Annals of Botany 120: 709–723, 2017
doi:10.1093/aob/mcx056, available online at www.academic.oup.com/aob
© The Author 2017. Published by Oxford University Press on behalf of the Annals of Botany Company.
All rights reserved. For Permissions, please email: journals.permissions@oup.com
Phylogeny of the ‘orchid-like’ bladderworts (gen. Utricularia sect. Orchidioides
and Iperua: Lentibulariaceae) with remarks on the stolon–tuber system
Fernanda Gomes Rodrigues1, Néstor Franco Marulanda1, Saura R. Silva2, Bartosz J. Płachno3, Lubom�ır
Adamec4 and Vitor F. O. Miranda1,*
1Universidade Estadual Paulista (Unesp), Faculdade de Ciências Agr�arias e Veterin�arias, Jaboticabal, Departamento de
Biologia Aplicada �a Agropecu�aria, S~ao Paulo, Brazil, 2Universidade Estadual Paulista (Unesp), Instituto de Biociências,
Botucatu, S~ao Paulo, Brazil, 3Department of Plant Cytology and Embryology, Jagiellonian University in Krak�ow, 9
Gronostajowa St., 30-387 Krak�ow, Poland and 4Institute of Botany of the Czech Academy of Sciences, T�rebo�n, Czech Republic
*For correspondence. E-mail vmiranda@fcav.unesp.br
Received: 25 February 2017 Returned for revision: 22 March 2017 Editorial Decision: 2 April 2017 Accepted: 13 April 2017
� Background and Aims The ‘orchid-like’ bladderworts (Utricularia) comprise 15 species separated into two sec-
tions: Orchidioides and Iperua. These robust and mostly epiphytic species were originally grouped within the sec-
tion Orchidioides by the first taxonomical systems. These species were later split into two sections when sect.
Iperua was proposed. Due to the lack of strong evidence based on a robust phylogenetic perspective, this study
presents a phylogenetic proposal based on four different DNA sequences (plastid and nuclear) and morphology to
test the monophyly of the two sections.
� Methods In comparison with all previous phylogenetic studies, the largest number of species across the sections
was covered: 11 species from sections Orchidioides and Iperua with 14 species as an external group. Maximum
likelihood and Bayesian inferences were applied to DNA sequences of rps16, trnL-F, matK, the internal transcribed
spacer (ITS) and three morphological characters: (1) the crest of the corolla; (2) the primary organs in the embryo;
and (3) tubers. Additionally, a histochemical analysis of the stolons and tubers is presented from an evolutionary
perspective.
� Key Results The analyses showed the paraphyly of sect. Iperua, since Utricularia humboldtii is more related to
the clade of sect. Orchidioides. Utricularia cornigera is grouped in the sect. Iperua clade based on chloroplast
DNA sequences, but it is nested to sect. Orchidioides according to ITS dataset. Morphological characters do not
support the breaking up of the ‘orchid-like’ species into two sections, either. Moreover, the stolon–tuber systems of
both sections serve exclusively for water storage, according to histological analyses.
� Conclusions This study provides strong evidence, based on DNA sequences from two genomic compartments
(plastid and nucleus) and morphology to group the Utricularia sect. Orchidioides into the sect. Iperua. The tubers
are important adaptations for water storage and have been derived from stolons at least twice in the phylogenetic
history of ‘orchid-like’ bladderworts.
Key words:Molecular phylogeny, Utricularia, anatomy, morphology, section Orchidioides, section Iperua, tuber.
INTRODUCTION
Lentibulariaceae is a cosmopolitan family presenting the great-
est diversity in species, habit and life form among carnivorous
plants. Around 350 species are distributed across the three gen-
era Pinguicula, Genlisea and Utricularia; Pinguicula is the sis-
ter group of the clade formed by Genlisea and Utricularia
(Jobson et al., 2003; Müller et al., 2004; Guisande et al., 2007;
Veleba et al., 2014). The genus Utricularia forms traps from
little vesicles called utricles or bladders, which have an active
suction mechanism triggered when the trichomes near
their entrance are stimulated by small organisms (Poppinga
et al., 2016). Based on the vegetative morphology, Taylor
(1989) split the genus Utricularia in into two subgenera:
Polypompholyx and Utricularia. Nevertheless there are interest-
ing, controversial proposals regarding the classification of the
two very close infrageneric taxa: sections Orchidioides and
Iperua.
The nine species of Utricularia sect. Orchidioides A.DC. are
distributed in central America, the Antilles and South America
and are orchid-like bladderworts (Fig. 1A-B, F). Moreover,
they are perennial epiphytes or terrestrials, with a tuber ensem-
ble in the peduncle basis (Fig. 9B, C). On the other hand,
Utricularia sect. Iperua P.Taylor has six species distributed in
South America (Fig. 1C-E, G). These are lithophytes and terres-
trial or aquatic epiphytes (U. nelumbifolia and U. humboldtii
are examples of the latter) and the majority of them form fleshy
stolons (Fig. 9A), except for U. geminiloba, which forms tubers
(Fig. 9B, C) similar to Orchidioides. The two sections have
very similar trap and calyx morphologies (Taylor, 1989).
De Candolle (1844) created sect. Orchidioides, which
included species with tubers. Kamie�nski (1895) later expanded
this section and included non-tuberous species such as
Utricularia nelumbifolia and Utricularia reniformis. Barnhart
(1916), on the other hand, proposed the new genus Orchyllium to
aggregate the species, with U. alpina (as Orchyllium alpinum) as
VC The Author 2017. Published by Oxford University Press on behalf of the Annals of Botany Company.
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Annals of Botany Page 1 of 15
doi:10.1093/aob/mcx056, available online at https://academic.oup.com/aob
PART OF A SPECIAL ISSUE ON MORPHOLOGY AND ADAPTATION
Received: 25 February 2017 Returned for revision: 22 March 2017 Editorial Decision: 2 April 2017 Accepted: 13 April 2017
Published electronically: 30 June 2017
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Rodrigues et al. — Phylogeny of the ‘orchid-like’ bladderworts (Lentibulariaceae)710
A
B
C
D
F
G
E
FIG. 1. Orchid-like Utricularia species. Sect. Orchidioides: (A) U. quelchii N.E.Br.; (B) U. asplundii P.Taylor; (C) U. humboldtii Schomb.; (D) U. reniformis A.St.-
Hil.; (E) U. nephrophylla Benj.; (F) U. alpina Jacq.; Sect. Iperua; (G) U. geminiloba Benj. Arrows denote the crest of the corolla. Photo credits: (A) Martin Hingst;
(B) Nicole Rebbert (utricularien.de); (C) Barry Rice; (F) Ron Lane.
Page 2 of 15 Rodrigues et al. — Phylogeny of the ‘orchid-like’ bladderworts (Lentibulariaceae)
the type species. Huynh (1968) questioned sect. Orchidioides,
since it included species of different groups based on pollen char-
acters. Taylor (1986), based on the morphological differences of
the corolla, seeds and pollen, split sect. Orchidioides and pro-
posed sect. Iperua, with Utricularia humboldtii Schomb. as the
type species.
There are interesting discussions concerning the generic mor-
phology of Utricularia and whether or not the species have a
clear bauplan and delimitated organs as in other angiosperms or
whether they fit within the Fuzzy Arberian Morphology (FAM)
concept (Rutishauser and Isler, 2001; Rutishauser, 2016). Thus,
it is not an easy task to classify species by means of morpholog-
ical characters. Taylor (1989), in his monograph, raises some
doubts about Utricularia morphology. In the Orchidioides and
Iperua sections specifically, U. reniformis was questioned as it
has thick, tuber-like stolons.
Recent molecular studies have also considered these sections
using a phylogenetic approach. Jobson et al. (2003) show the
monophyly of the Iperua and Orchidioides sections with the
plastid sequences rps16 and trnL-F; Müller and Borsch (2005),
on the other hand, proposed the exclusion of sect. Iperua on the
basis of the plastid intron trnK with the matK gene.
Thus, our aim was to test the hypothesis of grouping the sec-
tions Orchidioides and Iperua by studying a broad range of spe-
cies; we evaluated 11 of the 15 that have been described. A
large number of molecular markers from two genomic compart-
ments [plastid DNA sequences rps16, trnL-F and matK and
nuclear internal transcribed spacer (ITS) region] and relevant
morphological characters were evaluated. We also included the
recently described Utricularia cornigera Studni�cka, a species
with a morphological similarity to sect. Iperua (Studni�cka,
2009). We also conducted a phylogenetic analysis of the
following morphological characteristics: (1) the crest on the
lower lip of the corolla; (2) primary organs in the embryo; and
(3) the presence of tubers. We conducted a histochemical analy-
sis, with a discussion of the function and evolution of these
organs.
MATERIALS AND METHODS
Plant samples and DNA markers
Plant samples from 20 species were obtained from both natural
populations and cultivated plants and DNA sequences were
also obtained from GenBank/NCBI (Table 1). In accordance
with previous studies, four DNA sequences were selected as
markers due to their phylogenetic signal: (1) rps16 (Oxelman
et al., 1997; Jobson and Albert 2002; Jobson et al., 2003); (2)
trnL-F (Taberlet et al., 1991; Jobson and Albert 2002; Jobson
et al., 2003); (3) matK (Müller and Borsch, 2005; Silva et al.,
2016); and (4) the ITS region (Hillis and Dixon, 1991). We
therefore obtained a total of 76 sequences, of which 26 were
produced in this study, with sequences for five species from
Utricularia sect. Orchidioides (which has a total of nine
described species) and six for sect. Iperua, including
Utricularia cornigera, thus representing all known species of
this section. In addition, 14 species from other sections were
used as an external group (Table 1).
TABLE 1. Utricularia species included in this study, their origin and GenBank access numbers, by molecular marker
Section Species (voucher) GenBank access number
rps16 trnL-F matK ITS
Orchidioides U. asplundii1 (TS000261) AF482558�1 AF482631�1 KY68970 KY689711
U. quelchii1 (TS000260) – KY689702 AF531846�1 –
U. endresii1 (TS000262) – AF482642�1 KY799062 KY689709
U. alpina1 (TS000263) AF482556�1 AF482629�1 AF531822�1 KY689712
U. praetermissa1 (TS000264) – KY689703 KY689698 KY689705
Iperua U. humboldtii2 (TS000199) – KY689704 AF531836�1 –
U. geminiloba1 (VFOM2045) – AF482646�1 KX604216�1 KY689716
U. nephrophylla1 (VFOM2047) AF482588�1 AF482664�1 AF531827�1 KY689707
U. reniformis (VFOM2044) AF482595�1 AF482671�1 KX604218�1 KY689706
U. nelumbifolia1 (VFOM2055) AF482586�1 AF482662�1 KX604217�1 KY689708
U. cornigera1 (TS000265) – KY689701 KY689699 KY689710
Utricularia U. aurea3 (TS000267) AF482559�1 AF482632�1 KX604176�1 KY689714
U. australis AF482560�1 AF482633�1 AF531823�1 –
U. intermedia AF482575�1 AF482651�1 AF531839�1 –
U. macrorhiza3 (TS000266) AF482581�1 AF482657�1 AF531835�1 KY689719
U. minor3 (TS000268) – GU169706�1 JN894028�1 KY689721
U. vulgaris3 (TS000269) – JQ728994�1 JN894054�1 KY689722
Psyllosperma U. huntii AF482574�1 AF482650�1 – –
U. praelonga AF482591�1 AF482667�1 AF531843�1 –
U. longifolia1 (VFOM1680) AF482580�1 AF482656�1 AF531834�1 KY689718
U. hispida1 (VFOM1637) – – AF531829�1 KY689717
U. calycifida – – AF531824�1 –
Foliosa U. tricolor1 (VFOM2043) AF482600�1 AF482677�1 KX604210�1 KY689720
U. tridentata – – AF531825�1 –
U. amethystina1 (VFOM1644) AF482557�1 AF482630�1 – KY689713
1–3Samples sequenced in this study: 1Carnivorous Plants Collection – Carlos Rohrbacher; 2Carnivorous Plant Collection – Barry Rice; 3Carnivorous Plant
Collection – Institute of Botany of the Czech Academy of Sciences, T�rebo�n, Czech Republic (vouchers deposited in Herbarium JABU – University of Sao Paulo
State – UNESP/FCAV).
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Rodrigues et al. — Phylogeny of the ‘orchid-like’ bladderworts (Lentibulariaceae) 711
A
B
C
D
F
G
E
FIG. 1. Orchid-like Utricularia species. Sect. Orchidioides: (A) U. quelchii N.E.Br.; (B) U. asplundii P.Taylor; (C) U. humboldtii Schomb.; (D) U. reniformis A.St.-
Hil.; (E) U. nephrophylla Benj.; (F) U. alpina Jacq.; Sect. Iperua; (G) U. geminiloba Benj. Arrows denote the crest of the corolla. Photo credits: (A) Martin Hingst;
(B) Nicole Rebbert (utricularien.de); (C) Barry Rice; (F) Ron Lane.
Page 2 of 15 Rodrigues et al. — Phylogeny of the ‘orchid-like’ bladderworts (Lentibulariaceae)
the type species. Huynh (1968) questioned sect. Orchidioides,
since it included species of different groups based on pollen char-
acters. Taylor (1986), based on the morphological differences of
the corolla, seeds and pollen, split sect. Orchidioides and pro-
posed sect. Iperua, with Utricularia humboldtii Schomb. as the
type species.
There are interesting discussions concerning the generic mor-
phology of Utricularia and whether or not the species have a
clear bauplan and delimitated organs as in other angiosperms or
whether they fit within the Fuzzy Arberian Morphology (FAM)
concept (Rutishauser and Isler, 2001; Rutishauser, 2016). Thus,
it is not an easy task to classify species by means of morpholog-
ical characters. Taylor (1989), in his monograph, raises some
doubts about Utricularia morphology. In the Orchidioides and
Iperua sections specifically, U. reniformis was questioned as it
has thick, tuber-like stolons.
Recent molecular studies have also considered these sections
using a phylogenetic approach. Jobson et al. (2003) show the
monophyly of the Iperua and Orchidioides sections with the
plastid sequences rps16 and trnL-F; Müller and Borsch (2005),
on the other hand, proposed the exclusion of sect. Iperua on the
basis of the plastid intron trnK with the matK gene.
Thus, our aim was to test the hypothesis of grouping the sec-
tions Orchidioides and Iperua by studying a broad range of spe-
cies; we evaluated 11 of the 15 that have been described. A
large number of molecular markers from two genomic compart-
ments [plastid DNA sequences rps16, trnL-F and matK and
nuclear internal transcribed spacer (ITS) region] and relevant
morphological characters were evaluated. We also included the
recently described Utricularia cornigera Studni�cka, a species
with a morphological similarity to sect. Iperua (Studni�cka,
2009). We also conducted a phylogenetic analysis of the
following morphological characteristics: (1) the crest on the
lower lip of the corolla; (2) primary organs in the embryo; and
(3) the presence of tubers. We conducted a histochemical analy-
sis, with a discussion of the function and evolution of these
organs.
MATERIALS AND METHODS
Plant samples and DNA markers
Plant samples from 20 species were obtained from both natural
populations and cultivated plants and DNA sequences were
also obtained from GenBank/NCBI (Table 1). In accordance
with previous studies, four DNA sequences were selected as
markers due to their phylogenetic signal: (1) rps16 (Oxelman
et al., 1997; Jobson and Albert 2002; Jobson et al., 2003); (2)
trnL-F (Taberlet et al., 1991; Jobson and Albert 2002; Jobson
et al., 2003); (3) matK (Müller and Borsch, 2005; Silva et al.,
2016); and (4) the ITS region (Hillis and Dixon, 1991). We
therefore obtained a total of 76 sequences, of which 26 were
produced in this study, with sequences for five species from
Utricularia sect. Orchidioides (which has a total of nine
described species) and six for sect. Iperua, including
Utricularia cornigera, thus representing all known species of
this section. In addition, 14 species from other sections were
used as an external group (Table 1).
TABLE 1. Utricularia species included in this study, their origin and GenBank access numbers, by molecular marker
Section Species (voucher) GenBank access number
rps16 trnL-F matK ITS
Orchidioides U. asplundii1 (TS000261) AF482558�1 AF482631�1 KY68970 KY689711
U. quelchii1 (TS000260) – KY689702 AF531846�1 –
U. endresii1 (TS000262) – AF482642�1 KY799062 KY689709
U. alpina1 (TS000263) AF482556�1 AF482629�1 AF531822�1 KY689712
U. praetermissa1 (TS000264) – KY689703 KY689698 KY689705
Iperua U. humboldtii2 (TS000199) – KY689704 AF531836�1 –
U. geminiloba1 (VFOM2045) – AF482646�1 KX604216�1 KY689716
U. nephrophylla1 (VFOM2047) AF482588�1 AF482664�1 AF531827�1 KY689707
U. reniformis (VFOM2044) AF482595�1 AF482671�1 KX604218�1 KY689706
U. nelumbifolia1 (VFOM2055) AF482586�1 AF482662�1 KX604217�1 KY689708
U. cornigera1 (TS000265) – KY689701 KY689699 KY689710
Utricularia U. aurea3 (TS000267) AF482559�1 AF482632�1 KX604176�1 KY689714
U. australis AF482560�1 AF482633�1 AF531823�1 –
U. intermedia AF482575�1 AF482651�1 AF531839�1 –
U. macrorhiza3 (TS000266) AF482581�1 AF482657�1 AF531835�1 KY689719
U. minor3 (TS000268) – GU169706�1 JN894028�1 KY689721
U. vulgaris3 (TS000269) – JQ728994�1 JN894054�1 KY689722
Psyllosperma U. huntii AF482574�1 AF482650�1 – –
U. praelonga AF482591�1 AF482667�1 AF531843�1 –
U. longifolia1 (VFOM1680) AF482580�1 AF482656�1 AF531834�1 KY689718
U. hispida1 (VFOM1637) – – AF531829�1 KY689717
U. calycifida – – AF531824�1 –
Foliosa U. tricolor1 (VFOM2043) AF482600�1 AF482677�1 KX604210�1 KY689720
U. tridentata – – AF531825�1 –
U. amethystina1 (VFOM1644) AF482557�1 AF482630�1 – KY689713
1–3Samples sequenced in this study: 1Carnivorous Plants Collection – Carlos Rohrbacher; 2Carnivorous Plant Collection – Barry Rice; 3Carnivorous Plant
Collection – Institute of Botany of the Czech Academy of Sciences, T�rebo�n, Czech Republic (vouchers deposited in Herbarium JABU – University of Sao Paulo
State – UNESP/FCAV).
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Rodrigues et al. — Phylogeny of the ‘orchid-like’ bladderworts (Lentibulariaceae)712
Amplification and sequencing
The DNA was extracted by the CTAB method (Doyle and
Doyle, 1987), modified by Lodhi et al. (1994). Amplification
reactions of the nuclear markers were conducted in 25lL of a
solution containing 20 mM MgCl2, 100 mM dNTPs, 10 mM of
each primer, 1U of Dream Taq Polymerase (Fermentas), and
on average 50 ng of DNA template. For the ITS region, the pri-
mers and dimethyl sulphoxide adjuvant (DMSO) were used as
recommended by Miranda et al. (2010). For matK and trnL-F
the primers employed were according Lim et al. (2012) and
Taberlet et al. (1991), respectively.
The thermal profile of the amplification reactions for the
intergenic spacer trnL-F was 95 �C for 3min; 30 cycles of
1min at 94 �C, 45 s at 52 �C and 1min at 72 �C, and 5min of
final extension at 72 �C. For the gene matK, it was 94 �C for
1min, 35 cycles of 40 s at 94 �C, 20 s at 52 �C and 50 s at 72 �C,
and 10min of final extension at 72 �C. For the nuclear ITS, it
was 95 �C for 3min, 35 cycles of 30 s at 95 �C, 30 s at 54 �C
and 1min at 72 �C, and 5min of final extension at 72 �C.
The amplicons were verified by 0�8 % agarose gel electro-
phoresis, precipitated with 100 % isopropanol, purified with
70 % ethanol and sequenced by the method developed by
Sanger et al. (1975) in an automatic sequencer, model 3730xl
ABI (Applied Biosystems).
Sequences and phylogenetic analyses
The identity of each sequence was determined with the
BLASTN application (Altschul et al., 1990). Using Geneious
v10.0 (Kearse et al., 2012), their Phred quality was verified and
visual and manual adjustments were made with the program
BioEdit v7.5.0.2 (Hall, 1999). MAFFT v7 (Katoh et al., 2002)
was used to align the sequences, and datasets (matrices) were
created with BioEdit v7.5.0.2 (Hall, 1999) with the addition of
masks (missing data ¼ ‘?’) (Wiens, 2006).
We produced five datasets: four for each isolated marker and
one for a combined (total evidence) analysis. For the matK frag-
ment, the sequences obtained from GenBank (Table 1) were
trimmed to achieve a homologue region according to the ampli-
fied sequences by the 3F_KIM and 1R_KIM primers (Lim
et al., 2012).
In each dataset generated, a Bayesian inference on platform
CIPRES (Miller et al., 2010) was performed, in which the best
fit was employed, as selected by the Akaike information crite-
rion (AIC) (Akaike, 1973), generated by the program
jModelTest v2.1.1 (Darriba et al., 2012). The best-fit model for
the plastid markers was the GTR þ G model (Tavaré, 1986) and
that for the nuclear spacer was the TIM3þ IþG model.
Additionally, we performed maximum likelihood (ML) analysis
with the CIPRES platform (Miller et al., 2010) and the
PAUP*4.0 program (Swofford, 2003) to get bootstrap values
(2000 pseudoreplicates and heuristic search with 1000 replicates
with random addition of sequences and the branch swapping
algorithm TBR). The trees obtained were edited with TreeGraph
2 (Stöver, 2010) and FigTree v1.3.1 (Rambaut, 2009).
Morphological characters
We conducted phylogenetic testing of the three morphologi-
cal characteristics that supposedly emphasize the differences
between the sections and have therefore been employed histori-
cally in the taxonomic circumscription of Orchidioides and
Iperua. These were: (1) a crest on the protuberance of the lower
lip of the corolla (Fig. 1C, E); (2) the presence of tubers
(Taylor, 1986, 1989); and (3) the primary organs in the embryo,
according to Płachno and �Swią tek (2010). A morphological
matrix was generated using the NDE Nexus Data Editor pro-
gram (Page, 2001), which overlapped the combined (total evi-
dence) Bayesian inference tree, for which the Mesquite
program (Maddison and Maddison, 2010) was used.
Histochemistry of storage organs and tissues
To achieve better comprehension and comparison of storage
tissues, we conducted histochemical analyses of the stolons of
Utricularia reniformis and Utricularia nelumbifolia (sect.
Iperua) and the tubers of Utricularia geminiloba (sect. Iperua)
and Utricularia alpina (sect. Orchidioides). The stolons of U.
reniformis and U. nelumbifolia and tubers of U. geminiloba
were collected from natural populations in December 2015 or
were taken from a collection in the Botanic Gardens of
Jagiellonian University in Krak�ow, Poland, and the vouchers
were deposited in Herbarium JABU (V.F.O. de Miranda et al.,
2044, 2055 and 2045, respectively).
The following reagents were used to show the general anat-
omy and the storage components: IKI (iodine–potassium iodide)
for starch þ proteins; saturated ethanolic solutions of Sudan III
and Sudan IV for lipids; alum carmine and iodine green for lig-
nin þ cellulose; and 0�1 % (w/v) ruthenium red for pectin and
mucilage (Filutowicz and Ku_zdowicz 1951; Ruzin 1999).
The material (thick stolons of U. reniformis and U. nelumbifo-
lia and tubers of U. geminiloba and U. alpina) was fixed in
2�5 % (v/v) glutaraldehyde/2�5 % (v/v) formaldehyde in 0�05 M
sodium cacodylate buffer (pH 7�0) for several days, washed
three times in the same buffer and postfixed in 1% (w/v)
osmium tetroxide solution for 1�5 h at 0 �C. This was followed
by dehydration using a graded ethanol series and infiltration and
embedding using an epoxy embedding medium kit (Fluka).
Semithin sections (0�9–1�0 mm) prepared for light microscopy
were stained for general histology using aqueous methylene
blue/azure II (MB/AII) for 1–2min (Humphrey and Pittman,
1974) and examined with an Olympus BX60 optical microscope.
Photosynthetic function of stolons and tubers
To verify the presence of chloroplasts and possible photosyn-
thetic activity in stolons and tubers, these organs were used in a
glasshouse experiment. Three stolon fragments (�2 cm) of U.
reniformis and three tubers of U. geminiloba were placed in
Petri dishes with moist absorbent paper and stored at 25 �C
under natural light conditions for 30 d. Photographs of the frag-
ments and tubers were then taken.
RESULTS
Phylogeny of Utricularia sections Orchidioides and Iperua
The phylogenetic trees from the Bayesian inference and ML
criteria are congruent in their general topology (Figs 2 and 3)
Page 4 of 15 Rodrigues et al. — Phylogeny of the ‘orchid-like’ bladderworts (Lentibulariaceae)
and showed that most groups were supported by posterior prob-
abilities (PPs) and ML bootstraps (> = 50%) (Table 2).
In the analysis of intron rps16, in which the sequences of the
studied sections taken from the study of Jobson and Albert
(2002) were used, a similar result, congruent to Taylor’s classi-
fication (1989), was found (Fig. 2A). The other plastid markers
(trnL-F and matK; Fig. 2B, C) show sect. Iperua as a paraphy-
letic group, by the inclusion of U. humboldtii (taxonomically
recognized as belonging to sect. Iperua) in sect. Orchidioides.
Furthermore, the sequences trnL-F and matK placed U. corni-
gera in sect. Iperua. The tree obtained with the spacer trnL-F
(Fig. 2B) revealed that U. humboldtii is a sister group to the
other species in sect. Orchidioides and that U. cornigera should
be included in sect. Iperua as a sister group to the clade
U. nelumbifolia–U. reniformis. With this marker, the ML analy-
sis showed the species U. nelumbifolia, U. reniformis, U.
cornigera and U. geminiloba as a monophyletic group (Fig.
2B). U. humboldtii has a similar position, being nested as an
external branch of the clade formed by U. praetermissa, U.
endresii, U. quelchii, U. asplundii and U. alpina. The matK tree
(Fig. 2C) revealed that U. humboldtii is closely related to sect.
Orchidioides as a sister group, because a similar topology to
the trnL-F dataset was found (Fig. 2B).
Additionally, while the plastid markers trnL-F and matK
showed U. cornigera nested in the sect. Iperua clade, the
ITS dataset (Fig. 2D) showed this species grouped in
Orchidioides
Iperua
Psyllosperma
Foliosa
Utricularia
Orchidioides
Iperua
Psyllosperma
Foliosa
Utricularia
Orchidioides
Iperua
Psyllosperma
Foliosa
Utricularia
Orchidioides
Iperua
Orchidioides
Iperua
Psyllosperma
Foliosa
Utricularia
U. alpina100/100
A rps16
100/100
100/100
100/100
100/100
90/69
70/-
100/99
80/81
90/100
90/100
U. asplundii
U. nelumbifolia
U. reniformis
U. nephrophylla
U. longifolia
U. praelonga
U. huntii
U. amethystina
U. tricolor
U. australis
U. macrorhiza
U. intermedia
U. aurea
C matK 80/55
60/60
80/60
80/87
90/95
100/100
100/100
100/69 100/97
100/97
100/100
100/88
100/94
100/56
100/100
70/-
50/-
100/-
70/54
U. quelchii
U. alpina
U. praetermissa
U. endresii
U. asplundii
U. humboldtii
U. geminiloba
U. nelumbifolia
U. reniformis
U. cornigera
U. nephrophylla
U. longifolia
U. hispida
U. praelonga
U. calycifida
U. tricolor
U. tridentata
U. minor
U. intermedia
U. macrorhiza
U. australis
U. vulgaris
U. aurea
50/71
100/76
80/79
100/98
100/100
100/100
90/94
100/81
50/81
70/-
100/-
90/87
100/98
50/-
50/-
70/90
B trnL-F
U. praetermissa
U. endresii
U. quelchii
U. asplundii
U. alpina
U. humboldtii
U. nelumbifolia
U. reniformis
U. cornigera
U. geminiloba
U. nephrophylla
U. praelonga
U. longifolia
U. huntii
U. amethystina
U. tricolor
U. australis
U. vulgaris
U. macrorhiza
U. minor
U. intermedia
U. aurea
60/-
50/-
90/97
100/100
50/56
70/55
90/61
100/100
100/100
100/100
100/100
100/99
D ITS
U. alpina
U. endresii
U. praetermissa
U. cornigera
U. asplundii
U. geminiloba
U. nelumbifolia
U. reniformis
U. nephrophylla
U. longifolia
U. hispida
U. amethystina
U. tricolor
U. minor
U. macrorhiza
U. vulgaris
U. aurea
FIG. 2. Bayesian inference trees for (A) rps16, (B) trnL-F, (C) matK and (D) ITS. Numbers above the branches are posterior probabilities followed by maximum like-
lihood bootstraps. �, branches with support value <50.
Rodrigues et al. — Phylogeny of the ‘orchid-like’ bladderworts (Lentibulariaceae) Page 5 of 15
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Rodrigues et al. — Phylogeny of the ‘orchid-like’ bladderworts (Lentibulariaceae) 713
Amplification and sequencing
The DNA was extracted by the CTAB method (Doyle and
Doyle, 1987), modified by Lodhi et al. (1994). Amplification
reactions of the nuclear markers were conducted in 25lL of a
solution containing 20 mM MgCl2, 100 mM dNTPs, 10 mM of
each primer, 1U of Dream Taq Polymerase (Fermentas), and
on average 50 ng of DNA template. For the ITS region, the pri-
mers and dimethyl sulphoxide adjuvant (DMSO) were used as
recommended by Miranda et al. (2010). For matK and trnL-F
the primers employed were according Lim et al. (2012) and
Taberlet et al. (1991), respectively.
The thermal profile of the amplification reactions for the
intergenic spacer trnL-F was 95 �C for 3min; 30 cycles of
1min at 94 �C, 45 s at 52 �C and 1min at 72 �C, and 5min of
final extension at 72 �C. For the gene matK, it was 94 �C for
1min, 35 cycles of 40 s at 94 �C, 20 s at 52 �C and 50 s at 72 �C,
and 10min of final extension at 72 �C. For the nuclear ITS, it
was 95 �C for 3min, 35 cycles of 30 s at 95 �C, 30 s at 54 �C
and 1min at 72 �C, and 5min of final extension at 72 �C.
The amplicons were verified by 0�8 % agarose gel electro-
phoresis, precipitated with 100 % isopropanol, purified with
70 % ethanol and sequenced by the method developed by
Sanger et al. (1975) in an automatic sequencer, model 3730xl
ABI (Applied Biosystems).
Sequences and phylogenetic analyses
The identity of each sequence was determined with the
BLASTN application (Altschul et al., 1990). Using Geneious
v10.0 (Kearse et al., 2012), their Phred quality was verified and
visual and manual adjustments were made with the program
BioEdit v7.5.0.2 (Hall, 1999). MAFFT v7 (Katoh et al., 2002)
was used to align the sequences, and datasets (matrices) were
created with BioEdit v7.5.0.2 (Hall, 1999) with the addition of
masks (missing data ¼ ‘?’) (Wiens, 2006).
We produced five datasets: four for each isolated marker and
one for a combined (total evidence) analysis. For the matK frag-
ment, the sequences obtained from GenBank (Table 1) were
trimmed to achieve a homologue region according to the ampli-
fied sequences by the 3F_KIM and 1R_KIM primers (Lim
et al., 2012).
In each dataset generated, a Bayesian inference on platform
CIPRES (Miller et al., 2010) was performed, in which the best
fit was employed, as selected by the Akaike information crite-
rion (AIC) (Akaike, 1973), generated by the program
jModelTest v2.1.1 (Darriba et al., 2012). The best-fit model for
the plastid markers was the GTR þ G model (Tavaré, 1986) and
that for the nuclear spacer was the TIM3þ IþG model.
Additionally, we performed maximum likelihood (ML) analysis
with the CIPRES platform (Miller et al., 2010) and the
PAUP*4.0 program (Swofford, 2003) to get bootstrap values
(2000 pseudoreplicates and heuristic search with 1000 replicates
with random addition of sequences and the branch swapping
algorithm TBR). The trees obtained were edited with TreeGraph
2 (Stöver, 2010) and FigTree v1.3.1 (Rambaut, 2009).
Morphological characters
We conducted phylogenetic testing of the three morphologi-
cal characteristics that supposedly emphasize the differences
between the sections and have therefore been employed histori-
cally in the taxonomic circumscription of Orchidioides and
Iperua. These were: (1) a crest on the protuberance of the lower
lip of the corolla (Fig. 1C, E); (2) the presence of tubers
(Taylor, 1986, 1989); and (3) the primary organs in the embryo,
according to Płachno and �Swią tek (2010). A morphological
matrix was generated using the NDE Nexus Data Editor pro-
gram (Page, 2001), which overlapped the combined (total evi-
dence) Bayesian inference tree, for which the Mesquite
program (Maddison and Maddison, 2010) was used.
Histochemistry of storage organs and tissues
To achieve better comprehension and comparison of storage
tissues, we conducted histochemical analyses of the stolons of
Utricularia reniformis and Utricularia nelumbifolia (sect.
Iperua) and the tubers of Utricularia geminiloba (sect. Iperua)
and Utricularia alpina (sect. Orchidioides). The stolons of U.
reniformis and U. nelumbifolia and tubers of U. geminiloba
were collected from natural populations in December 2015 or
were taken from a collection in the Botanic Gardens of
Jagiellonian University in Krak�ow, Poland, and the vouchers
were deposited in Herbarium JABU (V.F.O. de Miranda et al.,
2044, 2055 and 2045, respectively).
The following reagents were used to show the general anat-
omy and the storage components: IKI (iodine–potassium iodide)
for starch þ proteins; saturated ethanolic solutions of Sudan III
and Sudan IV for lipids; alum carmine and iodine green for lig-
nin þ cellulose; and 0�1 % (w/v) ruthenium red for pectin and
mucilage (Filutowicz and Ku_zdowicz 1951; Ruzin 1999).
The material (thick stolons of U. reniformis and U. nelumbifo-
lia and tubers of U. geminiloba and U. alpina) was fixed in
2�5 % (v/v) glutaraldehyde/2�5 % (v/v) formaldehyde in 0�05 M
sodium cacodylate buffer (pH 7�0) for several days, washed
three times in the same buffer and postfixed in 1% (w/v)
osmium tetroxide solution for 1�5 h at 0 �C. This was followed
by dehydration using a graded ethanol series and infiltration and
embedding using an epoxy embedding medium kit (Fluka).
Semithin sections (0�9–1�0 mm) prepared for light microscopy
were stained for general histology using aqueous methylene
blue/azure II (MB/AII) for 1–2min (Humphrey and Pittman,
1974) and examined with an Olympus BX60 optical microscope.
Photosynthetic function of stolons and tubers
To verify the presence of chloroplasts and possible photosyn-
thetic activity in stolons and tubers, these organs were used in a
glasshouse experiment. Three stolon fragments (�2 cm) of U.
reniformis and three tubers of U. geminiloba were placed in
Petri dishes with moist absorbent paper and stored at 25 �C
under natural light conditions for 30 d. Photographs of the frag-
ments and tubers were then taken.
RESULTS
Phylogeny of Utricularia sections Orchidioides and Iperua
The phylogenetic trees from the Bayesian inference and ML
criteria are congruent in their general topology (Figs 2 and 3)
Page 4 of 15 Rodrigues et al. — Phylogeny of the ‘orchid-like’ bladderworts (Lentibulariaceae)
and showed that most groups were supported by posterior prob-
abilities (PPs) and ML bootstraps (> = 50%) (Table 2).
In the analysis of intron rps16, in which the sequences of the
studied sections taken from the study of Jobson and Albert
(2002) were used, a similar result, congruent to Taylor’s classi-
fication (1989), was found (Fig. 2A). The other plastid markers
(trnL-F and matK; Fig. 2B, C) show sect. Iperua as a paraphy-
letic group, by the inclusion of U. humboldtii (taxonomically
recognized as belonging to sect. Iperua) in sect. Orchidioides.
Furthermore, the sequences trnL-F and matK placed U. corni-
gera in sect. Iperua. The tree obtained with the spacer trnL-F
(Fig. 2B) revealed that U. humboldtii is a sister group to the
other species in sect. Orchidioides and that U. cornigera should
be included in sect. Iperua as a sister group to the clade
U. nelumbifolia–U. reniformis. With this marker, the ML analy-
sis showed the species U. nelumbifolia, U. reniformis, U.
cornigera and U. geminiloba as a monophyletic group (Fig.
2B). U. humboldtii has a similar position, being nested as an
external branch of the clade formed by U. praetermissa, U.
endresii, U. quelchii, U. asplundii and U. alpina. The matK tree
(Fig. 2C) revealed that U. humboldtii is closely related to sect.
Orchidioides as a sister group, because a similar topology to
the trnL-F dataset was found (Fig. 2B).
Additionally, while the plastid markers trnL-F and matK
showed U. cornigera nested in the sect. Iperua clade, the
ITS dataset (Fig. 2D) showed this species grouped in
Orchidioides
Iperua
Psyllosperma
Foliosa
Utricularia
Orchidioides
Iperua
Psyllosperma
Foliosa
Utricularia
Orchidioides
Iperua
Psyllosperma
Foliosa
Utricularia
Orchidioides
Iperua
Orchidioides
Iperua
Psyllosperma
Foliosa
Utricularia
U. alpina100/100
A rps16
100/100
100/100
100/100
100/100
90/69
70/-
100/99
80/81
90/100
90/100
U. asplundii
U. nelumbifolia
U. reniformis
U. nephrophylla
U. longifolia
U. praelonga
U. huntii
U. amethystina
U. tricolor
U. australis
U. macrorhiza
U. intermedia
U. aurea
C matK 80/55
60/60
80/60
80/87
90/95
100/100
100/100
100/69 100/97
100/97
100/100
100/88
100/94
100/56
100/100
70/-
50/-
100/-
70/54
U. quelchii
U. alpina
U. praetermissa
U. endresii
U. asplundii
U. humboldtii
U. geminiloba
U. nelumbifolia
U. reniformis
U. cornigera
U. nephrophylla
U. longifolia
U. hispida
U. praelonga
U. calycifida
U. tricolor
U. tridentata
U. minor
U. intermedia
U. macrorhiza
U. australis
U. vulgaris
U. aurea
50/71
100/76
80/79
100/98
100/100
100/100
90/94
100/81
50/81
70/-
100/-
90/87
100/98
50/-
50/-
70/90
B trnL-F
U. praetermissa
U. endresii
U. quelchii
U. asplundii
U. alpina
U. humboldtii
U. nelumbifolia
U. reniformis
U. cornigera
U. geminiloba
U. nephrophylla
U. praelonga
U. longifolia
U. huntii
U. amethystina
U. tricolor
U. australis
U. vulgaris
U. macrorhiza
U. minor
U. intermedia
U. aurea
60/-
50/-
90/97
100/100
50/56
70/55
90/61
100/100
100/100
100/100
100/100
100/99
D ITS
U. alpina
U. endresii
U. praetermissa
U. cornigera
U. asplundii
U. geminiloba
U. nelumbifolia
U. reniformis
U. nephrophylla
U. longifolia
U. hispida
U. amethystina
U. tricolor
U. minor
U. macrorhiza
U. vulgaris
U. aurea
FIG. 2. Bayesian inference trees for (A) rps16, (B) trnL-F, (C) matK and (D) ITS. Numbers above the branches are posterior probabilities followed by maximum like-
lihood bootstraps. �, branches with support value <50.
Rodrigues et al. — Phylogeny of the ‘orchid-like’ bladderworts (Lentibulariaceae) Page 5 of 15
Orchidioides
Iperua
Psyllosperma
Foliosa
Utricularia
Orchidioides
Iperua
Psyllosperma
Foliosa
Utricularia
Orchidioides
Iperua
Psyllosperma
Foliosa
Utricularia
Orchidioides
Iperua
Orchidioides
Iperua
Psyllosperma
Foliosa
Utricularia
U. alpina100/100
A rps16
100/100
100/100
100/100
100/100
90/69
70/-
100/99
80/81
90/100
90/100
U. asplundii
U. nelumbifolia
U. reniformis
U. nephrophylla
U. longifolia
U. praelonga
U. huntii
U. amethystina
U. tricolor
U. australis
U. macrorhiza
U. intermedia
U. aurea
C matK 80/55
60/60
80/60
80/87
90/95
100/100
100/100
100/69 100/97
100/97
100/100
100/88
100/94
100/56
100/100
70/-
50/-
100/-
70/54
U. quelchii
U. alpina
U. praetermissa
U. endresii
U. asplundii
U. humboldtii
U. geminiloba
U. nelumbifolia
U. reniformis
U. cornigera
U. nephrophylla
U. longifolia
U. hispida
U. praelonga
U. calycifida
U. tricolor
U. tridentata
U. minor
U. intermedia
U. macrorhiza
U. australis
U. vulgaris
U. aurea
50/71
100/76
80/79
100/98
100/100
100/100
90/94
100/81
50/81
70/-
100/-
90/87
100/98
50/-
50/-
70/90
B trnL-F
U. praetermissa
U. endresii
U. quelchii
U. asplundii
U. alpina
U. humboldtii
U. nelumbifolia
U. reniformis
U. cornigera
U. geminiloba
U. nephrophylla
U. praelonga
U. longifolia
U. huntii
U. amethystina
U. tricolor
U. australis
U. vulgaris
U. macrorhiza
U. minor
U. intermedia
U. aurea
60/-
50/-
90/97
100/100
50/56
70/55
90/61
100/100
100/100
100/100
100/100
100/99
D ITS
U. alpina
U. endresii
U. praetermissa
U. cornigera
U. asplundii
U. geminiloba
U. nelumbifolia
U. reniformis
U. nephrophylla
U. longifolia
U. hispida
U. amethystina
U. tricolor
U. minor
U. macrorhiza
U. vulgaris
U. aurea
D
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Deleted Text:
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Deleted Text:
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Deleted Text: of the section Orchidioides
Deleted Text:
Deleted Text:
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Deleted Text: section
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Deleted Text: similar
Rodrigues et al. — Phylogeny of the ‘orchid-like’ bladderworts (Lentibulariaceae)714
sect. Orchidioides as a sister group of U. praetermissa, with
maximum support values from both analyses (100 % for PP
and ML bootstrap). Sections Iperua and Orchidioides were
shown as paraphyletic (Fig. 2D), and U. humboldtii is missing
in this analysis.
The tree resulting from the concatenated datasets presents
sect. Iperua as a paraphyletic group, which shows U. humboldtii
as an external branch of sect. Orchidioides (Fig. 3, clade A).
Utricularia cornigera is shown in the clade of sect.
Orchidioides, as a monophyletic group with U. asplundii,
U. quelchii and U. praetermissa, but this clade is not strongly
supported (PP 53% and ML bootstrap <50%). Despite this low
support, the clade of sect. Orchidioides, which includes U. cor-
nigera, is strongly supported by the Bayesian inference and ML
bootstrap, with 100 and 99% confidence, respectively (Fig. 3).
Distribution of morphological characters
The characteristics of both the crests on the corolla lower
lip (Fig. 4A) and the tubers (Fig. 4C) do no support as synapo-
morphies to the sections Orchidioides and Iperua. According to
U. asplundiiClade A
Clade B
Taylor (1989)
77/-
53/-
66/-
100/99
100/99
100/100
100/100
100/100
100/100
100/100
100/100
100/99
79/89
92/63
100/100
100/90
91/94
100/100
100/100
100/100
Orchidioides
Orchidioides
O
rc
h
id
io
id
es
Iperua
Iperua
Psyllosperma
Foliosa
Utricularia
This study
U. quelchii
U. praetermissa
U. cornigera
U. endresii
U. alpina
U. humboldtii
U. geminiloba
U. reniformis
U. nelumbifolia
U. nephrophylla
U. praelonga
U. hispida
U. longifolia
U. huntii
U. calycifida
U. tridentata
U. amethystina
U. tricolor
U. australis
U. macrorhiza
U. vulgaris
U. intermedia
U. minor
U. aurea
FIG. 3. Bayesian inference for the combined analysis (rps16 þ trnL-F þ matKþ ITS). Numbers above the branches are the posterior probabilities followed by maxi-
mum likelihood bootstraps. �, branches with support value <50.
TABLE 2. Matrices and statistical analyses of alignments and cla-
dograms inferred by maximum likelihood (ML) and Bayesian
inference (BI)
Dataset Genome Terminals,
n
Characters
considering
gaps, bp
Clades with
support �50 %,
n (%)1
Posterior
probability (BI)
Bootstraps
(ML)
rps16 Plastid 14 926 11 (84) 10 (77)
trnL-trnF Plastid 22 1�091 16 (76) 12 (57)
matK2 Plastid 23 883 19 (86) 16 (72)
ITS Nucleus 17 962 12 (75) 10 (62)
Combined Nucleus þ
Plastid
25 3�864 20 (83) 17 (71)
1Percentage of clades was calculated from the total of possible tree clades
(¼ terminal numbers�1).
2Sequences obtained from GenBank (see Table 1) were trimmed to achieve
a homologous region according to the fragment amplified by the 3F_KIM and
1R_KIM primers (Lim et al., 2012).
Page 6 of 15 Rodrigues et al. — Phylogeny of the ‘orchid-like’ bladderworts (Lentibulariaceae)
the combined analysis, the crest on the corolla has possibly
appeared at least twice. It was once present in the ancestral line-
age of Orchidioides–Iperua complex (clade A þ clade B),
which was lost by an ancestor of the clade Iperua only to arise
again as a reversion in U. cornigera. The character of the tubers
was also shown to be homoplastic, with two independent ori-
gins: one in sect. Orchidioides, with the reversion to U.
cornigera, which lacks tubers, and the other in U. geminiloba.
The characteristic primary embryo organs (Fig. 4B) occur in U.
cornigera (Studni�cka, 2009), U. humboldtii, U. nelumbifolia
and species of sect. Utricularia (Płachno and �Swią tek, 2010
and references therein). The lack of information regarding the
embryology of several species makes it difficult to trace a
robust and well-supported hypothesis for the evolution of this
character.
Histological analysis of stolons and tubers
In our analyses, the thick stolons of U reniformis (Figs 5 and
9A) and U. nelumbifolia (Fig. 7) and the tubers of U. gemini-
loba (Figs 6 and 9B, C) and U. alpina (Fig. 8) have similar
anatomy. In general, as shown in the transverse sections, the
epidermis and parenchymatous cortex surround the ectophloic
central cylinder. Only the stolons of U. nelumbifolia have many
lacunae (Fig. 7A–D). The cortex consists of large parenchyma
cells with large vacuoles; the xylem and phloem elements are
separated from each other. In both organs small epidermal
trichomes occur, each consisting of one basal cell, one short,
central cell and a long-headed cell (Fig. 7E).
Very small and infrequent lipid droplets were observed in the
cytoplasm of the cortical cells (Fig. 5C). Cell walls stained with
the ruthenium red (Figs 5E and 6G) showed no mucilage and
those stained with IKI showed no starch or storage proteins
(Figs 5D, 6F, 7F and 8C). Paracrystalline protein inclusions
were occasionally present in the nuclei of various cell types
(Figs 5F and 6H). The cell walls of the trichome barrier cell
stained selectively with Sudan (Figs 6D and 7E).
DISCUSSION
Phylogeny of Orchidioides–Iperua complex: one section is
enough
Our results support the paraphyly of Utricularia sect. Iperua
and Orchidioides, considering U. cornigera (Figs 2 and 3). The
key species that makes sect. Iperua paraphyletic is U. humbold-
tii, which is the type species assigned to this section (Taylor,
1986, 1989). Similar results were obtained by Müller et al.
(2004) based on matK and the flanking trnK intron, and later
Müller and Borsch (2005) suggested acceptance of only sect.
Orchidioides, underpinning De Candolle (1844) and Kamie�nski
(1895). De Candolle based his system on only three species of
the complex: U. alpina (as U. montana) and U. unifolia, classi-
fied as sect. Orchidioides and, curiously, U. humboldtii, which
was maintained in his ‘Species dubiae’ section.
Utricularia humboldtii (Fig. 1C), a perennial and one of the
largest terrestrial species of the genus, produces spectacular
flowers, which is a common trait of the Orchidioides–Iperua
complex. This species is found as a terrestrial or even as an
aquatic epiphyte, since the plants can project aerial horizontal
stolons that reach and grow between bromeliad leaves (Taylor,
1989). While the species of sect. Iperua are distributed in South
America, mainly in Southern and Southeastern Brazil (BFG,
2015; Miranda et al., 2015), U. humboldtii is the only species
of this section that occurs in the Guiana Highlands. Thus, clades
A and B (Fig. 3) are also supported by species distribution
(with the exception of U. cornigera, which is endemic to
Southeastern Brazil) (BFG, 2015; Miranda et al., 2015).
Utricularia cornigera Studni�cka
In our analysis with the plastid markers trnL-F (Fig. 2B) and
matK (Fig. 2C), U. cornigera is nested into sect. Iperua and
related to the clade U. reniformis–U. nelumbifolia and also
nested with U. geminiloba, despite the incongruence found with
this species when considering both markers. The hypothesis of
a natural hybrid, originating from the crossing between U. reni-
formis and U. nelumbifolia (Fleischmann, 2012), has been
refuted by Studni�cka (2013, 2015) by an interbreeding
experiment.
However, in the ITS analysis U. cornigera is included in
sect. Orchidioides, with maximum statistical support in both
phylogenetic analyses (PP and ML), and it also remains in the
Orchidioides in the concatenated tree (Fig. 3). While the mater-
nal DNA (chloroplast DNA in this case) supports a close rela-
tionship with the species of sect. Iperua, the ITS region, which
is of biparental origin, suggests that U. cornigera has the same
common ancestral species related to the Orchidioides clade.
The multiple copies of the ITS region found in eukaryotic
genomes may be a problem when using these data for phyloge-
netic inferences, since it is not certain that the sequences
achieved are orthologues (Miranda et al., 2010). Nonetheless,
despite the multiple copies, concerted evolution occurs when
sequence differences among copies in the same genome
become homogenized to the same sequence by mechanisms
such as high-frequency unequal crossing-over and gene conver-
sion (�Alvarez and Wendel, 2003). Considering that the impor-
tance of the ITS region for phylogenetic inferences is mainly
due to its high signal, which can be used to solve the phylogeny
of closely related taxa (Hillis and Dixon, 1991), the results pre-
sented in this study are very important. However, further analy-
ses with the use of different nuclear markers should address this
issue. The topologies presented by the ITS analysis and the
combined analysis provide phylogenetic support for the recog-
nition of U. cornigera as a species.
Distribution of morphological characters
Taylor (1986, 1989) used three main characters for justify-
ing the splitting of sect. Orchidioides and for the creation of
sect. Iperua: the crest on the lower lip of the corolla and the
morphology of the seeds and pollen. The pollen has already
been studied in the species of these sections by Huynh
(1968), who placed U. alpina, U. praetermissa, U. jamesoni-
ana and U. humboldtii into one group, similar to our molecu-
lar results, which included U. humboldtii in sect. Orchidioides
(Fig. 3, clade A).
The characteristic crest on the lower lip of the corolla
(Fig. 4A) is exclusive to sect. Iperua (Taylor, 1989). Since our
Rodrigues et al. — Phylogeny of the ‘orchid-like’ bladderworts (Lentibulariaceae) Page 7 of 15
U. asplundiiClade A
Clade B
Taylor (1989)
77/-
53/-
66/-
100/99
100/99
100/100
100/100
100/100
100/100
100/100
100/100
100/99
79/89
92/63
100/100
100/90
91/94
100/100
100/100
100/100
Orchidioides
Orchidioides
O
rc
h
id
io
id
es
Iperua
Iperua
Psyllosperma
Foliosa
Utricularia
This study
U. quelchii
U. praetermissa
U. cornigera
U. endresii
U. alpina
U. humboldtii
U. geminiloba
U. reniformis
U. nelumbifolia
U. nephrophylla
U. praelonga
U. hispida
U. longifolia
U. huntii
U. calycifida
U. tridentata
U. amethystina
U. tricolor
U. australis
U. macrorhiza
U. vulgaris
U. intermedia
U. minor
U. aurea
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niversidade Estadual Paulista J�
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esquita Filho user on 23 April 2019
Deleted Text: the m
Deleted Text: in each analysis
Deleted Text: &hx0025;
Deleted Text: &hx0025;
Deleted Text: &hx0025;
Deleted Text:
Deleted Text: &hx0025;
Deleted Text: &hx2013;
Deleted Text: .
Deleted Text: &hx2013;
Deleted Text:
Deleted Text: Utricularia
Deleted Text: the section
Deleted Text: The s
Deleted Text: of
Deleted Text: &hx0025;
Rodrigues et al. — Phylogeny of the ‘orchid-like’ bladderworts (Lentibulariaceae) 715
sect. Orchidioides as a sister group of U. praetermissa, with
maximum support values from both analyses (100 % for PP
and ML bootstrap). Sections Iperua and Orchidioides were
shown as paraphyletic (Fig. 2D), and U. humboldtii is missing
in this analysis.
The tree resulting from the concatenated datasets presents
sect. Iperua as a paraphyletic group, which shows U. humboldtii
as an external branch of sect. Orchidioides (Fig. 3, clade A).
Utricularia cornigera is shown in the clade of sect.
Orchidioides, as a monophyletic group with U. asplundii,
U. quelchii and U. praetermissa, but this clade is not strongly
supported (PP 53% and ML bootstrap <50%). Despite this low
support, the clade of sect. Orchidioides, which includes U. cor-
nigera, is strongly supported by the Bayesian inference and ML
bootstrap, with 100 and 99% confidence, respectively (Fig. 3).
Distribution of morphological characters
The characteristics of both the crests on the corolla lower
lip (Fig. 4A) and the tubers (Fig. 4C) do no support as synapo-
morphies to the sections Orchidioides and Iperua. According to
U. asplundiiClade A
Clade B
Taylor (1989)
77/-
53/-
66/-
100/99
100/99
100/100
100/100
100/100
100/100
100/100
100/100
100/99
79/89
92/63
100/100
100/90
91/94
100/100
100/100
100/100
Orchidioides
Orchidioides
O
rc
h
id
io
id
es
Iperua
Iperua
Psyllosperma
Foliosa
Utricularia
This study
U. quelchii
U. praetermissa
U. cornigera
U. endresii
U. alpina
U. humboldtii
U. geminiloba
U. reniformis
U. nelumbifolia
U. nephrophylla
U. praelonga
U. hispida
U. longifolia
U. huntii
U. calycifida
U. tridentata
U. amethystina
U. tricolor
U. australis
U. macrorhiza
U. vulgaris
U. intermedia
U. minor
U. aurea
FIG. 3. Bayesian inference for the combined analysis (rps16 þ trnL-F þ matKþ ITS). Numbers above the branches are the posterior probabilities followed by maxi-
mum likelihood bootstraps. �, branches with support value <50.
TABLE 2. Matrices and statistical analyses of alignments and cla-
dograms inferred by maximum likelihood (ML) and Bayesian
inference (BI)
Dataset Genome Terminals,
n
Characters
considering
gaps, bp
Clades with
support �50 %,
n (%)1
Posterior
probability (BI)
Bootstraps
(ML)
rps16 Plastid 14 926 11 (84) 10 (77)
trnL-trnF Plastid 22 1�091 16 (76) 12 (57)
matK2 Plastid 23 883 19 (86) 16 (72)
ITS Nucleus 17 962 12 (75) 10 (62)
Combined Nucleus þ
Plastid
25 3�864 20 (83) 17 (71)
1Percentage of clades was calculated from the total of possible tree clades
(¼ terminal numbers�1).
2Sequences obtained from GenBank (see Table 1) were trimmed to achieve
a homologous region according to the fragment amplified by the 3F_KIM and
1R_KIM primers (Lim et al., 2012).
Page 6 of 15 Rodrigues et al. — Phylogeny of the ‘orchid-like’ bladderworts (Lentibulariaceae)
the combined analysis, the crest on the corolla has possibly
appeared at least twice. It was once present in the ancestral line-
age of Orchidioides–Iperua complex (clade A þ clade B),
which was lost by an ancestor of the clade Iperua only to arise
again as a reversion in U. cornigera. The character of the tubers
was also shown to be homoplastic, with two independent ori-
gins: one in sect. Orchidioides, with the reversion to U.
cornigera, which lacks tubers, and the other in U. geminiloba.
The characteristic primary embryo organs (Fig. 4B) occur in U.
cornigera (Studni�cka, 2009), U. humboldtii, U. nelumbifolia
and species of sect. Utricularia (Płachno and �Swią tek, 2010
and references therein). The lack of information regarding the
embryology of several species makes it difficult to trace a
robust and well-supported hypothesis for the evolution of this
character.
Histological analysis of stolons and tubers
In our analyses, the thick stolons of U reniformis (Figs 5 and
9A) and U. nelumbifolia (Fig. 7) and the tubers of U. gemini-
loba (Figs 6 and 9B, C) and U. alpina (Fig. 8) have similar
anatomy. In general, as shown in the transverse sections, the
epidermis and parenchymatous cortex surround the ectophloic
central cylinder. Only the stolons of U. nelumbifolia have many
lacunae (Fig. 7A–D). The cortex consists of large parenchyma
cells with large vacuoles; the xylem and phloem elements are
separated from each other. In both organs small epidermal
trichomes occur, each consisting of one basal cell, one short,
central cell and a long-headed cell (Fig. 7E).
Very small and infrequent lipid droplets were observed in the
cytoplasm of the cortical cells (Fig. 5C). Cell walls stained with
the ruthenium red (Figs 5E and 6G) showed no mucilage and
those stained with IKI showed no starch or storage proteins
(Figs 5D, 6F, 7F and 8C). Paracrystalline protein inclusions
were occasionally present in the nuclei of various cell types
(Figs 5F and 6H). The cell walls of the trichome barrier cell
stained selectively with Sudan (Figs 6D and 7E).
DISCUSSION
Phylogeny of Orchidioides–Iperua complex: one section is
enough
Our results support the paraphyly of Utricularia sect. Iperua
and Orchidioides, considering U. cornigera (Figs 2 and 3). The
key species that makes sect. Iperua paraphyletic is U. humbold-
tii, which is the type species assigned to this section (Taylor,
1986, 1989). Similar results were obtained by Müller et al.
(2004) based on matK and the flanking trnK intron, and later
Müller and Borsch (2005) suggested acceptance of only sect.
Orchidioides, underpinning De Candolle (1844) and Kamie�nski
(1895). De Candolle based his system on only three species of
the complex: U. alpina (as U. montana) and U. unifolia, classi-
fied as sect. Orchidioides and, curiously, U. humboldtii, which
was maintained in his ‘Species dubiae’ section.
Utricularia humboldtii (Fig. 1C), a perennial and one of the
largest terrestrial species of the genus, produces spectacular
flowers, which is a common trait of the Orchidioides–Iperua
complex. This species is found as a terrestrial or even as an
aquatic epiphyte, since the plants can project aerial horizontal
stolons that reach and grow between bromeliad leaves (Taylor,
1989). While the species of sect. Iperua are distributed in South
America, mainly in Southern and Southeastern Brazil (BFG,
2015; Miranda et al., 2015), U. humboldtii is the only species
of this section that occurs in the Guiana Highlands. Thus, clades
A and B (Fig. 3) are also supported by species distribution
(with the exception of U. cornigera, which is endemic to
Southeastern Brazil) (BFG, 2015; Miranda et al., 2015).
Utricularia cornigera Studni�cka
In our analysis with the plastid markers trnL-F (Fig. 2B) and
matK (Fig. 2C), U. cornigera is nested into sect. Iperua and
related to the clade U. reniformis–U. nelumbifolia and also
nested with U. geminiloba, despite the incongruence found with
this species when considering both markers. The hypothesis of
a natural hybrid, originating from the crossing between U. reni-
formis and U. nelumbifolia (Fleischmann, 2012), has been
refuted by Studni�cka (2013, 2015) by an interbreeding
experiment.
However, in the ITS analysis U. cornigera is included in
sect. Orchidioides, with maximum statistical support in both
phylogenetic analyses (PP and ML), and it also remains in the
Orchidioides in the concatenated tree (Fig. 3). While the mater-
nal DNA (chloroplast DNA in this case) supports a close rela-
tionship with the species of sect. Iperua, the ITS region, which
is of biparental origin, suggests that U. cornigera has the same
common ancestral species related to the Orchidioides clade.
The multiple copies of the ITS region found in eukaryotic
genomes may be a problem when using these data for phyloge-
netic inferences, since it is not certain that the sequences
achieved are orthologues (Miranda et al., 2010). Nonetheless,
despite the multiple copies, concerted evolution occurs when
sequence differences among copies in the same genome
become homogenized to the same sequence by mechanisms
such as high-frequency unequal crossing-over and gene conver-
sion (�Alvarez and Wendel, 2003). Considering that the impor-
tance of the ITS region for phylogenetic inferences is mainly
due to its high signal, which can be used to solve the phylogeny
of closely related taxa (Hillis and Dixon, 1991), the results pre-
sented in this study are very important. However, further analy-
ses with the use of different nuclear markers should address this
issue. The topologies presented by the ITS analysis and the
combined analysis provide phylogenetic support for the recog-
nition of U. cornigera as a species.
Distribution of morphological characters
Taylor (1986, 1989) used three main characters for justify-
ing the splitting of sect. Orchidioides and for the creation of
sect. Iperua: the crest on the lower lip of the corolla and the
morphology of the seeds and pollen. The pollen has already
been studied in the species of these sections by Huynh
(1968), who placed U. alpina, U. praetermissa, U. jamesoni-
ana and U. humboldtii into one group, similar to our molecu-
lar results, which included U. humboldtii in sect. Orchidioides
(Fig. 3, clade A).
The characteristic crest on the lower lip of the corolla
(Fig. 4A) is exclusive to sect. Iperua (Taylor, 1989). Since our
Rodrigues et al. — Phylogeny of the ‘orchid-like’ bladderworts (Lentibulariaceae) Page 7 of 15
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Deleted Text: section
Deleted Text: the
Deleted Text: &hx2013;
Deleted Text: section
Deleted Text: Utricularia
Deleted Text: section
Deleted Text: ion
Deleted Text:
Deleted Text: crossing
Deleted Text: be assured
Deleted Text: cannot
Deleted Text:
Deleted Text: p
Deleted Text: remained
Deleted Text: section
Deleted Text: Utricularia
Deleted Text:
Deleted Text:
Deleted Text: -
Deleted Text: the section
Deleted Text: Utricularia
Deleted Text: ial
Deleted Text:
Deleted Text:
Deleted Text: -
Deleted Text: -
Deleted Text: &hx201D;
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Deleted Text: -
Deleted Text:
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Deleted Text: to
Deleted Text: as
Deleted Text: -
Rodrigues et al. — Phylogeny of the ‘orchid-like’ bladderworts (Lentibulariaceae)716
U. asplundii
Character present
A
C
B
Character absent
Equivocal
U. quelchii
U. praetermissa
U. cornigera
U. endresii
U. alpina
U. humboldtii
U. geminiloba
U. reniformis
U. nelumbifolia
U. nephrophylla
U. praelonga
U. hispida
U. longifolia
U. calycifida
U. huntii
U. tridentata
U. amethystina
U. tricolor
U. australis
U. macrorhiza
U. vulgaris
U. intermedia
U. minor
U. aurea
U. asplundii
U. quelchii
U. praetermissa
U. cornigera
U. endresii
U. alpina
U. humboldtii
U. geminiloba
U. reniformis
U. nelumbifolia
U. nephrophylla
U. praelonga
U. hispida
U. longifolia
U. huntii
U. calycifida
U. tridentata
U. amethystina
U. tricolor
U. australis
U. macrorhiza
U. vulgaris
U. intermedia
U. minor
U. aurea
FIG. 4. Distribution of morphological characters based on the Bayesian combined tree. (A) Crest on the lower lip of the corolla. Primary organs in the embryo (B)
and tubers (C). Photo credit: Barry Rice, detail in (B).
Page 8 of 15 Rodrigues et al. — Phylogeny of the ‘orchid-like’ bladderworts (Lentibulariaceae)
molecular results showed that U. humboldtii is closely related
to the clade of sect. Orchidioides (clade A), this character
becomes worthless as a diagnostic feature for this section.
For the seeds and embryos, Taylor (1989) observed that
those from sect. Orchidioides are more uniform, which is
markedly different from the situation in sect. Iperua: in the lat-
ter, U. humboldtii and U. nelumbifolia form a thin and
transparent testa, with chlorophyllose embryos having numer-
ous primary organs (Goebel, 1891; Merl, 1915; Lloyd, 1942;
and see arrow in Fig. 4B). The primary organs of the embryos
of U. nelumbifolia and U. reniformis A.St.-Hil. ‘Enfant
Terrible’ were studied by Płachno and �Swią tek (2010). In U.
nelumbifolia, the primary organs are homologous to those
found in the seedlings of sect. Utricularia; similar to the ‘leaf’
A
100 µm
50 µm 100 µm
100 µm
200 µm 20 µm
B
C D
E F
FIG. 5. Anatomy and histochemistry of Utricularia reniformis stolons. (A, B) General stolon anatomy. Note the parenchymatous cortex (Pc), which surrounds the
ectophloic central cylinder (CC). pf, phloem; x, xylem. Scale bar ¼ 100 mm. (C) Reaction for lipids (Sudan IV). Arrows indicate small lipid droplets. Scale bar ¼
50 mm. (D) Section after IKI treatment. Note the lack of starch grains. Scale bar ¼ 100 mm. (E) Section treated with ruthenium red for pectins and mucilage. Note
the positive pectin reaction in cell walls. Scale bar ¼ 200 mm. (F) Semithin section. Note the giant vacuoles and the nucleus with paracrystalline protein inclusions.
Scale bar ¼ 20 mm.
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Rodrigues et al. — Phylogeny of the ‘orchid-like’ bladderworts (Lentibulariaceae) 717
U. asplundii
Character present
A
C
B
Character absent
Equivocal
U. quelchii
U. praetermissa
U. cornigera
U. endresii
U. alpina
U. humboldtii
U. geminiloba
U. reniformis
U. nelumbifolia
U. nephrophylla
U. praelonga
U. hispida
U. longifolia
U. calycifida
U. huntii
U. tridentata
U. amethystina
U. tricolor
U. australis
U. macrorhiza
U. vulgaris
U. intermedia
U. minor
U. aurea
U. asplundii
U. quelchii
U. praetermissa
U. cornigera
U. endresii
U. alpina
U. humboldtii
U. geminiloba
U. reniformis
U. nelumbifolia
U. nephrophylla
U. praelonga
U. hispida
U. longifolia
U. huntii
U. calycifida
U. tridentata
U. amethystina
U. tricolor
U. australis
U. macrorhiza
U. vulgaris
U. intermedia
U. minor
U. aurea
FIG. 4. Distribution of morphological characters based on the Bayesian combined tree. (A) Crest on the lower lip of the corolla. Primary organs in the embryo (B)
and tubers (C). Photo credit: Barry Rice, detail in (B).
Page 8 of 15 Rodrigues et al. — Phylogeny of the ‘orchid-like’ bladderworts (Lentibulariaceae)
molecular results showed that U. humboldtii is closely related
to the clade of sect. Orchidioides (clade A), this character
becomes worthless as a diagnostic feature for this section.
For the seeds and embryos, Taylor (1989) observed that
those from sect. Orchidioides are more uniform, which is
markedly different from the situation in sect. Iperua: in the lat-
ter, U. humboldtii and U. nelumbifolia form a thin and
transparent testa, with chlorophyllose embryos having numer-
ous primary organs (Goebel, 1891; Merl, 1915; Lloyd, 1942;
and see arrow in Fig. 4B). The primary organs of the embryos
of U. nelumbifolia and U. reniformis A.St.-Hil. ‘Enfant
Terrible’ were studied by Płachno and �Swią tek (2010). In U.
nelumbifolia, the primary organs are homologous to those
found in the seedlings of sect. Utricularia; similar to the ‘leaf’
A
100 µm
50 µm 100 µm
100 µm
200 µm 20 µm
B
C D
E F
FIG. 5. Anatomy and histochemistry of Utricularia reniformis stolons. (A, B) General stolon anatomy. Note the parenchymatous cortex (Pc), which surrounds the
ectophloic central cylinder (CC). pf, phloem; x, xylem. Scale bar ¼ 100 mm. (C) Reaction for lipids (Sudan IV). Arrows indicate small lipid droplets. Scale bar ¼
50 mm. (D) Section after IKI treatment. Note the lack of starch grains. Scale bar ¼ 100 mm. (E) Section treated with ruthenium red for pectins and mucilage. Note
the positive pectin reaction in cell walls. Scale bar ¼ 200 mm. (F) Semithin section. Note the giant vacuoles and the nucleus with paracrystalline protein inclusions.
Scale bar ¼ 20 mm.
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Deleted Text: &hx201D;
Deleted Text: &hx201C;
Deleted Text: section
Deleted Text: &hx2013;
Deleted Text: Utricularia
Deleted Text: ion
Deleted Text: of the
Deleted Text: os
Deleted Text: ab
Deleted Text: re
Deleted Text: ion
Deleted Text: the
Deleted Text: section
Rodrigues et al. — Phylogeny of the ‘orchid-like’ bladderworts (Lentibulariaceae)718
A
100 µm 100 µm
100 µm 100 µm
100 µm
100 µm 20 µm
100 µm
B
C D
E F
G H
FIG. 6. Anatomy and histochemistry of Utricularia geminiloba tubers. (A–C) General tuber anatomy. Note the parenchymatous cortex (Pc), which surrounds the
ectophloic central cylinder (CC). pf, phloem; x, xylem. Scale bar ¼ 100 mm. (D) Reaction for lipids (Sudan IV). Note positive reaction in barrier cell of the trichome
(arrow). Scale bar ¼ 100 mm. (E) Reaction for lipids (Sudan IV). Scale bar ¼ 100 mm. (F) Section after IKI treatment. Note the lack of starch grains, but the positive
reaction for protein in the nuclei and trichome. Scale bar¼ 100 mm. (G) Section treated with ruthenium red for pectins and mucilage. Note (arrow) the positive pectin
reaction in cell walls. Scale bar¼ 100 mm. (H) Semithin section. Note the giant vacuoles and the nucleus with paracrystalline protein inclusion. Scale bar ¼ 20 mm.
Page 10 of 15 Rodrigues et al. — Phylogeny of the ‘orchid-like’ bladderworts (Lentibulariaceae)
structure, but with the likely function of nutrient absorption
from the environment. These numerous primary embryo organs
are adaptations for germination in bromeliad tanks (Studni�cka,
2009, 2011). Płachno and �Swią tek (2010) also disagreed with
sect. Iperua, as they verified that the embryos of U. nelumbifo-
lia and U. reniformis sensu stricto had similar structures, unlike
U. humboldtii. In the last species, the basal part of the embryo
is not so prominent and the primary organs dominate (Goebel,
1893). In any case, the germination pattern of U. reniformis
‘Enfant Terrible’ is different from that of U. reniformis sensu
stricto (Goebel, 1893; Merl, 1925), U. humboldtii and U.
nelumbifolia. The embryo in U. nephrophylla does not have
A B
100 µm
100 µm
100 µm
100 µm
100 µm
C D
E F
FIG. 7. Anatomy and histochemistry of Utricularia nelumbifolia stolons. (A, B) General stolon anatomy. Note the parenchymatous cortex (Pc), which surrounds the
ectophloic central cylinder (CC). pf, phloem; x, xylem; La, lacunae. Scale bar ¼ 100 mm. (C) Autofluorescence of tissues under UV light. Pc, parenchymatous cor-
tex; CC, central cylinder (CC). xylem. Arrow indicates xylem. Scale bar ¼ 100 mm. (D) Negative reaction for lipids (Sudan III). Scale bar ¼ 100 mm. (E) Reaction
for lipids (Sudan IV). Note the positive reaction in cuticle of epidermal cells and the barrier cell of the trichome. tc, terminal cell; bc, basal cell. Arrow indicates a
barrier cell. Scale bar ¼ 50 mm. (F) Section after IKI treatment. Note the lack of starch grains. Scale bar ¼ 100 mm.
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Rodrigues et al. — Phylogeny of the ‘orchid-like’ bladderworts (Lentibulariaceae) 719
A
100 µm 100 µm
100 µm 100 µm
100 µm
100 µm 20 µm
100 µm
B
C D
E F
G H
FIG. 6. Anatomy and histochemistry of Utricularia geminiloba tubers. (A–C) General tuber anatomy. Note the parenchymatous cortex (Pc), which surrounds the
ectophloic central cylinder (CC). pf, phloem; x, xylem. Scale bar ¼ 100 mm. (D) Reaction for lipids (Sudan IV). Note positive reaction in barrier cell of the trichome
(arrow). Scale bar ¼ 100 mm. (E) Reaction for lipids (Sudan IV). Scale bar ¼ 100 mm. (F) Section after IKI treatment. Note the lack of starch grains, but the positive
reaction for protein in the nuclei and trichome. Scale bar¼ 100 mm. (G) Section treated with ruthenium red for pectins and mucilage. Note (arrow) the positive pectin
reaction in cell walls. Scale bar¼ 100 mm. (H) Semithin section. Note the giant vacuoles and the nucleus with paracrystalline protein inclusion. Scale bar ¼ 20 mm.
Page 10 of 15 Rodrigues et al. — Phylogeny of the ‘orchid-like’ bladderworts (Lentibulariaceae)
structure, but with the likely function of nutrient absorption
from the environment. These numerous primary embryo organs
are adaptations for germination in bromeliad tanks (Studni�cka,
2009, 2011). Płachno and �Swią tek (2010) also disagreed with
sect. Iperua, as they verified that the embryos of U. nelumbifo-
lia and U. reniformis sensu stricto had similar structures, unlike
U. humboldtii. In the last species, the basal part of the embryo
is not so prominent and the primary organs dominate (Goebel,
1893). In any case, the germination pattern of U. reniformis
‘Enfant Terrible’ is different from that of U. reniformis sensu
stricto (Goebel, 1893; Merl, 1925), U. humboldtii and U.
nelumbifolia. The embryo in U. nephrophylla does not have
A B
100 µm
100 µm
100 µm
100 µm
100 µm
C D
E F
FIG. 7. Anatomy and histochemistry of Utricularia nelumbifolia stolons. (A, B) General stolon anatomy. Note the parenchymatous cortex (Pc), which surrounds the
ectophloic central cylinder (CC). pf, phloem; x, xylem; La, lacunae. Scale bar ¼ 100 mm. (C) Autofluorescence of tissues under UV light. Pc, parenchymatous cor-
tex; CC, central cylinder (CC). xylem. Arrow indicates xylem. Scale bar ¼ 100 mm. (D) Negative reaction for lipids (Sudan III). Scale bar ¼ 100 mm. (E) Reaction
for lipids (Sudan IV). Note the positive reaction in cuticle of epidermal cells and the barrier cell of the trichome. tc, terminal cell; bc, basal cell. Arrow indicates a
barrier cell. Scale bar ¼ 50 mm. (F) Section after IKI treatment. Note the lack of starch grains. Scale bar ¼ 100 mm.
Rodrigues et al. — Phylogeny of the ‘orchid-like’ bladderworts (Lentibulariaceae) Page 11 of 15
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lio de M
esquita Filho user on 23 April 2019
Deleted Text: latter
Deleted Text: ion
Deleted Text: the
Rodrigues et al. — Phylogeny of the ‘orchid-like’ bladderworts (Lentibulariaceae)720
A B
C D
100 µm
100 µm 100 µm
100 µm
FIG. 8. Anatomy and histochemistry of Utricularia alpina tubers. (A, B) General tuber anatomy. Note the parenchymatous cortex (Pc), which surrounds the ecto-
phloic central cylinder (CC). Scale bar ¼ 100 mm. (C) Section after IKI treatment, showing positive reaction for protein in nuclei. Scale bar ¼ 100 mm. (D) Reaction
for lipids (Sudan IV). Scale bar ¼ 100 mm.
A
B
C
FIG. 9. Habit of Utricularia reniformis (A) and tuber of Utricularia geminiloba before (B) and after (C) a 30-d experiment to verify the photosynthetic function. L,
leaf; E, stolon; T, tuber. Photo credit for (A): David Banks. Scale bar ¼ 5mm.
Page 12 of 15 Rodrigues et al. — Phylogeny of the ‘orchid-like’ bladderworts (Lentibulariaceae)
primordial ‘leaves’ (Merl, 1925) and/or, as in U. geminiloba,
they are not well enough developed (Taylor, 1989) or are not
seen (Płachno and �Swią tek, 2010). The newly described spe-
cies U. cornigera (Studni�cka, 2009) has green embryos with
numerous primary organs, unlike the ‘true’ U. reniformis.
Tubers occur as an important, specialized water storage
organ in all species of sect. Orchidioides as these species are
epiphytes (Taylor, 1989). In sect. Iperua tubers are only present
in U. geminiloba, which is found as a lithophyte or a terrestrial.
Despite the differences between these life forms, the epiphytic
and lithophytic habitats may represent similar restrictions, with
both having harsh and highly variable seasonal conditions.
Utricularia geminiloba is commonly found on granitic walls
with little organic matter (Taylor, 1989; V.F.O. Miranda, pers.
observ.) and thus the tubers are an important water source in
dry seasons.
Taylor (1989) observed great similarity between the tubers,
and the stolons of U. reniformis (Fig. 7), which are thick, with-
out constriction and almost like very elongated tubers. The
author did not, however, obtain any further information about
these structures.
Function and evolution of the stolon–tuber system: are there
differences between stolons and tubers?
The anatomy of the thick stolons of U. reniformis and U. gem-
iniloba tubers resembles the anatomy of the stolons of U. alpina
(Brugger and Rutishauser, 1989) and U. longifolia (Rutishauser
and Isler, 2001), but in this case with a more developed cortex.
Unlike our observation of U. reniformis and U. geminiloba, the
thick stolons of U. humboldtii (Brugger and Rutishauser, 1989)
and U. nelumbifolia have many lacunae (Fig. 7). Our observation
agrees with that of Adlassnig et al. (2005), in that the tubers of
U. alpina are constituted of developed parenchyma cells forming
a giant vacuole. Darwin (1875) also analysed the tubers of U.
alpina and did not find starch, and suggested their function was
water storage (Juniper et al., 1989).
Taylor (1989) recognized that the tubers in the species of
sect. Orchidioides worked like orchid pseudobulbs, which
ensures the species’ survival in dry periods. Due to this hydric
stress and the species growing far above the soil, the acquisition
and storage of water are the main abiotic factors for the growth
of epiphytic species, whereas the availability of nutrients and
solar irradiance remain secondary concerns (Zotz and Hietz,
2001; Laube and Zotz, 2003). Thus, in most epiphytes there are
adaptive structures and pseudobulbs (Orchidaceae), velamen
(Orchidaceae, Araceae, Bromeliaceae), and also leaf trichomes
(Bromeliaceae), which facilitate the absorption of water
(Benzing and Sheemann, 1978). Also, when exposed to light,
the tubers and stolons can play an important role as photosyn-
thetic organs (Fig. 9B, C).
In sect. Iperua, U. geminiloba is a terrestrial and lithophytic
species that is often found on moist walls and can survive dry
periods, like U. reniformis, which is terrestrial, epiphytic and
also lithophytic. According to Taylor (1989), it is not always
certain whether an Utricularia species is a holoepiphyte or a
facultative or accidental epiphyte, as some species were rarely
observed in their natural habitat. For example, U. alpina,
although usually an epiphyte, occasionally grows on the ground
(Taylor, 1989).
From this study it is possible to infer the close relationship
between tubers and thick stolons as a crucial survival strategy
for epiphytes and lithophytes. Despite this, the stolon–tuber
system is linked with the species of sections Iperua and
Orchidioides; other sections within the genus Utricularia also
have species with tubers or tuber-like thick stolons (e.g. in sec-
tions Aranella, Chelidon, Phyllaria, Pleiochasia and
Utricularia), and there is one species of Genlisea (Rivadavia
et al., 2013). In these taxa there may also be a nutrient storage
function, particularly for species found in poor and seasonally
stressful environments. For example, U. menziesii has a dor-
mancy period throughout the hot, dry Australian summer
(Taylor, 1989). The tubers of this species may play an impor-
tant role in carbohydrate storage (Rice, 2011), but further stud-
ies with histological analysis are necessary to prove this
assumption. In this way, it is possible that the stolon–tuber sys-
tem, with its primary function being water storage, originated
independently (as homoplasies) within the genus Utricularia as
an adaptation to the hydric deficits, with a common occurrence
in the Orchidioides–Iperua complex.
Conclusions
Our phylogenetic analyses included U. humboldtii and U.
cornigera in sect. Orchidioides and therefore do not support the
separation of these sections made by Taylor (1986, 1989). In
addition, the stolons and tubers of the species in sect. Iperua
have anatomical patterns similar to those of the Orchidioides
tubers and perform the same primary function as water storage
organs, with a potentially common origin. Our results therefore
support the joining of these sections into one section called
Orchidioides, as suggested by Müller and Borsch (2005).
ACKNOWLEDGEMENTS
We dedicate our paper to Dr Miloslav Studni�cka (Liberec
Botanic Garden, Czech Republic), who has been involved in
studying Utricularia in the Orchidioides and Iperua sections
for many years. Thanks are due to Drs Carlos Rohrbacher
(Brazil) and Barry Rice (USA), who provided part of the plant
material, and to Martin Hingst, Nicole Rebbert, Barry Rice,
David Banks and Ron Lane for kindly providing the pictures
of some species. Thanks to Drs Nilber Gonçalves da Silva and
Yani Cristina Aranguren D�ıaz for helping during the field
trips, and for fruitful discussions. Thanks are also due to horti-
culturist Lucyna Kurleto for her conscientious care of the col-
lection of carnivorous plants in the Botanical Garden of
Jagiellonian University in Krak�ow, Poland. Special thanks are
due to Dr Brian G. McMillan (University of Strathclyde,
Glasgow) for the correction of the language. This work was
supported by CAPES (Coordenaç~ao de Aperfeiçoamento de
Pessoal de N�ıvel Superior) (fellowships for F.G.R., N.F.M.
and S.R.S.); Conselho Nacional de Desenvolvimento
Cient�ıfico e Tecnol�ogico (CNPq) (Bolsa de Produtividade,
grant number 309040/2014-0 for V.F.O.M.); and partly (for
L.A.) by the Research Programme of the Czech Academy of
Sciences (RVO 67985939).
Rodrigues et al. — Phylogeny of the ‘orchid-like’ bladderworts (Lentibulariaceae) Page 13 of 15
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esquita Filho user on 23 April 2019
Rodrigues et al. — Phylogeny of the ‘orchid-like’ bladderworts (Lentibulariaceae) 721
A B
C D
100 µm
100 µm 100 µm
100 µm
FIG. 8. Anatomy and histochemistry of Utricularia alpina tubers. (A, B) General tuber anatomy. Note the parenchymatous cortex (Pc), which surrounds the ecto-
phloic central cylinder (CC). Scale bar ¼ 100 mm. (C) Section after IKI treatment, showing positive reaction for protein in nuclei. Scale bar ¼ 100 mm. (D) Reaction
for lipids (Sudan IV). Scale bar ¼ 100 mm.
A
B
C
FIG. 9. Habit of Utricularia reniformis (A) and tuber of Utricularia geminiloba before (B) and after (C) a 30-d experiment to verify the photosynthetic function. L,
leaf; E, stolon; T, tuber. Photo credit for (A): David Banks. Scale bar ¼ 5mm.
Page 12 of 15 Rodrigues et al. — Phylogeny of the ‘orchid-like’ bladderworts (Lentibulariaceae)
primordial ‘leaves’ (Merl, 1925) and/or, as in U. geminiloba,
they are not well enough developed (Taylor, 1989) or are not
seen (Płachno and �Swią tek, 2010). The newly described spe-
cies U. cornigera (Studni�cka, 2009) has green embryos with
numerous primary organs, unlike the ‘true’ U. reniformis.
Tubers occur as an important, specialized water storage
organ in all species of sect. Orchidioides as these species are
epiphytes (Taylor, 1989). In sect. Iperua tubers are only present
in U. geminiloba, which is found as a lithophyte or a terrestrial.
Despite the differences between these life forms, the epiphytic
and lithophytic habitats may represent similar restrictions, with
both having harsh and highly variable seasonal conditions.
Utricularia geminiloba is commonly found on granitic walls
with little organic matter (Taylor, 1989; V.F.O. Miranda, pers.
observ.) and thus the tubers are an important water source in
dry seasons.
Taylor (1989) observed great similarity between the tubers,
and the stolons of U. reniformis (Fig. 7), which are thick, with-
out constriction and almost like very elongated tubers. The
author did not, however, obtain any further information about
these structures.
Function and evolution of the stolon–tuber system: are there
differences between stolons and tubers?
The anatomy of the thick stolons of U. reniformis and U. gem-
iniloba tubers resembles the anatomy of the stolons of U. alpina
(Brugger and Rutishauser, 1989) and U. longifolia (Rutishauser
and Isler, 2001), but in this case with a more developed cortex.
Unlike our observation of U. reniformis and U. geminiloba, the
thick stolons of U. humboldtii (Brugger and Rutishauser, 1989)
and U. nelumbifolia have many lacunae (Fig. 7). Our observation
agrees with that of Adlassnig et al. (2005), in that the tubers of
U. alpina are constituted of developed parenchyma cells forming
a giant vacuole. Darwin (1875) also analysed the tubers of U.
alpina and did not find starch, and suggested their function was
water storage (Juniper et al., 1989).
Taylor (1989) recognized that the tubers in the species of
sect. Orchidioides worked like orchid pseudobulbs, which
ensures the species’ survival in dry periods. Due to this hydric
stress and the species growing far above the soil, the acquisition
and storage of water are the main abiotic factors for the growth
of epiphytic species, whereas the availability of nutrients and
solar irradiance remain secondary concerns (Zotz and Hietz,
2001; Laube and Zotz, 2003). Thus, in most epiphytes there are
adaptive structures and pseudobulbs (Orchidaceae), velamen
(Orchidaceae, Araceae, Bromeliaceae), and also leaf trichomes
(Bromeliaceae), which facilitate the absorption of water
(Benzing and Sheemann, 1978). Also, when exposed to light,
the tubers and stolons can play an important role as photosyn-
thetic organs (Fig. 9B, C).
In sect. Iperua, U. geminiloba is a terrestrial and lithophytic
species that is often found on moist walls and can survive dry
periods, like U. reniformis, which is terrestrial, epiphytic and
also lithophytic. According to Taylor (1989), it is not always
certain whether an Utricularia species is a holoepiphyte or a
facultative or accidental epiphyte, as some species were rarely
observed in their natural habitat. For example, U. alpina,
although usually an epiphyte, occasionally grows on the ground
(Taylor, 1989).
From this study it is possible to infer the close relationship
between tubers and thick stolons as a crucial survival strategy
for epiphytes and lithophytes. Despite this, the stolon–tuber
system is linked with the species of sections Iperua and
Orchidioides; other sections within the genus Utricularia also
have species with tubers or tuber-like thick stolons (e.g. in sec-
tions Aranella, Chelidon, Phyllaria, Pleiochasia and
Utricularia), and there is one species of Genlisea (Rivadavia
et al., 2013). In these taxa there may also be a nutrient storage
function, particularly for species found in poor and seasonally
stressful environments. For example, U. menziesii has a dor-
mancy period throughout the hot, dry Australian summer
(Taylor, 1989). The tubers of this species may play an impor-
tant role in carbohydrate storage (Rice, 2011), but further stud-
ies with histological analysis are necessary to prove this
assumption. In this way, it is possible that the stolon–tuber sys-
tem, with its primary function being water storage, originated
independently (as homoplasies) within the genus Utricularia as
an adaptation to the hydric deficits, with a common occurrence
in the Orchidioides–Iperua complex.
Conclusions
Our phylogenetic analyses included U. humboldtii and U.
cornigera in sect. Orchidioides and therefore do not support the
separation of these sections made by Taylor (1986, 1989). In
addition, the stolons and tubers of the species in sect. Iperua
have anatomical patterns similar to those of the Orchidioides
tubers and perform the same primary function as water storage
organs, with a potentially common origin. Our results therefore
support the joining of these sections into one section called
Orchidioides, as suggested by Müller and Borsch (2005).
ACKNOWLEDGEMENTS
We dedicate our paper to Dr Miloslav Studni�cka (Liberec
Botanic Garden, Czech Republic), who has been involved in
studying Utricularia in the Orchidioides and Iperua sections
for many years. Thanks are due to Drs Carlos Rohrbacher
(Brazil) and Barry Rice (USA), who provided part of the plant
material, and to Martin Hingst, Nicole Rebbert, Barry Rice,
David Banks and Ron Lane for kindly providing the pictures
of some species. Thanks to Drs Nilber Gonçalves da Silva and
Yani Cristina Aranguren D�ıaz for helping during the field
trips, and for fruitful discussions. Thanks are also due to horti-
culturist Lucyna Kurleto for her conscientious care of the col-
lection of carnivorous plants in the Botanical Garden of
Jagiellonian University in Krak�ow, Poland. Special thanks are
due to Dr Brian G. McMillan (University of Strathclyde,
Glasgow) for the correction of the language. This work was
supported by CAPES (Coordenaç~ao de Aperfeiçoamento de
Pessoal de N�ıvel Superior) (fellowships for F.G.R., N.F.M.
and S.R.S.); Conselho Nacional de Desenvolvimento
Cient�ıfico e Tecnol�ogico (CNPq) (Bolsa de Produtividade,
grant number 309040/2014-0 for V.F.O.M.); and partly (for
L.A.) by the Research Programme of the Czech Academy of
Sciences (RVO 67985939).
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esquita Filho user on 23 April 2019
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Rodrigues et al. — Phylogeny of the ‘orchid-like’ bladderworts (Lentibulariaceae)722
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