Annals of Botany 120:

Page 709–723,
1 of 15 2017
doi:10.1093/aob/mcx056, available online at www.academic.oup.com/aob
https://academic.oup.com/aob

PART OF A SPECIAL ISSUE ON MORPHOLOGY AND ADAPTATION

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,*
1
Universidade Estadual Paulista (Unesp), Faculdade de Ci^ encias Agr�arias e Veterin�
arias, Jaboticabal, Departamento de
Biologia Aplicada a� Agropecu� ao Paulo, Brazil, 2Universidade Estadual Paulista (Unesp), Instituto de Bioci^
aria, S~ encias,
ao Paulo, Brazil, 3Department of Plant Cytology and Embryology, Jagiellonian University in Krak�
Botucatu, S~ 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 [email protected]

Received: 25 February 2017 Returned for revision: 22 March 2017 Editorial Decision: 2 April 2017 Accepted: 13 April
April 2017
2017
Published electronically: 30 June 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 The nine species of Utricularia sect. Orchidioides A.DC. are

distributed in central America, the Antilles and South America
Lentibulariaceae is a cosmopolitan family presenting the great- and are orchid-like bladderworts (Fig. 1A-B, F). Moreover,
est diversity in species, habit and life form among carnivorous they are perennial epiphytes or terrestrials, with a tuber ensem-
plants. Around 350 species are distributed across the three gen- ble in the peduncle basis (Fig. 9B, C). On the other hand,
era Pinguicula, Genlisea and Utricularia; Pinguicula is the sis- Utricularia sect. Iperua P.Taylor has six species distributed in
ter group of the clade formed by Genlisea and Utricularia South America (Fig. 1C-E, G). These are lithophytes and terres-
(Jobson et al., 2003; Müller et al., 2004; Guisande et al., 2007; trial or aquatic epiphytes (U. nelumbifolia and U. humboldtii
Veleba et al., 2014). The genus Utricularia forms traps from are examples of the latter) and the majority of them form fleshy
little vesicles called utricles or bladders, which have an active stolons (Fig. 9A), except for U. geminiloba, which forms tubers
suction mechanism triggered when the trichomes near (Fig. 9B, C) similar to Orchidioides. The two sections have
their entrance are stimulated by small organisms (Poppinga very similar trap and calyx morphologies (Taylor, 1989).
et al., 2016). Based on the vegetative morphology, Taylor De Candolle (1844) created sect. Orchidioides, which
(1989) split the genus Utricularia in into two subgenera: included species with tubers. Kamie�nski (1895) later expanded
Polypompholyx and Utricularia. Nevertheless there are interest- this section and included non-tuberous species such as
ing, controversial proposals regarding the classification of the Utricularia nelumbifolia and Utricularia reniformis. Barnhart
two very close infrageneric taxa: sections Orchidioides and (1916), on the other hand, proposed the new genus Orchyllium to
Iperua. aggregate the species, with U. alpina (as Orchyllium alpinum) as

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Rodrigues et al. —
— Phylogeny
Phylogeny of the ‘orchid-like’ bladderworts
bladderworts (Lentibulariaceae)
(Lentibulariaceae)

A D E

C G

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.

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 Rodrigues et al. — Phylogeny
Phylogeny of
of the
the ‘orchid-like’
‘orchid-like’bladderworts
bladderworts(Lentibulariaceae)
(Lentibulariaceae) Page 3 of711
15

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

1
Orchidioides U. asplundii (TS000261) AF4825581 AF4826311 KY68970 KY689711
U. quelchii1 (TS000260) – KY689702 AF5318461 –
U. endresii1 (TS000262) – AF4826421 KY799062 KY689709
U. alpina1 (TS000263) AF4825561 AF4826291 AF5318221 KY689712
U. praetermissa1 (TS000264) – KY689703 KY689698 KY689705
Iperua U. humboldtii2 (TS000199) – KY689704 AF5318361 –
U. geminiloba1 (VFOM2045) – AF4826461 KX6042161 KY689716
U. nephrophylla1 (VFOM2047) AF4825881 AF4826641 AF5318271 KY689707
U. reniformis (VFOM2044) AF4825951 AF4826711 KX6042181 KY689706
U. nelumbifolia1 (VFOM2055) AF4825861 AF4826621 KX6042171 KY689708
U. cornigera1 (TS000265) – KY689701 KY689699 KY689710
Utricularia U. aurea3 (TS000267) AF4825591 AF4826321 KX6041761 KY689714
U. australis AF4825601 AF4826331 AF5318231 –
U. intermedia AF4825751 AF4826511 AF5318391 –
U. macrorhiza3 (TS000266) AF4825811 AF4826571 AF5318351 KY689719
U. minor3 (TS000268) – GU1697061 JN8940281 KY689721
U. vulgaris3 (TS000269) – JQ7289941 JN8940541 KY689722
Psyllosperma U. huntii AF4825741 AF4826501 – –
U. praelonga AF4825911 AF4826671 AF5318431 –
U. longifolia1 (VFOM1680) AF4825801 AF4826561 AF5318341 KY689718
U. hispida1 (VFOM1637) – – AF5318291 KY689717
U. calycifida – – AF5318241 –
Foliosa U. tricolor1 (VFOM2043) AF4826001 AF4826771 KX6042101 KY689720
U. tridentata – – AF5318251 –
U. amethystina1 (VFOM1644) AF4825571 AF4826301 – KY689713

1–3
Samples 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, Trebo
n, Czech Republic (vouchers deposited in Herbarium JABU – University of Sao Paulo
State – UNESP/FCAV).

the type species. Huynh (1968) questioned sect. Orchidioides, recently described Utricularia cornigera Studnicka, a species
since it included species of different groups based on pollen char- with a morphological similarity to sect. Iperua (Studnicka,
acters. Taylor (1986), based on the morphological differences of 2009). We also conducted a phylogenetic analysis of the
the corolla, seeds and pollen, split sect. Orchidioides and pro- following morphological characteristics: (1) the crest on the
posed sect. Iperua, with Utricularia humboldtii Schomb. as the lower lip of the corolla; (2) primary organs in the embryo; and
type species. (3) the presence of tubers. We conducted a histochemical analy-
There are interesting discussions concerning the generic mor- sis, with a discussion of the function and evolution of these
phology of Utricularia and whether or not the species have a organs.
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, MATERIALS AND METHODS
it is not an easy task to classify species by means of morpholog-
ical characters. Taylor (1989), in his monograph, raises some Plant samples and DNA markers
doubts about Utricularia morphology. In the Orchidioides and Plant samples from 20 species were obtained from both natural
Iperua sections specifically, U. reniformis was questioned as it populations and cultivated plants and DNA sequences were
has thick, tuber-like stolons. also obtained from GenBank/NCBI (Table 1). In accordance
Recent molecular studies have also considered these sections with previous studies, four DNA sequences were selected as
using a phylogenetic approach. Jobson et al. (2003) show the markers due to their phylogenetic signal: (1) rps16 (Oxelman
monophyly of the Iperua and Orchidioides sections with the et al., 1997; Jobson and Albert 2002; Jobson et al., 2003); (2)
plastid sequences rps16 and trnL-F; Müller and Borsch (2005), trnL-F (Taberlet et al., 1991; Jobson and Albert 2002; Jobson
on the other hand, proposed the exclusion of sect. Iperua on the et al., 2003); (3) matK (Müller and Borsch, 2005; Silva et al.,
basis of the plastid intron trnK with the matK gene. 2016); and (4) the ITS region (Hillis and Dixon, 1991). We
Thus, our aim was to test the hypothesis of grouping the sec- therefore obtained a total of 76 sequences, of which 26 were
tions Orchidioides and Iperua by studying a broad range of spe- produced in this study, with sequences for five species from
cies; we evaluated 11 of the 15 that have been described. A Utricularia sect. Orchidioides (which has a total of nine
large number of molecular markers from two genomic compart- described species) and six for sect. Iperua, including
ments [plastid DNA sequences rps16, trnL-F and matK and Utricularia cornigera, thus representing all known species of
nuclear internal transcribed spacer (ITS) region] and relevant this section. In addition, 14 species from other sections were
morphological characters were evaluated. We also included the used as an external group (Table 1).

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Rodrigues et al. —
— Phylogeny
Phylogeny of the ‘orchid-like’ bladderworts
bladderworts (Lentibulariaceae)
(Lentibulariaceae)

Amplification and sequencing between the sections and have therefore been employed histori-
The DNA was extracted by the CTAB method (Doyle and cally in the taxonomic circumscription of Orchidioides and
Doyle, 1987), modified by Lodhi et al. (1994). Amplification Iperua. These were: (1) a crest on the protuberance of the lower
reactions of the nuclear markers were conducted in 25 lL of a lip of the corolla (Fig. 1C, E); (2) the presence of tubers
solution containing 20 mM MgCl2, 100 mM dNTPs, 10 mM of (Taylor, 1986, 1989); and (3) the primary organs in the embryo,
� ˛tek (2010). A morphological
according to Płachno and Swia
each primer, 1 U of Dream Taq Polymerase (Fermentas), and
on average 50 ng of DNA template. For the ITS region, the pri- matrix was generated using the NDE Nexus Data Editor pro-
mers and dimethyl sulphoxide adjuvant (DMSO) were used as gram (Page, 2001), which overlapped the combined (total evi-
recommended by Miranda et al. (2010). For matK and trnL-F dence) Bayesian inference tree, for which the Mesquite
the primers employed were according Lim et al. (2012) and program (Maddison and Maddison, 2010) was used.
Taberlet et al. (1991), respectively.
The thermal profile of the amplification reactions for the
intergenic spacer trnL-F was 95 � C for 3 min; 30 cycles of Histochemistry of storage organs and tissues
1 min at 94 � C, 45 s at 52 � C and 1 min at 72 � C, and 5 min of To achieve better comprehension and comparison of storage
final extension at 72 � C. For the gene matK, it was 94 � C for tissues, we conducted histochemical analyses of the stolons of
1 min, 35 cycles of 40 s at 94 � C, 20 s at 52 � C and 50 s at 72 � C, Utricularia reniformis and Utricularia nelumbifolia (sect.
and 10 min of final extension at 72 � C. For the nuclear ITS, it Iperua) and the tubers of Utricularia geminiloba (sect. Iperua)
was 95 � C for 3 min, 35 cycles of 30 s at 95 � C, 30 s at 54 � C and Utricularia alpina (sect. Orchidioides). The stolons of U.
and 1 min at 72 � C, and 5 min of final extension at 72 � C. reniformis and U. nelumbifolia and tubers of U. geminiloba
The amplicons were verified by 0�8 % agarose gel electro- were collected from natural populations in December 2015 or
phoresis, precipitated with 100 % isopropanol, purified with were taken from a collection in the Botanic Gardens of
70 % ethanol and sequenced by the method developed by Jagiellonian University in Krak�ow, Poland, and the vouchers
Sanger et al. (1975) in an automatic sequencer, model 3730xl were deposited in Herbarium JABU (V.F.O. de Miranda et al.,
ABI (Applied Biosystems). 2044, 2055 and 2045, respectively).
The following reagents were used to show the general anat-
Sequences and phylogenetic analyses omy and the storage components: IKI (iodine–potassium iodide)
for starch þ proteins; saturated ethanolic solutions of Sudan III
The identity of each sequence was determined with the and Sudan IV for lipids; alum carmine and iodine green for lig-
BLASTN application (Altschul et al., 1990). Using Geneious nin þ cellulose; and 0�1 % (w/v) ruthenium red for pectin and
v10.0 (Kearse et al., 2012), their Phred quality was verified and mucilage (Filutowicz and Ku_zdowicz 1951; Ruzin 1999).
visual and manual adjustments were made with the program The material (thick stolons of U. reniformis and U. nelumbifo-
BioEdit v7.5.0.2 (Hall, 1999). MAFFT v7 (Katoh et al., 2002) lia and tubers of U. geminiloba and U. alpina) was fixed in
was used to align the sequences, and datasets (matrices) were 2�5 % (v/v) glutaraldehyde/2�5 % (v/v) formaldehyde in 0�05 M
created with BioEdit v7.5.0.2 (Hall, 1999) with the addition of sodium cacodylate buffer (pH 7�0) for several days, washed
masks (missing data ¼ ‘?’) (Wiens, 2006). three times in the same buffer and postfixed in 1% (w/v)
We produced five datasets: four for each isolated marker and osmium tetroxide solution for 1�5 h at 0 � C. This was followed
one for a combined (total evidence) analysis. For the matK frag- by dehydration using a graded ethanol series and infiltration and
ment, the sequences obtained from GenBank (Table 1) were embedding using an epoxy embedding medium kit (Fluka).
trimmed to achieve a homologue region according to the ampli- Semithin sections (0�9–1�0 mm) prepared for light microscopy
fied sequences by the 3F_KIM and 1R_KIM primers (Lim were stained for general histology using aqueous methylene
et al., 2012). blue/azure II (MB/AII) for 1–2 min (Humphrey and Pittman,
In each dataset generated, a Bayesian inference on platform 1974) and examined with an Olympus BX60 optical microscope.
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 Photosynthetic function of stolons and tubers
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 To verify the presence of chloroplasts and possible photosyn-
that for the nuclear spacer was the TIM3 þ I þ G model. thetic activity in stolons and tubers, these organs were used in a
Additionally, we performed maximum likelihood (ML) analysis glasshouse experiment. Three stolon fragments (�2 cm) of U.
with the CIPRES platform (Miller et al., 2010) and the reniformis and three tubers of U. geminiloba were placed in
PAUP*4.0 program (Swofford, 2003) to get bootstrap values Petri dishes with moist absorbent paper and stored at 25 � C
(2000 pseudoreplicates and heuristic search with 1000 replicates under natural light conditions for 30 d. Photographs of the frag-
with random addition of sequences and the branch swapping ments and tubers were then taken.
algorithm TBR). The trees obtained were edited with TreeGraph
2 (Stöver, 2010) and FigTree v1.3.1 (Rambaut, 2009).
RESULTS
Morphological characters Phylogeny of Utricularia sections Orchidioides and Iperua
We conducted phylogenetic testing of the three morphologi- The phylogenetic trees from the Bayesian inference and ML
cal characteristics that supposedly emphasize the differences criteria are congruent in their general topology (Figs 2 and 3)

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 Rodrigues et al. — Phylogeny
Phylogeny of
of the
the ‘orchid-like’
‘orchid-like’bladderworts
bladderworts(Lentibulariaceae)
(Lentibulariaceae) Page 5 of713
15

A
A rps16
rps16 B
B trnL-F
trnL-F
50/71 U. praetermissa
U. praetermissa
100/100
100/100 U. alpina
U. alpina 50/71
Orchidioides
Orchidioides U. endresii
U. endresii
U. asplundii
U. asplundii 100/76 70/90
100/76 Orchidioides
Orchidioides
90/100
90/100 70/90 U. quelchii
U. quelchii
100/100
100/100 U. nelumbifolia
U. nelumbifolia 80/79
80/79 U. asplundii
U. asplundii
100/100
100/100 Iperua
Iperua U. alpina
U. alpina
U. reniformis
U. reniformis
U. humboldtii
U. humboldtii
90/100
90/100 U. nephrophylla
nephrophylla
U. 100/98
100/98 50/81 U. nelumbifolia
U. nelumbifolia
50/81
100/99 70/-
70/- U. reniformis
U. reniformis
100/99 U. longifolia
U. longifolia
100/81
100/81 U. cornigera
U. cornigera Iperua
Iperua
U. praelonga
U. praelonga Psyllosperma
Psyllosperma 100/100
100/100
80/81
80/81 U. geminiloba
U. geminiloba
U. huntii
U. huntii U. nephrophylla
U. nephrophylla
100/100
100/100
U. amethystina
amethystina 100/100
100/100 U. praelonga
U. praelonga
70/-
70/- U.
Foliosa
Foliosa 90/94
90/94 U. longifolia
U. longifolia
U. tricolor
U. tricolor Psyllosperma
Psyllosperma
100/- U. huntii
U. huntii
100/-
100/100
100/100 U. australis
U. australis 50/-
50/- U. amethystina
U. amethystina
U. tricolor
tricolor
Foliosa
Foliosa
90/69
90/69 U.
U. macrorhiza
U. macrorhiza
50/-
50/- U. australis
U. australis
U. intermedia
U. intermedia Utricularia
Utricularia 90/87
90/87 U. vulgaris
U. vulgaris
U. aurea
U. aurea 100/98
100/98 U. macrorhiza
U. macrorhiza
U. minor
minor
Utricularia
Utricularia
U.
U. intermedia
U. intermedia
U. aurea
U. aurea

C
C matK
matK 80/55
80/55 U. quelchii
U. quelchii D
D ITS
ITS
60/-
60/- U. alpina
U. alpina
U. alpina
U. alpina
60/60
60/60 U. endresii
U. endresii Orchidioides
Orchidioides
U. praetermissa
U. praetermissa Orchidioides
Orchidioides 90/97
90/97
80/60
80/60 100/100
100/100 U. praetermissa
U. praetermissa
U. endresii
U. endresii
80/87
80/87 50/56
50/56
U. asplundii
U. asplundii U. cornigera
U. cornigera Iperua
Iperua
U. humboldtii
U. humboldtii 50/-
50/- U. asplundii
U. asplundii Orchidioides
Orchidioides
90/95
90/95 U. geminiloba
U. geminiloba 70/55
70/55 U. geminiloba
U. geminiloba
70/54
70/54
U. nelumbifolia
U. nelumbifolia 90/61
90/61
100/97
100/97 U. nelumbifolia
U. nelumbifolia
100/69
100/69 Iperua
Iperua 100/100 Iperua
Iperua
U. reniformis
U. reniformis 100/100
100/100
100/100 U. reniformis
U. reniformis
U. cornigera
U. cornigera
100/100
100/100 U. nephrophylla
U. nephrophylla
U. nephrophylla
U. nephrophylla
100/100
100/100 U. longifolia
U. longifolia
U. longifolia
U. longifolia
100/97
100/97 Psyllosperma
Psyllosperma
U. hispida
hispida U. hispida
U. hispida
100/100
100/100 U.
100/88
100/88 U. praelonga
U. praelonga Psyllosperma
Psyllosperma 100/100
100/100 U. amethystina
U. amethystina
U. calycifida
U. calycifida U. tricolor
U. tricolor Foliosa
Foliosa
100/94
100/94
100/100
100/100 U. tricolor
U. tricolor U. minor
U. minor
Foliosa
Foliosa 100/99
100/99
U. tridentata
U. tridentata U. macrorhiza
macrorhiza
100/100
100/100 U.
70/-
70/- U. minor
U. minor
U. vulgaris
U. vulgaris Utricularia
Utricularia
U. intermedia
U. intermedia
100/56
100/56 U. aurea
U. aurea
50/-
50/- U. macrorhiza
U. macrorhiza Utricularia
Utricularia
100/-
100/- U. australis
U. australis
U. vulgaris
U. vulgaris
U. aurea
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.

and showed that most groups were supported by posterior prob- be included in sect. Iperua as a sister group to the clade
abilities (PPs) and ML bootstraps (> = 50%) (Table 2). U. nelumbifolia–U. reniformis. With this marker, the ML analy-
In the analysis of intron rps16, in which the sequences of the sis showed the species U. nelumbifolia, U. reniformis, U.
studied sections taken from the study of Jobson and Albert cornigera and U. geminiloba as a monophyletic group (Fig.
(2002) were used, a similar result, congruent to Taylor’s classi- 2B). U. humboldtii has a similar position, being nested as an
fication (1989), was found (Fig. 2A). The other plastid markers external branch of the clade formed by U. praetermissa, U.
(trnL-F and matK; Fig. 2B, C) show sect. Iperua as a paraphy- endresii, U. quelchii, U. asplundii and U. alpina. The matK tree
letic group, by the inclusion of U. humboldtii (taxonomically (Fig. 2C) revealed that U. humboldtii is closely related to sect.
recognized as belonging to sect. Iperua) in sect. Orchidioides. Orchidioides as a sister group, because a similar topology to
Furthermore, the sequences trnL-F and matK placed U. corni- the trnL-F dataset was found (Fig. 2B).
gera in sect. Iperua. The tree obtained with the spacer trnL-F Additionally, while the plastid markers trnL-F and matK
(Fig. 2B) revealed that U. humboldtii is a sister group to the showed U. cornigera nested in the sect. Iperua clade, the
other species in sect. Orchidioides and that U. cornigera should ITS dataset (Fig. 2D) showed this species grouped in

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— Phylogeny
Phylogeny of the ‘orchid-like’ bladderworts
bladderworts (Lentibulariaceae)
(Lentibulariaceae)

Taylor (1989) This study

Clade A 77/- U. asplundii
53/- U. quelchii Orchidioides
100/100 U. praetermissa
100/99
U. cornigera Iperua

Orchidioides
100/99 66/- U. endresii
Orchidioides
U. alpina

100/100 U. humboldtii

Clade B 100/100 U. geminiloba

100/100 U. reniformis Iperua
100/100
U. nelumbifolia
100/100 U. nephrophylla
100/100 U. praelonga
100/100 U. hispida
U. longifolia Psyllosperma
100/90
U. calycifida
91/94 U. huntii
100/100 U. tridentata
U. amethystina Foliosa

U. tricolor
92/63 U. australis
100/99 U. macrorhiza
100/100 U. vulgaris
Utricularia
79/89 U. intermedia
U. minor
U. aurea

FIG. 3. Bayesian inference for the combined analysis (rps16 þ trnL-F þ matK þ 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- sect. Orchidioides as a sister group of U. praetermissa, with
dograms inferred by maximum likelihood (ML) and Bayesian maximum support values from both analyses (100 % for PP
inference (BI) and ML bootstrap). Sections Iperua and Orchidioides were
shown as paraphyletic (Fig. 2D), and U. humboldtii is missing
Dataset Genome Terminals, Characters Clades with in this analysis.
n considering support �50 %, The tree resulting from the concatenated datasets presents
gaps, bp n (%)1
sect. Iperua as a paraphyletic group, which shows U. humboldtii
Posterior Bootstraps as an external branch of sect. Orchidioides (Fig. 3, clade A).
probability (BI) (ML) Utricularia cornigera is shown in the clade of sect.
rps16 Plastid 14 926 11 (84) 10 (77)
Orchidioides, as a monophyletic group with U. asplundii,
trnL-trnF Plastid 22 1�091 16 (76) 12 (57) U. quelchii and U. praetermissa, but this clade is not strongly
matK2 Plastid 23 883 19 (86) 16 (72) supported (PP 53 % and ML bootstrap <50 %). Despite this low
ITS Nucleus 17 962 12 (75) 10 (62) support, the clade of sect. Orchidioides, which includes U. cor-
Combined Nucleus þ 25 3�864 20 (83) 17 (71) nigera, is strongly supported by the Bayesian inference and ML
Plastid
bootstrap, with 100 and 99 % confidence, respectively (Fig. 3).
1
Percentage of clades was calculated from the total of possible tree clades
(¼ terminal numbers�1).
2
Sequences obtained from GenBank (see Table 1) were trimmed to achieve Distribution of morphological characters
a homologous region according to the fragment amplified by the 3F_KIM and
1R_KIM primers (Lim et al., 2012). 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

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Phylogeny of
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the ‘orchid-like’
‘orchid-like’bladderworts
bladderworts(Lentibulariaceae)
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the combined analysis, the crest on the corolla has possibly stolons that reach and grow between bromeliad leaves (Taylor,
appeared at least twice. It was once present in the ancestral line- 1989). While the species of sect. Iperua are distributed in South
age of Orchidioides–Iperua complex (clade A þ clade B), America, mainly in Southern and Southeastern Brazil (BFG,
which was lost by an ancestor of the clade Iperua only to arise 2015; Miranda et al., 2015), U. humboldtii is the only species
again as a reversion in U. cornigera. The character of the tubers of this section that occurs in the Guiana Highlands. Thus, clades
was also shown to be homoplastic, with two independent ori- A and B (Fig. 3) are also supported by species distribution
gins: one in sect. Orchidioides, with the reversion to U. (with the exception of U. cornigera, which is endemic to
cornigera, which lacks tubers, and the other in U. geminiloba. Southeastern Brazil) (BFG, 2015; Miranda et al., 2015).
The characteristic primary embryo organs (Fig. 4B) occur in U.
cornigera (Studnicka, 2009), U. humboldtii, U. nelumbifolia
and species of sect. Utricularia (Płachno and Swia  ˛tek, 2010 Utricularia cornigera Studni
cka
and references therein). The lack of information regarding the
In our analysis with the plastid markers trnL-F (Fig. 2B) and
embryology of several species makes it difficult to trace a
matK (Fig. 2C), U. cornigera is nested into sect. Iperua and
robust and well-supported hypothesis for the evolution of this
related to the clade U. reniformis–U. nelumbifolia and also
character.
nested with U. geminiloba, despite the incongruence found with
this species when considering both markers. The hypothesis of
Histological analysis of stolons and tubers a natural hybrid, originating from the crossing between U. reni-
formis and U. nelumbifolia (Fleischmann, 2012), has been
In our analyses, the thick stolons of U reniformis (Figs 5 and refuted by Studnicka (2013, 2015) by an interbreeding
9A) and U. nelumbifolia (Fig. 7) and the tubers of U. gemini- experiment.
loba (Figs 6 and 9B, C) and U. alpina (Fig. 8) have similar However, in the ITS analysis U. cornigera is included in
anatomy. In general, as shown in the transverse sections, the sect. Orchidioides, with maximum statistical support in both
epidermis and parenchymatous cortex surround the ectophloic phylogenetic analyses (PP and ML), and it also remains in the
central cylinder. Only the stolons of U. nelumbifolia have many Orchidioides in the concatenated tree (Fig. 3). While the mater-
lacunae (Fig. 7A–D). The cortex consists of large parenchyma nal DNA (chloroplast DNA in this case) supports a close rela-
cells with large vacuoles; the xylem and phloem elements are tionship with the species of sect. Iperua, the ITS region, which
separated from each other. In both organs small epidermal is of biparental origin, suggests that U. cornigera has the same
trichomes occur, each consisting of one basal cell, one short, common ancestral species related to the Orchidioides clade.
central cell and a long-headed cell (Fig. 7E). The multiple copies of the ITS region found in eukaryotic
Very small and infrequent lipid droplets were observed in the genomes may be a problem when using these data for phyloge-
cytoplasm of the cortical cells (Fig. 5C). Cell walls stained with netic inferences, since it is not certain that the sequences
the ruthenium red (Figs 5E and 6G) showed no mucilage and achieved are orthologues (Miranda et al., 2010). Nonetheless,
those stained with IKI showed no starch or storage proteins despite the multiple copies, concerted evolution occurs when
(Figs 5D, 6F, 7F and 8C). Paracrystalline protein inclusions sequence differences among copies in the same genome
were occasionally present in the nuclei of various cell types become homogenized to the same sequence by mechanisms
(Figs 5F and 6H). The cell walls of the trichome barrier cell such as high-frequency unequal crossing-over and gene conver-
stained selectively with Sudan (Figs 6D and 7E). 
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
DISCUSSION
of closely related taxa (Hillis and Dixon, 1991), the results pre-
Phylogeny of Orchidioides–Iperua complex: one section is sented in this study are very important. However, further analy-
enough ses with the use of different nuclear markers should address this
issue. The topologies presented by the ITS analysis and the
Our results support the paraphyly of Utricularia sect. Iperua
combined analysis provide phylogenetic support for the recog-
and Orchidioides, considering U. cornigera (Figs 2 and 3). The
nition of U. cornigera as a species.
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.
Distribution of morphological characters
(2004) based on matK and the flanking trnK intron, and later
Müller and Borsch (2005) suggested acceptance of only sect. Taylor (1986, 1989) used three main characters for justify-
Orchidioides, underpinning De Candolle (1844) and Kamienski ing the splitting of sect. Orchidioides and for the creation of
(1895). De Candolle based his system on only three species of sect. Iperua: the crest on the lower lip of the corolla and the
the complex: U. alpina (as U. montana) and U. unifolia, classi- morphology of the seeds and pollen. The pollen has already
fied as sect. Orchidioides and, curiously, U. humboldtii, which been studied in the species of these sections by Huynh
was maintained in his ‘Species dubiae’ section. (1968), who placed U. alpina, U. praetermissa, U. jamesoni-
Utricularia humboldtii (Fig. 1C), a perennial and one of the ana and U. humboldtii into one group, similar to our molecu-
largest terrestrial species of the genus, produces spectacular lar results, which included U. humboldtii in sect. Orchidioides
flowers, which is a common trait of the Orchidioides–Iperua (Fig. 3, clade A).
complex. This species is found as a terrestrial or even as an The characteristic crest on the lower lip of the corolla
aquatic epiphyte, since the plants can project aerial horizontal (Fig. 4A) is exclusive to sect. Iperua (Taylor, 1989). Since our

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— Phylogeny
Phylogeny of the ‘orchid-like’ bladderworts
bladderworts (Lentibulariaceae)
(Lentibulariaceae)

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. calycifida
U. huntii
U. tridentata
U. amethystina
U. tricolor
U. australis
U. macrorhiza
U. vulgaris
U. intermedia
A U. minor B
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. calycifida
U. huntii
U. tridentata
U. amethystina Character present
U. tricolor
Character absent
U. australis
U. macrorhiza Equivocal
U. vulgaris
C 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).

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A B

100 µm 100 µm

C D

50 µm 100 µm

E F

200 µm 20 µm

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.

molecular results showed that U. humboldtii is closely related transparent testa, with chlorophyllose embryos having numer-
to the clade of sect. Orchidioides (clade A), this character ous primary organs (Goebel, 1891; Merl, 1915; Lloyd, 1942;
becomes worthless as a diagnostic feature for this section. and see arrow in Fig. 4B). The primary organs of the embryos
For the seeds and embryos, Taylor (1989) observed that of U. nelumbifolia and U. reniformis A.St.-Hil. ‘Enfant
those from sect. Orchidioides are more uniform, which is 
Terrible’ were studied by Płachno and Swia˛tek (2010). In U.
markedly different from the situation in sect. Iperua: in the lat- nelumbifolia, the primary organs are homologous to those
ter, U. humboldtii and U. nelumbifolia form a thin and found in the seedlings of sect. Utricularia; similar to the ‘leaf’

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(Lentibulariaceae)

A B

100 µm 100 µm

C D

100 µm 100 µm

E F

100 µm
100 µm

G H

100 µm 20 µm

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 bar ¼ 100 mm. (G) Section treated with ruthenium red for pectins and mucilage. Note (arrow) the positive pectin
reaction in cell walls. Scale bar ¼ 100 mm. (H) Semithin section. Note the giant vacuoles and the nucleus with paracrystalline protein inclusion. Scale bar ¼ 20 mm.

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A B

100 µm 100 µm

C D

100 µm 100 µm

E F

100 µm

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.

structure, but with the likely function of nutrient absorption U. humboldtii. In the last species, the basal part of the embryo
from the environment. These numerous primary embryo organs is not so prominent and the primary organs dominate (Goebel,
are adaptations for germination in bromeliad tanks (Studnicka, 1893). In any case, the germination pattern of U. reniformis

2009, 2011). Płachno and Swia˛tek (2010) also disagreed with ‘Enfant Terrible’ is different from that of U. reniformis sensu
sect. Iperua, as they verified that the embryos of U. nelumbifo- stricto (Goebel, 1893; Merl, 1925), U. humboldtii and U.
lia and U. reniformis sensu stricto had similar structures, unlike nelumbifolia. The embryo in U. nephrophylla does not have

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(Lentibulariaceae)

A B

100 µm 100 µm

C D

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 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 5 mm.

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15

primordial ‘leaves’ (Merl, 1925) and/or, as in U. geminiloba, although usually an epiphyte, occasionally grows on the ground
they are not well enough developed (Taylor, 1989) or are not (Taylor, 1989).
�
seen (Płachno and Swia˛tek, 2010). The newly described spe- From this study it is possible to infer the close relationship
cies U. cornigera (Studni�cka, 2009) has green embryos with between tubers and thick stolons as a crucial survival strategy
numerous primary organs, unlike the ‘true’ U. reniformis. for epiphytes and lithophytes. Despite this, the stolon–tuber
Tubers occur as an important, specialized water storage system is linked with the species of sections Iperua and
organ in all species of sect. Orchidioides as these species are Orchidioides; other sections within the genus Utricularia also
epiphytes (Taylor, 1989). In sect. Iperua tubers are only present have species with tubers or tuber-like thick stolons (e.g. in sec-
in U. geminiloba, which is found as a lithophyte or a terrestrial. tions Aranella, Chelidon, Phyllaria, Pleiochasia and
Despite the differences between these life forms, the epiphytic Utricularia), and there is one species of Genlisea (Rivadavia
and lithophytic habitats may represent similar restrictions, with et al., 2013). In these taxa there may also be a nutrient storage
both having harsh and highly variable seasonal conditions. function, particularly for species found in poor and seasonally
Utricularia geminiloba is commonly found on granitic walls stressful environments. For example, U. menziesii has a dor-
with little organic matter (Taylor, 1989; V.F.O. Miranda, pers. mancy period throughout the hot, dry Australian summer
observ.) and thus the tubers are an important water source in (Taylor, 1989). The tubers of this species may play an impor-
dry seasons. tant role in carbohydrate storage (Rice, 2011), but further stud-
Taylor (1989) observed great similarity between the tubers, ies with histological analysis are necessary to prove this
and the stolons of U. reniformis (Fig. 7), which are thick, with- assumption. In this way, it is possible that the stolon–tuber sys-
out constriction and almost like very elongated tubers. The tem, with its primary function being water storage, originated
author did not, however, obtain any further information about independently (as homoplasies) within the genus Utricularia as
these structures. an adaptation to the hydric deficits, with a common occurrence
in the Orchidioides–Iperua complex.

Function and evolution of the stolon–tuber system: are there Conclusions

differences between stolons and tubers? Our phylogenetic analyses included U. humboldtii and U.
The anatomy of the thick stolons of U. reniformis and U. gem- cornigera in sect. Orchidioides and therefore do not support the
iniloba tubers resembles the anatomy of the stolons of U. alpina separation of these sections made by Taylor (1986, 1989). In
(Brugger and Rutishauser, 1989) and U. longifolia (Rutishauser addition, the stolons and tubers of the species in sect. Iperua
and Isler, 2001), but in this case with a more developed cortex. have anatomical patterns similar to those of the Orchidioides
Unlike our observation of U. reniformis and U. geminiloba, the tubers and perform the same primary function as water storage
thick stolons of U. humboldtii (Brugger and Rutishauser, 1989) organs, with a potentially common origin. Our results therefore
and U. nelumbifolia have many lacunae (Fig. 7). Our observation support the joining of these sections into one section called
agrees with that of Adlassnig et al. (2005), in that the tubers of Orchidioides, as suggested by Müller and Borsch (2005).
U. alpina are constituted of developed parenchyma cells forming
a giant vacuole. Darwin (1875) also analysed the tubers of U. ACKNOWLEDGEMENTS
alpina and did not find starch, and suggested their function was
water storage (Juniper et al., 1989). We dedicate our paper to Dr Miloslav Studni�cka (Liberec
Taylor (1989) recognized that the tubers in the species of Botanic Garden, Czech Republic), who has been involved in
sect. Orchidioides worked like orchid pseudobulbs, which studying Utricularia in the Orchidioides and Iperua sections
ensures the species’ survival in dry periods. Due to this hydric for many years. Thanks are due to Drs Carlos Rohrbacher
stress and the species growing far above the soil, the acquisition (Brazil) and Barry Rice (USA), who provided part of the plant
and storage of water are the main abiotic factors for the growth material, and to Martin Hingst, Nicole Rebbert, Barry Rice,
of epiphytic species, whereas the availability of nutrients and David Banks and Ron Lane for kindly providing the pictures
solar irradiance remain secondary concerns (Zotz and Hietz, of some species. Thanks to Drs Nilber Gonçalves da Silva and
2001; Laube and Zotz, 2003). Thus, in most epiphytes there are Yani Cristina Aranguren D�ıaz for helping during the field
adaptive structures and pseudobulbs (Orchidaceae), velamen trips, and for fruitful discussions. Thanks are also due to horti-
(Orchidaceae, Araceae, Bromeliaceae), and also leaf trichomes culturist Lucyna Kurleto for her conscientious care of the col-
(Bromeliaceae), which facilitate the absorption of water lection of carnivorous plants in the Botanical Garden of
(Benzing and Sheemann, 1978). Also, when exposed to light, Jagiellonian University in Krak�ow, Poland. Special thanks are
the tubers and stolons can play an important role as photosyn- due to Dr Brian G. McMillan (University of Strathclyde,
thetic organs (Fig. 9B, C). Glasgow) for the correction of the language. This work was
In sect. Iperua, U. geminiloba is a terrestrial and lithophytic supported by CAPES (Coordenaç~ao de Aperfeiçoamento de
species that is often found on moist walls and can survive dry Pessoal de N�ıvel Superior) (fellowships for F.G.R., N.F.M.
periods, like U. reniformis, which is terrestrial, epiphytic and and S.R.S.); Conselho Nacional de Desenvolvimento
also lithophytic. According to Taylor (1989), it is not always Cient�ıfico e Tecnol�ogico (CNPq) (Bolsa de Produtividade,
certain whether an Utricularia species is a holoepiphyte or a grant number 309040/2014-0 for V.F.O.M.); and partly (for
facultative or accidental epiphyte, as some species were rarely L.A.) by the Research Programme of the Czech Academy of
observed in their natural habitat. For example, U. alpina, Sciences (RVO 67985939).

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Phylogeny of the ‘orchid-like’ bladderworts
bladderworts (Lentibulariaceae)
(Lentibulariaceae)

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