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Is Drosera meristocaulis a pygmy sundew? Evidence of a long-distance dispersal
between Western Australia and northern South America
F. Rivadavia1, V. F. O. de Miranda2, G. Hoogenstrijd3, F. Pinheiro4, G. Heubl 5and A. Fleischmann5,*
1
Daniel Burnham Ct., San Francisco, CA 94109, USA,
2
University of Sa
˜o Paulo State, Department of Applied Biology,
FCAV-DBAA, Via de Acesso Prof. Paulo Donato Castellane s/n, 14884-900, Jaboticabal, SP, Brazil,
3
H. Ronnerstraat 50,
1073 KR Amsterdam, The Netherlands,
4
Instituto de Bota
ˆnica, 04301-012, Sa
˜o Paulo, SP, Brazil and
5
Institute of
Systematic Botany, University of Munich, Menzinger Strasse 67, D-80638 Munich, Germany
* For correspondence. E-mail fleischmann@lrz.uni-muenchen.de
Received: 3 January 2012 Returned for revision: 13 February 2012 Accepted: 7 March 2012 Published electronically: 28 May 2012
†Background and aims South America and Oceania possess numerous floristic similarities, often confirmed by
morphological and molecular data. The carnivorous Drosera meristocaulis (Droseraceae), endemic to the Neblina
highlands of northern South America, was known to share morphological characters with the pygmy sundews of
Drosera sect. Bryastrum, which are endemic to Australia and New Zealand. The inclusion of D. meristocaulis in
a molecular phylogenetic analysis may clarify its systematic position and offer an opportunity to investigate char-
acter evolution in Droseraceae and phylogeographic patterns between South America and Oceania.
†Methods Drosera meristocaulis was included in a molecular phylogenetic analysis of Droseraceae, using
nuclear internal transcribed spacer (ITS) and plastid rbcL and rps16 sequence data. Pollen of D. meristocaulis
was studied using light microscopy and scanning electron microscopy techniques, and the karyotype was inferred
from root tip meristem.
†Key Results The phylogenetic inferences (maximum parsimony, maximum likelihood and Bayesian
approaches) substantiate with high statistical support the inclusion of sect. Meristocaulis and its single
species, D. meristocaulis, within the Australian Drosera clade, sister to a group comprising species of sect.
Bryastrum. A chromosome number of 2n¼approx. 32–36 supports the phylogenetic position within the
Australian clade. The undivided styles, conspicuous large setuous stipules, a cryptocotylar (hypogaeous) germin-
ation pattern and pollen tetrads with aperture of intermediate type 7– 8 are key morphological traits shared
between D. meristocaulis and pygmy sundews of sect. Bryastrum from Australia and New Zealand.
†Conclusions The multidisciplinary approach adopted in this study (using morphological, palynological, cyto-
taxonomic and molecular phylogenetic data) enabled us to elucidate the relationships of the thus far unplaced
taxon D. meristocaulis. Long-distance dispersal between southwestern Oceania and northern South America is
the most likely scenario to explain the phylogeographic pattern revealed.
Key words: Droseraceae, Drosera sect. Bryastrum, America– Oceania disjunction, carnivorous plants, ITS, rbcL,
rps16, phylogeny, pollen morphology, germination pattern, chromosome numbers.
INTRODUCTION
The carnivorous plants known as sundews of the genus
Drosera (Droseraceae) comprise nearly 200 species spread
worldwide, mostly in the Southern Hemisphere and especially
in southwestern Australia (Diels, 1906;Schlauer, 2007;
McPherson, 2010). Species of the most distinctive groups of
Drosera, known as the pygmy sundews – because of their
usually diminutive size – are all endemic to the southwestern
tip of Western Australia, except for D. pygmaea which is
also found in southeastern Australia and New Zealand
(Lowrie, 1989).
The pygmy sundews make up sect. Bryastrum (following
the sectional classification of Seine and Barthlott, 1994), con-
sisting of approx. 50 species (Lowrie, 1989, 1998; Lowrie and
Carlquist, 1992;Lowrie and Conran, 2007;Mann, 2007), and
are characterized not only by their relatively diminutive size,
but also by large translucent papery stipules which are
arranged as a dense stipule bud in the centre of the rosette,
three to five undivided styles, long fibrous roots and their
unique capability to reproduce vegetatively by small leaf-
derived propagules known as gemmae. The gemmae are modi-
fied leaves, which are chlorophyllous and rich in starch
(Goebel, 1908;Karlsson and Pate, 1992). Recent molecular
phylogenetic data (Rivadavia et al., 2003) showed the
pygmy sundews to be a well supported monophyletic group,
which is part of a large clade containing mostly Australian
species, and sister to a clade including mostly taxa native to
the New World and southern Africa.
Botanical expeditions in the 1950s to the isolated highlands
known as the Neblina massif on the Brazilian– Venezuelan
border in the Amazonas lowlands of northern South America
resulted in the description of numerous endemic species, in-
cluding Drosera meristocaulis (Maguire and Wurdack, 1957)
(Fig. 1). Because this species has only three undivided
styles, a unique character among New World Drosera taxa, a
monotypic sect. Meristocaulis was created for this taxon
(Maguire and Wurdack, 1957;Seine and Barthlott, 1994),
#The Author 2012. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved.
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Annals of Botany 110: 11–21, 2012
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which was raised to subgeneric level by Schlauer (1996).
Other conspicuous characters of this taxon include long
stems up to 40 cm in length and nearly sessile flowers
nested among the leaves and stipules (Fig. 1). Nonetheless,
D. meristocaulis also presents characteristics reminiscent of
pygmy sundews, such as diminutive leaves, large translucent
papery stipules and long fibrous roots. The extreme isolation
of the remote Neblina massif kept D. meristocaulis from
being studied in more depth, thus heightening scientists’ curi-
osity about the relationship of this species to other members of
Drosera.Maguire and Wurdack (1957) were well aware of the
similarities of D. meristocaulis to the pygmy Drosera of sect.
Bryastrum from Australia (Fig. 1). Due to the undivided styles,
however, they supposed a close relationship to the single
South American member of sect. Thelocalyx,D. sessilifolia.
Duno de Stefano (1995) studied the pollen morphology
of D. meristocaulis for the first time and proposed a close
relationship of sect. Meristocaulis with sect. Drosera.
The Neblina massif is a huge sandstone formation reaching
nearly 3000 m above sea level and is covered in part by low
vegetation (‘Neblinaria scrub’; Brewer-Carı
´as, 1988;Huber,
1995) composed of species not found in the hot surrounding
lowlands. Several expeditions to Neblina and other mountains
of the Guayana Highlands (known as tepuis) documented an
AB
CD
FIG.1. Drosera meristocaulis (A, C) from the Neblina massif in the Amazon and the Western Australian pygmy sundew Drosera gibsonii (B, D) show a re-
markable similarity in overall habit and in flower morphology.
Rivadavia et al. — Is Drosera meristocaulis of Australian origin?12
at Instituto de Botânica on July 18, 2012http://aob.oxfordjournals.org/Downloaded from
impressive number of endemic taxa and contributed to the idea
of a diverse and unique flora with a high degree of endemism
(Steyermark, 1979). In an attempt to explain this unique flora,
the idea of ‘Lost Worlds’ was created, postulating that the
origin of local biota was relictual as a result of a long
history of evolution in isolation on the mountain summits
(Rull, 2004). On the other hand, the ‘Vertical Displacement’
hypothesis assumes the lack of total geographical isolation
among tepui summits, with extensive valleys and gentle
slopes possibly being important paths connecting highlands
with lowlands, thus providing hypothetical migrational path-
ways (Huber, 1988;Rull, 2004).
Long-distance dispersal (LDD) was accepted and rejected
many times as a good theory to explain floristic similarities
among continents since Darwin’s experiments (1859).
Besides the fact that LDD was accepted as a natural process
that occurred on recent volcanic islands (Carlquist, 1966,
2010), the plate tectonics theory provided vicariance explana-
tions for many cases of disjunctions (de Queiroz, 2005).
Molecular clock techniques have revealed that many plant
lineages have a recent origin, with radiation events occurring
after continental splits (Givnish and Renner, 2004;Mun
˜oz
et al., 2004;Sytsma et al., 2004;Dick et al., 2007). Now
many dispersion routes are corroborated by multiple taxa in
the Southern Hemisphere (de Queiroz, 2005), and LDD can
explain the disjunction patterns of many groups.
In the present study, a multidisciplinary investigation was
carried out in order to clarify the phylogenetic position of
D. meristocaulis in Droseraceae and to test the hypothesis of
a putative common ancestry with species from sect.
Bryastrum. The pattern of seed germination, pollen morph-
ology, chromosome counts and a molecular approach based
on nuclear and plastid DNA sequences were investigated.
MATERIALS AND METHODS
Seed germination
Seeds of Drosera meristocaulis and D. capillaris were
obtained from a commercial carnivorous plant seed source
(A. Lowrie, Duncraig, Australia) and were sown on pure
peat and on milled long fibre sphagnum in a greenhouse,
and kept moist at 20– 25 8C.
Chromosome counts
Root tips of greenhouse-grown seedlings were used for
karyotype analysis. In addition, in vitro raised plants of
D. meristocaulis were obtained from a commercial nursery
(bestcarnivorousplants.com). For mitotic chromosome counts,
root tips of in vitro and ex vitro plants were collected and pre-
treated with 0.002 M8-hydroxyquinoline for 3 h to achieve
mitotic arrest, and then fixed in ethanol:acetic acid (3:1) and
stored at 4 8C. Fixed root tips were hydrolysed in 2 Mhydro-
chloric acid at 60 8C for 10 min, and then enzymatically
macerated with 5 % cellulase (Roth, Germany) at 37 8C for
20 min. Root tips were rinsed with distilled water, squashed
on glass slides and the prepared root tip meristems were
orcein stained (Orcein: Roth, Germany). Chromosome counts
were made using a light microscope (Leitz, Germany), and
slides were documented photographically using a digital
camera (Nikon D5000, Germany).
Pollen analysis
Dried anthers were taken from herbarium specimens of
D. meristocaulis deposited in SPF (voucher F. Rivadavia
et al. 1881). The anthers were soaked in 10 % KOH overnight
and then prepared by acetolysis following Erdtman (1960).
After a final washing step, the acetolysed pollen grains were
stored in acetone for light microscopy (LM) and scanning elec-
tron microscopy (SEM) analysis. Photomicrographs of pollen
grains in LM were obtained with a video camera (Olympus)
connected to a PC. SEM analyses were made using acetolysed
pollen grains, which were washed in pure water at several steps
to remove residual acetone, and then put on lightstub carbon
plates. The samples were gold coated in a vacuum at 36 mA
for 2 min using an SCD 050 sputter coater (BAL-TEC,
Liechtenstein) and analysed with a 438VP scanning electron
microscope (LEO, Germany).
Plant material and DNA extraction
Voucher specimens of D. meristocaulis were deposited at
the University of Sa
˜o Paulo Herbarium SPF (F. Rivadavia
et al. 1881). DNA from dried leaves was extracted using the
cetyltrimethylammonium bromide (CTAB) buffer protocol
(Doyle and Doyle, 1987). Genomic DNA of species of sect.
Bryastrum and of Drosera glanduligera,Drosera regia and
the outgroup taxon Dionaea muscipula (Droseraceae) (see
Table 1) was extracted from fresh leaf tissue of greenhouse-
grown plants from the private collection of A. Fleischmann,
using a NucleoSpin
w
Plant Kit (Macherey-Nagel, Du
¨ren,
Germany), following the manufacturer’s protocol (Macherey-
Nagel, 2007). Voucher specimens are listed in Table 1.
PCR conditions/DNA amplification and sequencing
Amplification of the plastid molecular marker rbcL was per-
formed using the primers and protocol of Hasebe et al. (1994).
The rps16 intron was amplified and sequenced using the
primers rpsF and rps2R and the protocol of Oxelman et al.
(1997). The nuclear internal transcribed spacer (ITS) region
was amplified using the PCR primers Leu1 (Walker and
Sytsma, 2007) and ITS4 (White et al., 1990), following the
PCR protocol published in White et al. (1990). ITS amplifica-
tion of D. muscipula and D. regia followed the protocol of
Miranda et al. (2010).
PCR-amplified sequences were purified using a GFXTM
PCR DNA and Gel Purification Kit (GE Healthcare, USA).
Both strands of the spacer region were sequenced by the
dideoxy chain terminator method in a thermal cycler
(GeneAmp
w
PCR System 9700, Applied Biosystems, Foster
City, CA, USA). The sequencing reactions were performed
in a total volume of 10 mL containing 30 – 50 ng of DNA,
5mMof each primer, 2 mL of the ABI Prism BigDye
Terminator v3.1 cycle sequencing ready reaction kit
(Applied Biosystems) and 1 mLof5×Sequencing Buffer
(Applied Biosystems). The thermal cycling parameters were
as follows: one cycle of 4 min at 94 8C, 40 cycles at 94 8C
Rivadavia et al. — Is Drosera meristocaulis of Australian origin? 13
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for 40 s, 52 8C for 40 s and 72 8C for 1 min. Electrophoresis
and fluorescence detection were carried out on an ABI Prism
3100 Genetic Analyzer (Applied Biosystems).
Phylogenetic reconstruction
The sequences were aligned using ClustalW 1.4(Thompson
et al., 1994) followed by manual examination using BioEdit
(Hall, 1999). Some of the ITS and rbcL sequences used here
were obtained from previous studies (Rivadavia et al., 2003;
V. Miranda et al., unpubl. res.) and are available from NCBI
GenBank (accession numbers for all nucleotide sequences
are listed in Table 1). Indels were treated as missing data. As
a strategy of tree rooting, several taxa were initially employed
as outgroups, most of them representatives from various
families of Caryophyllales known to be closely related to
Droseraceae (i.e. Ancistrocladaceae, Dioncophyllaceae and
Nepenthaceae). Nevertheless most of these sequences resulted
in pairwise similarity ,75 %, compared with the sequences of
the Drosera ingroup, a scenario that could increase noise in the
analyses. Therefore, we chose to employ only the monotypic
Dionaea as an outgroup in all the analyses, because of the
higher values of pairwise similarities gained. The phylogenetic
analyses were performed for each individual matrix (ITS,
rps16 and rbcL) and as combined matrices (ITS +rps16 +
rbcL). An additional analysis was carried out with the com-
bined ITS +rps16 data set, because of an incongruent position
of D. meristocaulis compared with the topology of the rbcL
data set. An analysis with a more complete rbcL data set of
Drosera spp. was also performed (Table 2; all rbcL sequence
data for Drosera from Rivadavia et al., 2003 from GenBank).
Further outgroup taxa were added to this rbcL analysis, based
on sequences available in GenBank: Armeria bottendorfensis,
Limonium sinense (Plumbaginaceae), Drosophyllum lusitani-
cum (Drosophyllaceae), and Polygonum capitatum and
Rheum delavayi (Polygonaceae) (Tables 1and 2).
Maximum parsimony
Phylogenetic analysis based on maximum parsimony (MP)
of the sequence data was performed using PAUP* version
4b10 (Swofford, 2002). The phylogenetic trees were obtained
by heuristic search through random addition with 5000 replica-
tions. The branch swapping followed the tree bisection–
reconnection (tbr) algorithm. The robustness of the inferred
trees was evaluated using decay indices (Bremer, 1988) and
bootstrap resampling (Felsenstein, 1985) through 2000
replicates (pseudomatrices) with 40 heuristic search replicates
and random taxon addition. Decay indices were calculated
using TNT version 1.1 (Goloboff et al., 2008) and only
absolute values ≤50 were considered.
Maximum likelihood and Bayesian analyses
The likelihood ratio test as implemented in ModelTest
version 3.7(Posada and Crandall, 1998), with the help of
MrMTgui version 1.0 (P. Nuin, GNU General Public
License), was employed to determinate the best-fit model of
DNA substitution for each data set (individual and combined
data sets) under the Akaike information criterion (AIC;
TABLE 1. List of the Drosera species and outgroup taxa used for the combined phylogenetic analysis, including voucher data and GenBank accession numbers of the
sequence data generated for this study
Species Source Distribution GenBank number rbcL GenBank number ITS GenBank number rps16
D. meristocaulis Neblina, F.Rivadavia et al. 1881 (SPF) Neblina massif, Brazil–Venezuela border JN388035 JN388038 JN388044
D. glanduligera cult. Fleischmann (M; photo voucher) SW Australia AB072511* JN388039 JN388045
D. barbigera cult. Fleischmann (M; photo voucher) SW Australia JQ712489 JQ712490 JQ712488
D. nitidula cult. Fleischmann (M; photo voucher) SW Australia JN388036 JN388040 JN388046
D. scorpioides cult. Fleischmann (M; photo voucher) SW Australia AB072509* JN388041 JN388047
D. occidentalis cult. Fleischmann (M; photo voucher) SW Australia AB072506* JN388042 JN388048
D. paradoxa cult. Fleischmann (M; photo voucher) Northern Australia D. petiolaris: L01913 JN388043 JN388049
D. ordensis cult. Fleischmann (M; photo voucher) Northern Australia JN388037 JN388075 JN388050
D. regia V.F.O. de Miranda 218 (HUMC) South Africa AB072566* JN388077 JN388051
Dionaea muscipula V.F.O. de Miranda 208 (HUMC) SE USA AB072558* JN388078 JN388052
Photographic vouchers are given as Supplementary Data, available online.
* Sequences published in Rivadavia et al. (2003).
Rivadavia et al. — Is Drosera meristocaulis of Australian origin?14
at Instituto de Botânica on July 18, 2012http://aob.oxfordjournals.org/Downloaded from
Akaike, 1974) to estimate the parameters. We used maximum
likelihood (ML) and a Bayesian framework (BA) with
Metropolis-coupled Markov chain Monte Carlo (MCMCMC;
Geyer, 1991) inference to estimate the phylogenetic hypoth-
eses to each data set. The ML analyses were run in PAUP*
version 4b10, using individual models, and estimated para-
meters to each matrix and clade support were calculated
with 2000 replicates (with 40 heuristic search replicates and
random addition). MCMCMC analyses were performed in
MrBayes version 3.1.2(Huelsenbeck and Ronquist, 2001;
Ronquist and Huelsenbeck, 2003) for each data set with 9 ×
10
6
generations sampled every 100 generations, using the
default parameters. For each analysis, four separate runs
were carried out starting from random trees. The sample
points prior to reaching stationarity were discarded as
burn-in. The posterior probabilities (PPs) for each clade
obtained from individual analyses were compared for congru-
ence and combined for evaluating a 50 % majority-rule con-
sensus tree.
RESULTS
Germination pattern
Seed germination occurred after approx. 3– 4 weeks at 20 – 25
8C. Drosera meristocaulis exhibits a cryptocotylar (hypoga-
eous) germination pattern, with the cotyledons remaining in
the testa (Fig. 5).
Pollen morphology
Drosera meristocaulis has pollen tetrads with the intermedi-
ate aperture type 7–8, following the terminology of Takahashi
and Sohma (1982) (Fig. 6). The size measurements are based
on our own LM observations and SEM micrographs, and on
Duno de Stefano (1995): tetrahedral or frequently tetragonal
tetrad, 90 – 130 mm in diameter (confirming Duno de
Stefano, 1995), exine spicate, pollen inoperculate, aperture:
one single central pore per grain (aperture type 7– 8), with
approx. 5–8 large channel openings with a thick exinous
wall surrounding one proximal central pore, radial plaits
poorly developed. Single grain 35 – 43 mm in diameter (45 –
55 mmbyDuno de Stefano, 1995). Channel openings
approx. 10 ×5 ( – 10) mm, standing alternate or opposite to
those of adjoining grains. Spines up to 4 mm long, density of
the spines 1.0–1.5mm
22
(confirming Duno de Stefano,
1995), spinules absent (confirming Duno de Stefano, 1995).
Chromosome counts
In total, ten meristematic root tips were prepared, and nu-
merous counts were made. However, due to the small chromo-
some size, and overall small size of the meristematic root cells
of D. meristocaulis of about 10 mm in diameter, an evaluation
of the exact karyotype was not possible. The chromosome
counts for D. meristocaulis revealed numbers of 32, 34 and
36 with equal frequency of occurrence. Therefore, a karyotype
of 2n¼approx. 32–36 is given for D. meristocaulis here.
Molecular data
All three markers used in this study revealed D. meristocaulis
in the Australian Drosera clade (sensu Rivadavia et al., 2003),
although the exact phylogenetic position differs between rbcL
and the other two markers (Fig. 4; see Supplementary Data for
sequence alignments). The plastid marker rbcL shows
D. meristocaulis nested within the pygmy Drosera clade, as
sister to the two sister pairs D. occidentalis and D. nitidula,
and D. barbigera and D. scorpioides (Fig. 4). In both the ITS
and rps16 data sets, and the combined phylogenetic reconstruc-
tion using all three markers, D. meristocaulis is revealed as sister
to the pygmy clade (Fig. 3), and the two are sister to the
TABLE 2. List of the Drosera species and outgroup taxa
additionally used for the enlarged rbcL data set from GenBank
Species GenBank number
Aldrovanda vesiculosa L. AB072550
Armeria bottendorfensis A.Schulz Z97640
Drosera adelae F.Muell. AY096107
D. alba E.Phillips AB072515
D. aliciae Raym.-Hamet AB072516
D. anglica Huds. AB072517
D. arcturi Hook. AB072512
D. ascendens A.St.-Hil. AB072542
D. brevifolia Pursh AB072519
D. burkeana Planch. AB072520
D. burmannii Vahl L01908
D. caduca Lowrie AB072510
D. capensis L. L01909
D. capillaris Poir. AB072521
D. chrysolepis Taub. AB072522
D. cistiflora L. AB072523
D.collinsiae N.E.Br. in Burtt Davy AB072524
D. cuneifolia L.f. AB072525
D. omissa Diels (as D. ericksoniae N.Marchant) AB072507
D. felix Steyerm. & L.B.Smith AB072527
D. filiformis Raf. L01911
D. gigantea Lindl. L19528
D. graminifolia A.St.-Hil. AB072528
D. graomogolensis T.R.S.Silva AB072529
D. hamiltonii C.R.P.Andrews AB072921
D. hilaris Cham. & Schlechtd. AB072530
D. hirtella A.St.-Hil. AB072531
D. indica L. L19529
D. macrantha Endl. subsp. planchonii N.G.Marchant AB072549
D. longiscapa Debbert (as D. madagascariensis DC) AB072533
D. montana A.St.-Hil. AB072534
D. natalensis Diels AB072537
D. pauciflora Banks ex DC. AB072552
D. peltata Thunb. L01912
D. pygmaea DC. AB072505
D. rotundifolia L. AB072538
D. schwackei (Diels) Rivadavia AB072535
D. sessilifolia A.St.-Hil. AB072551
D. spatulata Labill. L19530
D. stenopetala Hook.f. AB072539
D. stolonifera Endl. L19531
D. trinervia Spreng. AB072548
D. tomentosa A.St.-Hil. AB072536
D. uniflora Willd. AB072540
D. villosa A.St.-Hil. AB072541
Drosophyllum lusitanicum Link L01907
Limonium sinense Kuntze FJ872106
Polygonum capitatum Korth. ex Meisn. HM850243
Rheum delavayi Franch. FJ872104
Rivadavia et al. — Is Drosera meristocaulis of Australian origin? 15
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petiolaris clade (represented by D. paradoxa,D. petiolaris and
D. ordensis in the present study). All nodes get high statistical
support, both in the trees resulting from each single marker
data set (see Fig. 2for rbcL) and in the combined tree (Fig. 3).
DISCUSSION
Recently collected material of D. meristocaulis has revealed
surprising new data supporting the placement of this
species in the Australian Drosera clade, at the base of sect.
Bryastrum. The most outstanding pattern recovered by the
joint investigation of different biological aspects (seed germin-
ation pattern, pollen morphology, chromosome count and
molecular phylogenetics) was the strong affinity of
D. meristocaulis for the species from sect. Bryastrum.
Phylogenetic significance of germination pattern
Cryptocotylar germination in Droseraceae was thus far ex-
clusively known from taxa belonging to a phylogenetic clade
containing predominantly Australian Drosera spp., including
D. adelae
D. hamiltonii
D. indica
D. alba
D. burkeana
D. capensis
D. cistiflora
D. collinsiae
D. cuneifolia
D. hilaris
D. madagascariensis
D. pauciflora
D. trinervia
D. aliciae
D. natalensis
D. ascendens
D. tomentosa
D. villosa
D. chrysolepis
D. graminifolia
D. graomogolensis
D. montana
D. schwackei
D. spatulata
D. anglica
D. capillaris
D. filiformis
D. hirtella
D. felix
D. brevifolia
D. rotundifolia
D. stenopetala
D. uniflora
D. burmannii
D. sessilifolia
D. caduca
D. petiolaris
D. ordensis
D. omissa
D. nitidula
D. occidentalis
D. pygmaea
D. scorpioides
D. gigantea
D. macrantha
D. peltata
D. stolonifera
D. glanduligera
D. arcturi
D. regia
Aldrovanda
Dionaea
Drosophyllum
Armeria
Limonium
Polygonum
Rheum
D. meristocaulis
Drosera sections
Arachnopus,
Stelogyne,
Ptycnostigma,
Psychophila,
Thelocalyx
‘petiolaris-complex’
(section Lasiocephala)
‘pygmy sundews’
(section Bryastrum)
‘Australian Clade’ Outgroup
‘tuberous sundews’
(section Erythrorhiza,
Stolonifera, Ergaleium)
section Coelophylla
71/97 <50/82
31
58/100
1
<50/87
1
1
1
1
1
1
1
<50/85
63/97
65/97
92/100
4
4
4
10
100/100
77/100
2
3
88/100
77/100
76/100
<50/96 <50/98
58/90
89/100
91/99
2
2
11
100/100
100/100
91/100
66/87
10
100/100
5
98/100
3
4
97/100
100/100
875/87
2
6
95/100
100/100
17 53/94
1
2
<50/74
100/100
99/100
10 100/100
18
23
9
5
92/100
66/98
5
2
2
54/67
2
65/78
99/100
10
FIG. 2. Strict consensus from 576 most parsimonious trees (754 steps) of Droseraceae from Bayesian analysis of the rbcL data set. Numbers above branches
show bootstrap (left), and Bayesian posterior probability (right) support values; numbers below branches are decay index values. The position of Drosera mer-
istocaulis is highlighted. Taxonomic groups and clades are indicated, following Rivadavia et al. (2003).
>50
100/100/100
100/100/100
100/100/100
29 19
100/100/100
100/100/100
>50
Dionaea
D. ordensis
D. paradoxa/D. petiolari
s
D. nitidula
D. occidentalis
D. scorpioides
D. barbigera
D. meristocaulis
D. glanduligera
D. regia
44
12
100/82/99
16
96/100/100
0·03
FIG. 3 . Single most parsimonious tree (1331 steps) of the combined ITS,
rps16 and rbcL data sets. Numbers above branches show MP bootstrap
(left), ML bootstrap (middle) and Bayesian posterior probability (right);
numbers below branches are decay index values. Branch lengths represent
genetic distance based on the scale at the bottom.
Rivadavia et al. — Is Drosera meristocaulis of Australian origin?16
at Instituto de Botânica on July 18, 2012http://aob.oxfordjournals.org/Downloaded from
the pygmy sundews of sect. Bryastrum (Conran et al., 1997,
2007). Thus, D. meristocaulis is the only New World
Drosera species with this type of germination (Fig. 5), with
all other species showing phanerocotyly (see D. capillaris in
Fig. 5). Cryptocotylar germination in small-seeded plants
like Droseraceae is rare (Clifford, 1984) and is usually asso-
ciated with fluctuating ecological conditions and therefore
interpreted as an adaptation to long-term seed dormancy
which requires induced germination. Cryptocotyly has only
evolved once in Drosera; it is a synapomorphy for the
Australian clade (sensu Rivadavia et al., 2003), but was lost
in the monotypic sect. Phycopsis consisting of D. binata.
Thus, it is most likely that this germination pattern evolved
among Drosera in Australia as the continent moved north-
wards and became drier (Yesson and Culham, 2006), as an
adaptation to the seasonal Mediterranean climate with a
pronounced dry season, occasional summer fires and cool
moist winters. The seed remains dormant until germination
is triggered by changing seasonal conditions, an ecological
strategy followed by a range of Australian plants, including
numerous Drosera spp. (Bell et al., 1993).
Although the summits of the Neblina massif are usually
regarded as stable, wet tropical Amazonian habitats,
D. meristocaulis occurs on the drier northern plateaus of
these highlands, from where occasional fires have been
reported (Givnish et al., 1986;McPherson, 2006). At least a
few endemic plants from this area seem to present morpho-
logical adaptations to avoid fire damage (Givnish et al.,
1986;Judziewicz, 1998). Seasonal droughts and wildfires are
conditions reminiscent of the habitats occupied by Drosera
sect. Bryastrum in Oceania, which may explain why cryptoco-
tyly is maintained in D. meristocaulis.
D. occidentalis D. microscapa
D. nitidula
D. nitidula
D. barbigera
D. barbigera
D. meristocaulis
D. meristocaulis
D. scorpioides
D. scorpioides
D. ordensis D. ordensis
D. petiolaris
D. paradoxa
D. glanduligera D. glanduligera
D. regia
D. regia
Dionaea Dionaea
rbcL
ITS+rps16
FIG. 4. Incongruence of the phylogenetic position of Drosera meristocaulis between ITS and rps16 topology (left) and rbcL (right). Identical phylogenetic
positions are indicated by dashed lines; a slash in the dashed line is for taxa equivalents used in the different data sets (see also Table 1).
sc
c
pl
c
c
pl
pl
pl
Ir
ec
sc
hc
pr
AB
FIG. 5 . Comparison of germination patterns of two different South American Drosera species. (A) Cryptocotyly in Drosera meristocaulis. (B) Phanerocotyly in
D. capillaris. Abbreviations: c, cotelydons (hidden inside the testa in D. meristocaulis); ec, epicotyl; hc, hypocotyl; lr, lateral root; pl, primary leaf; pr, primary
root; sc, seed coat (testa). Scale bars ¼1 mm.
Rivadavia et al. — Is Drosera meristocaulis of Australian origin? 17
at Instituto de Botânica on July 18, 2012http://aob.oxfordjournals.org/Downloaded from
Phylogenetic significance of pollen morphology
Further morphological similarities between D. meristocaulis
and members of sect. Bryastrum can be found in pollen. Duno
de Stefano (1995) observed one single central pore as an aperture
in pollen tetrads of D. meristocaulis and therefore assigned it to
aperture type 7, which is confined to sect. Drosera (Takahashi
and Sohma, 1982). However, he did not recognize that a single
proximal pore is also found in aperture type 8 and the intermedi-
ate type 7–8 (Takahashi and Sohma, 1982).
The pollen tetrad of D. meristocaulis shares common features
of aperture type (one proximal central pore in each pollen grain)
and pollen structure (radial channel plaits poorly developed and
channel openings surrounded by a thick exinous wall) with
pollen known as type 8 or intermediate type 7 – 8, respectively
(Takahashi and Sohma, 1982). These two pollen types are con-
fined to species of the Australian Drosera clade (sensu
Rivadavia et al., 2003), except for D. glanduligera of the mono-
typic sect. Coelophylla, which exhibits a unique pollen tetrad of
type 5 (Takahashi and Sohma, 1982). The ornamentation of the
exinous wall of D. meristocaulis pollen is also distinct from that
of all other South American Drosera spp. (Duno de Stefano,
1995), as it has no spinules and few rather large spines. This or-
namentation is commonly found in Australian Drosera spp., es-
pecially in members of sect. Bryastrum and sect. Lasiocephala
(Takahashi and Sohma, 1982).
Phylogenetic significance of leaf trichome characters
The leaves of members of the Bryastrum clade (including
sect. Lasiocephala and sect. Bryastrum,sensu Seine and
Barthlott, 1994) are characterized by the presence of biseriate
sessile trichomes (‘microglands’) with elongated basal cells,
which represent a synapomorphic character for this monophy-
letic group. These trichomes were called ‘Rorella-type glands’
by Seine and Barthlott (1993) and classified as ‘type 4 and 5
glands’ by La
¨nger et al. (1995).Drosera meristocaulis has
type T2 biseriate and T11–12 multiseriate sessile trichomes
(Seine and Barthlott, 1993;La
¨nger et al., 1995). Conran
et al. (2007) stated that the trichome patterns found in
D. meristocaulis are ambiguous, as they can be observed in
members of both the Drosera and the Bryastrum clade
(sensu Rivadavia et al., 2003), and that only in combination
with the germination pattern could the phylogenetic position
of sect. Meristocaulis be verified. However, the stout, short
yellow gland-like trichomes on the adaxial and abaxial
petiole surface of D. meristocaulis (Seine and Barthlott,
1993) do also occur in some pygmy Drosera spp. (e.g.
Drosera nitidula and related species, A. Fleischmann, pers.
obs.). These trichomes have a four-celled peduncle and a glan-
dular head consisting of about 20 cells. This type of glandular
trichome produces a sub-cuticular yellow secretion and occurs
on the leaf surface and also on the emergences (Fig. 7).
Members of sect. Bryastrum all share a special, eight-celled
biseriate type of microgland, so-called ‘Rorella-trichomes’
(Seine and Barthlott, 1993), which are usually found on the
abaxial surface of the petiole and lamina. Seine and
Barthlott (1993) did not observe these Rorella-trichomes in
the specimens of D. meristocaulis they studied, and we did
not detect them in our study material. The absence of
Rorella-trichomes is a morphological character that supports
the phylogenetic position of D. meristocaulis as sister to
sect. Bryastrum, not as a member of this section, and rejection
of subgenus Meristocaulis sensu Schlauer (1996).
Phylogenetic significance of karyology
Chromosome numbers in Drosera range from 2n¼6to2n¼
80, and are in strong phylogenetic accordance with the clades
FIG. 7 . Yellow glandular trichomes on a leaf of Drosera meristocaulis. Scale
bar ¼1 mm. Photograph by Daniel Olschewski, with kind permission.
cp
co
rp
rp
cp
co
AB
FIG. 6. Pollen of Drosera meristocaulis. (A) LM photograph, (B) SEM photograph. Abbreviations: co, channel opening; cp, central pore; rp, radial plait. Scale bars ¼10 mm.
Rivadavia et al. — Is Drosera meristocaulis of Australian origin?18
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revealed by Rivadavia et al. (2003). The Australian clade exhi-
bits the greatest variability of karyotypes and forms extensive
aneuploid and polyploid series, with relatively low chromosome
numbers, ranging from 2n¼6to2n¼40, resulting from base
numbers x¼3, 5, 6, 7, 8, 9, 10, 11, 13, 14 and 23 (Kondo and
Lavarack, 1984;Sheikh and Kondo, 1995;Rivadavia et al.,
2003;Rivadavia, 2005;Lowrie and Conran, 2007). In contrast,
all New World species of Drosera (belonging to sect. Drosera
and sect. Thelocalyx) form a homogeneous group, with relative-
ly conserved chromosome numbers of 2n¼20 or 40 (i.e. poly-
ploid series of the base number x¼10), suggesting at least two
independent colonization events of a diploid and tetraploid
group (Rivadavia et al., 2003;Rivadavia, 2005). Although
representing an approximate range, the newly inferred karyotype
of 2n¼approx. 32–36 for D. meristocaulis contrasts with the
polyploid series found in all other South American species,
but fits the aneuploid series found in Drosera spp. of the
Australian clade.
Karyology can be a useful character in Drosera taxonomy
for both species delimitation and infrageneric classification.
An example for the latter was shown with the proposal to
remove the enigmatic northern Australian D. banksii from
sect. Ergaleium and to place it in sect. Lasiocephala (Kondo
and Lavarack, 1984). This suggestion was later confirmed by
further morphological (Seine and Barthlott, 1994) and molecu-
lar phylogenetic data (A. Fleischmann, unpubl. res.), which
revealed that this species grouped with sect. Lasiocephala.
CONCLUSIONS
Molecular phylogenetic data and morphological characters, in-
cluding germination pattern, pollen anatomy, karyotype and
leaf trichome characters, support the placement of
D. meristocaulis in the Australian clade in a monotypic
section (sect. Meristocaulis), as sister to sect. Bryastrum
(Figs 3and 4), or even in this section in the case of the
rbcL data set (Fig. 2). In contrast to the Drosera of sect.
Bryastrum,D. meristocaulis does not reproduce asexually by
gemmae. It is possible that the ancestors of D. meristocaulis
lost the ability to produce gemmae after reaching South
America, but it is also probable that gemmae production
evolved in pygmy sundews after this lineage split from
D. meristocaulis. Gemmae are a synapomorphy of the
pygmy sundews and are likely to have evolved as an adaptation
to a seasonal climate as the Australian continent became drier
(Yesson and Culham, 2006). The fact that gemmae are found
in all species of sect. Bryastrum suggests that it is not only a
successful means of asexual reproduction, but also an essential
ecological survival strategy in the Mediterranean climate of
southwestern Australia. The production of gemmae requires
a considerable allocation of resources (Karlsson and Pate,
1992) and is possibly an important mechanism for rapid
clonal colonization of seasonally available habitats.
Cryptocotylar germination may represent an adaptation to
fire, a common phenomenon in both regions (Oceania and
the Neblina massif ), playing an important role in the mainten-
ance of morphological similarities between D. meristocaulis
and species of sect. Bryastrum.
Any explanation for the presence on the Neblina massif of a
plant species descended from an Australian lineage is sure to be
at least controversial. A recent study estimated that sect.
Bryastrum began its diversification about 13– 12 Mya (Yesson
and Culham, 2006), and therefore contradicts a Gondwanan
origin for D. meristocaulis. Despite the lack of information on a
dispersal route from Australia to northern South America, the evi-
dence that this did in fact occur cannot be rejected. As a vector for
this rare LDD event, birds or wind seem most conceivable, al-
though no avian migratory pathways from Australia to northern
South America have been reported (Lomolino et al., 2006). An
Australian to temperate South America disjunction is also
known from a few plant families (Thorne, 1972), including
Winteraceae. A strikingly similar biogeographic pattern is found
in the three earliest branching members of Loranthaceae (showy
mistletoes), namely the terrestrial monotypic genera Nuytsia flori-
bunda from south-western Western Australia, Atkinsonia ligus-
trina from eastern Australia and Gaiadendron from Central and
South America (also occurring on Neblina) (Vidal-Russell and
Nickrent, 2008). In the case of Loranthaceae, a Gondawanan
origin is assumed, which would explain the biogeography of the
three taxa, which are successive sister taxa to all remaining
Loranthaceae (Vidal-Russell and Nickrent, 2008).
Recent LDD from Australia (or southeast Asia) to South
America has previously been proposed for the species sister
pair Drosera burmannii and D. sessilifolia of sect.
Thelocalyx (Rivadavia et al., 2003). In accordance with phylo-
genetic and other evidence presented above, D. meristocaulis
is most probably also descended from an LDD event from
Australia to South America, and is probably not a supposed
palaeoendemic species that descended from pygmy sundew-
like plants previously widespread in Gondwana, and which
also led to extant sect. Bryastrum.
SUPPLEMENTARY DATA
Supplementary data are available online at www.aob.oxford
journals.org and consist of photographic vouchers for Drosera
barbigera,D. glanduligera,D. nitidula,D. occidentalis,
D. ordensis,D. paradoxa and D. scorpioides, and sequence
alignments (.txt files) for rbcL,ITS and rps16.
ACKNOWLEDGEMENTS
We thank Allen Lowrie for providing plant material for ger-
mination studies and for useful discussions on Australian
pygmy Drosera species; Ivan Snyder and Matt Hochberg for
helping with the seed germination experiments; Kamil Pasek
for sending in vitro material of Drosera meristocaulis;
Susanne Renner for helpful input on long-distance dispersal
events; Daniel Olschewski, Bochum, for providing photos of
the glands from cultivated plants; Tanja Ernst, Munich, and
M. V. A. Sluys, Sa
˜o Paulo, for laboratory support; and Eva
Facher, Munich, for assistance with SEM photography.
Michael Fay and two anonymous reviewers are thanked for
helpful comments on the manuscript.
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