American Journal of Botany 99(10): 1592–1608. 2012.

F LOWERS AND OF THE SEAGRASS P OSIDONIA (POSIDONIACEAE, ) 1

M ARGARITA V. R EMIZOWA 2,5 , D MITRY D. SOKOLOFF 2 , S EBASTIANO C ALVO 3 , A GOSTINO T OMASELLO 3 , AND P AULA J. RUDALL 4

2 Department of Higher , Faculty of Biology, Lomonosov Moscow State University, Moscow 119234, Russia; 3 Dipartimento di Scienze della Terra e del Mare, Viale delle Scienze, Edifi cio 16 90128 Palermo, Italy; and 4 Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond, Surrey, TW9 3AB, UK

• Premise of the study: The predominantly aquatic order Alismatales displays a highly variable fl ower groundplan associated with a diverse range of developmental patterns. We present the fi rst detailed description of fl ower anatomy and development in Posidonia , the sole genus of the seagrass family Posidoniaceae. Existing accounts provide confl icting interpretations of fl oral and infl orescence structure, so this investigation is important in clarifying morphological evolution within this early-divergent monocot order. • Methods: We investigated two species of Posidonia using light microscopy and scanning electron microscopy. Our observa- tions are interpreted in the framework of a recent molecular phylogeny. • Key results: Partial infl orescences are bracteate spikes, which are arranged into a botryoid or a panicle. The fl owers are perian- thless. The is monomerous with the ventral carpel side oriented abaxially. The carpel contains a single pendent bitegmic ovule with a nucellus and long chalaza, both extending along the carpel wall. The ovule develops an integumentary outgrowth. Each fl ower is supplied by a vascular bundle, whereas the fl ower-subtending bracts are nonvascularized. • Conclusions: Our data support a racemose interpretation for the partial infl orescence of Posidonia and the presence of fl ower- subtending bracts. In common with some other Alismatales, Posidonia has simultaneous development of the fl ower and its subtending bract and loss of the bract vascular supply accompanied by innervation of the fl ower by a single vascular strand. The unusual carpel orientation could be an evolutionary reduction of a formerly tricarpellate gynoecium. The ovule of Posidonia is campylotropous and unusual within Alismatales in possessing an integumentary outgrowth.

Key words: anatomy; Alismatales; embryology; fl ower; infl orescence; morphology; ovule; Posidonia ; vasculature.

The predominantly aquatic order Alismatales (sensu APG explained by these two factors (early-divergent placement and III, 2009 ) displays a diverse range of fl ower morphology and adaptation to aquatic habitats). Alismatales is one of very few development (reviewed by Posluszny and Charlton, 1993; monocot lineages in which gynoecia with free carpels are pres- Posluszny et al., 2000 ). In contrast with most other monocot ent in some members. This feature, previously considered orders, Alismatales is characterized by a highly variable fl ower primitive (e.g., Takhtajan, 1987; Cronquist, 1988) is now com- groundplan (Endress, 1995; Remizowa et al., 2010). The near- monly regarded as derived at the level of monocots (Doyle and basal phylogenetic placement of the order in monocots (Chase Endress, 2000; Chen et al., 2004; Endress and Doyle, 2009; et al., 2000, 2006 ; Davis et al., 2004; Graham et al., 2006; APG Remizowa et al., 2010 ; Sokoloff et al., in press ). The core Alis- III, 2009; Iles et al., in press), makes Alismatales potentially matales include the seagrasses, which are unique in possessing important in inferring early monocot evolution and the putative underwater pollination and fi liform pollen grains (the latter re- ancestral morphology of the monocot fl ower. On the other hand, viewed by Ducker et al., 1978 and Furness and Banks, 2010 ). specialized aquatic members of “core” Alismatales (i.e., ex- The seagrasses are adapted to permanently saline underwater cluding Araceae and Tofi eldiaceae) possess characteristically habitats, where they can occupy large areas, forming under- reduced fl owers that are adapted for underwater pollination water meadows that play a signifi cant role in the functioning of ( Posluszny and Charlton, 1993 ; Endress, 1995 ). However, marine ecosystems. the high fl oral diversity of core Alismatales cannot be fully Of 13 families of Alismatales recognized by the Angiosperm Phylogeny Group (APG III, 2009), 12 have been well inves- 1 Manuscript received 13 May 2012; revision accepted 10 September tigated with respect to fl ower development ( Table 1 ). The only 2012. family for which such data are hitherto lacking is the monoge- The authors thank Peter Endress, Will Iles, and Usher Posluszny for neric seagrass family Posidoniaceae, which is distributed in the suggestions on the manuscript, Will Iles and Sean Graham for discussion Mediterranean and the southern coast of Australia. This excep- of the phylogeny of Alismatales, Ivan I. Shamrov for discussion on ovule tion is primarily due to technical diffi culties in obtaining suit- morphology, and Evgeny Mavrodiev for providing some literature. This able material; reproductive development of Posidonia K. D. work was supported by an International Joint Project grant from the Royal Koenig occurs at some depth in the sea ( Campey et al., 2002 ). Society (to M.V.R., D.D.S., P.J.R.) and grants from the Russian Foundation for Basic Research and Ministry of Education and Science of Russia (FCP Furthermore, in the Mediterranean species P. oceanica (L.) ‘Kadry’, NK-541P/P314) (to M.V.R., D.D.S.). Delile, fl owering is infrequent, and clonal growth is important 5 Author for correspondence (e-mail: [email protected]) in maintenance of populations (Procaccini and Mazzella, 1998; Balestri and Cinelli, 2003 ; Calvo et al., 2006 , 2010 ; Diaz-Almela doi:10.3732/ajb.1200227 et al., 2006). Large, vegetative clones of P. oceanica are

American Journal of Botany 99(10): 1592–1608, 2012; http://www.amjbot.org/ © 2012 Botanical Society of America 1592 October 2012] REMIZOWA ET AL.— AND INFLORESCENCES OF THE SEAGRASS POSIDONIA 1593

T ABLE 1. Summary of literature on fl oral development in families of season. Because young infl orescences of Posidonia are not vis- Alismatales sensu APG (2009). Note that fl ower development is still ible externally on the , a developmental series is obtain- unknown in Maundia , for which a monogeneric family Maundiaceae able only by collection of ample material and careful laboratory can be adopted ( von Mering and Kadereit, 2010 ). dissection to reveal relatively few infl orescences. Flowers of Family Reference Posidonia are morphologically unusual and lack close ana- logues in monocots, especially in possessing with large Alismataceae Kaul, 1967a , b ; Singh and Sattler, 1972 , 1973 ; -like outgrowths. As Tomlinson (1982) noted, the various Charlton and Ahmed, 1973 ; Sattler and Singh, 1973 ; interpretations of fl ower and infl orescence morphology of Posi- Leins and Stadler, 1973 ; van Heel, 1988 ; Ronse De Craene and Smets, 1995 ; Charlton, 2004 donia are highly controversial, and information from anatomy Aponogetonaceae Singh and Sattler, 1977 and particularly development are urgently needed to provide Araceae Lehmann and Sattler, 1992 ; Scribailo and Tomlinson, useful interpretative evidence. Kamelina (1990 , 2011 ) noted 1992 ; Barabé, et al., 2000 , 2002 , 2011 ; Buzgo, 2001 that the embryology of Posidoniaceae is almost unknown, a gap Butomaceae Singh and Sattler, 1974 that was only partially fi lled by a recent paper on megasporo- Cymodoceaceae Tomlinson and Posluszny, 1978 ; McConchie et al., 1982 genesis and embryo development in Australian Posidonia ( Ma Hydrocharitaceae Kaul, 1969 ; Scribailo and Posluszny, 1985 et al., 2012 ). In particular, no detailed data are available on Lieu, 1979 ; Charlton, 1981 ; Posluszny et al., 1986 ; Buzgo et al., 2006 ; Remizowa et al., in press ovule morphology in Posidoniaceae, even though this feature Posidoniaceae This study represents an important taxonomic marker in Alismatales. Sattler, 1965 ; Posluszny and Sattler, 1973 , 1974a , 1976 ; Molecular phylogenetic data indicate that Posidoniaceae Posluszny and Tomlinson, 1977 ; Posluszny, 1981 ; are close relatives of Cymodoceaceae and Ruppiaceae (Les Sun et al., 2000 et al., 1997; Iles et al., in press). These results contradict the Ruppiaceae Posluszny and Sattler, 1974b ; Kaul, 1993 ; traditional views that Cymodoceaceae are most closely related Lock et al., 2011 ; Lock, 2012 Scheuchzeriaceae Posluszny, 1983 to Zannichelliaceae and Ruppiaceae to Potamogetonaceae Tofi eldiaceae Remizova and Sokoloff, 2003 ; Remizowa et al., 2005 , (Eckardt, 1964; Dahlgren et al., 1985; Takhtajan, 1987). Thus, 2006 , in press detailed morphological data on Posidonia are needed to rein- Zosteraceae Soros-Pottruff and Posluszny, 1995a , b vestigate the evolutionary history of Alismatales in the new phylogenetic context. estimated to be hundreds to thousands of years old ( Arnaud- Haond et al., 2012 ). MATERIALS AND METHODS Here, we present the fi rst detailed description of fl ower anat- omy and development in Posidonia , including specimens spe- Specimens were obtained from three sources. (1) Material of the Mediter- cifi cally collected by underwater diving several times during a ranean species Posidonia oceanica (L.) Delile including developmental fl oral

Fig. 1. Posidonia oceanica . (A) Scheme of shoot and infl orescence morphology. (B) Diagram of a lateral spikelet. The third fl ower is shown as male on the diagram, but sometimes it is bisexual. f1, f2, f3 = sequentially numbered fl owers in a spikelet; l1, l2 = sequentially numbered foliage located below a spikelet; lsa = lateral spikelet axis; lssl = lateral spikelet-subtending ; mia = main infl orescence axis; pr = prophyll; rb = renovation bud; vl = vegetative leaves on creeping . Black arcs = foliage leaves; open arcs = fl ower-subtending bracts (short scales); gray areas on the diagram = connective; black incomplete circles adjacent to it = thecae. 1594 AMERICAN JOURNAL OF BOTANY [Vol. 99

Fig. 2. Posidonia oceanica . (A) Entire post-anthetic spike consisting of two bisexual fl owers, side view. (B) Post-anthetic fl ower from the same spike, front view. (C) Detail of the same fl ower as in (B), side view. br = fl ower-subtending bract; c = connective; g = gynoecium; ia = infl orescence axis; sc = supraconnective; st = stamen; t = area of theca attachment (the thecae abscise after anthesis); w = wing-like extensions of connective forming late during fl oral development. Scale bar = 1000 µm. stages was collected by two of us (S. Calvo, A. Tomasello) by scuba diving at were embedded in Technovit (Heraeus-Kulzer, Wehrheim, Germany) 7100 and 5 m depth near Palermo (Sicily, Italy) on 13 July, 5 August, 20 August, and sectioned using a Leica rotary microtome. Sections were stained in toluidine 1 October 2009 (voucher specimens deposited at SC under the following col- blue (0.5% in distilled water) and mounted in DPX. Photomicrographs were lection numbers: specimen on 13/07/09, # 7-01; specimen on 05/08/09, # 7-02; taken using a Zeiss Axioplan microscope fi tted with a digital camera. Images specimen on 20/08/09, # 7-03; specimen on 01/10/09, # 7-04). Since very were processed and assembled using Adobe (San Jose, California, USA) young fl oral stages in situ are hidden between the leaves, a probabilistic ap- Photoshop. proach was used to calculate the sample size needed. Every year on average Character optimizations were performed using the program WinClada 7.2 fl owers/1000 shoots are produced along the Sicily coasts ( Tomasello et al., (Nixon, 2002). Multistate characters were treated as unordered. The tree topol- 2009). Thus, 200 shoots were collected randomly to obtain at least one shoot ogy used was based on Iles et al. (in press) . bearing a young infl orescence at the appropriate stage and fi xed in 70% ethanol for further laboratory analysis. (2) Anthetic and post-anthetic material of P. oce- anica was collected by M. V. Remizowa and D. D. Sokoloff on the seashore of RESULTS Kalamaria (Greece) on 25 December 2010 (voucher herbarium specimen de- posited at MW, accession MW230001) and fi xed in 70% ethanol. (3) Material of P. australis Hook. f. was obtained from the Spirit Collection at the Royal Organography— The partial infl orescences are few-fl owered Botanic Gardens, Kew (K), from two collections by A. M. Baird in the Perth spikes ( Figs. 1, 2A, 3A–C ). The spikes terminate both main and region of Western Australia in September 1953 (specimen number 34668) and lateral branches (sometimes of different orders), so the whole October 1953 (specimen number 34670) had been fi xed in FAA (40% formal- infl orescence could be described as either a botryoid of spikes dehyde solution–glacial acetic acid–70% ethanol, 10 : 5 : 85) and stored in 70% (Fig. 1A) or a panicle of spikes (not shown). Each spike is sur- ethanol. For scanning electron microscopy (SEM) at the Royal Botanic Gardens rounded by two leaves (l1 and l2 on Fig. 1), which resemble the Kew ( Figs. 3, 7, 9, 10 ), material was dissected in 96% ethanol, dehydrated vegetative leaves but are much smaller. The fl owers are ar- through absolute ethanol and critical-point dried using an Autosamdri-815B ranged distichously along the spike axis (Figs. 1B, 2A, 3B, 3C). CPD (Tousimis Research, Rockville, Maryland, USA). Material was mounted Within the spike, the plane of distichy changes so that the plane on SEM stubs, coated with platinum using an Emitech (Hailsham, UK) K550 of fl ower insertion is at 90 ° to the plane of the leaves (Fig. 1A). sputter coater and examined using a Hitachi cold fi eld emission SEM S-4700-II at 1 kV. For SEM at Moscow State University ( Fig. 2 ), fi xed material was de- Each fl ower is located in the axil of a fl ower-subtending bract, hydrated through absolute acetone and critical-point dried using a Hitachi which is thin and fi lmy ( Figs. 2, 3B, 3C, 3D, 3H ). In many HCP-2 CPD, coated with gold and palladium using a Eiko IB-3 ion-coater cases, the tip of the infl orescence axis extends above the attach- (Tokyo, Japan), and observed using CamScan 4DV (CamScan, UK). ment of the uppermost fl ower ( Figs. 3B, 3C, 3I, 10B ). For light microscope observations, material was sectioned using standard The fl owers are mostly bisexual. In the observed infl orescences Paraplast embedding and serial sectioning at 10–15 µm thickness at the Royal of P. australis, all fl owers were bisexual, whereas the uppermost Botanic Gardens, Kew and Moscow State University. Sections were stained in safranin and alcian blue or in picroindigocarmine and carbolic fuchsine fl owers in spikes of P. oceanica were either bisexual ( Figs. 2A, (Axenov, 1967) and mounted in DPX (Agar Scientifi c, Stansted, UK) mounting 9F, 10B ) or male ( Fig. 1B, 9E ). In the latter case, they consisted medium or BioMount (BioOptica, Italy). For ovule anatomy, dissected carpels of only two (lateral) stamens. Each bisexual fl ower consists of October 2012] REMIZOWA ET AL.—FLOWERS AND INFLORESCENCES OF THE SEAGRASS POSIDONIA 1595

Fig. 3. Posidonia australis . and infl orescence morphology (SEM), some images artifi cially colored. (A) Entire anthetic spike consisting of three bisexual fl owers (only two are visible), front view. (B) Front and (C) lateral views of another spike, with two upper fl owers removed; in the removed fl ower no. 3 (f3), the fl ower-subtending bract and the bases of the removed carpel and stamens are visible. (D) Detail of fl ower-subtending bract. (E) Opened thecae of two adjacent stamens (detail of A), releasing fi liform pollen. (F) Entire stamen, adaxial view. (G) Anthetic carpel demonstrating irregu- larly lobed stigma. (H) Young fruit with abscised stigma, surrounded by persistent stamens. (I) Flower after fruit abscission. br = fl ower-subtending bract; c = connective; ca = conical appendages on stigma; f1, f2, f3 = fl owers of a spike numbered sequentially; ia = infl orescence axis; sc = supraconnective; t = theca; w = wing-like extension of connective. In artifi cially colored images, green = fl ower-subtending bract; orange = thecae; yellow = the rest of the stamen; red = gynoecium. Scale bars: in A–C, F, H = 1000 µm; in D, E, G = 500 µm; in I = 2000 µm. 1596 AMERICAN JOURNAL OF BOTANY [Vol. 99 three stamens and a single carpel, but lacks a perianth ( Figs. 1B, by a degenerating nucellus, and the endosperm is eventually 2A, 2B, 3A–C). The stamens form a single whorl, with one sta- consumed ( Fig. 6 ). The nucellus represents a nutritive tissue for men being median-abaxial and the other two stamens inserted the embryo. In addition, the embryo receives nutrients via the transversely ( Figs. 1B, 2B, 3B ). Stamens are narrow and not hypostase, which develops along the chalaza above the vascu- overlapping at the very base, but are much wider in their middle lar supply. The hypostase consists of transfer tissue (labeled and distal parts where the median−abaxial stamen occupies an “tt” in Fig. 6A) containing cells of two distinct types: smaller outer position with respect to the two transversal stamens ( Figs. cells located near the vascular bundle and radially elongated 2A, 2B, 3A ). The anthers are extrorse ( Fig. 3A–C ). The two the- cells that protrude into the nucellus (labeled “smc” and “ec” in cae are widely gaping on a very broad shield-like connective, Fig. 6D ). In the mature fruit, the seed occupies the entire fruit which extends upward into a supraconnective ( Figs. 2B, 3F ). In cavity. In our material of P. australis, many seeds commenced P. australis, the supraconnective is massive and triangular germination viviparously, while the fruit was still attached to ( Fig. 3F, 3H ), while the supraconnective of P. oceanica possesses a the plant. In these cases, the radicle protruded through the mi- long, narrow tip ( Fig. 2A, 2B ). The fl at stamen resembles a peri- cropyle ( Fig. 7). anth organ. Superfi cially, the thecae appear to be inserted on the abaxial side of this fl at structure ( Figs. 2A, 2B, 3H ). However, Infl orescence vasculature in Posidonia australis — The spike their insertion is morphologically marginal, as is evident from axis contains only two vascular strands, which are situated on their development ( Figs. 9G, 9H, 10D, 10E ); the physical mar- the radii of the fl owers (Fig. 8). Along the infl orescence axis, gins of the fl at structure develop by unequal growth of the adaxial these bundles give off branches to supply the fl owers and do not side of the connective (forming wing-like extensions, labeled extend above the levels of attachment of the two uppermost “w” in Figs. 2B, 3H ) and represent a secondary margin, whereas fl owers (each of the two strands terminates in a fl ower). In the the primary margin is occupied by the thecae. Each theca has two proximal part of the infl orescence, slightly below the level of pollen sacs (microsporangia), that are fi lled with fi liform pollen fl ower insertion, the bundle that is closest to the fl ower gives off grains (Figs. 3A, upper fl ower; 3E, 8B). The pollen sacs are sepa- a branch (Fig. 8A, E). This branch immediately enters the rated by a septum (Fig. 8B), which disappears before anther fl ower receptacle, where it divides into four narrow veins, of opening (so that no septum is present in the open theca that has which three diverge outward to supply the stamens, and the released fi liform pollen grains in Fig. 3E ). The fi liform pollen fourth remains in the center of the receptacle ( Fig. 8F, G ). Thus, grains are oriented perpendicularly to the long axis of the theca the stamens are each supplied by a single bundle. Within the ( Fig. 3E ) and at an angle of about 45 ° to the septum ( Fig. 8B ). stamen, the vascular bundle does not extend to the tip, but ter- After anthesis, the thecae drop off, while the remaining parts of minates in the connective base. The remaining bundle in the the stamens remain attached to protect the young fruit ( Figs. 2, receptacle center enters the carpel base, where it divides into 3H, 3I ). ventral and dorsal carpellary bundles ( Fig. 8H, I ). The fl ower- In both species, the carpel is ascidiate and massive with a subtending bracts are not vascularized. thick ovary wall and large capitate stigma (Figs. 2A, 3G). The ovary contains a single ovule, which is attached to the long, Organogenesis — Along the coast of Sicily, infl orescences of broad abaxial placenta. The chalaza is extended along the pla- P. oceanica can be seen by scuba divers in September (shallow centa (Figs. 4, 5). The ovule is sessile and bitegmic. Superfi - meadows < −15 m) and October−November (deeper meadows). cially, the nucellus appears to be straight, but morphologically Among four samples of P. oceanica from shallow meadows it can be regarded as basally curved with the chalaza extending used in this study, only the collection made on 1 October con- along one side of the nucellus. Because the attachment area tained young stages of fl ower development. In total, seven (chalaza) is perpendicular to the rest of the nucellus, the ovule young infl orescences were found in this sample. These were at can be described as campylotropous. slightly different developmental stages, covering events from The micropyle is formed by the inner integument ( Fig. 4D ). initiation of fl owers ( Fig. 9A, B) to ovule initiation (Fig. 10C, D) In the micropylar region, the outer integument is shorter and and closure of the carpel mouth (Fig. 10F). Thus, fl ower ini- thinner than the inner integument. The integuments are tightly tiation apparently occurs somewhat asynchronously in Septem- appressed to each other and to the nucellus. The micropyle is ber, and anthesis occurs in the September of a subsequent year, directed toward the ovary bottom. Opposite the micropyle, both at least in shallow meadows of P. oceanica along the coast of integuments (which are fused with each other but free from the Sicily. nucellus at this point) form an outgrowth, which extends into The fl owers are initiated acropetally along the infl ores- the hollow style (Figs. 4, 5A) and elongates extensively after cence axis (Fig. 9). A terminal fl ower is absent, and a residual fertilization, sealing the stylar canal ( Fig. 6A ). The style is rela- meristem is often present at the infl orescence apex. The sub- tively short and not clearly distinguishable from the ovary in tending bract and fl ower are initiated simultaneously by two young carpels. The broad, massive stigma is differentiated separate primordia (Fig. 9). The primordium of the fl ower- around the carpel mouth. The stigma surface is unevenly cov- subtending bract is much smaller than the corresponding fl o- ered by conical appendages ( Figs. 2A, 3G ). ral primordium and surrounds its lower part like a belt. During The fertilized ovule grows rapidly at the same time as the further development, the subtending bract develops into a carpel walls. In the maturing fruit, the seed remains oriented membranous structure with fi mbriate margins ( Figs. 9, 10 ). along the fruit axis, parallel to the carpel walls ( Figs. 5–7 ). The The bract becomes bent outward by the developing massive embryo passes through a globular stage ( Fig. 5 ) and ultimately fl ower. The stamens are initiated as three separate rounded develops a plumule, a primary root, and a massive hypocotyl primordia, and the remainder of the fl oral meristem forms (Figs. 6, 7). At the globular stage, the embryo is surrounded by the carpel (Figs. 9, 10). The primordium of the median sta- a nuclear endosperm ( Fig. 5 ). In mature embryos, an adventi- men is slightly smaller than the primordia of the lateral sta- tious root arises at the boundary between the plumule and the mens. The stamens are triangular with an acute tip. The thecae hypocotyl (Figs. 6, 7). The fully developed embryo is surrounded form on the lateral sides of each stamen. As the young stamens October 2012] REMIZOWA ET AL.—FLOWERS AND INFLORESCENCES OF THE SEAGRASS POSIDONIA 1597

Fig. 4. Posidonia australis. (A–C) Serial longitudinal sections at different levels through the same fertilized ovule. (D) Line drawing based on section illustrated in (A). Note degenerating nucellus cells close to embryo sac. White curved arrow in (A) shows extent of chalaza. Black arrowhead in (A) indi- cates micropyle; black arrowhead in (B) and (C) indicates small and large cells of embryo sac (presumably zygote and primary endosperm cell respec- tively); asterisk indicates the integumentary outgrowth. es = embryo sac; ii = inner integument; n = nucellus; oi = outer integument; vb = vascular bundle. Scale bar = 100 µm. grow, the thecae gradually shift outward on the abaxial side. According to Cronquist (1981) and Takhtajan (2009) , the fl owers While the thecae curve outward, a fold forms behind each are arranged in a modifi ed cymose infl orescence that appears to theca, establishing a secondary stamen margin. This process be a terminal, pedunculate, compound spike. The most plausible yields the leafy appearance of the stamen. Finally, the supra- interpretation, which we support in this paper, was adopted by connective enlarges to reach its full length (Figs. 9, 10). Ostenfeld (1916) and further supported by Tomlinson (1982) and During early development, the carpel grows as a hemispheri- Kuo and McComb (1998) . The ultimate infl orescence units rep- cal structure ( Figs. 9, 10 ). Subsequently, a rim is formed at the resent spikes; they never possess a terminal fl ower, and there is a periphery of the developing carpel (Fig. 10). The ovule appears sterile continuation of the spike axis above the uppermost fl ower. on the abaxial side of the carpel (Fig. 10). The carpel becomes The primary difference between the interpretations of Cronquist ascidiate, and the ovary, style, and stigma are formed. During (1981), Takhtajan (2009), and Tomlinson (1982) is in the pres- late fl oral development, the carpel mouth represents a narrow ence of fl ower-subtending bracts. Cronquist (1981) and Takhtajan slit oriented along the median plane of the fl ower and hidden by (2009) reported that the fl owers are bractless. stigma appendages ( Figs. 9, 10 ). In agreement with Tomlinson (1982) , our study supports The developing spike is protected by two distichously arranged the presence of true fl ower-subtending bracts in Posidonia . surrounding leaves. At fl ower initiation, the plane of distichy The structures that we interpret as fl ower-subtending bracts changes, and fl owers are initiated perpendicular to the protective are inserted below each fl ower on the infl orescence axis. Other leaves (see Fig. 9A–C , where the left and right margins of the interpretations of these membranous structures below each last-formed protective leaf are visible). This phyllotactic transi- fl ower are questionable. Some authors have interpreted them tion, which allows more space for fl owers to develop as they es- as connective outgrowths (see discussion in Tomlinson, 1982 ). cape the pressure of the broad leaf bases, is typically abrupt. Den Hartog (1970) hypothesized that they represent a group However, in one infl orescence, the transition was more gradual, of fused squamules, which are small scale-like structures that as the lowermost fl ower occupied an intermediate position. occur in the leaf axil of many Alismatales. The main argu- ments against this theory are that there is no leaf to which DISCUSSION these squamules belong and that squamules never become fused together in other Alismatales. Our own observations Infl orescence structure — Some authors have interpreted the and a literature review suggest that the fl ower-subtending infl orescence of Posidonia as cymose throughout (den Hartog, bracts of Alismatales lack intravaginal squamules (e.g., in 1970 ; see also Dahlgren et al., 1985 ; den Hartog and Kuo, 2006 ). ), in contrast with their vegetative leaves, which 1598 AMERICAN JOURNAL OF BOTANY [Vol. 99

Fig. 5. Posidonia australis. Longitudinal sections through fertilized ovule with globular embryo. (A) Entire ovule. (B) Micropylar area with globular embryo surrounded by coenocytic endosperm. (C) Detail of endosperm with numerous nuclei. Asterisk indicates the integumentary outgrowth; arrowheads indicate endosperm nuclei. emb = embryo; en = endosperm; ii = inner integument; n = nucellus; oi = outer integument; tt = transfer tissue; vb = vascular bundle. Scale bars: in A = 300 µm, in B = 100 µm, in C = 50 µm. possess squamules. One could speculate that the structure we pronounced (often as basal fusion between a stamen and a call a fl ower-subtending bract in fact represents a perianth tepal) in the “tepaloid” clade of Alismatales, to which Posido- member, whereas the fl ower-subtending bract is lacking. We nia belongs. No association is present between the structure reject this hypothesis on the following grounds. (1) of that we interpret as a fl ower-subtending bract and any of the other Alismatales, especially those phylogenetically closely stamens, whereas a strong association with the median stamen related to Posidonia (e.g., Potamogeton, Juncaginaceae), have would be expected if it were a tepal. Furthermore, the absence narrow bases, whereas the structure that we interpret as a of the median stamen in male fl owers of Posidonia ( Fig. 1B ) fl ower-subtending bract in Posidonia has an extremely wide does not affect the presence or position of the fl ower-subtending base. (2) In monocots, tepals are closely associated with sta- bract. (3) The tepals are vascularized in all members of Alis- mens occurring on the same radii in the fl ower (e.g., Endress, matales that bear indisputable tepals (see later regarding 1995; Remizowa et al., 2010), and this feature is especially Cymodoceaceae). October 2012] REMIZOWA ET AL.—FLOWERS AND INFLORESCENCES OF THE SEAGRASS POSIDONIA 1599

Fig. 6. Posidonia australis. Longitudinal sections through an ovule (seed) with fully developed embryo. (A) Entire ovule. (B) Apical region of embryo. (C, D) Transfer tissue at different magnifi cations. Asterisk indicates the integumentary outgrowth. ar = adventitious root; emb = embryo; cot = cotyle- don; ec = elongated cells of transfer tissue protruding into the nucellus; hyp = hypocotyle; lf = fi rst leaf; n = nucellus; rad = primary root; smc = smaller cells of transfer tissue located near the vascular bundle; tt = transfer tissue; vb = vascular bundle. Scale bars: in A = 1000 µm; in B, C = 300 µm; in D = 100 µm.

In most angiosperms, fl oral primordia arise in the axils et al., in press). In many tepaloid Alismatales with bracteate of bracts that are already initiated (e.g., Payer, 1857), as for racemose infl orescences, reduction in bract size is accompa- example in Scheuchzeria (see fi gures in Tomlinson, 1982 ; nied by loss of bract vasculature and innervation of the fl ower Posluszny, 1983). In Posidonia , the fl ower-subtending bracts by a single strand that divides in the receptacle to supply the are initiated simultaneously with their fl oral primordia. This fl oral organs ( Uhl, 1947 ; Singh, 1965a ; Gamerro, 1968 ; fact does not create a problem for the fl ower-subtending bract Posluszny and Sattler, 1974b ; Remizowa and Lock, 2011 ; homology assessment in Posidonia , because similar develop- Remizowa et al., in press ). Similarly, in Posidonia , small-sized mental dynamics also occur in lineages that are phylogenetically bracts lack any conductive tissue, and the fl ower is supplied by closely related. In some Alismatales with racemose infl orescences, a single conductive strand. In Posidonia, the vascular bundle the fl owers and their subtending bracts are initiated simulta- that supplies the fl ower departs directly from the infl orescence neously, either from separate primordia (Sattler, 1965; Posluszny axis, leaving no gap in the axis stele. This condition also occurs and Sattler, 1974a; Posluszny, 1981; Sun et al., 2000; Remi zowa in some other tepaloid Alismatales with racemose infl ores- et al., in press ) or sometimes from common primordia, as in cences and distichous phyllotaxy, but it is apparently absent some species of Tofi eldia and some species of Potamogeton from other monocot groups. The bundle entering the pedicel ( Remizowa et al., 2006 , in press ). In Potamogeton , the primor- arises as a result of radial division of the stele bundles (or pos- dium of the fl ower-subtending bract can be initiated simulta- sibly radial fusion of the stele bundles, though the developmen- neously with (Sun et al., 2000) or slightly later than the lateral tal processes are currently unknown in Posidonia ). In Posidonia , tepals or even considerably later in cases of separate bract and the bundle entering the pedicel originates from two strands that fl ower initiation ( Remizowa et al., in press ). run parallel to the plane of distichy. In Ruppia , the axis of the Tepaloid Alismatales with racemose infl orescences have a infl orescence (which is a spike of 2−3 fl owers) is very thin and general (homoplastic) tendency to bract reduction (Posluszny contains only a single bundle that gives off branches on the and Charlton, 1993; Remizowa and Lock, 2011; Remizowa radii of fl owers ( Singh, 1965a ; Gamerro, 1968 ; Posluszny and et al., in press ). The fl ower-subtending bracts can be reduced Sattler, 1974b; Lock, 2012). In Zosteraceae, the fl oral boundaries either by suppression or by formation of hybrid organs (Remi zowa are less obvious, and the infl orescence is a highly modifi ed, 1600 AMERICAN JOURNAL OF BOTANY [Vol. 99

Fig. 7. Posidonia australis. SEMs of ovules (seeds) with fully developed embryo. (A) Carpel dissected to show seed. (B) Dissected carpel with seed dissected to show embryo. (C) Front and (D) lateral views of viviparously germinated seed with protruding primary root. Note that the embryo has grown signifi cantly in the seeds illustrated in images C and D compared with that in B. (E) Entire isolated embryo. (F) Lateral and (G) front views of apical region of the same embryo in image E. White asterisk indicates the integumentary outgrowth. ar = adventitious root; emb = embryo; cot = cotyledon; hyp = hypocotyl; lf = fi rst leaf; n = nucellus; rad = primary root. Scale bars: in A–E = 1000 µm; in F, G = 500 µm.

fl attened spadix. In all genera of Zosteraceae, the spadix axis In tepaloid Alismatales, even if fl ower-subtending bracts contains three parallel bundles but only the lateral ones produce are present, they often do not perform a protective function, branches to supply the fl oral organs ( Uhl, 1947 ) either before or after anthesis. In many cases, the developing October 2012] REMIZOWA ET AL.—FLOWERS AND INFLORESCENCES OF THE SEAGRASS POSIDONIA 1601

Fig. 8. Posidonia australis . Infl orescence vasculature. (A) Longitudinal section showing the two vascular bundles of the infl orescence axis. (B) Transverse section of pre-anthetic theca showing a septum between pollen sacs and densely packed fi liform pollen. (C–I) Serial transverse sections of pre-anthetic infl orescence. (C) surrounded by two leaves; note two vascular bundles. (D) Level just below fl ower insertion. (E) Level of fl ower insertion and origin of fl ower vascular supply. (F) Level of fl ower receptacle and division of single fl ower bundle into branches supplying gynoecium and stamens. (G, H) Levels of stamen and gynoecium separation. (I) Level of ovule insertion. br = fl ower-subtending bract; d = dorsal carpellary bundle; fp = fi liform pollen; g = gynoecium and its vascular bundle; s1 and s2 = bundles of infl orescence axis; st = stamen connective and its vascular bundle; t = theca; v = ventral carpellary bundle. Scale bars: in A, C–I = 1000 µm; in B = 300 µm.

fl owers are covered by the tepals ( Fig. 11 ). Alternatively, if in the generative axes that ultimately develop fl owers. For the perianth is absent or delayed in development, the entire example, fl owers of Triglochin are arranged spirally along the developing infl orescence is protected by the surrounding raceme axis, whereas in Potamogeton the distichy of the veg- leaves, as in Zosteraceae, Potamogeton , Ruppia , and Posido- etative leaves is followed by a whorled fl oral arrangement nia (Sattler, 1965; Posluszny and Sattler, 1973, 1974a , ( Sattler, 1965 ; Posluszny and Sattler, 1974a , b ; Posluszny, b ; Posluszny, 1981; Tomlinson, 1982; Soros-Pottruff and 1981; Charlton, 1981; Tomlinson, 1982; Sun et al., 2000; Posluszny, 1995a , b ; Sun et al., 2000 ; Remizowa et al., in Lock et al., 2009 ; Remizowa et al., in press ). press ). In most tepaloid Alismatales, the vegetative leaves are Posidonia represents a transitional case, because it has disti- arranged distichously (e.g., Tomlinson, 1982 ). In Ruppia , chous phyllotaxy on vegetative axes and within the infl orescence phyllotaxy is consistent throughout, and the fl owers maintain itself, but the plane of distichy changes to place the fl owers the distichy of the vegetative regions (e.g., Posluszny and Sattler, perpendicular to the leaves. In Posidonia , this switch allows 1974b ). In contrast, the phyllotaxy of some other genera changes more space for development of the massive fl owers. However, 1602 AMERICAN JOURNAL OF BOTANY [Vol. 99

Fig. 9. Posidonia oceanica . SEMs of developing infl orescences in lateral view. (A) Incipient infl orescence. (B) Infl orescence with lowermost initiated fl ower and its subtending bract. (C) Infl orescence consisting of two fl owers. The thecae are not yet apparent, but the gynoecium has commenced differentia- tion. (D, E) Infl orescences consisting of three fl owers. The upper fl ower is bisexual in (D) and male in (E). (F) Infl orescence consisting of two bisexual fl owers. The thecae have started to differentiate on the lateral stamen sides. (G–I) Infl orescences with male upper fl owers. The thecae gradually shift abaxi- ally during development. ap = infl orescence apex; br = fl ower-subtending bract; fl = fl ower primordium; g = gynoecium; ia = infl orescence axis; -lf, lf- = left and right margins of the last-formed protective leaf; sq = intravaginal squamules of the removed foliage leaf surrounding the spikelet (l2 on Fig. 1B); st = stamen; t = theca. Scale bars: in A, B, E = 150 µm; in C, D = 250 µm; in F = 200 µm; in G–I = 500 µm. there is no obvious functional explanation for the retention of that can branch further. Posidonia is intermediate, with several phyllotaxy in Ruppia, whereas spiral or whorled phyllotaxy in branches bearing few-fl owered spikes. The condition found in Potamogeton and Triglochin apparently allows the develop- Posidonia could equally be regarded as a modifi cation of either ment of more fl owers than does a distichous arrangement. The highly branched or less branched patterns. change in phyllotaxy and number of fl owers per (partial) infl o- rescence is negatively correlated with branching intensity Flower morphology and development — In most accounts, within a synfl orescence. A greater number of fl owers per infl o- fl owers of Posidonia are reported as perianthless ( Ostenfeld, rescence is correlated with fewer paracladia. This compensa- 1916; Tomlinson, 1982; Dahlgren et al., 1985; Kuo and tion mechanism is a general rule and does not always refl ect McComb, 1998; Takhtajan, 2009). However, Cronquist (1981) evolutionary transformations ( Khokhryakov, 1974; Kusnet zova, mentioned three vestigial tepals, without indicating which 1998). Thus, the synfl orescence of Triglochin , with its long, species he examined. We found no sign of a perianth, even many-fl owered primary infl orescence (which is a raceme) during early development. In Posidonia, the perianth is at develops no lateral branches (paracladia). On average, species least partially substituted by the persistent shield-like sta- of Potamogeton with relatively long spikes often possess only mens, which protect the fruit after anthesis. Superfi cially, two paracladia. The synfl orescence of Ruppia, with 2−3- structures that resemble the stamens of Posidonia are present fl owered spikes, is a panicle: it develops many lateral branches in Potamogeton. In Potamogeton , the extrorse thecae are October 2012] REMIZOWA ET AL.—FLOWERS AND INFLORESCENCES OF THE SEAGRASS POSIDONIA 1603

Fig. 10. Posidonia oceanica. SEMs of gynoecium development. (A, B) Early stage in which the gynoecium is represented by a solid primordium. (C, D) Early stages of carpel wall formation and ovule initiation. (E, F) Later stages of carpel wall formation. Note slit-like carpel mouth in (F). ap = continuation of the infl orescence axis; br = fl ower-subtending bract; g = gynoecium; ov = ovule primordium; st = stamen; t = theca. Scale bars: in A, C, F = 100 µm; in B, D = 200 µm; in E = 250 µm. widely gaping on the narrow base of a tepal-like structure that carpel side is most frequently oriented adaxially (toward the is sometimes interpreted as a connective outgrowth (e.g., As- infl orescence axis) ( Eichler, 1875 ; Swamy and Bailey, 1949 ; cherson and Graebner, 1907 ). The nature of this structure was Endress, 1989 ; Igersheim et al., 2001 ). In Posidonia , the ventral long debated in the literature, until developmental studies carpel surface faces the fl ower-subtending bract, while the demonstrated that the fl ower of Potamogeton possesses a true dorsal side is directed toward the spike axis. We interpret ovule perianth (Sattler, 1965; Posluszny and Sattler, 1973, 1974a ). attachment as ventral in Posidonia , based on a comparison In Potamogeton , the stamens and tepals are initiated indepen- with related Alismatales possessing multicarpellate gynoecia dently and become united late in development via formation (Ruppiaceae, Potamogetonaceae, Juncaginaceae), in which of a common stalk (i.e., late congenital fusion). In Posidonia , ovule attachment is ventral (Uhl, 1947; Singh, 1965a, b ; the tepal-like structures are clearly stamens. However, due Igersheim et al., 2001 ; Kamelina, 2011 ). to their unusual shape, their development is also unusual. The The unusual carpel orientation could be explained as an evo- stamens commence growth as laminar structures but soon lutionary reduction of a formerly tricarpellary gynoecium. In develop thecae on their lateral surfaces, then the secondary this scenario, an ancestral fl ower possessed three carpels alter- margins fold out and a long appendage (supraconnective) nating with the three stamens. All three carpels had their ventral forms on the stamen tip. Among the closest relatives of Posi- sides oriented inside the fl ower, as in typical monocot gynoecia. donia with racemose infl orescences, stamens with a supra- Two abaxial carpels were lost during evolution, while the connective are present in the perianthless genus Ruppia, but adaxial one retained its orientation and occupied the center they are relatively small and appear very late in fl oral devel- of the fl ower. A similar condition was reported for the female opment (Uhl, 1947; Singh, 1965a; Gamerro, 1968; Posluszny fl owers of the core eudicot Cercidiphyllum (Saxifragales) and Sattler, 1974b ). In both Ruppia and Posidonia , the stamen (Swamy and Bailey, 1949; Endress, 1986; Yan et al., 2007). bundle terminates in the connective and does not continue Among other tepaloid Alismatales with nonmonomerous fl ow- into the supraconnective. ers, only Potamogeton zosteriformis consistently develops a One unique and remarkable feature of Posidonia is its mono- single carpel ( Posluszny, 1981 ). In this species, the monocarpel- carpellate gynoecium with the ovule attached to the abaxial late condition is a consequence of reduction, because other side. In most angiosperms, the ovules are developed on the ven- species of Potamogeton typically possess four carpels. In Pota- tral carpel side, which faces inward in the fl ower (e.g., Eichler, mogeton zosteriformis , the monocarpellate condition has been 1875; Eames, 1961). In monocarpellate gynoecia, the ventral achieved via reduction of the space available for gynoecium 1604 AMERICAN JOURNAL OF BOTANY [Vol. 99

Fig. 11. Maximum parsimony optimizations of character evolution onto a phylogenetic tree of Alismatales. Tree topology is based on Iles et al. (in press). In the upper row, hash (#) indicates taxa with racemose bracteate infl orescences, and asterisk (*) indicates racemose nonbracteate infl orescences, the condition of this character in Maundia (?) requires further examination ( von Mering and Kadereit, 2010 ). In ecology coding, character state 0 implies that plants never grow as permanently submerged into saline water, state 1 implies that plants are always submerged marine, and state 2 implies that plants are tolerant to a wide range of salinity, including hypersaline and brackish waters. October 2012] REMIZOWA ET AL.—FLOWERS AND INFLORESCENCES OF THE SEAGRASS POSIDONIA 1605 development (compared with other species). In contrast with The ovule of Posidonia best fi ts the campylotropous type. Posidonia , carpel orientation is variable in Potamogeton zos- The ovule is bitegmic, endostomic, with a superfi cially straight teriformis—each of the four initial carpels has equal potential nucellus and chalaza extending along the nucellus. Characteris- to develop (Posluszny, 1981). By contrast, in Posidonia the car- tically, the nucellus and chalaza are not aligned, and this feature pel position is precisely fi xed. suggests campylotropy. The superfi cially straight appearance It is instructive to compare patterns of ovule insertion in of the nucellus is due to extreme curvature at the attachment Posidonia with those of Cymodoceaceae and Ruppiaceae, the area. This curvature is restricted to the base of the ovule and two families that are phylogenetically most closely related to does not affect the nucellus itself. Some grasses (Poaceae) dem- Posidoniaceae. In Ruppiaceae, each fl ower has a polymerous gynoecium, and ovule insertion is clearly ventral. In Cymod- onstrate similar ovule morphology to Posidonia ; the primary oceaceae, each female reproductive unit (commonly described difference is the absence of an outgrowth and the lack of one- as a “fl ower”) has two carpels, and each carpel has a dorsally sided fusion of the two integuments. The unusual ovule struc- inserted ovule ( McConchie et al., 1982 ; Tomlinson and Posluszny, ture of grasses has provoked an extensive literature dealing 1978 ). Based on comparison of infl orescence structure, Sokoloff mainly with issues of terminology (reviewed by Petrova et al., et al. (2006) proposed that the so-called fl owers of Cymod- 1985 ; Batygina and Yakovlev, 1990 ). oceaceae actually represent pseudanthia that are homologous Being sessile, the ovule of Posidonia is attached on the with the spikelets of Ruppia (and Posidonia ). If this interpreta- lateral ventral placenta by a very broad chalaza. Kuo and den tion is correct, the two carpels of Cymodoceaceae perfectly Hartog (2006: fi g. 11F) illustrated ovule insertion in P. australis fi t the carpel positions in a two-fl owered spikelet of Posidonia . as apparently basal, but in our interpretation their fi gure actually In this interpretation, ovule insertion is identical in Posidonia shows the transverse section of a carpel. and Cymodoceaceae. We also note that the involucral struc- All other members of the clade to which Posidonia belongs tures that surround the reproductive units of some Cymod- (Cymodoceaceae, Posidoniaceae, Potamogetonaceae, Ruppiaceae, oceaceae are homologous to the fl ower-subtending bracts of Zosteraceae) (Chen et al., 2004; Iles et al., in press) are charac- Posidonia , if a pseudanthial interpretation is accepted for terized by pendent ovules that are orthotropous (Fig. 11), at Cymodoceaceae. least before fertilization (Eber, 1934; Kudryashov and Savich, Evolution of fi liform pollen grains in seagrasses— Under- 1968; Kamelina, 1990, 2011 ; Kamelina and Teryokhin, 1990; water pollination by fi lamentous inaperturate pollen grains Teryokhin, 1990; Igersheim et al., 2001). Posidonia is the ( Fig. 11) occurs in only three families of seagrasses (Cymod- only representative whose ovules are campylotropous and de- oceaceae, Posidoniaceae, and Zosteraceae) and is absent from velop an outgrowth. This appendage is formed by both in- other Alismatales and all other angiosperms ( Ducker et al., teguments, which are fused together at this point and located 1978 ; Dahlgren et al., 1985 ; Takhtajan, 2009 ; Furness and opposite the micropyle. Posidonia shares some other gynoecial Banks, 2010 ; Kamelina, 2011 ). These three families do not and ovular features with its closest relatives, such as the type of form a clade in most molecular phylogenetic trees (e.g., Les placentation (ovules are attached to a nonbasal placenta) and et al., 1997; Chen et al., 2004; Iles et al., in press). Mapping the the orientation of the micropyles toward the base of the ovary. occurrence of fi liform pollen grains onto the molecular phylo- Compared with Posidonia , all other families of the clade pos- genetic tree of Iles et al. (in press) suggests two equally parsi- sess a relatively well-developed funiculus, and the attachment monious hypotheses: either (1) independent origins of fi liform area is rounded and relatively small (Kamelina, 1990; Kamelina pollen grains in Cymodoceaceae, Posidoniaceae, and Zoster- and Teryokhin, 1990 ; Teryokhin, 1990 ). Endress (2011) noted aceae, or (2) a single origin with two subsequent losses in Pota- an interesting phenomenon that also occurs in families related mogetonaceae and Ruppiaceae (Fig. 11). Possible support for the hypothesis of independent origins is provided by the differ- to Posidonia. If orthotropous ovules develop on a lateral pla- ing orientation of pollen grains within the anther in the dif- centa in a narrow ovary (as in ascidiate carpels bearing a single ferent groups. Orientation occurs along the anther length in large ovule), architectural constraints dictate that they are some- Zosteraceae ( Soros-Pottruff and Posluszny, 1995a ), spirally coiled what curved at their bases. Under such conditions, the ovules within the anther in Cymodoceaceae ( Ascherson and Graebner, can equally be described as anatropous or orthotropous. The 1907; Ducker et al., 1978), and straight and packed perpendicu- question is whether these ovules are fundamentally anatropous lar to the thecal locules in Posidonia (this study). Multiple ori- but cannot become curved due to space limitations in the ovary gins of fi liform pollen grains are also supported by considerable locule or whether they are fundamentally orthotropous but need differences in structural details of pollination in Posidonia and to curve slightly. A similar ambiguity was reported for Pota- other seagrasses ( McConchie and Knox, 1989 ). mogeton ( Kamelina, 1990 ; Igersheim at al., 2001 ). One possible way to avoid curvature during development is to insert ovules Ovule morphology — The ovule of Posidonia is character- obliquely. In this case, a rounded attachment area should be ized in the literature as orthotropous ( den Hartog and Kuo, changed to an elliptic one (extended along the carpel). Thus, in 2006 ). According to Endress (2011) , there are three major narrower locules we could expect more obliquely inserted types of ovule morphology: orthotropous, anatropous, and ovules; the shorter the funiculus, the more pronounced the campylotropous (however, see Batygina, 2002 ; Shamrov, 2008 ). Both orthotropous and anatropous ovules possess a straight obliquity. Compared with other genera with common carpel nucellus that lies along the same line as the micropyle and cha- morphology, Posidonia possesses a relatively narrow ovary laza. Campylotropous ovules possess a more or less curved nu- locule. Thus, we hypothesize that the unique ovule of Posido- cellus, so that the nucellus, micropyle and chalaza never lie along nia could have evolved from an orthotropous condition ( Fig. the same line. In some campylotropous ovules, the chalaza and 11) in a very narrow ovary locule via reduction of the funiculus funiculus (if present) are located parallel to the nucellus. and extreme elongation of the attachment area. 1606 AMERICAN JOURNAL OF BOTANY [Vol. 99

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