Structural Features of Carnivorous Plant (Genlisea, Utricularia) Tubers as Abiotic Stress Resistance Organs

Carnivorous plants from the Lentibulariaceae form a variety of standard and novel vegetative organs and survive unfavorable environmental conditions. Within Genlisea, only G. tuberosa, from the Brazilian Cerrado, formed tubers, while Utricularia menziesii is the only member of the genus to form seasonally dormant tubers. We aimed to examine and compare the tuber structure of two taxonomically and phylogenetically divergent terrestrial carnivorous plants: Genlisea tuberosa and Utricularia menziesii. Additionally, we analyzed tubers of U. mannii. We constructed phylogenetic trees using chloroplast genes matK/trnK and rbcL and used studied characters for ancestral state reconstruction. All examined species contained mainly starch as histologically observable reserves. The ancestral state reconstruction showed that specialized organs such as turions evolved once and tubers at least 12 times from stolons in Lentibulariaceae. Different from other clades, tubers probably evolved from thick stolons for sect. Orchidioides and both structures are primarily water storage structures. In contrast to species from section Orchidioides, G. tuberosa, U. menziesii and U. mannii form starchy tubers. In G. tuberosa and U. menziesii, underground tubers provide a perennating bud bank that protects the species in their fire-prone and seasonally desiccating environments.

Hibernacula occur in temperate species like Pinguicula vulgaris, P. grandiflora and P. alpina. The hibernaculum consists of an abbreviated stem-bearing scales and leaf primordia. Reserves (starch)

Genlisea tuberosa Rivadavia, Gonella & A.Fleischm.
Up to three tubers occur per plant. The tubers were white-yellow in color, ovoid-shaped ( Figure 1A-C). The tuber is attached to the plant by a stalk (Figure 2A). At the tuber pole, where there was stalk, the developing primordia of new organs were observed in the bud ( Figure 2B). The tuber surface was covered by mucilage, debris and fungal hyphae ( Figure 2C,D). Small epidermal trichomes occurred, each consisting of one basal cell, one short, pedestal cell and a head cell ( Figure 2C-E). The lateral wall of the pedestal cell was impregnated by cutin ( Figure 2E). The head cell was globular shaped. These trichomes produced mucilage. Stomata were present on the tuber surface ( Figure 2C,F). Both open and closed stomata were observed ( Figure 2F). As shown in the transverse sections, the tuber was round ( Figure 3A,B). The parenchymatous cortex was well developed. Vascular bundles (about 10) formed a ring, with centrally located additional vascular bundles ( Figure 3B-D) that showed evidence of branching within the tuber. Within these vascular bundles, there were two groups of phloem cells near the xylem. The xylem is composed of one or two tracheary elements with evident vessels. The vasculature associated with the central part of pith comprised a vessel surrounded by radially elongated parenchymatous cells ( Figure 3D). In the pith, there were intercellular spaces. Parenchyma cells of both cortex and pith were highly vacuolated and rich in large starch grains (SGs) ( Figure 3E). Smaller SGs also occurred in the epidermal cells ( Figure 3F, Table 1). Small SGs were in parenchyma cells of vascular bundles ( Figure 4A). SGs, in parenchyma cells of both cortex and pith, were simple ( Figure 4C), but in epidermal cells, mixed configurations comprising compound and simple grains co-occurred in the same cell ( Figure 4D). Protein storage vacuoles were recorded in the pith near the place where the new bud was formed ( Figure 4B). Ruthenium red stained cell walls of parenchyma ( Figure 4E), but also head cells of trichomes ( Figures 2D and 4E), indicating pectins and mucilage. The PAS (periodic acid-Schiff) reaction revealed that material secreted by trichomes was most likely a polysaccharide ( Figure 4F).

Utricularia menziesii R.Br.
The tubers were white, turgid, ovoid ( Figure 5B,C) attached to a highly abbreviated stem. Old tubers (from the previous season) were flattened and comprised no obvious intact storage cells ( Figures 5C and 6A). The tuber was attached to the plant with a stalk, at the other tuber pole no buds or other organs were found. The tuber surface was covered by mucilage, debris and fungal hyphae, nevertheless, no trichomes were recorded ( Figure 6B). As shown in the transverse sections, the tuber was round ( Figure 6C,D) with a well-developed parenchymatous cortex. There was no clear border between the cortex and pith. Vascular tissues: phloem and xylem were located centrally in the organ ( Figure 6C,D). Xylem with one tracheary element and a vessel element surrounded by phloem ( Figure 6E,F). Although the xylem and phloem elements were distant from each other, this structure may be treated as a single vascular bundle. Parenchyma cells were highly vacuolated and contained small SGs ( Figure 7A,B, Table 1). There were uniform simple and compound SGs ( Figure 7A). More SGs occurred in parenchyma which surrounded the vascular tissues ( Figure 7B). Mucilage in parenchyma cells was absent, and ruthenium red staining only occurred in the cell walls ( Figure 7C,D).      The tubers were white, turgid, ovoid (Figures 5B-C) attached to a highly abbreviated stem. Old tubers (from the previous season) were flattened and comprised no obvious intact storage cells ( Figures 6E-F). Although the xylem and phloem elements were distant from each other, this structure may be treated as a single vascular bundle. Parenchyma cells were highly vacuolated and contained small SGs ( Figures 7A-B, Table 1). There were uniform simple and compound SGs ( Figure  7A). More SGs occurred in parenchyma which surrounded the vascular tissues ( Figure 7B). Mucilage in parenchyma cells was absent, and ruthenium red staining only occurred in the cell walls ( Figures 7C-D).

Utricularia Mannii Oliv. (Figures 8-9)
The tubers were green in color, obovoid to globose ( Figure 8A). Epidermal trichomes were numerous ( Figure 8B), each consisting of one basal cell, one short, pedestal cell and a head cell ( Figure 8C). The lateral wall of the pedestal cell was impregnated by cutin ( Figures 8B-C). The head cell was strongly elongated ( Figure 8C). As shown in the transverse sections, the epidermis and parenchymatous cortex surrounded a large central cylinder ( Figure 8D). Vascular bundles formed a ring, with centrally located additional vascular bundles that were larger than the perimeter bundles ( Figures 8E-F). Xylem elements were surrounded by phloem ( Figure 8F). Chloroplasts occurred in epidermal cells and parenchyma ( Figure 9A). Intercellular spaces were well developed and were present both in the cortex and pith ( Figure 9B). Parenchyma cells were highly vacuolated and contained small SGs ( Figure 9C, Table 1). There were uniform simple SGs and also compound. The stolon had a similar vascular anatomy to the tuber ( Figure 9D) though restricted to just one vascular bundle ( Figure 9D).

Utricularia mannii Oliv.
The tubers were green in color, obovoid to globose ( Figure 8A). Epidermal trichomes were numerous ( Figure 8B), each consisting of one basal cell, one short, pedestal cell and a head cell ( Figure 8C). The lateral wall of the pedestal cell was impregnated by cutin ( Figure 8B,C). The head cell was strongly elongated ( Figure 8C). As shown in the transverse sections, the epidermis and parenchymatous cortex surrounded a large central cylinder ( Figure 8D). Vascular bundles formed a ring, with centrally located additional vascular bundles that were larger than the perimeter bundles ( Figure 8E,F). Xylem elements were surrounded by phloem ( Figure 8F). Chloroplasts occurred in epidermal cells and parenchyma ( Figure 9A). Intercellular spaces were well developed and were present both in the cortex and pith ( Figure 9B). Parenchyma cells were highly vacuolated and contained small SGs ( Figure 9C, Table 1). There were uniform simple SGs and also compound. The stolon had a similar vascular anatomy to the tuber ( Figure 9D) though restricted to just one vascular bundle ( Figure 9D).

Phylogenetic Analyses (Figure 10)
The concatenated matrix comprehends an alignment of 212 DNA sequences of different species of Lentibulariaceae: 20 of Genlisea, 126 of Utricularia, and 66 sequences of Pinguicula used as the outgroup. The analyses were performed in 4012 characters of which 1755 were parsimony-informative.
Tubers with carbohydrate content evolved at least five times independently in the Lentibulariaceae: in Genlisea tuberosa, Utricularia menziesii, U. mannii lineages (Figure 10, outer tree), U. brachiata and U. inflata. In addition, water storage is found as a synapomorphy, thus only derived once with thick stolons and tubers in sect. Orchidioides.
In addition, ancestral character tracing ( Figure 10) shows that turions have appeared as a novel adaptation at least once in Utricularia, within the species of sect Utricularia from temperate climates.

Phylogenetic Analyses
The concatenated matrix comprehends an alignment of 212 DNA sequences of different species of Lentibulariaceae: 20 of Genlisea, 126 of Utricularia, and 66 sequences of Pinguicula used as the outgroup. The analyses were performed in 4012 characters of which 1755 were parsimony-informative.
Tubers with carbohydrate content evolved at least five times independently in the Lentibulariaceae: in Genlisea tuberosa, Utricularia menziesii, U. mannii lineages (Figure 10, outer tree), U. brachiata and U. inflata. In addition, water storage is found as a synapomorphy, thus only derived once with thick stolons and tubers in sect. Orchidioides.
In addition, ancestral character tracing ( Figure 10) shows that turions have appeared as a novel adaptation at least once in Utricularia, within the species of sect Utricularia from temperate climates. Figure 10. Phylogenetic trees were reconstructed with the Maximum Likelihood method. The inner tree and outer tree indicate the ancestral character states reconstructed according to parsimony criteria. The inner tree shows the possible ancestral character states of tubers, thick stolons and turions (orange, purple and green branch color, respectively). Branch numbers correspond to ultrafast bootstrap values. The outer tree shows the possible ancestral character states of the tuber and thick stolons content: carbohydrate in red; water in blue; dashed lines correspond to unknown data. The names of species are colored according to life cycle: purple for perennial, orange for annual and green for annual and/or perennial species according to Taylor [14] and Fleischmann [34]. I and II denote potential points of tuber appearance. Pinguicula species were used as the outgroup.

Discussion
Darwin [30] examined tubers of Utricularia alpina (as Utricularia montana) and concluded that this species accumulates water that enable the plant to survive seasonal drought. This was later confirmed by Adlassnig [35] and Rodrigues et al. [21]. In addition, other species from sect. Orchidioides produce water-storing tubers or thick stolons [21]. Compton [36] studied tubers of U. brachiata (sect. Phyllaria) and found tuber parenchyma contained starch grains (SG). He concluded the water-storing function is a secondary with the primary function being carbohydrate storage. We found that Genlisea tuberosa, Utricularia menziesii and U. mannii form carbohydrate-rich tubers. SGs exhibited different morphologies and sizes depending on the species. The largest SGs occurred in parenchyma cells of Genlisea tuberosa tubers. The highest number of SGs was recorded in Utricularia menziesii. Accumulation of SGs was recorded also in other Lentibulariaceae perennating organs such as Pinguicula hibernacula ([9]; Płachno unp.) and Utricularia turions (e.g., [19]). Carbohydrate-rich tubers were recorded in the Lentibulariaceae in Genlisea tuberosa and four species of Utricularia that Figure 10. Phylogenetic trees were reconstructed with the Maximum Likelihood method. The inner tree and outer tree indicate the ancestral character states reconstructed according to parsimony criteria. The inner tree shows the possible ancestral character states of tubers, thick stolons and turions (orange, purple and green branch color, respectively). Branch numbers correspond to ultrafast bootstrap values. The outer tree shows the possible ancestral character states of the tuber and thick stolons content: carbohydrate in red; water in blue; dashed lines correspond to unknown data. The names of species are colored according to life cycle: purple for perennial, orange for annual and green for annual and/or perennial species according to Taylor [14] and Fleischmann [34]. I and II denote potential points of tuber appearance. Pinguicula species were used as the outgroup.

Discussion
Darwin [30] examined tubers of Utricularia alpina (as Utricularia montana) and concluded that this species accumulates water that enable the plant to survive seasonal drought. This was later confirmed by Adlassnig [35] and Rodrigues et al. [21]. In addition, other species from sect. Orchidioides produce water-storing tubers or thick stolons [21]. Compton [36] studied tubers of U. brachiata (sect. Phyllaria) and found tuber parenchyma contained starch grains (SG). He concluded the water-storing function is a secondary with the primary function being carbohydrate storage. We found that Genlisea tuberosa, Utricularia menziesii and U. mannii form carbohydrate-rich tubers. SGs exhibited different morphologies and sizes depending on the species. The largest SGs occurred in parenchyma cells of Genlisea tuberosa tubers. The highest number of SGs was recorded in Utricularia menziesii. Accumulation of SGs was recorded also in other Lentibulariaceae perennating organs such as Pinguicula hibernacula ( [9]; Płachno unp.) and Utricularia turions (e.g., [19]). Carbohydrate-rich tubers were recorded in the Lentibulariaceae in Genlisea tuberosa and four species of Utricularia that are not closely related (subgen. Polypompholyx: sect. Pleiochasia U. menziesii, subgen. Bivalvaria: sect. Phyllaria U. brachiata, sect. Chelidon U. mannii and subgenus Utricularia: sect. Utricularia U. inflata). Thus, these starch-rich tubers evolved at least five times independently in the Lentibulariaceae lineages. Nonetheless, further investigations are required across storage structures in the family, for example in another two species in sect. Phyllaria showing 1-2-mm-thick tubers, probably also serving as starch-rich vegetative propagules as found in U. christopheri and U. forrestii [14].
Regardless of whether the tubers accumulate starch grains or water, the tubers have well-developed parenchyma, which perform a storage function. Epidermal trichomes produce mucilage that may additionally protect the tuber surface. Mucilage may also interact with microflora and fungi though the function of such co-associating microorganisms is unclear.
Tubers exhibit different vascularization; however, in all species, phloem and xylem elements are evident. The morphological and anatomical tuber characters enable these species to occupy otherwise hostile ecological niches through perennation structures that survive periods of drought. However, tuber vascularization is most likely more related to evolutionary history of species than to organ specialization.
In seasonally dry environments such as the Brazilian Cerrado and kwongkan of south-western Australia the ability to sequester nutrients, moisture and energy in underground organs represents a key strategy for survival [37]. Carbohydrates are stored in various types of organs: tubers, corms, bulbs, rhizomes, rhizophores, tuberous roots, lignotubers, and xylopodia. Starch is the most abundant storage material, with combinations of insoluble and soluble carbohydrates occurring in underground storage organs [37][38][39].
In Genlisea tuberosa and Utricularia menziesii, the subterranean tubers act as a key structure and bud repository able to withstand seasonal drought, summer temperatures and the periodic passage of fire (soil is good heat insulator; see [40]). Several fire adaptations are found in endemic Cerrado flora, including functionally herbaceous or woody geoxylic suffrutices (the 'underground trees', see [41]) with enlarged underground xylopodia, lignotubers, thick corky bark, thick shoots, leaves congested at shoot tips and also specialized flowering and fruiting phenologies [42]. However, there is little known of the morphological and anatomical bases to ecological adaptation in tuberous structures (see [43]).
Utricularia menziesii has adopted a growth and development phenology that is similar to that found in most other herbaceous perennial geophytes that occur in the same habitat [22]. Dry season (summer) dormancy occurs from November to May, with sprouting occurring in concert with the onset of cooler and wetter winter conditions. Within a month of sprouting, tuber primordia are evident and these extend and enlarge over the growing season as the parent tubers shrivel and wither. The tuber lacks protective oversummering structures found in many other herbaceous perennial geophytes in the area where U. menziesii grows. For example, common geophytic Drosera accumulate around the tuber paper-like tissues from spent tubers that act to enclose the tuber and function to enhance water retention [22]. Though Utricularia menziesii grows in wet sandy swamps to moss aprons on granite rocks it is surprising that no other Utricularia in the southwest Australian region, a noted hotspot for carnivorous plants, have developed any form of vegetative perennation.
Tubers from sect. Orchidioides and secondary tubers of Utricularia mannii are tuberized (enlarged) stolons [14,21,44], which can continue apical growth to form the tuberous structures. In U. alpina, tubers may have also lateral branches-stolons bearing traps (see illustration in Darwin [30]). In contrast to that, tubers of Genlisea tuberosa and U. menziesii have reduced apical growth and no lateral additional organs. According to Taylor [14], U. mannii grows as an epiphyte on mossy tree trunks; however, this species may grow on rocks as a lithophyte ( Figure 8A; L'uboš Majeský personal observations). Thus, tubers of this species may also have a water-storing function. In addition, Compton [36] proposed both starch and water storage for tubers of U. brachiata. Therefore, the division into tubers which storage only water or only carbohydrates represents an artificial separation.
However, there are some similarities and differences in tuberous structures in the Lentibulariaceae. Epidermal trichomes occur in tubers of Genlisea tuberosa, U. mannii and tubers and thick stolons of species Utricularia from sect. Orchidioides [21]. Darwin [30] did not observe intercellular spaces in tubers of Utricularia alpine however in this study they were found in tubers of U. mannii. Well-developed lacunae were observed in Utricularia nelumbifolia stolons [21]. In the study species, there was no clear partitioning between cortex and pith, but this border is clearly seen in tubers and thick stolons of species Utricularia sect. Orchidioides [21]. There are also clear differences in vascularization in tubers of Genlisea tuberosa, Utricularia menziesii and U. mannii where phloem and xylem formed vascular bundles, but in the tubers and thick stolons of species Utricularia from sect. Orchidioides, [21] the xylem and phloem elements are separated. Vascular bundles with phloem groups flanking the xylem were observed in stolons of Utricularia dichotoma [6]. In tubers of Genlisea tuberosa and U. mannii, vascular bundles are branching in contrast to the tuber of Utricularia menziesii, with single vascular bundle in the tuber. With single, centrally located vascular bundles, tubers of Utricularia menziesii resembles the classical anatomy of root tubers (a ring of collateral bundles is typical for stem tubers in eudicots); however, Utricularia species do not produce roots [14]. Compton [36] also noted single vascular bundles in tubers of U. brachiata. This type of tuber originates from a stolon with one vascular bundle. Such stolons are known for species from section Pleiochasia, to which U. menziesii belongs [6].
For Utricularia mannii, the tubers are very similar to the tubers of species Utricularia from sect. Orchidioides, particularly the occurrence of chlorophyll and glandular trichomes. However, they differ in starch and vascular tissue development.
The phylogenetic hypothesis ( Figure 10) are in general congruent to previously published studies [12,27,33,[45][46][47][48]. Indeed, we used accessible public databases (mostly from Westermeier [46] and Silva [45]), and performed the analyses using a supermatrix approach with two concatenated chloroplast regions. One is a phylogenetically informative marker for Utricularia and Genlisea species, the matK gene with trnK intron, while the other is the rbcL gene, which is more conserved for these genera. Therefore, the polymorphic nature of the matK/trnK region and the conservation of rbcL [49,50] could provide greater resolution. Considering the two main clades with perennating organs, and ignoring the situation in which these organs are autapomorphic (Genlisea tuberosa, Utricularia menziesii, U. dichotoma, U. uliginosa, U. graminifolia, U. mannii, U. simulans, U. moniliformis and U. radiata), thick stolons and tubers for sect. Orchidioides (Figure 10, I and II) and turions for sect. Utricularia are present in lineages with relatively short branches in comparison with other groups (see the outer tree, Figure 10), even regarding the several factors that could reflect in mutation rates, as body size, population dynamics, and lifestyle among others [51,52]. Taking into account that we applied plastidial DNA sequences, the generation time effect and life history may also affect the rates of molecular evolution in flowering plants (e.g., [53]). This could explain this pattern for lineages of sect. Orchidioides and sect. Utricularia.

Materials and Methods
Material of Genlisea tuberosa Rivadavia, Gonella & A.Fleischm. was collected in the Serra da Canastra region, southern Minas Gerais State (Southeastern Brazil), in campos rupestres (rupestrian grasslands) of the Cerrado (collecting permits was ICMBio/MMA/SISBIO #74307-1). Utricularia menziesii R.Br. was collected from the Alison Baird Reserve (Yule Brook) in Western Australia, a privately owned and managed nature reserve. Utricularia mannii Oliv. (from Mount Cameroon, Cameroon, Africa) was cultivated in the Department of Plant Cytology and Embryology, Jagiellonian University in Kraków. Tubers were fixed as below for anatomical and histochemical studies.
The tubers were examined using light microscopy (LM) and scanning electron microscopy (SEM) as follows. Material was fixed in a mixture of 2.5% or 5% glutaraldehyde with 2.5% formaldehyde in a 0.05 M cacodylate buffer (Sigma-Aldrich, Sigma-Aldrich LLB, Poznan, Poland; pH 7.2) overnight or for several days, washed three times in a 0.1 M sodium cacodylate buffer and post-fixed in a 1% osmium tetroxide solution at room temperature for 1.5 h. Later material was treated as previously [54].
The semi-thin sections (0.9-1.0 µm thick) prepared for the LM were stained with aqueous methylene blue/azure II (MB/AII) for 1-2 min [55] and examined using an Olympus BX60, as well as Nikon Eclipse E400 light microscope for the general histology. The periodic acid-Schiff (PAS) reaction for the LM (semi-thin sections) was also used to reveal the presence of insoluble polysaccharides.
Materials were also embedded in Technovit 7100 (Kulzer, Germany) for further histological analysis. This material was fixed (as above), washed three times in a 0.1 M sodium cacodylate buffer, dehydrated in a graded ethanol series for 15 min at each concentration and kept overnight in absolute ethanol. Later, the samples were infiltrated for 1 h each in 3:1, 1:1 and 1:3 (v/v) mixtures of absolute ethanol and Technovit and then stored for 12 h in pure Technovit. The resin was polymerised by adding a hardener. Materials were also sectioned to 5 µm thick using a rotary microtome, stained with 0.1% toluidine blue O (TBO) and mounted in DPX (Sigma-Aldrich, Sigma-Aldrich LLB, Poznan, Poland). The selected Technovit sections were stained with naphthol blue black (NBB) for total protein staining or the periodic acid-Schiff (PAS) reaction was performed for starch visualization.
In order to identify the main classes of the chemical compounds that are present in the tubers, histochemical procedures with the fixed tubers using Lugol's solution were performed to detect the starch grains and proteins [56]. 0.1% ruthenium red was used for pectin and mucilage detection [57,58].
Tubers were cut using a razor blade and observed under UV light using an Olympus BX60, as well as Nikon Eclipse E400 light microscope to determine: cell walls impregnated with cutin and autofluorescence of chlorophyll.
For the SEM, tubers were fixed (as above) and later dehydrated and critical point dried using CO 2 . They were then sputter-coated with gold and examined at an accelerating voltage of 20 kV using a Hitachi S-4700 scanning electron microscope (Hitachi, Tokyo, Japan), which is housed in the Institute of Geological Sciences, Jagiellonian University in Kraków, Poland.
We measured starch grain diameter for each species (Table 1) as follows. For each species, one randomly chosen tuber section was selected. The number of starch grain measurements per cell type of a particular species was 100. Each variable was tested using the Shapiro-Wilk W-test for normality. The homogeneity of variance was estimated with Levene's test. Statistical differences in starch grains diameter in each species were assessed using one-way ANOVA, followed by Tukey's post-hoc comparison test. Statistical analyses were performed on the raw data using Statistica 13 software (StatSoft Inc., Oklahoma, USA). Data from measurements of starch grain diameter were expressed in µm as mean ± SD. Data were considered statistically significant at *** p < 0.001. We measured also number of starch grains per 100 µm 2 ( Table 2). For each species one randomly chosen tuber section was selected. To reconstruct the phylogenetic hypothesis, we aligned sequences of chloroplast regions matK/trnK and rbcL genes from GenBank Nucleotide database (Table S1) using MAFFT version 7 [59] with default parameters and generated a supermatrix with FASConCAT-G version 1.04 [60]. With the resulting supermatrix we calculated a Maximum Likelihood phylogenetic trees with IQ-TREE version 1.6.12 [61] using TVM+F+I+G4 model parameters chosen according with AIC criterion [62] using ModelFinder [63] implemented in IQ-TREE. Clade support was evaluated using ultrafast bootstrap [64] with 1000 replicates. Gaps were treated as missing data. The ancestral state reconstruction was performed using Maximum Parsimony criteria from a character matrix developed according to data from the present study and previous publications [12,14,20,21,34] to map the absence or presence of turions, tubers, thick stolons and the type of storage material into the molecular phylogeny using Mesquite version 3.61 [65]. The final tree was edited using Interactive Tree of Life (iTol) version 5.5.1 [66].

Conclusions
In contrast to species from section Orchidioides, which produce tubers storing mainly water, Genlisea tuberosa, Utricularia menziesii and U. mannii form starch-rich tubers. Such tubers evolved independently at least five times in the family Lentibulariaceae. In Genlisea tuberosa and Utricularia menziesii, underground tubers are a perennating structure that are key strategy to survive in summer fire-prone environments. In contrast to examined species here, in tubers of species from section Orchidioides the xylem and phloem elements are separated from each other which supports molecular studies from previous studies that this species is not related to species from Orchidioides. In contrast to most Utricularia and Genlisea lineages, where tubers evolved from regular stolons, sect. Orchidioides probably evolved through a continuum of transformation going from stolons, thick stolons to tubers. The occurrence of stomata on the underground tubers of G. tuberosa is unusual and point to a stem-based origin for the tuber tissue. Both tubers of G. tuberosa, and U. mannii had epidermal trichomes, which produce mucilage for protection. In G. tuberosa and U. mannii, vascular bundles formed a ring, but there was a centrally located additional vascular bundle. However, in U. menziesii, the single vascular bundle was located centrally in the tuber.  Table S1. Data used for the phylogenetic analyses. "-" denotes missing data. Pinguicula species were 20 used as outgroup.