Unusual embryo structure in viviparous Utricularia nelumbifolia, with remarks on embryo evolution in genus Utricularia

ORIGINAL ARTICLE

Unusual embryo structure in viviparous Utricularianelumbifolia, with remarks on embryo evolutionin genus Utricularia

Bartosz J. Płachno & Piotr Świątek

Received: 14 September 2009 /Accepted: 22 October 2009 /Published online: 18 November 2009# Springer-Verlag 2009

Abstract In most species of the Genlisea–Utriculariasister lineage, the organs arising directly after germinationcomprise a single leaf-like structure, followed by a bladder-trap/stolon, with the lack of an embryonic primary rootconsidered a synapomorphic character. Previous anatomicalwork suggests that the most common recent ancestor ofUtricularia possessed an embryo comprising storage tissueand a meristematic apical region minus lateral organs.Studies of embryogenesis across the Utricularia lineagesuggest that multiple primary organs have only evolved inthe viviparous Utricularia nelumbifolia, Utricularia reni-formis, and Utricularia humboldtii within the derivedIperua/Orchidioides clade. All three of these species arespecialized for growth as “aquatic epiphytes” in the tanks ofbromeliads, with recent phylogenetic evidence suggestingthe possibility that multiple primary organs may haveevolved twice independently within this clade. The primaryorgans of viviparous Utricularia also possess epidermalsurface glands, and our study suggests that these mayfunction as root hairs for uptake of solutes from the externalenvironment—a possible adaptation for the “aquatic–epiphytic” habitat.

Keywords Embryo evolution . Embryo ultrastructure .

Storage materials . Seed structure . Arabidopsis mutants .

Carnivorous plants . Lentibulariaceae

Introduction

A typical mature angiosperm seed contains a bipolarembryo consisting of cotyledon(s), an apical meristem,hypocotyls, and radicle (Natesh and Rau 1984; Czapik andIzmaiłow 2001). Proper development of the embryo isimportant for growth (Kaplan and Cooke 1997) andreproductive success in the adult plant. Studies of the earlydevelopmental stages of the young sporophyte not onlyprovide important information about the morphogeneticprogram, as shown for Arabidopsis mutants (e.g., Chandler2008), but also shed light on embryophyte evolution.

The number of cotyledons, either singular or paired,is a basis for the traditional classification of angio-sperms into two major classes: monocotyledons anddicotyledons. In some plants, however, mature seedscontain simplified embryos that may be arrested at theglobular stage (Natesh and Rau 1984), or two-celled pro-embryo stage, as observed in the micotrophic genusMonotropa (Olson 1980). For embryos that lack organs,specialization may occur at the cellular level, as previouslyshown for in orchids Cattleya and Vanda (Arditti 1992;Natesh and Rau 1984), and small and simplified embryosare common characters of parasitic plants (e.g., Teryokhinand Nikiticheva 1981; Natesh and Rau 1984). It alsoworth to mention that, in some species of Streptocarpus, atypical shoot apical meristem never forms and the entirevegetative plant body may consist of a single giantcotyledon (Möller and Cronk 2001; Mantegazza et al.2007).

B. J. Płachno (*)Department of Plant Cytology and Embryology,Jagiellonian University,ul. Grodzka 52,31-044 Krakow, Polande-mail: [email protected]

P. ŚwiątekDepartment of Animal Histology and Embryology,University of Silesia,ul. Bankowa 9,40-007 Katowice, Poland

Protoplasma (2010) 239:69–80DOI 10.1007/s00709-009-0084-1

Another example of embryo reduction is found in theLentibulariaceae family, in which is observed step-by-stepembryo simplification (Merl 1915): in Pinguicula, theembryo possesses a typical radicle, hypocotyl, one or twocotyledons, and shoot apex (e.g., Degtjareva et al. 2004,2006); in Genlisea, the embryo lacks a radicle (Merl 1915),and finally, in Utricularia, most species have embryos thatform a mass of barely differentiated cells without lateralorgans (Lloyd 1942). However, the ripe seeds of threeviviparous species from section Iperua, Utricularia renifor-mis, Utricularia humboldtii, and Utricularia nelumbifolia(Taylor 1989) possess embryos with multiple photosyntheticlateral organs that are octopus-like in shape (Merl 1915,1925; Goebel 1893a; Lloyd 1942; Taylor 1989). Thequestion of whether or not multiple lateral organs hadevolved just once within Utricularia (sect. Iperua) wasrecently challenged. Müller and Borsch (2005) providedphylogenetic evidence for placement of U. humboldtii withinthe Iperua sister section Orchidioidies, suggesting thepossibility that these organs had evolved twice independent-ly. In any case, the morphological/developmental character-ization of the viviparous embryo organs is unclear, and theyare often described as primary leaves, leaf segments, orcotyledon-like appendages (Lloyd 1942; Brugger andRutishauser 1989; Degtjareva et al. 2006).

In this study, we apply morphological, anatomical, andfunctional criteria to examine whether or not viviparousembryo organs share homology with the cotyledons ofPinguicula and Genlisea and with “typical” embryo organsof other Utricularia species. We also examine whetherthese organs may represent alternative/unique structuresand survey the literature on Utricularia embryos to identifyevolutionary trends of embryo structure within the genus.

Materials and methods

Seeds of U. nelumbifolia Gardner were provided by KamilPásek (Czech Republic). Other seeds of this species wereobtained from the Botanical Garden of the JagiellonianUniversity in Krakow, Poland. A plant of U. reniformis (St.Hil.) “Enfant Terrible” {B. Rice and M. Studnicka} (Riceand Studnička 2004) was given to us by the LiberecBotanical Garden, and later, this form was successfullycultivated in the Botanical Garden of the JagiellonianUniversity in Krakow. The plants flowered in 2008, andfruits with seeds were obtained after hand-pollination.Seeds of this species were planted in wet peat forgermination. Fruits of Utricularia intermedia Hayne (sect.Utricularia) were obtained from two localities in southernPoland: the Jeleniak–Mikuliny Nature Reserve [50°32.554N, 018°44.146 E, 247 m above sea level (asl)] near thetown of Lubliniec, and Tworóg village (50°37.087 N 018°

47.035 E, 261 m asl) near the town of Tarnowskie Góry(Płachno and Świątek 2008). Additional seeds of thisspecies and seedlings of Utricularia aurea were providedby Dr. Lubomir Adamec (Academy of Sciences of theCzech Republic, Třeboň). U. intermedia was collectedunder a permit from the Polish Ministry of the Environment(No. DLOPiK-op/ogiz-4211/I-29.2/8052/06/msz andDLOPiK-op/ogiz-4211/I-29.3/8052/06/msz).

Light and electron microscopy

For electron microscopy, seeds of U. nelumbifolia and fruitsof U. reniformis “Enfant Terrible” were fixed in 2.5%formaldehyde and 2.5% glutaraldehyde in 0.05 M cacody-late buffer (pH 7.0) overnight. The material was postfixedin 1% OsO4 in cacodylate buffer for 24 h at 4°C, rinsed inthe same buffer, treated with 1% uranyl acetate in distilledwater for 1 h, dehydrated with acetone, and embedded inEpon 812 (Fullam, Latham, NY, USA). Fruits of U.intermedia were fixed in a mixture of 4% formaldehydeand 2.5% glutaraldehyde in stabilizing buffer (SB; 50 mMPIPES, 1 mM MgCl2, 10 mM EGTA, pH 6.8) for 2 h atroom temperature. After fixation, the plant material wasrinsed in SB buffer and then dehydrated in a graded ethanolseries and embedded in LR White resin (which betterpenetrated the seeds than Epon). Semithin sections werestained with methylene blue and examined with anOlympus BX60 microscope. Ultrathin sections were cuton a Leica Ultracut UCT ultramicrotome. After contrastingwith uranyl acetate and lead citrate, the sections wereexamined with a Hitachi H500 electron microscope.

The procedures for preparing samples for SEM were asdescribed earlier (Płachno et al. 2005a, b). Briefly, seeds andfruits were fixed as for histological analysis or else naturallydry seeds were used. The dried tissues were sputter-coatedwith gold and viewed with a HITACHI S-4700 microscope(Scanning Microscopy Laboratory of Biological and Geo-logical Sciences, Jagiellonian University).

Results

Information regarding storage materials in Utriculariaembryo are provided in Table 1. General information aboutmorphology and germination patterns in Utricularia em-bryos is given in Table 2.

U. nelumbifolia

The seed was pyriform in shape and about 1.6 mm long(Fig. 1a). The seed pedestal connects the seed with theplacenta (not shown). Sometimes the exotesta cells are

70 B.J. Płachno, P. Świątek

disjunctive so that a broad space between two layers of theseed coat was observed, and the seed coat appeared to beinflated (Fig. 1b). The outer parts of the anticlinal walls ofadjacent exotesta cells were divided by a furrow (Fig. 1c).There were bolster-shaped thickenings (∼3 µm) on theanticlinal walls of the exocesta cells (Fig. 1d). The outerpericlinal wall was very thin, about 0.3 µm. The mesotestaconsists of crushed cells (Fig. 1d).

The embryo was straight, consisting of two distinct parts:the green apical part with primary organs and the yellow–whitebasal part (Fig. 1e). The embryo was about 1,140 µm long.The diameter of the embryo varies depending on the part: inthe terminal–basal part ∼150 µm, in the middle–basal part∼270 µm, in the middle part ∼213 µm, and in the apical partwhere primary organs occur was about 340 µm. There are 13primary organs in a spiral arrangement, with their apexesdichotomously forked (Fig. 1e, f). On the surface of theprimary organs are numerous small glands (glandular hairs;Figs. 1e, f, 2a). A single gland consisted of a basal cell,middle pedestal cell, and one terminal cell.

The basal part of the embryo consists of large (∼24.5 µm)parenchyma cells, regular in shape, and filled with lipiddroplets (Fig. 2b). Epidermis cells also contain lipids. Noprovascular tissue was observed in this part of the embryo.The apical part of the embryo consists mainly of largeirregularly shaped (∼26.7 µm) cells, which contain chlor-oplasts and also some starch grains. Provascular tissueoccurs in the central part (Fig. 2c); it branches to the primaryorgans (Fig. 2d), which are green and contain chloroplasts.Phloem elements were observed in the provascular tissue.

During germination, parasitic fungi developed on theseeds and inhibited completion of germination. Also, theseeds of plants with fruits were kept in a warm, drygreenhouse and showed very low viability.

U. reniformis “Enfant Terrible”

The seed was cylindrical and about 1 mm long (Fig. 3a). Theseed coat was thin and transparent and forms numerous multi-cellular outgrowths. The outer periclinal walls of the exotestacells are very thin, in contrast to the middle part of theanticlinal walls which was thickened. The embryo was moreor less egg-shaped, 450 µm long, and 214 µm wide. Most ofthe embryo body consists of parenchyma cells filled withstorage material, mainly lipid droplets, protein bodies, andalso starch grains (Fig. 3b, c). Epidermal cells were also filledwith storage materials. Meristematic tissue occurred in theapical part of the embryo which may extend a short distancetowards the embryo axis. During germination, the first organformed was a leaf, and the second was a stolon (Fig. 3d).

U. intermedia

The seed was discoid and flattened in shape (Fig. 4a). Theembryo was flattened, 640 µm wide, and about 350 µmlong. In the observed stage, the syncytium, which isthought to transport nutrients to the embryo, was absent;only a cavity was in its place (Fig. 4a). The outer periclinalwalls of the exotesta cells were very thin, in contrast to theinner part of the anticlinal walls and internal periclinal

Table 1 Storage materials in Utricularia embryo, sections according to Taylor (1989)

Subgenus Section Species Kind of storage materials Author/s

Utricularia Utricularia U. gibba subsp. gibba Starch Kondo et al. 1978

U. radiata Starch Kondo et al 1978

U. inflexa Starch, protein Swamy and Mohan Ram 1969

U. aurea (as U. flexuosa) Food grains Khan 1954

U. intermedia Protein, starch This paper

Iperua U. reniformis “Enfant Terrible” Lipids, protein, starch This paper

U. nelumbifolia Lipids, starch This paper

Setiscapella U. subulata Starch Kondo et al 1978

Bivalvaria Phyllaria U. striatula Starch Kondo et al 1978

Oligocista U. bifida Starch Kondo et al 1978

U. uliginosa Starch Farooq 1965

U. smithiana Starch Rajan and Kumar 1974

U. polygaloides as (U. stricticaulis) Starch Shivaramiah 1967

U. scandens Starch Farooq and Bilquis 1966

Stomoisia U. cornuta Starch Siddiqui 1979

Nigrescentes U. caerulea Starch Kausik 1938

Polypompholyx Pleiochasia U. dichotoma Food material Siddiqui 1978

Polypompholyx U. multifida Lipids, protein Lang 1901

Unusual embryo structure in viviparous Utricularia nelumbifolia 71

Table 2 Morphology and germination pattern of Utricularia embryos, sections according to Taylor (1989)

Subgenus Section Morphology of embryo+germination pattern Author/s

Utricularia Vesiculina Embryo ovate shaped with flattened broad end,during germination successively three primaryorgans arise (U. purpurea)

Lloyd 1942

Utricularia Embryo bun or disk shaped with a depression at thevegetative pole, where variable number of primordiamay occur (eg. U. vulgaris, U. stellaris, U. aurea,U. inflexa), during germination they form several“cotyledonoids”

Kondo et al 1978, Farooq and Siddiqui 1964,Khan 1954, Lloyd 1942, Merz, Kumazawa1967, Kamieński 1876, This paper

Embryo flattened by lateral compression, withgrowth pole on its edge, during germination two“cotyledonoids”/primary organs occur and later twoothers arise which become shoots (U. gibba)

Iperua andOrchidioides

Embryo in mature seed with c. 6–15 green identicalprimary organs well developed before germination(U. reniformis s. str., U. humboldtii, U. nelumbifolia)

Merl 1915, 1925, Goebel 1893a, Brugger andRutishauser 1989, this paper

Embryo without organs. During germination twoprimary organs: leaf+trap are formed (U. alpina)or during germination leaf+stolon are formed (U.reniformis “Enfant Terrible”, U. nephrophylla)

Psyllosperma Embryo compressed with meristematic apex inlateral position (U. hispida)

Kamieński 1876, Merl 1915

During germination two primary organs: leaf+stolonare formed (U. longifolia)

Setiscapella C. ellipsoidal shaped with meristematic apex,during germination two primary organs: leaf+stolonare formed (U. subulata)

Kamieński 1876, Lloyd 1942, Kondo et al 1978

Bivalvaria Phyllaria Embryo with two small primordia (U. striatula,U. brachiata). During germination two primordiaarise, one forms leaf, the second the trap/oneprimordium arises from the embryo, the nextprimordia are formed on the first, which finallyforms the leaf (U. striatula)

Kamieński 1876, Goebel 1893a, b, Compton 1909,Kondo et al 1978

Oligocista C. ellipsoidal shaped with meristematic apex(eg. U. albocaerulea, U. bifida, U. smithiana,U. polygaloides, U. uliginosa)

Kamieński 1876, Kondo et al 1978, Farooq 1965,Rajan and Kumar 1974, Shivaramiah 1967,Merz 1897, Farooq and Bilquis 1966

Calpidisca C. ellipsoidal shaped with meristematic apex(U. bisquamata, U. sandersonii). Duringgermination, two primary organs: leaf+stolonare formed (U. bisquamata)

Kamieński 1876, Rutishauser and Sattler 1989,Płachno 2002

Stomoisia C. ellipsoidal shaped with rounded ends andwith meristematic apex (U. cornuta). Duringgermination two primary organs: leaf+stolonare formed (U. cornuta, U. juncea)

Siddiqui 1979, Kondo 1971

Nigrescentes C. ellipsoidal shaped with meristematic apex(U. coerulea)

Kausik 1938

Australes C. ellipsoidal shaped with meristematic apex,during germination two primary organs: leaf+stolon are formed (U. lateriflora)

Kamieński 1876

Polypompholyx Pleiochasia Nearly oval with rounds ends, apical meristematicapex (U. dichotoma). During germination twoprimary organs/“cotyledonoids”: leaf+stolon areformed (U. monanthos). The embryo may persistthough the life of the plant (U. violacea,U. hookeri)

Siddiqui 1978, Lloyd 1942

Polypompholyx C. round with depression at the micropylar side,and meristematic part of opposite side. Duringgermination two primary organs: leaf+stolonare formed. The embryo may persist though the lifeof the plant. (U. tenella)

Lang 1901, Lloyd 1942

72 B.J. Płachno, P. Świątek

walls, which were thick and layered (Fig. 4b). Themesotesta consisted of obliterated cells. The outer periclinalwalls of external endosperm cells were thick and probablylignified or cutinized. These endosperm cells containedsmall starch grains. Most of the embryo body consisted oflarge parenchyma cells filled with storage material: proteinbodies and starch grains (Fig. 4c, d). The epidermal cellswere also filled with storage materials.

Discussion

Morphology of the embryo

We found that the embryos of U. nelumbifolia and U.reniformis sensu stricto (see Fig. 44 in Goebel 1893a) have

very similar structural designs, in contrast to U. humboldtiiin which the basal part of the embryo is not prominent andprimary organs dominate (see Tab. XIV, Fig. 3 in Goebel1893a). There is a small difference in the shape of theprimary organs between these three species: they arebroadly spatulate in U. reniformis (Goebel 1893a), dichot-omously forked in U. nelumbifolia (Fig. 1e, 2a; Merl 1915,Fig. 16), and tripartite in U. humboldtii (Rutishauser andSattler 1989). In U. reniformis, there are about 11 primaryorgans (Goebel 1893a), six to ten in U. humboldtii(Rutishauser and Sattler 1989), and from eight to 15 in U.nelumbifolia (Merl 1915), and in all these species, they arein a spiral arrangement. This arrangement of organ ischaracteristic of leaf structures, not cotyledons. Morpho-logically, these organs differ from the organs that areconsidered to be adult leaves in these species. In the

Fig. 1 Morphology and anato-my of a U. nelumbifolia seed.a General seed morphology bar500 µm. b Transversal sectionthrough the seed bar 100 µm. Eembryo, black arrow exotesta,white arrow mesotesta. c Mor-phology of exotesta cells bar50 µm. d A part of the trans-versal section through the seed,note bolster-shaped thickeningon the anticlinal walls of theexocesta cells bar 20 µm. Eembryo, black arrow exotesta,white arrow mesotesta. e Gen-eral morphology of the embryobar 500 µm. f Spiral arrange-ment of the embryo primaryorgans bar 300 µm

Unusual embryo structure in viviparous Utricularia nelumbifolia 73

seedlings of some other plants, however, juvenile leavesmay have a more simple contraction than in the adult plant.

Our finding that the embryo of U. reniformis (St. Hil.)“Enfant Terrible” has a germination pattern differing fromU. reniformis sensu stricto (see Figs 44, 45 in Goebel1893a and Merl 1925), U. humboldtii as well as U.nelumbifolia is especially interesting because, for Utricu-laria nephrophylla (Merl 1925, syn. Utricularia Dusenii), aphylogenetically related species of sect. Iperua (Jobson etal. 2003), the embryo does not possess leaf primordia, or, asin Utricularia geminiloba, the leaf primordia are not welldeveloped (Taylor 1989) or were not seen during germina-tion (see Fig. 129 in Studnička 2006).

However, the seed morphology of U. reniformis “EnfantTerrible” (our Fig. 3a) is similar to the seed shown byTaylor (1989) for U. reniformis, but differs from U.nephrophylla, and U. geminiloba. This is easy to imaginewhen we consider that the formation of leaves is induced bya local concentration of auxin (auxin accumulates at the siteof initiation; Benkova et al. 2003) and that organdevelopment is affected by auxin efflux inhibitors (Lomaxet al., 1995). Merl (1915) described a small variant of U.reniformis (similar to U. reniformis “Enfant Terrible”) andshowed seeds of this plant in which the embryo has onlyone primary leaf, but he thought that the embryo was notmature. To settle these questions, more molecular and

anatomical studies should be performed, including adetailed, step-by-step study of the development of the U.reniformis (large type) embryo (Płachno, Clivati, Mirandain prep.).

Anatomy of the embryo

The embryo of U. nelumbifolia showed polarity, with twofunctionally different poles: basal–micropylar, which func-tions as a storage magazine, and apical–chalazal, which isgreen and has primary organs. This is similar to the embryoof U. reniformis described by Merl (1915). Also, provas-cular strands occur in both species (our results, Merl 1915).Apart from these species, the formation of a provascularsystem in the mature Utricularia embryo was noted onlyin members of sect. Utricularia (Kondo et al. 1978).Kamieński (1876) found the primary organs in theUtricularia vulgaris embryo (“cotyledonoids”/primaryleaves) to have vascular tissue which is in contact withvascular tissue of the main body of the embryo. We suggestthat this anatomy is similar to that of primary organs of U.nelumbifolia and U. reniformis, both having a spiralarrangement with homologous organs. Goebel (1893a)noted the similarity of these organs and termed bothPrimärblätter (i.e., primary leaves). The number, anatomy,and morphology of primary organs in U. nelumbifolia and

Fig. 2 Morphology and anato-my of a U. nelumbifolia embryo.a Schema of general morpholo-gy of the embryo, note numer-ous glands on the primary organsurface bar 500 µm. b Trans-versal section through the basalpart of the embryo, note numer-ous lipids droplets bar 20 µm.c Transversal section throughthe apical part of the embryo,note pro-vascular strands in thecentral part (star) bar 50 µm.d A part of the longitudinalsection through the primaryorgans, note pro-vascularstrands in the central part (star)bar 50 µm

74 B.J. Płachno, P. Świątek

allied species are unlike the cotyledons of Pinguicula,which have a very simple structure (Degtjareva et al. 2004,2006).

Function of the primary organs in viviparous embryo

The primary function of these “leaf primordia” is the supplyof assimilates to the embryo via photosynthesis. The seedcoat of U. reniformis and U. nelumbifolia is a thin,transparent structure (Taylor 1989), and this is connectedto the cell wall of the seed coat via specialized cells and issimilar to that found in U. humboldtii, where the greenembryo is also visible through a transparent seed coat(Taylor 1989; Rivadavia 2001; Rice 2005). However, incontrast to the cotyledons of some other dicotyledons, theprimary embryo organs of U. nelumbifolia are not rich instorage products, as shown in Fig. 2b, the basal part of theU. nelumbifolia embryo performs the storage function.According to Studnička (2005), primary organs are alsovery persistent, remaining in place for ∼40 days aftergermination, and may act as stabilizers maintaining theseedling on the water surface in the water-filled tank of abromeliad rosette (its primary habitat).

We suggest a new function for the primary organs of U.reniformis, U. humboldtii, and U. nelumbifolia. Theirsurfaces are covered in glands that share some similarityto those described for Utricularia monanthos of sect.Pleiochasia (Taylor 1989; Fineran 1980). These glandsmay absorb solutes from the external environment andfulfills a role similar to that of root hairs. The primaryorgans of viviparous Utricularia could function like a root,the likes of which are aborted during embryogenesis in allspecies of Utricularia (Taylor 1989).

Benefits and disadvantages of viviparous embryony

According to Rivadavia (2001), the seeds of U. nelumbi-folia are short-lived and very fragile. In the current study,we also observed that they are neither resistant to hightemperature nor to low humidity and are often infected withfungi that may possibly inhibit germination. According toStudnička (2005, 2006), seeds of U. nelumbifolia and U.humboldtii have no dormancy and germination occurs veryquickly after contact with water—less than 24 h in the caseof U. humboldtii. For seed of these species, the shortviability is offset by rapid germination. It is possible that

Fig. 3 U. reniformis “EnfantTerrible”. a General seed mor-phology bar 500 µm. b, cSections through the embryocells, note numerous storagematerials bar 1 µm and bar0.85 µm. St starch grain, L lipiddroplet, G protein body. d Mor-phology of the seedling bar1 mm. S seed, white arrowstolon

Unusual embryo structure in viviparous Utricularia nelumbifolia 75

viviparous embryony is an adaptation to life in bromeliador Xyridaceae rosettes, with all three Utricularia specieswith viviparous embryos able to inhabit this environment:U. humboldtii in Brocchinia and Orectanthe, U. nelumbi-folia and U. reniformis in Vriesa (e.g., Goebel 1893a;Taylor 1989; Rivadavia 2001; Studnička 2003, 2004). Theevolution of highly specialized seeds in these viviparousspecies is probably an adaptation growth as “aquaticepiphytes” in the water-filled tanks of bromeliads andmay confer a dispersal and/or growth–survival advantage,although U. humboldtii and U. reniformis may also occupyother habitats (i.e., terrestrial and lithophytic; Taylor 1989).

Storage materials

According to Khan (1992), albuminous seeds occur inUtricularia, but this kind of seed—endospermous—haswell-developed endosperm with storage materials in matureseeds. In Utricularia, the endosperm is strongly reduced inmature seeds, and storage materials are located mainly inthe embryo cells (Figs. 2, 3, 4; Table 1). Therefore, seedstructure in Utricularia resembles that of exalbuminous(non-endospermic) seeds, but unlike typical exalbuminousseeds in which storage materials are in cotyledons, althoughall Utricularia are probably cotyledonless. In addition,Siddiqui (1978) only observed exalbuminous (or nearly so)seeds in a study of Utricularia dichotoma (sect. Pleiocha-

sia). Considering what is currently known about storagematerials in the Utricularia embryo (see Table 1), we findthat the majority of species utilize starch as storage material.Only a few species, especially those of subgenus Utricularia(Müller et al. 2006), also utilize proteins and lipids as storagematerial; both of these materials were also found in subgenusPolympompholyx (see Table 1), a lineage sister to all otherUtricularia (Jobson and Albert 2002; Jobson et al. 2003). InUtricularia, the embryo epidermis also acts as storage tissue.

The seed pedestal

“Seed pedestal” refers a structure that connects a seed with theplacenta and is developmentally placental in origin, not part ofthe ovule (Rebernig and Weber 2007). Płachno et al. (2009)described its occurrence in U. reniformis (both large andsmall types) and predicted that Utricularia species that havesimilar patterns of seed development would most likelypossess seed pedestals. Here, we show that the seed pedestalalso occurs in U. nelumbifolia, which is both morphologi-cally (Taylor 1989) and phylogenetically (Jobson et al. 2003)related to U. reniformis.

Evolution of the embryo in Lentibulariaceae

According to Jobson and Albert (2002), the clades thatcomprise sect. Polympompholyx and sect. Pleiochasia form

Fig. 4 Seed structure of U.intermedia. a The longitudinalsection through the seed andpart of the placenta bar 200 µm.E embryo, P placenta, star anempty place where syncytiumoccurred. b Sections through theseed coat bar 20 µm. Ex exo-testa, en endosperm. c Sectionsthrough the embryo cells, notenuclei and numerous storagematerials bar 20 µm. d A part ofthe section through the embryocell; note numerous storagematerial bar 1,5 µm. St starchgrain, G protein body

76 B.J. Płachno, P. Świątek

a monophyletic lineage which is sister to a clade thatincludes all other sections of Utricularia and based onmorphology, is thought to contain the most ancestral types,including organless embryos (Taylor 1989).

The previous anatomical and phylogenetic work sug-gests that the most common recent ancestor of Utriculariapossessed an embryo comprising storage tissue and ameristematic apical region minus lateral organs (Fig. 5).Studies of embryogenesis across the Utricularia lineagesuggest that multiple primary organs have only evolved inthe viviparous U. nelumbifolia, U. reniformis, and U.humboldtii within the derived Iperua/Orchidioides clade.

The absence of the embryonic primary root is asynapomorphy of Genlisea–Utricularia lineage (Taylor1989; Jobson et al. 2003; Müller et al. 2004). During theevolution of this lineage, there was a reduction in the sizeand number of cotyledons. In Genlisea, we can see differentsteps of cotyledon reduction; in some species, they are stilllarge, as in Genlisea violacea (see Fig. 29a in Merl 1915),and in others, they are strongly reduced, as in Genliseafiliformis (see Fig. 29b in Merl 1915). However, thisprocess was most dramatic in the Utricularia ancestorwhere, like “laterne” mutants of Arabidopsis (Treml et al.2005), the cotyledons are entirely aborted. In Arabidopsis,there are other mutants in which the cotyledons arereduced, malformed, or aborted (Chandler 2008). Treatmentof Brassica juncea embryos with exogenous auxin com-pletely inhibited morphogenesis (producing ball-shaped

embryos) or produced egg- and cucumber-shaped embryos;application of antiauxin inhibited development of thecotyledons, hypocotyls, and radicle (Hadfi et al. 1998).Since cotyledons are aborted in the most ancestral forms ofUtricularia, the cotyledon-like organs in the more derivedclades of subgenus Utricularia are unlikely to be truecotyledons, although it is also possible that reversal to thisdevelopmental condition may have occurred.

It should also be mentioned that seedlings of Arabidop-sis “laterne” mutants sometimes develop multiple-leafprimordia (Treml et al. 2005), a situation comparable tothe U. nelumbifolia, U. reniformis, and U. humboldtii(Degtjareva et al. 2006).

During the embryogenesis of most species of Utricu-laria, only initial histogenesis occurs: the epidermis,meristematic tissue at the apex, and storage parenchymaare formed. In contrast, U. reniformis, U. humboldtii, andU. nelumbifolia, along with several species of sect.Utricularia, initial organogenesis also occurs in the matureembryo (Table 2).

Unusual organogenesis may also have been responsiblefor the lack of provascular tissue in the embryos of mostUtricularia species, with normal development of this tissueoccurring in Pinguicula (see Fig. 5H and I in Degtjareva etal. 2006). For this reason, organogenesis and provasculartissue formation in U. reniformis, U. humboldtii, and U.nelumbifolia seems to be an evolutionary novelty.

Germination type

Lloyd (1942) observed three main types of germination inUtricularia: (1) Simple, where there are two cotyledonoids

Fig. 6 Seedling of the Genlisea repens. bar 1 mm. S seed, whitearrow leaf, black arrow trap

Fig. 5 Simplified cladogram of Lentibulariaceae evolution (based on themolecular work of Jobson et al. 2003) and possible embryo evolution. 1Ancestor of Lentibulariaceae with well-developed embryo: embryo withtwo cotyledons, root. 2A Embryo with two or one cotyledons and root.2B Common ancestor of the Gelisea+Utricularia clade, embryo withtwo cotyledons, and aborted root primordia. 3 Ancestor of Utricularia,simplification of embryo morphology and histology (an embryoconsisting of a mass of cells with storage materials, with a meristematicapical region, epidermis and without any lateral organs). 4A Matureembryo with well-developed primary organs (U. nelumbifolia, U.reniformis s. str., and U. humboldtii). 4B Embryo with numerousprimary organs during germination (U. vulgaris and allied species). 4CEmbryo with three primary shoots (U. purpurea, sect. Vesiculina)

Unusual embryo structure in viviparous Utricularia nelumbifolia 77

with one forming as a stolon/trap; (2) Complex, withgenerally many (6–13) cotyledonoids (suspended aquaticU. vulgaris and allied species) but occasionally, only two(e.g., Utricularia gibba); (3) Lacking cotyledonoids,where, instead, three primary shoots occur (e.g., sectVesiculina).

In Table 2, we present all available data on Utriculariagermination. The general pattern observed from this data isthat the simple type of germination is the most frequent,while the complex germination type is restricted to sect.Utricularia and some species of sect. Iperua (sensu Taylor1989). Lloyd (1942) considered that germination of U.gibba is related to the U. vulgaris type but of a simplertype. According to Jobson et al. (2003), this species is sisterto other species from sect. Utricularia and its germinationtype may be considered an initial step towards the evolutionof the complex type in the lineage.

Regarding the type 3 germination, the molecularphylogenies of Jobson et al. (2003) and Müller and Borsch(2005) suggest that the suspended aquatic sections Utricu-laria and Vesiculina do not form a monophyletic group andtherefore, species lacking cotyledonoids may have evolvedindependently to those of the complex type. This is inagreement with morphological data (i.e., members of sect.Utricularia have body architecture differing from membersof sect. Vesiculina), along with data on embryo structureand germination (Table 2, Taylor 1989; Rutishauser andSattler 1989).

Now, we examine the most probable germination type inthe most common recent ancestor of Utricularia. In view ofthe data on germination in Utricularia (Table 2) andmolecular work (Jobson and Albert 2002; Jobson et al.2003), we suggest that the hypothetical ancestor ofUtricularia had the simple type of germination sensu Lloyd(1942). In some Utricularia seedlings, there is a slightvariant of the simple type: a trap is produced in place of theusually formed stolon (e.g., U. monanthos, see Plate 22Fig. 4 in Lloyd 1942, Utricularia alpina Goebel 1893a).Surprisingly, this variation on the simple form of germina-tion was also found to occur in Genlisea (our Fig. 6, Goebel1893b), and we therefore predict that the simple type ofgermination was the ancestral type for the Genlisea–Utricularia lineage.

In terms of classical plant anatomy, the “leaf” inUtricularia could be considered a phylloclade rather than atrue leaf, but for plants such as Utricularia or Genlisea thatpossess an unusual vegetative body contraction not presentin other angiosperms, it is better to use a system other thanclassical morphology. One such proposal is the “FuzzyArberian Morphology” approach that accepts developmen-tal mosaics or rather, partial developmental homologybetween root, shoot, and leaf structures (Rutishauser andIsler 2001).

Final remarks

– The primary multiple organs of U. nelumbifolia, U.reniformis, and U. humboldtii embryos are homologouswith the primary multiple embryo organs of membersof sect. Utricularia but are not homologous with thecotyledons of Pinguicula.

– The primary multiple organs of viviparous U. nelumbi-folia and allied species are photosynthetic and some-what resemble typical “leaf” structures but may alsohave the ability to absorb nutrients from the externalenvironment, fulfilling a role similar to that of roothairs. These structures are an evolutionary novelty inUtricularia.

– We predict that the most recent common ancestor forUtricularia had an embryo which was a mass of cellscontaining storage materials and possessed a meriste-matic apical region with no lateral organs and pos-sessed the simple germination type where the firstorgan is the “leaf” while the second is the trap/stolon.

Acknowledgments This paper is dedicated to Charles Darwin tomark 200 years since his birth and 150 years since the publication ofhis “On the Origin of Species”. This study was funded by grant NN304 002536 from the Polish Ministry of Science and HigherEducation. The first author gratefully acknowledges the support ofan award from the Foundation for Polish Sciences (Start Programme).We thank our colleagues Kamil Pásek (Czech Republic, http://www.bestcarnivorousplants.com) and Dr. Lubomir Adamec for kindlyproviding seeds and seedlings for this study. We thank the rector ofthe Jagiellonian University, Professor Szczepan Biliński, for gener-ously supporting our projects, and Dr. Miroslav Studnička (Director ofthe Botanical Garden in Liberec, Czech Republic) for kindlyproviding U. reniformis “Enfant Terrible” for our study. Weparticularly thank reviewers for very helpful suggestions to makeour manuscripts more clear.

Conflict of interest The authors declare that they have no conflict ofinterest.

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