Complex patterns of morphogenesis, embryology,and reproduction in Triuris brevistylis, a speciesof Triuridaceae (Pandanales) closely related toLacandonia schismatica

Silvia Espinosa-Matías, Francisco Vergara-Silva, Sonia Vázquez-Santana,Eddy Martínez-Zurita, and Judith Márquez-Guzmán

Abstract: Triuris brevistylis Donn. Sm. (Triuridaceae: Pandanales), a mycoheterotrophic monocotyledon with populationsin Mexico and Guatemala, is closely related to Lacandonia schismatica E. Martínez et C. H. Ramos, a triurid speciesdisplaying a peculiar “inside-out” arrangement in its reproductive axes. Triuris brevistylis is a polygamodioecious speciesin which four types of bisexual ramets–genets coexist besides regular (i.e., unisexual) male and female flowers. Here, wecharacterize the embryology of three of the following different types of reproductive axes in T. brevistylis: male andfemale flowers and a bisexual floral type in which stamens develop at the base of the receptacle of an otherwise female-looking flower. Through this embryological characterization, we have detected that cleistogamy, a mechanism previouslyconsidered exclusive to L. schismatica, occurs in the bisexual flowers. Besides serving as characters for the systematics ofTriuridaceae and Pandanales, our data establish that morphogenetic and reproductive patterns in T. brevistylis are morecomplex than in L. schismatica. Therefore, we claim that our results contribute to a refined assessment of the controversyregarding morphological interpretation of reproductive axes in triurids.

Key words: embryology, cleistogamy, Lacandonia, polygamodioecy, Triuridaceae, Triuris.

Résumé : Le Triuris brevistylis Donn. Sm. (Triuridaceae : Pandanales), une espèce mycohétérotrophe avec despopulations au Mexique et au Guatémala, est étroitement reliée au Lacandonia schismatica E. Martínez et C. H. Ramos,une espèce de triuride montrant un arrangement particulier “interne–externe” de ses axes reproductifs. Le T. brevistylis estune espèce polygamodioïque chez laquelle coexistent quatre types de ramettes–genets bisexués, en plus des fleurs mâles etfemelles régulières. Les auteurs ont caractérisé l’embryologie de trois types différents d’axes reproductifs chez leT. brevistylis : fleurs mâles et femelles, et un type bisexué de fleur dans lequel les étamines se développement a la base duréceptacle d’une autre fleur d’allure femelle. Par cette caractérisation embryologique, les auteurs ont pu s’apercevoir que lacléistogamie, un mécanisme jusqu’ici considéré comme exclusif au L. schismatica, survient chez les fleurs bisexuées. Enplus de servir comme caractères pour la systématique des Triuridaceae et des Pandanales, ces données établissent que lespatrons morphogénétiques et reproductifs du T. brevistylis sont plus complexes que chez le L. schismatica.Conséquemment, les auteurs avancent que leurs résultats contribuent a une évaluation plus précise de la controverseconcernant l’interprétation morphologique des axes de reproduction chez les triurides.

Mots-clés : embryologie, cléistogamie, Lacandonia, polygamodioécie, Triuridaceae, Triuris.

[Traduit par la Rédaction]

Introduction

The monocotyledon family Triuridaceae Gardner is com-prised of 11 extant genera and approximately 50 species ofsmall, inconspicuous mycoheterotrophic plants, distributedmostly throughout the tropical and subtropical regions of theOld and New Worlds (Rübsamen-Weustenfeld 1991; Maas-van de Kamer and Weustenfeld 1998; Franke et al. 2000;

Cheek 2003; Cheek et al. 2003). In addition, Upper Cretaceousfossil genera have been assigned to Triuridaceae (Gandolfo etal. 2002; see also Doyle et al. 2008; Friis et al. 2011). Atpresent, Triuridaceae is placed within the order Pandanales(Angiosperm Phylogeny Group (APG) 2009), a systematicposition originally suggested by phylogenetic reconstructionsbased on molecular data (Chase et al. 2000; Davis et al. 2004).Besides Triuridaceae, the current circumscription of Pandan-

Received 13 January 2012. Accepted 9 May 2012. Published at www.nrcresearchpress.com/cjb on 28 October 2012.

S. Espinosa-Matías and E. Martínez-Zurita. Laboratorio de Microscopía Electrónica de Barrido, Facultad de Ciencias, UniversidadNacional Autónoma de México (UNAM), México D.F., 04510, Mexico.F. Vergara-Silva. Laboratorio de Sistemática Molecular (Jardín Botánico), Instituto de Biología, UNAM, México D.F., 04510, Mexico.S. Vázquez-Santana and J. Márquez-Guzmán. Laboratorio de Desarrollo en Plantas, Facultad de Ciencias, UNAM, México D.F.,04510, Mexico.

Corresponding author: F. Vergara-Silva (e-mail: fvs@ibiologia.unam.mx; f.vergarasilva@gmail.com).

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Botany 90: 1133–1151 (2012) Published by NRC Research Pressdoi:10.1139/b2012-060

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ales includes the autotrophic families Cyclanthaceae, Pandan-aceae, Stemonaceae, and Velloziaceae (APG 2009).

As a taxonomic assemblage, Pandanales (sensu APG 2009)is intriguing. The five subordinate monocotyledon groups thatconstitute the order show great disparity in growth forms andhabits. Whereas all genera in Cyclanthaceae and Pandanaceaeare either herbs or robust trees (Harling et al. 1998; Stone et al.1998), species of Stemonaceae and Velloziaceae are relativelysmaller, being either woody or herbaceous (Kubitzki 1998a,1998b; Stevens 2001). In turn, the overall morphology ofevery genus in Triuridaceae contrasts starkly with that of theremaining taxa in Pandanales; all triurids have highly reducedstructures, and anatomy of the vegetative organs is stronglyassociated with their mycoheterotrophic condition (Maas-vande Kamer and Weustenfeld 1998; Rudall 2008).

Floral morphology also differs considerably between thefive families of Pandanales. In fact, there is no consensus onstraightforward definitions of the nature of reproductive struc-tures in some of its members (Remizowa et al. 2010). In aformal cladistic analysis of a matrix of vegetative and repro-ductive morphological characters that included several generawithin Pandanales, Rudall and Bateman (2006) recommendedthat special attention be paid to reproductive character coding,particularly within the monophyletic Triuridaceae. This obser-vation was made in an attempt to decide between euanthial andpseudanthial morphological interpretations of the nature oftriurid reproductive axes (Rudall 2003; Vergara-Silva et al.2003; Ambrose et al. 2006). In the subsequent debate implicitin these contrasting views (see Rudall 2008, 2010; Álvarez-Buylla et al. 2010), the species Lacandonia schismaticaE. Martínez et C. H. Ramos has continued to play a major role,given its unusual heterotopic reproductive organ arrangementin which the androecium is central (i.e., distal) with respect tothe gynoecium (Martínez and Ramos 1989; Vergara-Silva etal. 2003; Ambrose et al. 2006). For a recent, nonpartisanassessment of the controversy over morphological interpreta-tions in Triuridaceae, see Cronk (2009).

A developmental genetics-based hypothesis aimed at ac-counting for the heterotopic arrangement of floral organs inL. schismatica complemented the default, euanthial morpho-logical interpretation of triurid reproductive axes (Vergara-Silva et al. 2000, 2003). Based on this hypothesis,characterization of the expression pattern of L. schismaticaMADS-box genes homologous to the B- and C-functions ofthe ABC model of flower development (Coen and Meyerowitz1991) and heterologous complementation experiments in Ara-bidopsis have refuted a strict version of the pseudanthialmorphological interpretation for Triuridaceae (Álvarez-Buyllaet al. 2010). From this perspective, assignment of the term“flower” to the reproductive axes of L. schismatica and otherTriuridaceae species is accepted to be correct. However, athird alternative morphological model involved in the afore-mentioned controversy, which suggests the absence of a de-fined inflorescence–flower boundary in reproductive axes inTriuridaceae (Rudall and Bateman 2006; Rudall 2008; Rem-izowa et al. 2010), has not been evaluated yet from a devel-opmental–genetic perspective. In this paper, we adopt theeuanthial nomenclature and refer to the reproductive structuresof Triuris brevistylis Donn. Sm. as either “flowers” or “reproduc-tive axes.” However, we remain open to future developmental–

genetic and (or) epigenetic evidence that completes the currentpicture of the mechanistic regulation of morphogenesis in triuridreproductive axes.

Mabberley (1997, 2008) considers Lacandonia as a ho-meotic form of the type genus of the family Triuris Miers. Eventhough species of Triuris have usually been described as dioe-cious (Giesen 1938; Jonker 1943; Maas and Rübsamen 1986),the occurrence of bisexual axes in this genus has also beendocumented (Rübsamen-Weustenfeld 1991). For instance, in acomparative study of morphogenesis in reproductive axes ofboth L. schismatica and T. brevistylis (Vergara-Silva et al.2003), it was shown that individual plants of T. brevistylis bearbisexual flowers in addition to “regular” (i.e., unisexual) maleand female flowers. The bisexual reproductive axes studied byVergara-Silva et al. (2003) displayed at least three of thefollowing different arrangements of stamens and carpels: (i)male-like axes with heterotopic carpels (labeled here as type“a” axes), (ii) female-like axes with intermingled stamens andcarpels (type “b”), and (iii) bisexual flowers with centralstamens and surrounding carpels (type “c”; see Vergara-Silvaet al. 2003, figs. 7–10). Because Mexican populations com-posing T. brevistylis occur near the L. schismatica localities inthe Lacandon rainforest (Chiapas State, Mexico), these authorssuggested these two triurid species are closely related phylo-genetically. This postulation is congruent with the results ofcladistic analyses based on morphological matrices in whichboth Lacandonia and Triuris are included, along with all othermembers of the family at the generic level (Gandolfo et al.2002; Rudall and Bateman 2006; Rudall 2008).

Developmental–genetic data related to the heterotopic phe-notypes in bisexual flowers of T. brevistylis might contributeto clarify evolutionary issues implicit in the conflicting mor-phological viewpoints already reviewed. In fact, one of thetypes of bisexual axes described by Vergara-Silva et al. (2003)actually resembles the characteristic “inside-out” arrangementof L. schismatica (type “c”, above; see Vergara-Silva et al.2003, fig. 10). However, the strictly embryological, nonge-netic aspects of morphogenesis in Triuris species should beconsidered equally important in this regard. Detailed studiesconducted so far on the embryology of L. schismatica(Márquez-Guzmán et al. 1989, 1993; Vázquez-Santana et al.1998) have already shown the presence of preanthesis cleis-togamy (Lord 1981; Culley and Klooster 2007), an earlydevelopmental event previously unknown in triurids. Prean-thesis cleistogamy and double fertilization in L. schismatica(Márquez-Guzmán et al. 1993) evidently indicate self-pollination, but repeated references to and (or) observations ofparthenogenetic or apogamous embryos in several differenttriurid taxa (Wirz 1910; Giesen 1938; Dahlgren et al. 1985;Tomlinson 1982; Maas and Rübsamen 1986) suggest thatreproductive strategies in the family might involve both cleis-togamy and chasmogamy.

In this paper, we provide a detailed description of theembryology of male and female flowers in T. brevistylis. Wealso characterize the embryological stages of bisexual flowerswith female appearance, which nonetheless have well-developed stamens at the base of the receptacle. Here, we labelthis additional bisexual flower class (originally observed byMartínez 1994) as type “d”, following our own labeling oftypes “a”, “b”, and “c” in reference to the three heterotopicbisexual flowers described in Vergara-Silva et al. (2003). At

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the same time, we use the term “polygamodioecious” (sensuSakai and Weller 1999) to describe the complexity of thereproductive system in T. brevistylis already hinted at inVergara-Silva et al. (2003). The flowers studied in this workwere part of individual plants collected from previouslyknown locations in the Chiapas Lacandon rainforest (Vergara-Silva et al. 2003).

Through the use of a fluorescence technique that comple-mented standard optical and electron microscope based obser-vations, we have assessed the functionality of reproductiveorgans (i.e., stamens and carpels) in type “d” bisexual flowers.We found that cleistogamy unequivocally occurs in theseflowers. We discuss the potential contribution that embryolog-ical character states, especially those related to pollen graingermination and ovule fertilization, could make for systematicstudies in Triuridaceae and place our finding of cleistogamy inT. brevistylis in the context of the set of reproductive modesthat might take place in this triurid species. Finally, we suggestthat the observed complexity of morphogenetic, embryologi-cal, and reproductive aspects in natural populations ofT. brevistylis justifies a reconsideration of the centrality thatL. schismatica has had so far in the controversy around thenature of reproductive axes in the family.

Materials and methods

FieldworkCollection of T. brevistylis materials for the present work

was carried out for a period of 10 years (from 1998 to 2008)during the rainy season at the Lacandon rainforest (ChiapasState, Southern Mexico). Most of the female reproductive axeshad one or more female flowers. We also found a smallnumber of individual stems exclusively bearing male flowers,as well as an even smaller set of individual stems with flowerscontaining both anthers and carpels. Noticeably, flowers in thelatter group have a female appearance, with anthers at the recep-tacle base that are only visible at anthesis upon closer inspection.In this work, we consider these flowers, as well as their corre-sponding stems, to be functionally bisexual, and designate themas a new class (type “d”) of bisexual ramets–genets.

Plant materialFloral buds, postanthetic and anthetic male, female, and

type “d” bisexual flowers were collected from the localityof Nahá (16°57.716=N, 91°35.583=W, and 16°57.723=N,91°35.736=W). This site belongs to the deciduous Lacandonrainforest, located in the state of Chiapas, southeastern Mex-ico. Vouchers of T. brevistylis were submitted to the Herbar-ium of the Universidad Nacional Autónoma de México(MEXU), labeled as E. Martínez S. 21864 (MEXU 917742)and E. Martínez S. 21920 (MEXU 917961).

Embedding, sectioning, and observation by lightmicroscopy (LM)

The collected materials were fixed in the field in FAA(formalin, acetic acid, 96% ethanol, water, 2:1:10:7 by vol-ume). After dehydration in a graded ethanol series, the sam-ples were embedded in paraplast and JB-4 resin (Polyscience,Inc., Niles, Ill., USA), and sectioned with an AO 820 rotatorymicrotome (American Optical Co., New York, N.Y., USA) orwith a Sorvall MT2-B ultramicrotome (Sorvall, Newtown,Conn., USA), respectively (Johansen 1940; Ruzin 1999).

Histochemical test and stainingSections were used for the following histochemical tests:

Schiff’s periodic acid reaction for nonsoluble polysaccharideinclusions, naphthol blue-black for proteins, oil red O reactionfor lipids, and 10% ferrous sulfate – HCL for tannins (Johan-sen 1940; O’Brien and McCully 1981).

Paraplast sections were stained and incubated for 5 min in0.1% decolorized aniline blue (Polyscience, Inc.) in a phosphatebuffer at 0.1 mol·L–1 and pH 8.5 (Ruzin 1999). Sections wereobserved using epifluorescence microscopy at UV excitationwavelength to localize callose of pollen mother cells andpollen tubes using an Olympus PROVIS AX 70 equipment(Olympus Co., Tokyo, Japan). Resin slides were stained with0.05% toluidine blue O (Polyscience, Inc.) and observed withan Olympus BH-2 photonic microscope (Olympus Co.).

Transmission electron microscopy (TEM)At the site of collection dissected flowers were fixed in 5%

glutaraldehyde � 4% paraformaldehyde � 0.1 mol·L–1

s-collidine buffer, pH 7.2 at 4 °C, dehydrated in a gradedacetone series, embedded in resin Epon 812 (Polyscience,Inc.), and sectioned with a Sorvall MT2-B ultramicrotome.Ultrathin sections (gold color) of the pollen grains werestained with uranyl acetate and lead citrate. Sections wereexamined with an EM-10 Zeiss transmission electron micro-scope (Carl Zeiss Co., Jena, Germany).

Scanning electron microscopy (SEM)Floral buds, flowers, fruits, and seeds already fixed in FAA

were dehydrated in a graded ethanol series and critical-pointdried using a CPD 030 critical-point dryer (Bal-Tec AG,Liechtenstein). The samples were mounted on aluminum stubsusing carbon double-sided tape and coated with gold by meansof a Denton Vacuum Desk II (Denton Vacuum, Moorestown,N.J., USA). Observations were carried out with Jeol JMS-35and Jeol JSM 5310-LV scanning electron microscopes (JEOL,Ltd., Tokyo, Japan).

Results

FieldworkThroughout the entire cycle of flowering and fructification in

T. brevistylis (i.e., October to December–January) during our 10years (from 1998 to 2008) of uninterrupted field collections, theproduction of male flowers, i.e., ramets bearing exclusively maleflowers, was estimated to be scarce, with a ratio of approximately100 female ramets to each male ramet. At the same time, approx-imately 15 individual ramets with bisexual flowers, typically withvariable numbers of stamens and carpels arranged irregularly,were observed, but never in co-occurrence with unisexualflowers. Added to the information currently at hand, new fieldobservations in populations of T. brevistylis might allow a quan-tification of the proportion in which the different type “d” bisex-ual flower, as well as the monoecious ramets, in which both maleand female flowers seemed well developed, are spontaneouslyproduced in the field every season.

In October and November of the years in which the studyperiod involved, most of the individual ramets bearing type“d” bisexual flowers and female flowers both have fruits atdifferent developmental stages. By the end of the fruitingseason, i.e., November and December, these ramets werescarce, and the small number that was still present had a few

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fruits still attached to the receptacle. The last fruits werecollected in January when male ramets were practicallygone.

Floral morphology

Male rametsThese ramets have exclusively male flowers. Their perianth

is formed by three triangular units, hereinafter referred to astepals. The tepals are arranged in a single cycle, and they arebasally connate, with a long, caudate apex, which remainsnarrowly upright with respect to the androphore during theentire flowering period (Fig. 1).

In male flowers, an androphore develops as a fleshy,hyaline, massive central column with faces alternate to the stamensand short wing-like tissue extensions at its apex (Fig. 2). Theandroecium, which is closely associated to the androphore,consists of three stamens with dithecate anthers, each one withvery short filaments (Fig. 3). The stamens alternate with thetepals and are situated within a cavity at the base of theandrophore (Fig. 4). The extrorse dehiscence of the anthers ofmale flowers occurs through longitudinal slits (Fig. 5). Incontrast, the anthers are indehiscent in type “d” bisexualflowers.

In male flowers, epidermal cells of the anthers, filaments,and the cavities of the androphore are glandular (Fig. 6), eachsecretory trichome being uni- or bi-cellular with dense vacu-olated cytoplasm (Fig. 7).The young anthers are dithecate andtetrasporangiate (Fig. 8), but the septum between the mi-crosporangia of a theca degenerates just prior to anthesis.Hence, mature anthers are dithecate and disporangiate (Fig. 9).The vascular bundle reaches the receptacle but does not enterthe filaments, tepals, or androphore. No pistillode or residualfloral apex of vascular bundle is observed.

Female and type “d” bisexual rametsThe perianth in female flowers and type “d” bisexual flow-

ers has three triangular tepals arranged in a single cycle and isbasally connate, each ending in a short, caudate apex. Once theflowers are opened, the caudal tips remain appressed to thepedicel until fruit maturation and dispersal take place. Thegynoecia is apocarpous, and the number of carpels in femaleand type “d” bisexual flowers are variable. The carpel number is aslow as 120 and as high as 500. In both cases, ripe fruits are formed(Fig. 10). The style is topographically subapical on the ovary,and there is no differentiated stigma (Fig. 11). The style isformed by a single row of parenchymatic cells, surrounded byepidermal cells; transmitting tissue is absent (Fig. 12). Nostaminode or residual floral apex is observed in the receptacle.However, in type “d” bisexual flowers, the indehiscent,monothecate, and monosporangiate functional stamens areobserved (see Figs. 54, 55).

In type “d” bisexual flowers, receptacle cells produce andaccumulate a mucilaginous secretion during floral develop-ment, which locates close to the vascular bundles. This secre-tion, which is incipient in female flowers, apparently hasnonsoluble polysaccharides and proteins and remains untillater stages in fruit maturation (Fig. 13). The floral vascularbundles reach the receptacle but do not enter the carpels or thetepals (Fig. 14).

Androecium development in male flowers and type “d”bisexual flowers

Early anther developmentYoung male flowers are enclosed within a bract that is

two to three cell layers thick, and the tepals develop syn-chronously and surround the androecium tissue (Fig. 15). Ina young anther, the archesporial tissue is delimited by anepidermis and a primary parietal layer (Fig. 16). Subsequently,the primary parietal layer divides periclinally, forming outerand inner secondary parietal layers. The former differentiatesinto the endothecium, whereas the latter divides again pericli-nally, giving rise to the middle layer and to the secretorytapetum (Fig. 17). Thus, anther wall development is of themonocotyledonous type, with four one-cell-wide layers: anepidermis, an endothecium, a middle layer (which disap-pears early), and a uni- to bi-nucleate secretory tapetum(Fig. 18).

Just before anthesis, anther walls comprise the epidermisand the endothecium, as the middle layer and tapetumbecome flattened and closely crushed against the endoth-ecium (Fig. 19). At anthesis, mature anther walls are theepidermis and the endothecium, with endothecial cell wallsshowing U-shaped thickenings (Fig. 20). In type “d” bisexualflowers, the anther wall cells develop similarly to those ofmale flowers.

Microsporogenesis and microgametogenesisIn floral buds, pollen mother cells are surrounded by callose

(Fig. 21). The anticlinal and internal walls of the tapetum aredegenerated and allow the formation of cytoplasmic bridgesamong these cells, which persist until early meiosis stages(Fig. 22). Secretory tapetum cell walls degenerate and theircytoplasmic content is released into the microsporangial cav-ity. Isobilateral microspore tetrads arise as a result of succes-sive cytokinesis (Fig. 23). Uninucleate free microsporesalready have exine ornaments at this point (Fig. 24).

The first mitotic division of the uninucleate microsporesgives rise to the generative and vegetative cells (Fig. 25), andmitotic divisions of the former give rise to the sperm cells(Fig. 26). Immediately afterwards, pollen grains are shed asmonads at the three-celled stage, through a longitudinal slit(Fig. 27). Germination stages of mature pollen grains, as wellas some exine remnants and collapsed pollen grains, are ob-served inside closed microsporangia in flowers of the maleramet (Fig. 28). However, fluorescence tests reveal that pollentubes are not present on the receptacle, anthers, filaments, orstems of these flowers.

Pollen grain structureMature pollen grains are spheroidal, inaperturate, and 20–

25 �m in diameter, and the exine is intectate, gemmate withsmall spines (spiny-gemmate) (Fig. 29). Some pollen grainsshow areas with exine detachment (Fig. 30). TEM micro-graphs show an exine and highly thickened intine (Fig. 31).

Gynoecium development in female and type “d” bisexualflowers

Megasporogenesis and megagametogenesisA bract of two to three cell layers thick encloses the floral

apex. A whorl of three tepals is formed synchronously

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Figs. 1–9. Morphology and anatomy of male ramets with male flower. Fig. 1. General view of the male flower showing three tepals withlong caudate apices (arrows). Scale bar � 2 mm. Fig. 2. Fleshy and hyaline androphore. The arrows indicate the wing-like tissueextensions at its apex. Scale bar � 700 �m. Fig. 3. Closeup of anther showing very short filament (arrow). The tepals were removed.SEM. Scale bar � 140 �m. Fig. 4. General view of male flower showing anthers alternating with tepals. The cavities are indicated byarrows. SEM. Scale bar � 700 �m. Fig. 5. Anther showing the extrorse dehiscence through a longitudinal slit (arrows); note the papillateepidermis. Scale bar � 180 �m. Fig. 6. Glandular epidermis in the filament base (asterisk); note the glandular epidermis with secretorytrichomes in the cavity of the androphore (arrow). Scale bar � 70 �m. Fig. 7. Closeup of glands in the cavities of androphore with densecytoplasm, vacuoles, and nucleus. Scale bar � 10 �m. Fig. 8. Longitudinal section of dithecate and tetrasporangiate young anther; theseptum between microsporangia is indicated by arrows. Scale bar � 140 �m. Fig. 9. Longitudinal section of dithecate and disporangiatemature anther; note that the septum between microsporangia has disappeared. The extrorse dehiscence is indicated by arrows. Scale bar �140. A, anther; AN, androphore; F, filament; TP, tepal.

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(Fig. 32). Carpels are formed asynchronously and centrifu-gally. In each young carpel, the ovule is curved in the samedirection as the carpel is involute (Fig. 33). The large arches-porial cell is conspicuous due to its denser cytoplasm andprominent nucleus. The archesporial cell acts directly as themegaspore mother cell, and shortly afterwards, the inner in-tegument primordium begin to develop from the nucellarepidermis (Fig. 34). As the megaspore mother cell enlarges, aswell the epidermal nucellus, both become surrounded by outerand inner teguments, each or which two-layered. Later, theinner integument forms the micropyle (Fig. 35).

The megaspore mother cell is divided meiotically andgives rise to a linear megaspore tetrad (Fig. 36). The threemicropylar megaspores degenerate, and the chalazal one re-mains as the functional megaspore (Fig. 37). The first mitoticdivision of the functional megaspore results in a binucleateembryo sac, with one nucleus migrating to the micropylarpole and the other to the chalazal pole (Fig. 38). Two mitoticcycles occur subsequently, forming a tetra- and octo-nucleatecoenocytic embryo sac with a central vacuole (Figs. 39, 40).

At the end of the mitotic phase, the nucellus is reabsorbed anda seven-celled embryo sac of the Polygonum type is formedwith three antipodal cells, a binucleate central cell, and an eggapparatus composed of an egg cell and two synergids(Fig. 41). The mature ovule is anatropous, tenuinucellate, andbitegmic, with outer and inner two-layered integuments.

Seed developmentThe remnants of pollen tubes observed in the micropyle of

female flowers suggest porogamic fertilization. The presenceof two- and three-celled linear proembryos indicates that di-visions at the beginning of embryogenesis are exclusivelytransverse (Figs. 42, 43). Afterwards, cell divisions occur inthe longitudinal and transverse planes until the embryo startsto develop. The basal cell of the proembryo represents the firstcell of the suspensor (Figs. 44, 45), which by transversedivisions becomes three to four celled (Fig. 46).

Endosperm development is of the free nuclear type. Duringthe first divisions, nuclei are attached to the vacuolate embryosac wall (Figs. 42, 43). Afterwards, the nuclei undergo free-

Figs. 10–14. Morphology and anatomy of the female ramets with female flower, and bisexual ramets with type “d” bisexual flower.Fig. 10. Female flower showing apocarpous gynoecium and tepals with short, caudate apex (arrow). Scale bar � 1 mm. Fig. 11. Closeupof carpels showing topographically subapical styles (arrows); note the absence of a receptive zone or stigma. SEM. Scale bar � 100 �m.Fig. 12. Single carpel showing topographically subapical style with parenchymatous cells; note the absence of a receptive zone. Scalebar � 80 �m. Fig. 13. Receptacle showing the mucilaginous tissue (arrow). Scale bar � 160 �m. Fig. 14. Closeup of receptacle showingvascular bundles (arrows) below mucilaginous tissue; note that the vascular bundles never reached carpels or tepals. Scale bar � 80 �m.C, carpel; ST, style; TP, tepal.

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nuclear divisions, and the endosperm appears as a large syn-cytium. At this stage, the cytoplasm-surrounded nuclei are themost prominent structures (Figs. 44, 45). When there are morethan 20 free nuclei (Fig. 45), endosperm cell wall formation isinitiated by the synthesis of incipient cell walls (Fig. 47).

The conspicuous cellular endosperm consists of large cells,each with a large nucleus and a dense cytoplasm with proteinbodies and nonsoluble polysaccharides as reserve materials.Neither starch bodies nor lipids are observed at any develop-

mental stage (Figs. 46, 48). Nonsoluble polysaccharides thatpotentially function as reserve materials are present in wallthickenings of the endosperm cells. Lipids are present in thenucellus remnants and inner integument cuticles. The cuticlesdivide the seed coat from the endosperm cells (Fig. 48).

The seed coat is developed only from the outer integument,as the inner integument degenerates after fertilization and thestart of seed differentiation (Fig. 47). Endotestal cells arelarger in the radial direction with respect to exotestal cells,

Figs. 15–20. Anther wall development. Fig. 15. Male floral bud showing an androecium meristem covered by two tepals (out of three) anda bract that is two to three cell layers thick. Scale bar � 65 �m. Fig. 16. Young anther showing primary parietal layer and sporogenoustissue. Scale bar � 30 �m. Fig. 17. Periclinal division of the inner secondary parietal layer (arrow); note the epidermis and endothecium.Scale bar � 15 �m. Fig. 18. Anther wall with an epidermis, an endothecium, a middle layer, and a uni- to bi-nucleate secretory tapetum;note the pollen mother cells. Scale bar � 10 �m. Fig. 19. Remnants of uni- to bi-nucleate secretory tapetum (arrow) flattened and adjacentto the endothecium; note the epidermis. Scale bar � 15 �m. Fig. 20. Mature anther wall showing epidermis and endothecium withU-shaped wall thickenings (arrows). Scale bar � 10 �m. B, bract; E, epidermis; ED, endothecium; M, middle layer; PMC, pollen mothercell; PP, primary parietal layer; SP, sporogenous tissue; T, tapetum; TP, tepal.

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Figs. 21–28. Microsporogenesis and microgametogenesis in male and type “d” bisexual flowers. Fig. 21. Pollen mother cells in meiosissurrounded by callose; note the uni- to bi-nucleate secretory tapetum. Scale bar � 45 �m. Fig. 22. Cytoplasmic bridges (arrow) amongtapetal cells; note that the pollen mother cells are surrounded by callose and are undergoing meiosis. Scale bar � 10 �m. Fig. 23.Isobilateral microspore tetrads. Scale bar � 25 �m. Fig. 24. Free uninucleate microspore. Scale bar � 7 �m. Fig. 25. Pollen grainshowing vegetative and generative cells. Scale bar � 6 �m. Fig. 26. Three-celled pollen grain showing vegetative cell and two sperm cells.Scale bar � 10 �m. Fig. 27. Mature pollen grains shedding through the extrorse longitudinal slit (arrow). Scale bar � 30 �m. Fig. 28.Pollen grain in germination stage (arrow) inside the closed microsporangium; note the remains of exine (arrowhead). Scale bar � 10 �m.CW, callose wall; E, epidermis; ED, endothecium; G, generative cell; M, middle layer; PMC, pollen mother cells; SC, sperm cells;T, tapetum; V, vegetative cell.

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especially at the micropylar region. The cytoplasm of endo-testal cells contains tannins and nonsoluble polysaccharideinclusions. In contrast, exotestal cells are smaller and flattenedand only some of them have nonsoluble polysaccharide inclu-sions. Additionally, none of exotestal cells has tannins(Figs. 48, 49).

Seeds are brown and ovoid with a round chalazal pole anda slightly attenuated micropylar pole. The surface of the seedcoat is composed of rectangular cells that form a reticulum.The outer periclinal walls and the cuticle are smooth (Fig. 50).

FruitThe dispersal units are achenes. Fruit development is asyn-

chronous and centrifugal. Achenes placed at the center of thereceptacle are the earliest to ripen and are also dispersed first(Fig. 51). Their shape is ovoid, with the epidermal cells muchelongated and with deeply depressed outer cell walls. Thepericarp appears reticulate (Fig. 52). The endocarp has elon-gated cells with thin walls. These cells have vacuoles ofconsiderable size, which restricted the cytoplasm to a thinlayer close to the cell wall (Fig. 53). The style persists untilachene dispersal (Figs. 52, 53).

Fertilization in type “d” bisexual flowersAndroecium and gynoecium development in type “d” bi-

sexual flowers (Figs. 54, 55) is similar to that observed innormal male and female flowers. The observation of severalstages of microsporogenesis, microgametogenesis, megas-porogenesis, and megagametogenesis in these bisexual flowerssuggests that their androecia and gynoecia are functional(Fig. 56).

In type “d” bisexual flowers, monothecate indehiscent an-thers are observed at the base of the receptacle. This observa-tion is made both in floral buds and in anthetic flowers. Stamendevelopment begins before the development of the latest car-pels (Figs. 54, 55, 57). Fluorescence tests indicate that, in thesebisexual flowers, pollen grains germinate inside the indehis-cent anthers during both early (bud stage) and late (anthesisstage) development. Anthers are indehiscent, despite the pres-ence of a developed endothecium (Fig. 59). Pollen tubes travelmainly through the mucilaginous tissue of the receptacle bothat the floral bud and at the anthesis stage (Figs. 57, 58). Pollentubes grow until they reach the base of carpels (Figs. 57–59).Finally, pollen tubes enter the ovule through the micropyle tocarry out porogamous fertilization (Figs. 60, 61). Pollen tubesare not observed growing through styles in type “d” bisexualflowers or in female flowers.

Monoecious ramets–genetsA small number of monoecious ramets–genets were ob-

served during the field collection phase of this study. To thenaked eye, both male and female flowers have normal mor-phology in these ramets–genets (Figs. 62, 63); however, em-bryological details for these flowers are currently unknown.

Discussion

Morphogenesis and embryology of male flowers,including anther and pollen development

In the Mexican and Guatemalan triurid T. brevistylis, theseptum between the microsporangia of each theca degeneratesat early stages in male flowers (see Figs. 8, 9). This commonlyoverlooked developmental feature may be the cause of incor-

Figs. 29–31. Pollen grains structure. Fig. 29. Spheroidalinaperturate pollen grain with intectate, spiny-gemmate exine.SEM. Scale bar � 6 �m. Fig. 30. Mature pollen grain showingpartially exineless region (arrow); note the intectate, spiny-gemmate exine. SEM. Scale bar � 2.5 �m. Fig. 31. Pollen grainshowing thin, intectate, spiny-gemmate exine and thick intine(arrowheads). TEM. Scale bar � 6.5 �m.

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Figs. 32–40. Megasporogenesis and megagametogenesis in female ramets with female flower and bisexual ramets with type “d” bisexualflower. Fig. 32. Gynoecium floral meristem surrounded by tepal primordia and a bract that is two to three cell layers thick. Scale bar �70 �m. Fig. 33. Receptacle showing asynchronous and centrifugal development of carpel primordia (arrows). Scale bar � 47 �m. Fig. 34.Carpel showing archesporial cell surrounded by nucellar tissue and inner integument primordium (arrowhead). Scale bar � 45 �m. Fig. 35.Ovule with elongate megaspore mother cell and outer and inner two-layered growing integuments; note the nucellus. Scale bar � 32 �m.Fig. 36. Ovule showing linear megaspore tetrad (arrows) surrounded by outer and inner two-layered integuments. Scale bar � 22 �m.Fig. 37. Ovule with functional megaspore in chalazal region and degenerated micropylar megaspores (arrowhead). Scale bar � 17 �m.Fig. 38. Binucleate embryo sac (arrow) and remnants of nucellus. Scale bar � 23 �m. Fig. 39. Tetranucleate embryo sac with a centralvacuole (arrow). Scale bar � 22 �m. Fig. 40. Ovule showing octonucleate embryo sac stage with a central vacuole; note the outer andinner two-layered integuments. Scale bar � 30 �m. AC, archesporial cell; B, bract; FM, functional megaspore; II, inner integument; MMC,megaspore mother cell; N, nucellus; OI, outer integument; TP, tepal.

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Figs. 41–49. Embryogenesis, endospermogenesis and seed morphology in female ramets with female flower, and bisexual ramets withtype “d” bisexual flower. Fig. 41. Seven-celled embryo sac with three antipodal cells, central cell with two polar nuclei fused and eggapparatus; note the egg cell. Scale bar � 27 �m. Fig. 42. Two-celled linear proembryo and free endosperm nuclei; note the endotesta withlarge cells towards the micropylar region. Scale bar � 50 �m. Fig. 43. Proembryo at the three-celled linear stage; note the free endospermnuclei. Scale bar � 20 �m. Fig. 44. Seed showing three-celled lineal proembryo and nuclei of endosperm; note the remnants of pollentubes in the micropyle. Scale bar � 15 �m. Fig. 45. Embryo with basal cell in mitosis (arrow), the nuclear-free stage is still observed inthe endosperm (arrowheads). Scale bar � 20 �m. Fig. 46. Embryo showing three-celled suspensor. The arrowheads indicate the cellularendosperm. Scale bar � 20 �m. Fig. 47. Seed showing incipient cellular stage of endosperm development, the thin cell walls are indicatedby arrows, and the arrowhead points to remnants of the tegmen. The endotesta and exotesta are also shown. Scale bar � 30 �m. Fig. 48.Seed showing cellular stage of endosperm. The arrowheads indicate the thick cell walls of endosperm; note the tannins and nonsolublepolysaccharides at the endotesta and exotesta. Scale bar � 20 �m. Fig. 49. Mature fruit showing a seed with reduced embryo; note thechalaza and copious endosperm. The endotesta shows nonsoluble polysaccharides in the micropylar region, and the exotesta is also shown.Scale bar � 70 �m. AT, antipodal cell; CC, central cell; CH, chalaza; EC, egg cell; EM, embryo; EN, endosperm; ET, endotesta; EX,exotesta; PE, proembryo; S, suspensor.

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rect interpretations of the number of microsporangia in themature anthers of Triuridaceae, where putative disporangiate,tetrasporangiate, and even trisporangiate conditions have beenreported (Tomlinson 1982; Dahlgren et al. 1985; Maas andRübsamen 1986; Maas-van de Kamer and Weustenfeld 1998).We therefore suggest that the number of microsporangia ineach theca in anthers of preanthetic flowers should be furtherinvestigated in detail for other triurids.

Anther wall development in T. brevistylis is of the monocoty-ledoneous type, with four cell layers, as in T. hexophthalmaMaas, T. hyalina Miers, and other species of tribe Sciaphileae

(Maas and Rübsamen 1986; Rübsamen-Weustenfeld 1991). Incorrespondence to the systematic scheme provided by Furnessand Rudall (2006), character states for anther development inT. brevistylis should be coded as follows: (i) secretory tapetumwhere cell walls remain until the microspore tetrad stage; (ii)successive microsporogenesis; (iii) medium-sized pollengrains; and (iv) pollen grains dispersed as monads.

A variety of tapetum types has been observed in Triuri-daceae, mainly periplasmodial and secretory, but also inter-mediate forms (Maas and Rübsamen 1986; Rübsamen-Weustenfeld 1991; Furness et al. 2002; Furness and Rudall

Figs. 50–53. Fruit and seed. Fig. 50. Ovoid mature seed with convex chalazal region and slender micropylar region (arrow); note therectangular cells of the seed coat. Pericarp was removed. SEM. Scale bar � 42 �m. Fig. 51. Receptacle of female flower showing fruits indifferent developmental stages; note that the ripe fruits are located in the center of the receptacle. SEM. Scale bar � 300 �m. Fig. 52.Achene showing the pericarp with reticulate and deeply depressed epidermal cells and persistent style. SEM. Scale bar � 44 �m. Fig. 53.Achene with pericarp showing thin cell walls; note the persistent style. Scale Bar � 85 �m. EN, endosperm; EX, exotesta; P, pericarp;ST, style.

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Figs. 54–61. Fertilization in bisexual ramets with type “d” bisexual flower Fig. 54. Apocarpous gynoeciun and one (of three) monothecateand monosporangiate indehiscent anthers. Tepals were removed. SEM. Scale bar � 90 �m. Fig. 55. Two basal stamens with monothecateand monosporangiate indehiscent anthers. Tepals were removed. Scale bar � 200 �m. Fig. 56. Closeup of an anther with pollen mothercells and a carpel with a megaspore mother cell. Scale bar � 55 �m. Fig. 57. Two basal stamens with monothecate and monosporangiateindehiscent anthers with germinating pollen grains. The pollen tubes inside the anthers growing on the receptacle are revealed with UVfluorescence test. No pollen tubes are observed in styles. Scale bar � 150 �m. Fig. 58. UV fluorescence test of a floral bud with pollentubes growing from the anthers to the carpels (arrows) crossing the receptacle through the mucilaginous tissue. Scale bar � 150 �m.Fig. 59. Closeup of UV fluorescence test of an anther (during anthesis) showing almost fully elongated pollen tubes passing through themucilaginous tissue of the receptacle (arrowheads). Note the secondary wall thickenings of the endothecium layer. Scale bar � 55 �m.Fig. 60. UV fluorescence test of carpel showing the entrance of a pollen tube inside the embryo sac through the micropyle (arrow). Scalebar � 25 �m. Fig. 61. Another view of a different carpel showing the entrance of the pollen tube into the embryo sac (arrow). Scalebar � 35 �m. A, anther; C, carpel.

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2006). The distribution of these character states is somewhatcorrelated with taxonomic grouping; although, the plasmodialtype is predominantly present in Sciaphileae, a secretory tapetumis common in Triurideae (Rübsamen-Weustenfeld 1991). As amember of Triurideae, T. brevistylis has an exclusively secre-tory tapetum, but other species of the genus have intermediateforms, e.g., T. hexophthalma, T. hyalina, and Andruris cf.andajensis (Sciaphila arfakiana) Becc.; Maas and Rübsamen(1986). Potential cytoplasmic bridges among tapetal cells,described for L. schismatica (Márquez-Guzmán et al. 1993) asa novel character state, were also observed in T. brevistylis (seeFig. 22). The commonality of this character state further suggests aclose phylogenetic affinity between the two triurid species.

Successive microsporogenesis in T. brevistylis occurs inassociation with isobilateral microspore tetrads. This char-acter state is also present in Sciaphila purpurea Benth.,Sciaphila rubra Maas, and T. hexophthalma (Rübsamen-Weustenfeld 1991) and in L. schismatica (Márquez-Guzmánet al. 1993). In contrast, decussate microspore tetrads havebeen observed in Seychellaria madagascariensis C. H.Wright. Observations regarding the tetrad stages for otherTriuris species are currently missing. Such information wouldbe necessary to complete a full list of possible character statesfor microsporogenesis in the family.

Some aspects of our palynological results for T. brevistylisdo not coincide with previous reports for other triurids, inparticular with regard to the presence of apertures and featuresof the intine. For instance, our observation of a uniformlythickened intine in T. brevistylis is in clear contrast withstatements by Rübsamen-Weustenfeld (1991), who recordedthat intine in Triuridaceae is thickened only towards the zoneoccupied by the vegetative cell. The presence of a very thickintine covered by an irregularly sculpted thinner exine inSeychellaria africana Vollensen and some species ofSciaphila has been interpreted as an aperturoid zone(Rübsamen-Weustenfeld 1991). In other triurid species suchas Andruris cf. andajensis (Sciaphila arfakiana), it is im-possible to distinguish an evident aperture zone (Wirz 1910;Erdtman 1952). Pollen grains showing this feature are consid-ered functionally monoaperturate (Furness and Rudall 1999)and are common in the families Cyclanthaceae (Dianthoveus,Evodianthus) (Tomlinson and Wilder 1984) and Stemonaceae(Pentastemona, Stichoneuron). Taking into account records ofpollen grains without a well-defined sulcus in Triuris (Maasand Rübsamen 1986), inaperturate pollen grains might beconsidered common in triurids (Furness and Rudall 1999;2006). Previous reports of monosulcate pollen grains in An-druris japonica (Makino) Giesen (Chuma 1990) and

Figs. 62–63. Photographs of specimens of monoecious ramets–genets. Fig. 62. Young monoecious ramet–genet with male and femaleflowers. Fig. 63. Mature monoecious ramet–genet with male and female flowers. Ripe fruits ready for dispersion can be observed over thereceptacle of the female flower. FE, female flower; MA, male flower.

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T. hexophthalma (Rübsamen-Weustenfeld 1991) were inter-preted as a technical artifact (Furness et al. 2002) or as aproduct of contamination (Furness and Rudall 2006), andolder reports on prolate monosulcate pollen grains in membersof Sciaphileae may also be invalid (Dahlgren et al. 1985; Maasand Rübsamen 1986). Our finding of inaperturate pollengrains, with highly thickened intine and less dense exine proneto easy detachment, therefore agree with the more recentevaluations of pollen morphology.

The pollen grain surface in T. brevistylis is spiny-gemmate,a character state that is common for Triuris (Rübsamen-Weustenfeld 1991) and Triuridaceae as a whole (Chuma 1990;Sahashi et al. 1991; Grayum 1992; Márquez-Guzmán et al.1993; Maas-van de Kamer and Weustenfeld 1998; Rudall et al.2007). Furness and Rudall (2006) state that spiny-gemmateexine is unusual in Pandanales, but along with Stemonaceae(Ham 1991), this exine type is putatively apomorphic anddiagnostic for Triuridaceae, although it is not so evident inSciaphila (Furness et al. 2002) and Andruris (Sahashi et al.1991). In other Pandanales, the exine commonly has agranular-verrucose surface and columellae of variable height(Furness and Rudall 2006). Exine character states observed inT. brevistylis are also found in L. schismatica (Márquez-Guzmán et al. 1993), once again suggesting a likely sister-group relationship.

Morphogenesis and embryology of female flowersExcept for the study of Rudall (2003), a comparative treat-

ment of female embryology in Pandanales that would corre-spond to the framework available for male aspects is currentlylacking. The type of embryo sac development present in triu-rids is a case in point. Rübsamen-Weustenfeld (1991) andMaas-van de Kamer (1995) have suggested that embryo sacdevelopment of the tribe Triurideae is of the Fritillaria type,where the megaspore tetrad forms the embryo sac; this is thecharacter state present in T. hyalina and T. hexophthalma.Even though T. brevistylis belongs to Triurideae, ourobservations clearly indicate that embryo sac development ismonosporic of the Polygonum type, where three megasporesdegenerate and only the chalazal megaspore forms the embryosac. This mode of development is also found in Sciaphilaalbescens Benth. and Soridium spruceanum Miers (Maas andRübsamen 1986; Rübsamen-Weustenfeld 1991), both mem-bers of the tribe Sciaphileae. This unexpected character state inT. brevistylis is of interest with regard to the type of embryosac development found in L. schismatica, in which the micro-pylar megaspore gives rise to the embryo sac (i.e., the so-called “Lacandonia type”; Vázquez-Santana et al. 1998). Suchevaluation might allow further insight into the causes of theinstability for this character in triurids and potentially withinPandanales. A parallel search for developmental–genetic con-trols of these embryological features might also contribute tothe same end.

As with most triurids, T. brevistylis has one anatropousovule per carpel. A contrasting comparison for these statescan be established, however, between Triurideae and mem-bers of Sciaphila, e.g., the recently discovered Kupea mar-tinetugei Cheek & S. A. Williams (Rudall et al. 2007). Asin other triurids, the seed coat differentiates from the outerintegument, whereas the inner integument disappears (Dahl-gren et al. 1985; Maas and Rübsamen 1986; Maas-van de

Kamer 1995; Maas-van de Kamer and Weustenfeld 1998). InT. brevistylis, the inner epidermis of the outer integument isthicker than the outer epidermis.

Mature seeds of some Triuridaceae have cell remnants fromthe inner integument, characterized by the presence of tanninsin the chalazal region (Maas and Rübsamen 1986). However,neither tannins nor lipids in the cytoplasm of endosperm cellswere observed in T. brevistylis, as reported for other triurids(Rübsamen-Weustenfeld 1991). Instead, tannins were onlyrecorded in the endotesta and pericarp, whereas lipids werepresent in the remains of the nucellar tissue and inner integ-ument cuticles. In this work, starch was not detected at anydevelopmental stage, as mentioned by Dahlgren et al. (1985).However, Maas and Rübsamen (1986) and Rübsamen-Weustenfeld (1991) had reported that starch is present only inimmature seeds and that it disappears during seed maturation.The location of nonsoluble polysaccharide inclusions and pro-tein bodies in T. brevistylis follows the pattern describedelsewhere for the family. Nonsoluble polysaccharide inclu-sions are found in the thickened endosperm walls and itscytoplasm, whereas protein bodies are located in the cyto-plasm of the same tissue (Maas and Rübsamen 1986; Maas-van de Kamer 1995). We assume that the major reservematerials in mature seeds of T. brevistylis are proteins andnonsoluble polysaccharides.

The presence of well-formed stamens and carpels, maturefruits, and mucilaginous tissue in the receptacle suggests that asimilar embryological–reproductive process might take place intypes “a”, “b”, and “c” bisexual flowers, i.e., the previouslydescribed bisexual ramets–genets in which the arrangement oforgans is irregular (Vergara-Silva et al. 2003). We now interpretmucilaginous tissue within the receptacle of female and type“d” bisexual flowers as a source of nutritional substances tothe growing pollen tubes during their growth trajectory to theegg cell. We assume that this interpretation supersedes earliersuggestions of the involvement of these substances in animal-mediated dispersion of mature fruits (Maas-van de Kamer1995; Maas-van de Kamer and Weustenfeld 1998).

Inference of reproductive strategies from morphogeneticand embryological data

Wirz (1910) and Dahlgren et al. (1985) pointed out that theembryo in Triuridaceae develops parthenogenetically throughapogamy. In turn, sexual reproduction in triurids was firstmentioned for Soridium spruceanun (Rübsamen-Weustenfeld1991; Espinosa 2009) and demonstrated in the bisexual flow-ers of L. schismatica (Márquez-Guzmán et al. 1993). Accord-ing to our fluorescence microscopy data, the mechanism ofsexual reproduction known as cleistogamy, a presumably au-tapomorphic feature of L. schismatica (Márquez-Guzmán etal. 1993), has been observed in type “d” bisexual flowers ofT. brevistylis. However, there is an important difference in thisembryological–reproductive character between the two spe-cies. Whereas pollination and self-fertilization occur strictly inclosed floral buds in L. schismatica, pollen tubes of bisexualflowers of T. brevistylis grow through the mucilaginous tissueof the receptacle, both in closed floral buds and in antheticflowers. An explanation for this difference in character statesis likely related to the centrifugal and asynchronous develop-ment of the sexual organs in T. brevistylis, which contrasts

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with the synchronous stamen and carpel development inL. schismatica.

Pollination in female flowers is still an unsolved question,not only in T. brevistylis but across Triuridaceae. Accepting aeuanthial morphological interpretation in Triuridaceae, cleis-togamy in T. brevistylis should not be considered strictlyhomologous to the phenomenon observed in taxa such asCallitriche (Philbrick 1984; Philbrick and Anderson 1992),where pollen tubes have been found to move through vegeta-tive tissues of inflorescences. Even within a euanthial frame-work, though, it is not known where pollen grains would bedeposited in some Triuridaceae, because carpels lack a recep-tive zone. For Maas-van de Kamer (1995), the high number offruits formed and the absence of unfertilized ovules indicatethat successful pollinators (e.g., small flies; Rudall and Bate-man 2006) are present. Emission of strong smells produced bypapillate tepal cells with glandular roles, which has beendocumented in Sciaphila albescens and Soridium spruceanum,supports this suggestion (Maas and Rübsamen 1986; Maas-van de Kamer 1995; Maas-van de Kamer and Weustenfeld1998). Within Pandanales as a whole, variation in pollinatorsis noticeable (Cox 1990; Franz 2004). However, despite thepresence of anatomical and morphological attributes in stami-nate flowers of Triuris as mentioned by Maas-van de Kamerand Weustenfeld (1998), which would suggest the existence ofcross pollination, we have been unable to detect the presenceof pollinators in T. brevistylis during several years of contin-uous fieldwork.

Seeds and fruits at different developmental stages wereobserved in T. brevistylis, mainly in female flowers. Theobservation of flowers at these ontogenetic stages by the endof flowering season, when male flowers are gone, has sug-gested the existence of some type of asexual reproduction,most likely apogamy–apomixis, in natural populations of thistriurid species. Asexual reproduction might take place in reg-ular female flowers of T. brevistylis by the end of the floweringseason, given that a majority of carpels still produce ripe fruitdespite the almost complete absence of male flowers in thefield during such stages of the life cycle. Theoretically, seeddevelopment through cleistogamy or apomixis might lowerresource expenditure, whereas chasmogamy might favor out-breeding (Lord 1981; Ruiz de Clavijo and Jiménez 1993; Díazand Macnair 1998). Given the different advantages that thesemodes of reproduction might have, it is worth asking which ofthem is dominant and (or) responsible for the production offruits and seeds observed during the entire cycle of floweringand fructification in T. brevistylis (i.e., October to December–January). For this purpose, detailed field observations in otherpopulations of the species would be needed to determine notonly the frequency of cleistogamous and chasmogamous flow-ers, but also the extent to which apomixis might occur, spe-cifically in female flowers, and perhaps particularly at the endof the flowering season.

As emphasized in other sections of this paper, the type “d”bisexual flower described in detail in the present study has aclear female appearance, in contrast to the bisexual flowerspreviously documented by Vergara-Silva et al. (2003). Givenour observations on stamens and pollen functionality, though,it is likely that triurid floral organs previously considered asstaminodes (Jonker 1943) in fact correspond to functionalstamens. Taking together the existence of unisexual ramets

with either male or female flowers, the presence of a smallnumber of monoecious ramets–genets, and the four types ofbisexual flowers that exist in T. brevistylis, we strongly sug-gest that the species should be classified as polygamodioe-cious, following the standardized terminology for gender andsexual dimorphism compiled by Sakai and Weller (1999). Inturn, attending to the classification for breeding systems pro-vided by Geber (1999), we also recommend that T. brevistylisshould be seen as a polygamous taxon. To place T. brevistyliswithin current classification schemes for gender–sexual dimor-phism and breeding systems, it would be useful to revisitpreviously published, related statements for other species ofTriuris, e.g., trioecy in T. hyalina (Rübsamen-Weustenfeld1991). Such classifications might allow a discussion of thepotentially “transitory status” that the reproductive strategyapparently displayed by T. brevistylis, i.e., polygamodioecysensu Sakai and Weller (1999), has in the context of its closephylogenetic vicinity with L. schismatica. Vergara-Silva et al.(2003) had already demonstrated that natural populations ofthe latter triurid are not exclusively composed of bisexual,homeotic flowers, but that there is a small proportion ofunisexual flowers occurring naturally.

Consideration of this evidence is enough to suggest thatL. schismatica is also polygamous. Moreover, if the diverse setof morphogenetic variants represented by the mature floralphenotypes observed in T. brevistylis in this work, as well asMartínez (1994) and Vergara-Silva et al. (2003), represents atransitional polygamodioecious stage that is closer to fulldioecy, and if such a stage is considered advanced (Burke et al.2010), it is logically valid to postulate that a triurid specieswith a reproductive strategy similar to that of L. schismaticamight have been the ancestor of both T. brevistylis andL. schismatica. Even though this statement can only remain asa hypothesis now, its formulation highlights the need to con-sider ecological aspects (i.e., “ultimate causes” sensu modernsynthesis evolutionary biology) and the proximal mechanismsputatively underlying meristem development and organ differ-entiation in triurid reproductive axes.

Concluding remarksOur long-term interest in the biology of Triuris, the type

genus of the monocotyledon family Triuridaceae (Maas-vande Kamer and Weustenfeld 1998), has been largely related toa controversy between euanthial and noneuanthial perspec-tives on the morphological interpretation of reproductive axesin this taxonomic group. This controversy, considered to besettled by some authors but not by others, has mainly involvedthe Mexican species L. schismatica (Rudall 2003, 2008; Am-brose et al. 2006; Cronk 2009), but the role that T. brevistylislegitimally has in it has been unfortunately downplayed inrecent work (e.g., in the plant development mathematicalmodeling exercises of Barrio et al. 2010).

Publication of the expression patterns of B- and C-functionMADS-box genes in L. schismatica (Álvarez-Buylla et al. 2010)has concluded a stage in a Triuridaceae “evo-devo” researchproject, first envisioned by Vergara-Silva et al. (2000). Inter-pretation of such results has falsified the pseudanthial alterna-tive initially offered by Rudall (2003) and, in turn, contributedto support the default, euanthial scheme that had been alreadydefended by Ambrose et al. (2006). However, the morphoge-netic and embryological evidences presented here for T. brevi-

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stylis suggest that the developmental–genetic data availablefor L. schismatica (Álvarez-Buylla et al. 2010) do not suffi-ciently account for the mechanistic (i.e., molecular–genetic)causes that might underlie the reproductive systems of thispresumably closely related pair of triurids taken together.

At the same time, we consider that the information on themorphogenesis, embryology, and reproduction strategies ofT. brevistylis provided here demonstrates that structural andfunctional complexity in this species is greater than in L. schis-matica. For this reason, such information should not be ig-nored as a guide to further research within Triuridaceae,particularly within the tribe Triurideae (Cheek 2003; Rudall2008), that could potentially clarify additional issues implicitin the controversy over morphological interpretations in thisfamily of the order Pandanales. A genomic approach involv-ing comparisons of whole genome based transcription pro-files in L. schismatica, T. brevistylis, and triurid speciesbelonging to other genera might point towards both geneticand epigenetic components that have been overlooked so farin the mechanistic framework initially chosen (Vergara-Silva et al. 2000, 2003) to explain the development andevolution of triurid reproductive morphology, i.e., the ABCmodel of floral development (Coen and Meyerowitz 1991).Evidently, such comparisons could be strengthened by jointstudies of the developmental genetics of reproductive morpho-genesis in Cyclanthaceae, Stemonaceae, Pandanaceae, andVelloziaceae. Ultimately, this research prospect might alsocontribute to a robust inference of phylogenetic relationshipswithin Pandanales. While this paper was under review, a briefdescription of a Brazilian species of Lacandonia was pub-lished (Melo and Alves 2012), as well as a review on thedevelopmental genetics of L. schismatica (Garay-Arroyo et al.2012). Information provided in the latter publication demon-strates the feasibility of the genomic experiments suggestedhere, in order to extend the evo-devo research project origi-nally presented by Vergara-Silva et al. (2000). The implica-tions that arguments presented in these two recent papers havefor such project will be discussed elsewhere.

AcknowledgementsWe appreciate the hospitality from the communities of

Nahá and El Censo, Chiapas, Mexico, during our visits tothe Chiapas rainforest, and thank J. Hernández-Guzmán,A. López Chan Kin, and K. García-Paniagua for theirguidance, advice, and sympathetic involvement during ourfieldwork. In Mexico City, A. Martínez-Mena and A. Gam-boa helped with photomicrographs and photographic pro-cessing, J.A. Hernández and A.I. Bieler-Antolín assistedwith image digitalization and plate design, and Y. Hornelasassisted with scanning electron microscopy. V. Rubiohelped us translate German texts, and S. Magallón com-mented on an early version of the manuscript. We thankE. Meyerowitz (California Institute of Technology, Calif.,USA), P. Engström (Uppsala University, Sweden), andMaría Elena Álvarez-Buylla Roces (Instituto de Ecología,UNAM) for their generous support over the years for de-velopmental– genetic research in L. schismatica andT. brevistylis and acknowledge their intellectual authorshipin that research area. We are also grateful to P. Rudall(Royal Botanic Gardens, Kew, UK) for her critical obser-vations regarding several aspects of our research on Mex-

ican Triuridaceae, as well as her creative contributions toalternative viewpoints on the morphology and evolution ofmonocotyledons, and to E. Martínez for his thorough de-scriptive work on the Lacandón rainforest flora. Finally, weappreciate the detailed reviews that we received from twoanonymous reviewers and the constructive advice from theJournal’s Editors. Direct or indirect financial support forthis project was provided by grants from the followingorganizations: Dirección General de Asuntos del PersonalAcadémico (DGAPA, UNAM, Mexico), Consejo Nacionalde Ciencia y Tecnología (CONACyT, Mexico), InstitutoNacional de Ecología (INE, Mexico), and Secretaría deDesarrollo Social (SEDESOL, Mexico).

ReferencesÁlvarez-Buylla, E.R., Ambrose, B.A., Flores-Sandoval, E., Englund,

M., Garay-Arroyo, A., García-Ponce, B., de la Torre-Bárcena, E.,Espinosa-Matías, S., Martínez, E., Piñeyro-Nelson, A., Engström,P., and Meyerowitz, E.M. 2010. B-Function expression in theflower center underlies the homeotic phenotype of Lacandoniaschismatica (Triuridaceae). Plant Cell, 22(11): 3543–3559. doi:10.1105/tpc.109.069153. PMID:21119062.

Ambrose, B.A., Espinosa-Matías, S., Vázquez-Santana, S., Vergara-Silva, F., Martínez, E., Márquez-Guzmán, J., and Álvarez-Buylla,E.R. 2006. Comparative developmental series of the Mexicantriurids support a euanthial interpretation for the unusual repro-ductive axes of Lacandonia schismatica (Triuridaceae). Am. J.Bot. 93(1): 15–35. doi:10.3732/ajb.93.1.15.

Angiosperm Phylogeny Group (APG). 2009. An update of the An-giosperm Phylogeny Group classification for the orders and fam-ilies of flowering plants: APG III. Bot. J. Linn. Soc. 161: 399–436.

Barrio, R.A., Hernández-Machado, A., Varea, C., Romero-Arias,J.R., and Álvarez-Buylla, E. 2010. Flower development as aninterplay between dynamic physical fields and genetic networks.PLoS ONE, 5(10): e13523. doi:10.1371/journal.pone.0013523.PMID:21048956.

Burke, J.M., Sanchez, A., Kron, K., and Luckow, M. 2010. Placingthe woody tropical genera of Polygonaceae: a hypothesis of char-acter evolution and phylogeny. Am. J. Bot. 97(8): 1377–1390.doi:10.3732/ajb.1000022. PMID:21616890.

Chase, M.W., Soltis, D.S., Soltis, P.S., Rudall, P.J., Fay, M.F., Hahn,W.H., Sullivan, S., Joseph, J., Givnish, T.J., Sytsma, K.J., andPires, J.C. 2000. Higher-level systematics of the monocotyledons:an assessment of current knowledge and a new classification. InMonocots: Systematics and Evolution. Proceedings of the SecondInternational Conference on the Comparative Biology of Mono-cotyledons, CSIRO, Sydney, Australia. Edited by K. Wilson andD. Morrison. CSIRO Publishing, Collingwood, Australia.pp. 3–16.

Cheek, M. 2003. Kupeaeae, a new tribe of Triuridaceae from Africa.Kew Bull. 58(4): 939–949. doi:10.2307/4111207.

Cheek, M., Williams, S., and Etuge, M. 2003. Kupea martinetugei, anew genus and species of Triuridaceae from western Cameroon.Kew Bull. 58(1): 225–228. doi:10.2307/4119366.

Chuma, C. 1990. On the pollen morphology of Andruris japonica(Triuridaceae). J. Jpn. Bot. 65: 349–351.

Coen, E.S., and Meyerowitz, E.M. 1991. The war of the whorls:genetic interactions controlling flower development. Nature,353(6339): 31–37. doi:10.1038/353031a0. PMID:1715520.

Cox, P.A. 1990. Pollination and the evolution of breeding systems in

Espinosa-Matías et al. 1149

Published by NRC Research Press

Bot

any

Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

UN

IVE

RSI

TY

OF

MIC

HIG

AN

on

03/0

8/13

For

pers

onal

use

onl

y.

Pandanaceae. Ann. Mo. Bot. Gard. 77(4): 816–840. doi:10.2307/2399673

Cronk, Q.C.B. 2009. The molecular organography of plants. OxfordUniversity Press. Oxford, UK.

Culley, T.M., and Klooster, M.R. 2007. The cleistogamous breedingsystem: a review of its frequency, evolution, and ecology inangiosperms. Bot. Rev. 73(1): 1–30. doi:10.1663/0006-8101(2007)73[1:TCBSAR]2.0.CO;2.

Dahlgren, R.M.T., Clifford, H.T., and Yeo, P.F. 1985. The families ofthe monocotyledons: structure, evolution and taxonomy. Springer-Verlag, New York.

Davis, J.I., Stevenson, D.W., Petersen, G., Seberg, O., Campbell,L.M., Freudenstein, J.V., Goldman, D.H., Hardy, C.R., Michelan-geli, F.A., Simmons, M.P., Specht, C.D., Vergara-Silva, F., andGandolfo, A.M. 2004. A phylogeny of the monocots, as inferredfrom rbcL and atpA sequence variation, and a comparison ofmethods for calculating jackknife and bootstrap values. Syst. Bot.29(3): 467–510. doi:10.1600/0363644041744365.

Díaz, A., and Macnair, M.R. 1998. The effect of plant size on theexpression of cleistogamy in Mimulus nasutus. Funct. Ecol. 12(1):92–98. doi:10.1046/j.1365-2435.1998.00170.x.

Doyle, J.A., Endress, P.K., and Upchurch, G.R., Jr. 2008. EarlyCretaceous monocots: a phylogenetic evaluation. Acta Mus. Nat.Pragae, Ser. B, Hist. Nat. 64: 59–87. [Praha. ISSN 0036-5343.]

Erdtman, G. 1952. Pollen morphology and plant taxonomy. Angio-sperms. Almqvist and Wiksell, Stockholm, Sweden.

Espinosa, S.M.Y. 2009. Embriología de Soridium spruceanum Miers(Triuridaceae). B.Sc. thesis, Facultad de Ciencias. UniversidadNacional Autónoma de México, México, D.F.

Franke, T., Beenken, L., and Hahn, C. 2000. Triuridopsis intermediaspec. nov. (Triuridaceae), a new myco-heterotrophic plant fromBolivia. Plant Syst. Evol. 225(1–4): 141–144. doi:10.1007/BF00985464.

Franz, N.M. 2004. Analysing the history of the derelomine flowerweevil – Carludovica association (Coleoptera: Curculionidae; Cy-clanthaceae). Biol. J. Linn. Soc. 81(4): 483–517. doi:10.1111/j.1095-8312.2003.00293.x.

Friis, E.M., Crane, P.R., and Pedersen, K.R. 2011. Early flowers andangiosperm evolution. Cambridge University Press, Cambridge,UK.

Furness, C.A., and Rudall, P.J. 1999. Inaperturate pollen in mono-cotyledons. Int. J. Plant Sci. 160(2): 395–414. doi:10.1086/314129.

Furness, C.A., and Rudall, P.J. 2006. Comparative structure anddevelopment of pollen and tapetum in Pandanales. Int. J. Plant Sci.167(2): 331–348. doi:10.1086/499503.

Furness, C.A., Rudall, P.J., and Eastman, A. 2002. Contribution ofpollen and tapetal characters to the systematics of Triuridaceae.Plant Syst. Evol. 235(1–4): 209–218. doi:10.1007/s00606-002-0232-y.

Gandolfo, M.A., Nixon, K.C., and Crepet, W.L. 2002. Triuridaceaefossil flowers from the Upper Cretaceous of New Jersey. Am. J.Bot. 89(12): 1940–1957. doi:10.3732/ajb.89.12.1940. PMID:21665623.

Garay-Arroyo, P., Piñeyro-Nelson, A., García-Ponce, B., Sánchez,M. de la P., and Álvarez-Buylla, E.R. 2012. When ABC becomesACB. J. Exp. Bot.63: 2377–2395.

Geber, M.A. 1999. Theories of the evolution of sexual dimorphism.In Gender and sexual dimorphism in flowering plants. Edited byM.A. Geber, T.E. Dawson, and L.F.Delph. Springer-Verlag, Ber-lin, pp. 97–122.

Giesen, H. 1938. Triuridaceae. In Das Pflanzenreich. Band IV. Teil18. Edited by A. Engler. Engelmann, Leipzig. pp. 1–84.

Grayum, M.H. 1992. Comparative external pollen ultrastructure ofthe Araceae and putatively derived taxa. Monogr. Syst. Bot. Mis-souri Bot. Gard. Vol. 43.

Ham, R.W.J.M. 1991. Pollen morphology of the Stemonaceae.Blumea, 36: 127–159.

Harling, G., Wilder, G.J., and Eriksson, R. 1998. Cyclanthaceae. InThe families and genera of vascular plants. Vol. III. Floweringplants, monocotyledons, Lilianae (except Orchidaceae). Edited byK. Kubitzki. Springer-Verlag, Berlin. pp. 202–215.

Johansen, D.A. 1940. Plant microtechnique. McGraw-Hill, NewYork.

Jonker, F.P. 1943. Triuridaceae. In Flora of Suriname (NetherlandsGuyana). Vol. 1, Part 1. Edited by A.A. Pulle. J.H. de Bussy,Amsterdam. pp. 461–466.

Kubitzki, K. (Editor). 1998a. Stemonaceae. In The families andgenera of vascular plants. Vol. III. Flowering plants, monocotyle-dons, Lilianae (except Orchidaceae). Springer-Verlag, Berlin.pp. 422–424.

Kubitzki, K. (Editor). 1998b. Velloziaceae. In The families andgenera of vascular plants. Vol. III. Flowering plants, monocotyle-dons, Lilianae (except Orchidaceae). Springer-Verlag, Berlin.pp. 459–466.

Lord, E.M. 1981. Cleistogamy: a tool for the study of floral morpho-genesis, function and evolution. Bot. Rev. 47(4): 421–449. doi:10.1007/BF02860538.

Maas, P.J.M., and Rübsamen, T. 1986. Triuridaceae. Flora neotro-pica, Monograph 40. Hafner, New York.

Maas-van de Kamer, H. 1995. Triuridiflorae: Gardner’s delight? InMonocotyledons: systematics and evolution. Edited by P.J. Rudall,P.J. Cribb, D.F. Cutler, and C.J. Humphries. Royal Botanic Gar-dens, Kew, UK. pp. 287–301.

Maas-van de Kamer, H., and Weustenfeld, T. 1998. Triuridaceae. InThe families and genera of vascular plants. Vol. III. Floweringplants, monocotyledons, Lilianae (except Orchidaceae). Edited byK. Kubitzki. Springer-Verlag, Berlin. pp. 452–458.

Mabberley, D.J. 1997. The plant book: a portable dictionary of plants,their classification, and uses. 2nd ed. Cambridge University Press,Cambridge, U.K.

Mabberley, D.J. 2008. Mabberley’s plant book: a portable dictionaryof plants, their classification, and uses. 3rd ed. Cambridge Univer-sity Press, Cambridge, U.K.

Márquez-Guzmán, J., Engleman, M., Martínez-Mena, A., Martínez,E., and Ramos, C. 1989. Anatomía reproductiva de Lacandoniaschismatica (Lacandoniaceae). Ann. Mo. Bot. Gard. 76(1): 124–127. doi:10.2307/2399345.

Márquez-Guzmán, J., Vázquez-Santana, S., Engleman, E.M.,Martínez-Mena, A., and Martínez, E. 1993. Pollen developmentand fertilization in Lacandonia schismatica (Lacandoniaceae).Ann. Mo. Bot. Gard. 80(4): 891–897. doi:10.2307/2399935.

Martínez, S.E. 1994. Triuridaceae. In Flora Mesoamericana. Vol. VI.Alismataceae and Cyperaceae. Edited by G. Davidse, M. Sousa,and A.D. Chaters. Universidad Nacional Autónoma de México,Mexico. pp. 18–19.

Martínez, S.E., and Ramos, C.H. 1989. Lacandoniaceae (Triuridales):una nueva familia de México. Ann. Mo. Bot. Gard. 76(1): 128–135. doi:10.2307/2399346.

Melo, A., and Alves, M. 2012. The discovery of Lacandonia (Triu-ridaceae) in Brazil. Phytotaxa 40: 21–25.

1150 Botany, Vol. 90, 2012

Published by NRC Research Press

Bot

any

Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

UN

IVE

RSI

TY

OF

MIC

HIG

AN

on

03/0

8/13

For

pers

onal

use

onl

y.

O’Brien, T.P., and McCully, M.E. 1981. The study of plant structure:principles and selected methods. Termarcarphi Pty. Ltd., Mel-bourne.

Philbrick, C.T. 1984. Pollen tube growth within vegetative tissues ofCallitriche (Callitrichaceae). Am. J. Bot. 71(6): 882–886. doi:10.2307/2443480.

Philbrick, C.T., and Anderson, G.J. 1992. Pollination biology in theCallitrichaceae. Syst. Bot. 17(2): 282–292. doi:10.2307/2419523.

Remizowa, M., Sokoloff, D.D., and Rudall, P.J. 2010. Evolutionaryhistory of the monocot flower. Ann. Mo. Bot. Gard. 97(4): 617–645. doi:10.3417/2009142.

Rübsamen-Weustenfeld, T. 1991. Morphologische, embryologischeund systematische Untersuchungen an Triuridaceae. BibliothecaBotanica Vol. 140. E. Schweizerbart’sche Verlagsbuchhandlung(Nägele u. Obermiller), Stuttgart, Germany.

Rudall, P.J. 2003. Monocot pseudanthia revisited: floral structureof the mycoheterotrophic family Triuridaceae. Int. J. Plant Sci.164(5 Suppl): S307–S320. doi:10.1086/376879.

Rudall, P.J. 2008. Fascicles and filamentous structures: comparativeontogeny of morphological novelties in Triuridaceae. Int. J. PlantSci. 169(8): 1023–1037. doi:10.1086/590476.

Rudall, P.J. 2010. All in a spin: centrifugal organ formation and floralpatterning. Curr. Opin. Plant Biol. 13(1): 108–114. doi:10.1016/j.pbi.2009.09.019. PMID:19853497.

Rudall, P.J., and Bateman, R.M. 2006. Morphological phylogeneticanalysis of Pandanales: testing contrasting hypotheses of floralevolut ion. Syst . Bot . 31(2): 223–238. doi :10.1600/036364406777585766.

Rudall, P.J., Strange, A., Cunniff, J., and Cheek, M. 2007 Floralanatomy of Kupea martinetugei. Kew Bull. 62: 287–292. Avail-able from www.jstor.org/stable/20443354.

Ruiz de Clavijo, E., and Jiménez, M.J. 1993. Cleistogamy and chas-mogamy in Ceratocapnos heterocarpa (Fumariaceae). Int. J. PlantSci. 154(2): 325–333. doi:10.1086/297113.

Ruzin, S.E. 1999. Plant microtechnique and microscopy. OxfordUniversity Press, Oxford, UK.

Sahashi, N., Ohmoto, T., Uehara, K., Ikuse, M., and Chuma, C. 1991.

Pollen morphology of Andruris japonica (Triuridaceae). Grana,30(3–4): 597–600. doi:10.1080/00173139109427818.

Sakai, A.K., and Weller, S.G. 1999. Gender and sexual dimorphismin flowering plants: a review of terminology, biogeographic pat-terns, ecological correlates, and phylogenetic approaches.In Gen-der and sexual dimorphism in flowering plants. Edited by M.A.Geber, T.E. Dawson, and L.F. Delph. Springer-Verlag, Berlin.pp. 1–32.

Stevens, P.F. (2001 onwards). Angiosperm phylogeny website. Ver-sion 9, June 2008 [and more or less continuously updated since].Available from http://www.mobot.org/MOBOT/research/APweb/.

Stone, B.C., Huyhn, K.-L., and Poppendieck, H.-H. 1998. Pandan-aceae. In The families and genera of vascular plants. Vol. III.Flowering plants, monocotyledons, Lilianae (except Orchidaceae).Edited by K. Kubitzki. Springer-Verlag, Berlin. pp. 397–403.

Tomlinson, P.B. 1982. Helobiae (Alismatidae). In Anatomy of themonocotyledons. Vol. VII. Edited by C.R. Metcalfe. ClarendonPress, Oxford, UK. pp. 466–475.

Tomlinson, P.B., and Wilder, G.J. 1984. Systematic anatomy ofCyclanthaceae (Monocotyledoneae): an overview. Bot. Gaz.145(4): 535–549. doi:10.1086/337489.

Vázquez-Santana, S., Engleman, M., Martínez-Mena, A., andMárquez-Guzmán, J. 1998. Ovule and seed development of Lacan-donia schismatica (Lacandoniaceae). Am. J. Bot. 85(3): 299–304.doi:10.2307/2446320. PMID:21715289.

Vergara-Silva, F., Martínez-Castilla, L.P., and Álvarez-Buylla, E.R.2000. MADS-box genes: development and evolution of plant bodyplans. J. Phycol. 36(5): 803–812. doi:10.1046/j.1529-8817.2000.03654.x.

Vergara-Silva, F., Espinosa-Matías, S., Ambrose, B.A., Vázquez-Santana, S., Martínez-Mena, A., Márquez-Guzmán, J., Martínez,E., Meyerowitz, E.M., and Álvarez-Buylla, E.R. 2003. Inside-outflowers characteristic of Lacandonia schismatica evolved at leastbefore its divergence from a closely related taxon, Triuris brevi-stylis. Int. J. Plant Sci. 164(3): 345–357. doi:10.1086/368235.

Wirz, H. 1910. Beiträge zur Entwicklungsgeschichte von Sciaphilaspec. und von Epirrhizanthes elongata Bl. Flora, 101: 395–446.

Espinosa-Matías et al. 1151

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