M13 tail, 0.16 μ M of M13
forward primer with fl uorescent 5 ′ -IRD700 modifi cation, 0.16 μ M unlabeled
reverse primer, 0.5 μ g/ μ L BSA, and 0.05 U Mango- Ta q DNA polymerase (Bioline).
All loci were amplifi ed using a standard PCR program with an initial denatur-
ation at 80 ° C for 5 min, followed by 30 cycles of denaturation at 94 ° C for 1
min, primer annealing at 52 ° C for 1 min, and elongation at 65 ° C for 2 min. Final
extension was performed at 65 ° C for 10 min. Samples were electrophoresed on
denaturing 6% polyacrylamide gels in 1 × TBE buffer, using an automated
sequencer (Li-Cor 4200 IR
2 ; Li-Cor Biosciences, Bad Homburg, Germany).
Fragment sizes were determined by visual examination with the help an external
size standard, as outlined by Wöhrmann et al. (2012a) . Allele sizes were validated
by repeated PCRs of subsets of samples using either Mango- Ta q polymerase or
a set of proofreading polymerases (Long High Fidelity Enzyme Mix; Rovalab,
Teltow, Germany), following the protocol supplied by the manufacturer.
The 12 most polymorphic cpSSR loci were used to genotype (1) population
samples from D. limae L. B. Sm., D. dissitifl ora , and D. pernambucana ; (2) single
accessions from 16 additional Dyckia species; and (3) one or two species each
of eight bromeliad genera belonging to fi ve subfamilies. Dyckia dissitifl ora was
chosen as an example of a Dyckia species with a relatively large distribution
range across Brazil, whereas D. limae and D. pernambucana were taken as a
typical example of two species that are not clearly distinguishable by morpho-
logical characters. Locus characteristics, primer sequences, and GenBank
accession numbers of these 12 markers are summarized in Table 1 , fragment
sizes for all samples and loci are compiled in Table 2 , and all plant materials
used in this study are listed in Appendix 1.
Three, fi ve, and six cpSSR loci were polymorphic among four individuals of
D. limae , 12 individuals from three populations of D. dissitifl ora , and 12 indi-
viduals from three populations of D. pernambucana , respectively ( Table 2 ).
Two to three alleles were observed per locus and species. All loci were highly
polymorphic at the species level, with three to 10 alleles per locus across 19
Dyckia species ( Table 2 ). Allele size distributions were generally compatible,
with a variable number of mononucleotide repeats being the molecular basis for
size variation. Overall, only six out of 540 individual PCRs performed with any
Dyckia species failed. All loci produced single PCR fragments within the ex-
pected size range in the closely related genera Encholirium and Deuterocohnia
Mez, and nine of the 12 primer pairs successfully cross-amplifi ed in six other
genera from fi ve subfamilies of Bromeliaceae ( Table 2 ).
CONCLUSIONS
The set of 12 novel cpSSR markers presented here provides
a promising toolbox for reconstructing plastid genealogies and
elucidating phylogeographical patterns within Dyckia . In con-
junction with nuclear SSR markers that are currently being devel-
oped in our group ( Wöhrmann et al., 2012b ), the cpSSRs are also
promising candidates for population genetic analyses in D. dis-
sitifl ora , D. limae , D. pernambucana , and probably many other
Dyckia species. Primer binding sites appear to be well-conserved
among Bromeliaceae, suggesting that the 12 cpSSR markers
may be applicable for genetic studies throughout the family.
LITERATURE CITED
D RUMMOND , A. J. , B. ASHTON , M . C HEUNG , J . HELED , M . K EARSE , R . M OIR ,
S . S TONES-HAVAS , ET AL . 2010 . Geneious v5.0. Website http://www.
geneious.com [accessed 3 December 2010].
E BERT , D. , AND R . P EAKALL . 2009 . Chloroplast simple sequence repeats
(cpSSRs): Technical resources and recommendations for expanding
cpSSR discovery and applications to a wide array of plant species.
Molecular Ecology 9 : 673 – 690 .
G UISINGER , M. M. , T. W. CHUMLEY , J. V. KUEHL , J. L. BOORE , AND R. K.
J ANSEN . 2010 . Implications of the plastid genome sequence of Typha
(Typhaceae, Poales) for understanding genome evolution in Poaceae.
Journal of Molecular Evolution 70 : 149 – 166 .
M ÜLLER , J . , K . M ÜLLER , AND D . QUANDT . 2010 . PhyDE: Phylogenetic data
editor. Version 0.9971. Program distributed by the author. Website
http://www.phyde.de/ [accessed 23 November 2010].
S CHUELKE , M. 2000 . An economic method for the fl uorescent labeling of
PCR fragments. Nature Biotechnology 18 : 233 – 234 .
S MITH , L. B. , AND R. J. DOWNS . 1974 . Pitcairnioideae (Bromeliaceae).
Flora Neotropica Monographs 14, part 1. New York Botanical Garden,
Bronx, New York, USA.
T EL-ZUR , N . , S . A BBO , D . M YSLABODSKI , AND Y . M IZRAHI . 1999 . Modifi ed
CTAB procedure for DNA isolation from epiphytic cacti of the genera
Hylocereus and Selenicereus (Cactaceae). Plant Molecular Biology
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W ÖHRMANN , T . , N . W AGNER , F . K RAPP , B . H UETTEL , AND K . W EISING .
2012a . Development of microsatellite markers in Fosterella rusbyi
(Bromeliaceae) using 454 pyrosequencing. American Journal of
Botany 99 : e160 – e163 .
W ÖHRMANN , T. , D. S. B. PINANGÉ , F . K RAPP , A. M. BENKO-ISEPPON ,
B . H UETTEL , AND K . W EISING . 2012b . Development of 15 nuclear micro-
satellite markers in the genus Dyckia (Pitcairnioideae; Bromeliaceae)
using 454 pyrosequencing. Conservation Genetics Resources DOI:
10.1007/s12686-012-9738-y.
e473
December 2012] AJB PRIMER NOTES & PROTOCOLS— DYCKIA CPSSRS
A PPENDIX 1 . Plant material used for this study.
Species Collector (Herbarium)
a Location
b GPS coordinates
Dyckia dissitifl ora Schult. f. A. M. Iseppon, Pinangé, D. & Cruz, G. 1605 (UFP) Cachoeira “Ferro Doido,” Bahia (BR) −11.6279; −41.0005
Pop. Cachoeira ( N = 4)
Dyckia dissitifl ora Schult. f. A. M. Iseppon, Pinangé, D. & Cruz, G. 1598 (UFP) Lajes, Bahia (BR) −11.6010; −41.1645
Pop. Lajes ( N = 4)
Dyckia dissitifl ora Schult. f. A. M. Iseppon, Pinangé, D. & Cruz, G. 1562 (UFP) Morrão, Bahia (BR) −11.5901; −41.2072
Pop. Morrão ( N = 4)
Dyckia limae L. B. Sm. A. M. Wanderley s.n. (UFP) Serra de Jerusalém, Pernambuco (BR) −8.5837; −37.2384
Pop. Jerusalém ( N = 4)
Dyckia pernambucana L. B. Sm. D. Pinangé et al. DCKB/09.2009 (UFP) Aldeia Couro d’Anta, Pernambuco (BR) −8.3254; −36.7562
Pop. Aldeia ( N = 4)
Dyckia pernambucana L. B. Sm. D. Pinangé et al. DKCA/09.2009 (UFP) Brejo da Madre de Deus, Pernambuco
(BR)
−8.1894; −36.3931
Pop. Brejo ( N = 4)
Dyckia pernambucana L. B. Sm. A. M. Wanderley s.n. (UFP) Pico do Papagaio, Pernambuco (BR) −7.8228; −38.0554
Pop. Papagaio ( N = 4)
Dyckia aff. brevifolia Baker P. Braun 840 (HD) Itacambira, Minas Gerais (BR) −17.0667; −43.3000
Dyckia estevesii Rauh P. Braun s.n. (HD) BR NA
Dyckia ferox Mez W. Rauh 64237 (HD) Cerro Colorado, Cordoba (RA) −30.1000; −63.9333
Dyckia goehringii E. Gross & Rauh W. Rauh 67622 (HD) Diamantina, Minas Gerais (BR) −18.2500; −43.6000
Dyckia granmogulensis Rauh W. Rauh 56484 (HD) Grão Mogol, Minas Gerais (BR) −16.5667; −42.9000
Dyckia aff.
ibiramensis Reitz L. Horst 1287 (HD) Diamantina, Minas Gerais (BR) −18.2500; −43.6000
Dyckia leptostachya Baker H. Amerhauser s.n. (WU) Caacupé, Cordillera (PY) −25.3833; −57.1500
Dyckia lindevaldae Rauh P. Braun BR 691 (HD) Alto Paraiso, Goiás (BR) −14.1167; −47.5167
Dyckia macedoi L. B. Sm. R. B. Louzada, Pinangé, D. & Medeiros, M. 151 (SP) Santana do Riacho, Minas Gerais (BR) −19.3539; −43.6237
Dyckia marnier-lapostollei var.
estevesii Rauh
L. Horst 5 (HD) Goiania, Goiás (BR) −16.6667; −49.2667
Dyckia marnier-lapostollei L. B. Sm. L. Horst 4 (HD) Cristalina, Goiás (BR) −16.7500; −47.6000
Dyckia microcalyx Baker W. Till 6020 (WU) Cerros Acahay, Paraguari (PY) −25.9167; −57.1500
Dyckia aff. pumila L. B. Sm. P. Braun BR 696 (HD) Ponte Branca, Mato Grosso (BR) −16.4500; −52.6667
Dyckia remotifl ora var. indet.
Otto & A. Dietr.
L. Horst s.n. (HD) BR NA
Dyckia tobatiensis Hassl. W. & S. Till 6050 (WU) Tobati, Cordillera (PY) −25.2500; −57.0667
Dyckia velascana Mez W. & S. Till 5012 (WU) Ascochinga, Cordoba (RA) −30.9500; −64.2667
Dyckia vestita Hassl. W. & S. Till 6018 (WU) Paraguari, Paraguari (PY) −25.6333; −57.1500
Encholirium horridum L. B. Sm. W. Schindhelm s.n. (HD) Pedra Azul, Minas Gerais (BR) −15.9867; −41.4069
Encholirium magalhaesii L. B. Sm. s.n. (BONN) BR NA
Deuterocohnia brevispicata
Rauh & L. Hrom.
N. Schütz 06/028 (FR) Florida, Santa Cruz (BOL) −18.0154; −64.1001
Deuterocohnia glandulosa E. Gross N. Schütz 06/019 (FR) Ipati, Santa Cruz (BOL) −19.7063; −63.6521
Fosterella villosula (Harms) L. B. Sm. J. Peters 06.0105 (HD) Cochabamba, Cochabamba (BOL) −17.0611; −65.6444
Fosterella weddelliana (Brongn. ex Baker)
L. B. Sm.
M. Miyagawa s.n. (HD) Solacana (BOL) NA
Pitcairnia feliciana (A. Chev.) Harms &
Mildbr.
I. Ebert & D. Bangoura s.n. ex coll. P. Bak (WU) RG NA
Pitcairnia heterophylla (Lindl.) Beer K. Senghas O-11230 (HD) Cruz de Ocotte, Guerrero (MEX) 17.5500; 99.8833
Puya ferruginea (Ruiz & Pav.) L. B. Sm. W. Rauh s.n. (HD) Rio Marañon (PE) NA
Puya herzogii Wittm. T. Krömer 6581 (HD) Carrasco, Cochabamba (BOL) −17.1933; −64.9731
Ananas ananassoides (Baker) L. B. Sm. P. Maas s.n. (HD) Est. Amazonas (BR) NA
Hechtia caerulea (Matuda) L. B. Sm. W. Rauh s.n. (HD) Est. Mexico (MEX) NA
Tillandsia usneoides (L.) L
Q6 . W. Rauh s.n. (HD) Yungas Cachi (RA) NA
Note : N = population size Q7 ; NA = not available Q8 .
a Herbaria: BONN = University of Bonn; FR = Senckenberg Research Institute, Frankfurt; HD = Botanical Garden of Heidelberg; SP = Instituto de
Botânica; São Paulo; UFP = Universidade Federal de Pernambuco; WU = University of Vienna.
b BOL = Bolivia; BR = Brazil; MEX = Mexico; PE = Peru; PY = Paraguay; RA = Argentina; RG = Guinea.
