Aliso: A Journal of Systematic and Floristic Botany Aliso: A Journal of Systematic and Floristic Botany Systematic Floral Anatomy of Pontederiaceae Systematic Floral Anatomy of Pontederiaceae

Twenty species of Pontederiaceae and six species of the outgroup families Haemodoraceae and Philydraceae were investigated with regard to floral anatomy, using standard histological methods and graphic reconstructions. Variation is described in several features, including functional carpel number, the distribution of floral aerenchyma, the presence of unusual floral epithelial cells, the presence, type, and distribution of tannin cells, crystal type and distribution, aspects of placentation, ovule number, number of ovule rows per carpel, and septal nectary presence. In order to better assess character homology, one of these features, placentation, was divided into three discrete characters: septal fusion, placenta position, and placenta evagination. Several of the floral characters were optimized onto a most parsimonious cladogram from a relatively recent study using combined chloroplast DNA data. Associations of character states with specific clades were observed in functional carpel number, aer enchyma distribution, epithelial cell presence, tannin cell distribution, septal nectary presence, and the three redefined aspects of placentation: septal fusion, placenta position, and placenta evagination. These floral data will be valuable in further elucidating relationships in Pontederiaceae in future studies of the group.


INTRODUCTION
Pontederiaceae Kunth are a pantropical family of herbaceous, annual or perennial, freshwater aquatic monocotyledonous plants (Hutchinson 1973;Cronquist 1981;Takhtajan 1997). A recent treatment of the family by Cook (1998) listed nine genera and approximately 33 species, although other treatments unite these nine genera into as few as four. Members of the family occur mainly in the neotropics, with a few species ranging as far north as southeastern Canada and as far south as southern Argentina. Other species are found in South Africa, Madagascar, Southeast Asia, and Australia. Eichhornia crassipes, the water hyacinth, is probably the most well-known member of the family, since it was introduced to many tropical and subtropical areas, perhaps originally as an aquatic ornamental. It is considered one of the world's most troublesome aquatic weeds (Gopal 1987;cited in Cook 1998).
In recent phylogenetic analyses, Pontederiaceae has been consistently allied in various combinations to Commelinaceae, Haemodoraceae, and Philydraceae (Duval et al. 1993;Chase et al. 2000). Morphological studies of monocots have also supported the close relationships among Haemodoraceae, Philydraceae, and Pontederiaceae, but the compiled evidence has been ambiguous about the exact sister group relationships. Simpson (1987) hypothesized that Pontederiaceae shares a most recent common ancestor with Haemodoraceae by inferring homology in pollen exine sculpturing; both taxa share a one-to two-layered nontectate-columellate exine architecture, and some a verrucate sculpturing (see also Eberhardt 1996). In addition, Simpson (1993) points out that some members of both Haemodoraceae and Pontederiaceae possess septal nectaries while all members of Philydraceae do not; however, septal nectaries are likely an ancestral character for the monocots. Tillich (1995), in an anal-ysis of monocot seedling morphology, noted that both Philydraceae and Pontederiaceae possess a bifacial, linear cotyledon, a potentially unique feature in angiosperms. Haemodoraceae, on the other hand, possess a variety of cotyledon forms, all of which are unifacial.
The intrafamilial systematics of Pontederiaceae has varied somewhat (see Eckenwalder and Barrett 1986;Graham and Barrett 1995). The family was divided by Schwartz (1930) into three tribes: Eichhornieae, Heteranthereae, and Pontederieae. In his treatment of Pontederiaceae, Cook (1998) cites the three tribes of Schwartz. However, Cook argues that Monochoria should be placed in Eichhornieae, not Heteranthereae (as placed by Schwartz), based on morphological features.
According to Cook (1998), tribe Pontederieae includes two genera, Pontederia L. (3-6 spp.) and Reussia Endl. (2 spp.), the latter sometimes recognized as a subgenus of Pontederia (Lowden 1973;Eckenwalder and Barrett 1986;Rosatti 1987;Graham and Barrett 1995). Members of Pontederieae have a zygomorphic, tubular perianth, and six stamens. The tribe is distinctive in having a uniovulate ovary with one functional carpel, the whole developing into a oneseeded fruit surrounded by a withered, accrescent, hardened perianth tube, a fruit type termed an "anthocarp." Reussia is distinguished from Pontederia by the presence of a spiny anthocarp. Pontederieae is perhaps the most well-supported intrafamilial grouping because of this distinctive ovary and fruit morphology. Members of tribe Eichhornieae, which includes Eichhornia Kunth (7 or 8 spp.) and Monochoria C. Presl (8 spp.), have a zygomorphic, tubular perianth, six stamens, and a multi-seeded, trilocular capsule. Although Monochoria resembles Eichhornia and Pontederia in having a geniculate infructescence, Monochoria is unique in the family in having a perianth fused only at the extreme base. Eichhornieae and Pontederieae are possibly linked via the zy-gomorphic perianth and a geniculate infructescence (Rosatti 1987). Eckenwalder and Barrett (1986). as well as Graham and Barrett ( 1995), noted that these taxa constitute a group of mainly tristylous species, suggesting that tristyly is ancestral for this complex. With the exception of one species (Eichhornia natans Solms, found in South Africa), these two tribes are native to the New World.
Tribe Heteranthereae is characterized by the presence of one to three dimorphic stamens with basifixed anthers, often cleistogamous, more or less actinomorphic flowers, and a largely enantiostylous floral syndrome (Eckenwalder and Barrett 1986;Graham and Barrett 1995). Within Heteranthereae, Cook chooses to recognize Heteranthera Ruiz & Pav. s.s., plus the four segregate genera of Eurystemon Alexander, Hydrothrix Hook f., Scholleropsis H. Perrier, and Zosterella Small, for a total of nine genera and approximately 33 species in the family. Following the convention of Graham et al. (1998), we merge both Eurystemon and Zosterella into Heteranthera in this study, but treat Hydrothrix and Scholleropsis as distinct genera.
A recent analysis of Pontederiaceae (Graham et al. 1998; see also Kohn et al. 1996) examined phylogenetic relationships using both chloroplast DNA data and morphological data, noting a significant discrepancy between the two. Although tree rooting was problematic, results of this study indicated monophyletic tribes Pontederieae and Heteranthereae (excluding Monochoria, as in Cook 1998), but with tribe Eichhornieae forming a grade at the base of Pontederieae. (See Discussion, below.) Because of the uncertainty of intrafamilial relationships in Pontederiaceae, additional characters are needed to assess these relationships more robustly. This is particularly important in tracing the evolution of adaptively significant features, e.g., those involving reproductive mechanisms such as heterostyly (Graham and Barrett 1995;Barrett and Graham 1997). The purpose of this study is to identify floral anatomical characters and to optimize character state changes, using recently generated cladistic hypotheses (Graham et al. 1998). The correlation of these features with putative monophyletic groups within the family is explored, with emphasis on their possible adaptive significance. This study expands that of Burton and Simpson (1996) and supplements a recent study by Strange et al. (2004). (See Discussion, below.)

MATERIALS AND METHODS
Mature buds or recently opened flowers were preserved in the field or greenhouse in FAA (formalin-acetic acid-alcohol), embedded in Paraplast, and sectioned serially, both transversely and longitudinally. Section thickness was generally 10 j.Lm, but occasionally ranged from 8-15 j.Lm depending on the flower size. Sections were stained with safranin, fast-green, and hematoxylin according to standard procedures (Johansen 1940;Sass 1958;Berlyn and Miksche 1976). Light microscope images were photographed using a Nikon Microphot-FX microscope with a Nikon Coolpix 990 digital camera ( Fig. 1-41).
Twenty species in Pontederiaceae, two of Haemodoraceae, and two of Philydraceae were examined. Voucher information is presented in Table 1.
Sections were studied with regard to presence, type, and distribution of crystals, tannin cells, aerenchyma, septal nectaries, and specialized anatomical features of cells and tissues (see Table 2). Descriptions of floral anatomical features were made for each species examined. In order to better visualize the spatial distribution of major floral components, we prepared a diagram showing the relative extent and position of the locular cavity, septal tissue (illustrating fused and unfused regions), ovule attachment, and septal nectaries of each species examined (Fig. 42). For three species a detailed graphical representation of a flower longitudinal section was prepared from sequential cross sections according to the procedure outlined in Simpson, 1998 (Fig. 43-45). A diagram was prepared illustrating various placentation features ( Fig. 46-50). Finally, in order to assess the evolutionary history of examined traits, several floral anatomical characters were optimized, onto one of the cladograms of Graham et al. ( 1998) using MacClade (Maddison and Maddison 1992;Fig. 51-58). Note that we chose one of the most parsimonious cladograms, derived from combined chloroplast ndhF, rbcL, and restriction-site variation data of Graham et al., 1998 (their Fig. 2, right side, with their undescribed "Eichhornia sp." omitted). We have also added two outgroups (Haemodoraceae and Philydraceae) on these phylogenies in order to better assess relative ancestral character states.

Eichhornia crassipes
Placentation is axile, with three carpels and three locules. The septa are fused throughout (synseptalous; Fig. 1-3). The septa are barely fused at the apex of the ovary, with a commissure not clearly present. The placentae consist of two evaginated flanges per carpel. Ovules are pleurotropous, numerous per carpel, in two to four rows per flange. The region of ovule attachment extends continuously from near the base to the apex of locule (Fig. 42). Septal nectaries are present, in the basal region of the ovary. Aerenchyma is present in tissues of the receptacle, perianth, ovary wall, and septa. Epithelial cells are absent. Calcium oxalate raphides are distributed in tissues of the receptacle, perianth, ovary wall, placentae, stamen filaments, and connective, especially concentrated in placentae. Tannin cells are mainly homogeneous, with some granular tannin cells; both are distributed in tissues of the receptacle, perianth, ovary wall, septa (very sparsely), placentae, and style. (See Fig. 1-4; Table 2).

Eichhornia diversifolia
Placentation is axile, with three carpels and three locules. The septa are fused throughout (synseptalous); commissures do not occur at the junction of the septa. The placentae consist of two evaginated flanges per carpel. Ovules are pleurotropous, numerous per carpel, in two rows per flange (becoming one row near the apex). The region of ovule attachment is continuous from near the base to the apex of the locule (Fig. 42). Septal nectaries are absent. Aerenchyma is present in tissues of the receptacle, perianth, and septa. Epithelial cells are absent. Calcium oxalate raphides are distributed sparsely in tissues of the receptacle, perianth, and ovary wall. Tannin cells are homogeneous and fibrillar and are distributed in tissues of the receptacle, perianth, ovary wall, and style, sparsely in septa. (See Fig. 8; Table 2).

Eic hho rnia heterosperma
Placentation is ax ile, with three carpe ls and three locules. The septa are fused throughout (sy nsepta lous). However, comm issures at the junctio n of the septa are present near the apex of th e ovary. The placentae consist of two evaginated fl anges per carpel. Ovules are pleurotropous, numerous per carpe l, in one row per flange. The region of ovul e attac hme nt exte nds continuo usly from near the base to the apex of th e locule (Fig. 42). Septal nectaries are present, in the basal regio n of the ovary. Aerenchyma is present in tissues of the receptacle, perianth, and septa. Epithe li a l cell s are absent. Calc ium oxalate raphides are distributed in ti ssues of the perianth , ovar y wall , and placentae. Tannin cell s are fibrillar (rarely homogeneous) and are distributed in tissues of the receptacle and sty le, sparsely in the perianth , ovary waJJ, and septa. (See Fig. 9, 10; Table 2).

E ic hhorn ia meyeri
Placentation is ax ile-parietal, with three carpe ls and three' locul es at ovary base, o ne locul e at the apex. The septa are fused from the basal to th e middle region of the locul e, unfused at the apex (herniseptalous). Commi ssures are not prese nt at the junction of the septa. The pl acentae consist of two evagi nated flanges per carpel. Ovules are pleurotropous, nume ro us per carpel, in two to three rows per flan ge. The reg ion of ovul e attachment is continuous fro m near the base to the apex of the locule (Fig. 42). Septal nectaries are present, in the basal region of the ovary. Aerenchyma is present in tissues of receptacle, perianth, ovary wall, and septa. Epithelial cells are absent. Calcium oxalate raphides are distributed in tissues of the receptacle, perianth, and ovary wall. Tannin cells are granular and very sparse in receptacle, placental, and stylar tissue, but otherwise absent. (See Fig. 11-13; Table 2).

Eichhornia paniculata
Placentation is axile-parietal, with three carpels and three locules at ovary base, one locule at the apex. The septa are fused from the base to the middle region of the locule (hemiseptalous). Commissures are present at the junction of the fused septa near the apex. The placentae consist of two evaginated flanges per carpel. Ovules are pleurotropous, numerous per carpel, in two rows per flange (becoming one row near apex). The region of ovule attachment is continuous from near the base to the apex of the locule (Fig. 42). Septal nectaries are present, in the basal region of the ovary (Fig. 42,43). Aerenchyma is present in tissues of receptacle, perianth, ovary wall, and septa. Epithelial cells are absent. Calcium oxalate raphides are distributed in tissues of the perianth, ovary wall, septa, placentae, and funiculus. Tannin cells are granular and sparse in receptacle tissue, but otherwise absent. 43; Table 2).

Heteranthera dubia [Zosterella dubia]
Placentation is parietal, with two carpels and one locule. The septa are unfused throughout (aposeptalous). The placentae consist of two slightly evaginated flanges. Ovules are epitropous and hypotropous, numerous, in one row per flange. The region of ovule attachment extends continuously from the base to the apex of the locule (Fig. 42). Septal nectaries are absent. Aerenchyma is present in tissues of the receptacle, perianth, ovary wall, and septa/placentae. Epithelial cells are absent. Calcium oxalate raphides are distributed in tissues of the receptacle and perianth. Tannin cells are homogeneous and granular and are distributed in tissues of the receptacle, ovary wall, septa, placentae, style, and androecium, very sparsely in the perianth. (See Fig. 14, 15; Table 2).

Heteranthera mexicana [Eurystemon mexicanum]
Placentation is parietal, with three carpels and one locule. The septa barely touch, with commissures at their junction and a large intercellular space at the center of the junction region. The placentae consist of two slightly evaginated flanges. Ovules are mostly pleurotropous, numerous, in two rows per flange. The region of ovule attachment extends from the base to the apex of the locule. Septal nectaries are absent. Aerenchyma is present in tissues of the receptacle, perianth, ovary wall, and septa. Epithelial cells are absent. Calcium oxalate raphides are distributed in tissues of the receptacle, perianth, ovary wall, and septa. Tannin cells are absent. (See Fig. 17, 18; Table 2).

Heteranthera multiflora
Placentation is parietal, with three carpels and one locule. The septa are unfused throughout (aposeptalous). Commissures at the junction of the septa are absent (septa not fused). The placentae consist of two evaginated flanges. Ovules are mostly pleurotropous, numerous, in two to three rows per flange. The region of ovule attachment is continuous from the base to the apex of the locule (Fig. 42). Septal nectaries are absent. Aerenchyma is present in tissues of the receptacle, perianth, ovary wall, and septa. Epithelial cells are absent. Calcium oxalate raphides are distributed in tissues of the receptacle, ovary wall, septa, and placentae, especially in aerenchyma spaces in the latter two regions. Tannin cells are homogeneous and are mostly absent, except for being sparse in the receptacular region. (See Table 2; not illustrated).

Heteranthera reniformis
Placentation is parietal, with three carpels and one locule. The septa are aposeptalous, but barely touch and are coherent at the center; commissures are present at the junction of the septa, typically with a large intercellular space in the center. The placentae consist of two evaginated flanges. Ovules are mostly pleurotropous, numerous, in two rows per flange. The region of ovule attachment extends continuously from the base to the apex of the locule (Fig. 42). Septal nectaries are absent. Aerenchyma is present in tissues of the receptacle, perianth, ovary wall, and septa. Epithelial cell s are absent. Calcium oxalate raphides are di stributed in ti ssues of the receptacle, ovary wall , septa, and pl acentae, especially in aerenchyma spaces in the latter two regions. Tannin cells are ho mogeneo us and are absent except for being very sparse in the receptacul ar region. (See Fig. 19, 20; Table 2).

Heteranthera rotundifolia
Pl acentati o n is axile-parietal with three carpels and three Jocul es at the ovary base, one locul e at th e apex. The septa are fused from base to middle region of loc ul e (he mi septalous). Commissures at the junction of the septa are present at the junction of the three fused septa. The placentae consist of two evaginated fl anges. Ovules are mostly pleurotropous, numerous, in two to three rows per fl ange. The region of ovule attac hment extend continuou ly from the base to the a pex of th e locul e. Septal nectaries are absent. Aerenchyma is present in tissues of the receptacle, pe ri anth , ovary wall , and septa. Epithelial cells are absent. Calcium oxalate raphides are di stributed in ti ssues of the receptacle, ovary wall , and septa/pl acentae junction, especial ly in aere nchyma spaces. Tannin cell s are absent. (See Fig. 2 1, 22; Table 2).

Heteranthe ra seuberti ana
Pl acentati o n is pari etal, with three carpe ls and one locul e. The septa are unfu sed throughout (aposepta lo us). Commissures at the j un ction of the septa are absent (septa not fused) . The placentae consist of two s li g htl y evaginated fl anges . Ovul es are mostly pleurotropous , nume rou s, in o ne to two rows per fl ange. The region of ovule attachment extends continuo usly fro m the base to the a pex of the locul e (Fig . 42). Septal nectaries a re absent (Fig . 45). Aerenc hyma is present in ti ss ues of the receptacle and septa . Epithelial ce ll s are absent. Calcium oxalate raphides are sparsely di stributed in ti ss ues of receptac le, pe ri a nth , ovary wall, and septa. Tannin ce ll s are ho mogeneous and a re sparse in the receptac ular reg io n, but otherwi se absent. (See Fig. 45; Table 2).

Hydrothri x gardneri
Flowers are paired, each with o ne stamen, the pa ir surro unded by bracts. Pl acentati on is parietal , w ith three carpels and one locul e. T he septa are unfu ed throug hout (aposeptalo us). Cornmissures at the juncti o n o f the septa are absent (septa not fused). The pl acentae cons ist of two sli ghtly evaginated fl anges. O vul es are mostly pl eurotropous, numerous, in one row per fl ange. T he regio n of ovul e attachment extends continuo usly fro m the base to the apex of the locul e (Fig. 42). Septal nectaries are absent. Aere nchyma is present in tissues of the receptacle, perianth, ovary wall, and septa. E pithe li a l cells are absent. Calc ium oxalate raphides are di stributed in ti ssues of the receptacle, sparse in pe ri anth and surrounding bracts, absent e lsewhere. Tannin cell s are ho mogeneous and granular and are distributed in tiss ues of the receptacle and style and in the surrounding bracts. (See F ig. 16; Table 2).

M onochori a has tata
Pl acentation is axile, with three carpels and three locules. The septa are fused thro ug ho ut (synsepta lous), na.J.Tow ly fused near the apex. Commi ssures at the juncti o n of the septa are not present. T he pl acentae consist of two evaginated fl anges. O vul es are mostl y pleurotropous, numerous, in two to three rows per fl a nge. T he regio n of ovul e attac hme nt extends continuo usly from the base to the apex of the locule. Septal nectaries are absent. Aere nchyma is present in tissues of th e receptacle, pe ri anth , ovary wall , and septa. E pi the li a l cells are present within the septal aerenchy ma. Calcium oxa late raphides are distribu ted in tissues of the receptacle, peri anth , and ovary wall . Tannin cell s are homogeneous and granular and are distribu ted sparsely in ti ssues of the receptacle and peri a nth. (See Fi g. 25; Table 2).

Mo nochori a korsikovii
Pl acentati on is ax ile, with three carpe ls and three locul es . ba e to the apex of th e locul e. Septa l nectaries are absent. Ae renchyma is present in ti ssues of the receptacle, peri anth , ovary wal l, and septa. E pitheli al cell s are present but reduced in septal aerenc hyma; structures resembling epi the li al ce ll s occur at the base of ovul es . Calcium oxalate raph.ides are di stributed in ti ssues of the receptacle, peri anth , ovar y wa ll , and sty le. Tannin cell s are ho mogeneous and are di stributed sparsely in ti ssues of the receptacle. (See Fig. 26; Table 2).

M onochori a vagina]j s
Place ntati on is ax ile, with three carpe ls and three locul es. The septa are fused th roughout (synsepta lo us) . Commi ssures at the junctio n of the septa are not present. The pl acentae consist of two evaginated fl a nges. O vul es are mostly ple urotro po us, numero us, in two to three rows per fl ange. The region of ovul e attachme nt exte nds continuo usly fro m the base to the a pex of the locul e. Septa l nectaries are absent. Aerenc hyma is present in ti ssues of th e receptacle, peri anth, ovary wa ll , a nd septa. Epithe li al cell s are present within septa l aere nchyma. Calcium oxalate raphides are di tributed in tissues of the peri anth , receptacle, and ovary waiJ . Tannin cell s are ho mogeneous and granul ar a nd are di stributed in tiss ues of the receptac le, peri anth , ovary wal l (s parsely), septa (central co lumn regio n), and sty le. (See F ig. 27, 28; Table   2) .

Pontede ria cordata va r. cordata
Placentati o n is apical-ax ile, w ith o ne fertile (median-anterior) carpe l, two sterile (latero-posterior) carpels and three locul es . T he septa are fused throug hout (synsepta lo us) . Co mmissures at the juncti o n of the septa are not present. The pl acentae are un expanded and effecti vely absent. The single ovul e is e pitropous, with the funiculu s curved upward 180°. The region of ovul e attachment is at the locul e apex onl y. Septal nectaries are present in th e lo wer po rtio n of th e locu le (Fig. 42, 44). Aerenc hy ma is present in ti ssues of the receptacle, perianth , and ovary wall. Epitheli al cell s are present in aere nchyma of ovar y wall s and at the junc ti on of ovary wall and septa. Calcium oxalate raphide, prismati c, and stylo id crysta ls are di stributed in ti ssues of th e receptacle, periandl , ovary wall , septa, and style; pri smati c and stylo id crysta ls are rare in ti ssues of the gynoecium . Tannin ceiJ s are ho mogeneous and granular and are di stributed in ti ssues of the receptac le, peri anth , o vary wall , septa, placentae, and style. (See Fig . 29, 30, 44; Table 2).

Pontederia sagittata
Pl acentati on is apica l-ax ile, with o ne fertile (medi an-a nteri or) carpe l, two sterile (l atero-posteri o r) carpels a nd three locul es. The septa are fused throughout (synseptalous). Co mmissures at the juncti o n of the septa are not present. The pl acentae are un ex panded a nd effecti vely absent. The sing le VOLUME 22  ovule is epitropous, with the funiculus curved upward 180°. The region of ovule attachment is at the locule apex only. Septal nectaries are present in the lower portion of the locule (Fig . 42). Aerenchyma is present in ti ssues of the receptac le, perianth , and ovary wall. Epitheli a l cells are present within the aerenchyma of ovary waJJs and at the junction of ovary wall and septa. Calcium oxalate raphide, prismatic, and styloid crystal s are distributed in ti ssues of th e receptacle, perianth , ovary wall, septa, and style. Prismatics and sty loids are common in inner cell layers of th e peri anth tube and the column/septa regions and are absent in stylar region . Raphides are more common in outer perianth tube tissues and in the ovary wall . Tannin cells are homogeneous, granular, and fibrillar and are distributed in ti ss ues of receptacle, perianth, ovary wall , septa, placentae, and style. In the perianth tube, the homogeneou s type makes up a hypodermi s in the outer layers; g ranular and fibrillar types compri se a hypodermi s of the inner layers. Granular tannin cells are more common in ti ssues of th e gynoecium. (See Fig. 31; Table 2).

Reussia subovata
Pl acentation is apical-axile, with one fertile (median-anterior) carpel, two sterile (latera-posterior) carpels and three loc ul es. The septa are fu sed throughout (synseptalous) . Commi ssures at the junction of th e septa are not present. The placentae are unexpanded and effective ly absent. The single ovule is e pitropous; the funiculu s is curved 180°. The region   of ovul e attachment is at the locul e apex o nl y. Septa l nectaries are present in the lower po rti o n of th e locul e (Fig. 42). Aere nchyma is present in tissues of the receptacle, peri a nth, ovary wall , and j unctio n of septa with ovary wall. Epitheli al cell s are absent. Calcium oxalate ra phide, pri smatic, and stylo id crystals are d istri buted in tissues of the receptac le, perianth , and ovary wall. Rap hides are especially common in aere nchyma spaces; pri smatic a nd stylo id cr ys ta l are rare in the gynoeci um. Tannin cell s are ho mogeneous a nd are d istributed in tissues of the receptacle, sparsely in th e ovary wa ll. (See F ig. 32, 33; Tabl e 2).

Scho lleropsis lutea
Placentati o n is ax ile-pari etal, with three carpels and three locul es at the ovary base, o ne at the apex, the septa fused from the base to midd le regio n of locule (hemi septalo us). T he presence or absence of co mmi ssures at the juncti o n of the septa is unk nown. T he pl acentae cons ist of two evaginated fl anges. O vul es are nume rous, the regio n of ovul e attachment extending continuo usly fro m the base to the apex of the locul e. Septal nectaries were not obser ved, the refore their presence or absence could not be confi rmed. The pres-ence and di stribu tio n of aere nchyma and e pithe li a l cell s a lso could not be determi ned . Calc ium oxalate ra phide crys ta ls are di stributed in tiss ues of the ovary wall and septa. The presence/absence of tannin cell s could not be determined. In gene ra l, the poor mate ri a l avail able for thi s species warrants a f uture reexan1inati on. (Not illustrated ; see Table 2).

Haemodorum spicatum
Pl acentati o n is ax ile, with three carpe ls and three locul es. The septa are fused thro ugho ut (synsepta lo us). Co mmi ssures at the junction of the septa are absent. The pl acentae are swo lle n but unevag inated, lac kin g fl anges . O vul es are hypotropous, two per carpe l, in two rows . The region of ovul e attachment is medi a l. Se pta l nectaries are present. Aerenc hyma and e pithe lia l cell s are absent. Only raphide crystals are present, di stributed in a ll ti ssues of the fl ower. Ta nnin cell s are absent. (Not illustrated ; see Tabl e 2).

Tribonanthes varia bili s
Pl acentati on is ax ile, with three carpels and three locules. The septa are fused thro ug ho ut (synseptal ous). Commi ssures at th e juncti on of the septa are absent. T he pl acentae are unevaginated, lacki ng fl anges. O vul es are pl eurotropous, in s ix to e ight rows per carpe l. The region of ovul e attac hment ex te nds continuously fro m near th e base to the apex of th e locul e. Septal nectaries are present. Aere nchyma and epitheli a l cell s are absent. Onl y raphide c rystals are present, di stributed in all ti ssues of the fl ower. Ta nnin cell s are ho mogeneous, g ranular, and fibrill ar, a nd are di stributed in the receptacle, ovary wall, septa, a nd pl acenta l ti ssues. (No t illustrated ; see Table 2).

He lmho ltzia acorifo lia
Placentati on is ax ile, with three carpe ls and three locul es. T he septa are fused throughout (sy nsepta lous). Commi ssures at th e juncti o n of th e septa are absent. The placentae consist of two evaginated flan ges per carpe l. O vul es are pleurotropous, numero us per carpe l, in three to fo ur rows per fl ange. The regio n of ovul e attachme nt exte nds continuo usly fro m near th e base to the apex of the locul e. Septal nectaries are absent. Aere nc hyma is present onl y in ti ssues of the septa. Epi the li a l cell s are absent. Pri smatic and styloid cr ystals o nl y are di stributed in all ti ssues of the fl ower. Tannin cells are ho mogeneous, gran ul ar, and fi brill ar, and are di stributed in al l ti ssues of the fl ower. (Not illu strated; see Table 2). P hil yd rum la nug inosum Pl acentati o n is pari etal, with three carpels and one locul e. The septa are unfused thro ugho ut (aposeptalou ). Commissures at the juncti o n of the septa are absent. T he pl acentae consist of two evaginated flanges per carpel. O vul es are ple urotropous, numerous per carpe l, in fo ur to eight rows per fl ange. T he regio n of ovul e attachment extends continuous ly fro m near the base to the apex of the locul e. Septal nectari es are absent. Aere nchyma is present on ly in ti ssues of the septa. Epithe li al cell s are absent. Prismatic and sty loid crysta ls o nl y are di stributed in a ll tissues of the fl ower. Tannin cell s are ho mogeneous, granul ar, and fi bri ll ar, and are di stributed in all tiss ues of the fl ower. (Not illustrated ; see Table 2).

D ISCUSS ION
T he above obser vati ons of the floral anatomy of Pontede ri aceae de mo nstrate considerable variation with in the famil y that may be val uable in systematic analyses . T he observa ti ons of Burto n a nd S im pson ( 1996) and of Strange et a l. (2004) were confi rmed from th e present study, but with add iti onal general characters and a hi gher taxo n sampling. In addi tio n, thi s stud y has led us to consider redefi ni tio n of some commonly used characters, particularly that of placentation (see below).
The following is a summary of the significant floral anatomical characters. As mentioned earlier, we use a most parsimonious tree derived from combined chloroplast DNA data of Graham et al. (1998), along with the two cited outgroups (Haemodoraceae and Philydraceae ), to evaluate character state evolution. However, some taxa investigated in our study were not investigated by Graham et al. ( 1998), and vice versa; the cladogram of Pontederiaceae portrays all taxa (with the exception of their unknown "Eichhornia sp. ") from the Graham et al. (1998) study. Although doubts were expressed in their study as to the rooting of Pontederiaceae, several concordances with their phylogenetic relationships are noted. (Note that for many of the characters below, we lack data for Scholleropsis lutea because of the poor quality of available material; character states are noted for this taxon only where observed.) (Fig. 51) All examined family members have three carpels. However, the two investigated members of Pontederia, P. cordata var. cordata, and P. sagittata, and the single investigated species of Reussia, R. subovata, all have a single functional median-anterior carpel with the other two latera-posterior carpels reduced and abortive (Fig. 29, 31, 33). All other species of Pontederia and Reussia are also known to have this condition (these plotted on Fig. 51; see Graham et al. 1998). This condition is sometimes referred to as "pseudomonomery" (e.g., Strange et al. 2004) because only one carpel and locule are obvious at floral maturity; however, it is evident from sections that all three are present, with only one being functional (i.e., containing an ovule).

Functional Carpel Number
Of the outgroups, only Barberetta Harv. of Haemodoraceae has a single functional carpel, but this is posterior in position (Simpson 1990). This difference in fertile carpel position plus the relatively terminal phylogenetic position of Barberetta support the notion that the single fertile carpel in members of the two families was independently derived. (Fig. 52) Aerenchyma tissue is found in all observed members of Pontederiaceae, the taxa differing only in the location within the flower. Within the family, all taxa examined have aerenchyma in the receptacle region (Table 2); the majority of taxa examined have aerenchyma in major floral parts: perianth (e.g., Fig. 17), ovary wall (e.g., Fig. 6), and septal/ placental regions (e.g., Fig. 9; see Table 2). However, two species of Eichhornia (E. diversifolia and E. heterosperma) lack aerenchyma in the ovary wall, having it only in the receptacle, perianth, and septa (rps). The two Pontederia species studied possess aerenchyma in the receptacle and perianth, and to a lesser extent in the ovary wall (rpo), but with no aerenchyma occurring in the septa and adjacent placental region. Reussia subovata, not studied by Graham et al. 1998, and not plotted in Fig. 52, is similar to these in having aerenchyma in the receptacle, perianth, and ovary wall and only very sparsely present in the septa (Table 2). Heteranthera seubertiana is distinctive in having aerenchy-rna only in the receptacle and septa/placental regions (rs) and lacking it in both the ovary wall and perianth (Fig. 52).

Floral Aerenchyma Distribution
In the outgroups, floral aerenchyma is found only in Philydraceae (two of four species investigated), restricted to the septa only and not found in the receptacular tissue. Aerenchyma is absent in all observed Haemodoraceae.
Aerenchyma is an anatomical characteristic of many aquatic plants (Esau 1965), presumably functioning in storage and diffusion of oxygen reserves and possibly in floatation. Because Pontederiaceae flowers are emergent, the presence of aerenchyma may not be functional, per se, but could be a developmental transfer from the formation of aerenchyma in vegetative parts of the plant. However, it is also possible that aerenchyma could be present in the fruits of family members, where it may function in fruit flotation, a likely aid in seed dispersal. (Fig. 53) Curious aggregations of cells termed "epithelial cells" (after Strange et al. 2004;see Fig. 40, 41) were observed inside aerenchymatous tissue in five taxa: three (of three observed) species of Monochoria-M. hastata, M. korsakovii (scantily present), and M. vaginalis-and the two observed species of Pontederia, P. cordata var. cordata, and P. sagittata. Interestingly, these epithelial cells were absent in Reussia subovata. The epithelial cells appear as laminar proliferations, which become convoluted during development. The adaptive significance of these structures is unknown and requires additional study. Epithelial cells were not present in any studied outgroup taxa. (Fig. 54) Distinctive floral tannin cells were observed (at least scantily) in almost all taxa of Pontederiaceae. The types of tannin cells vary, being either granular, in which the cells contain granular tannin contents adjacent to the inside of the cell wall (Fig. 34), homogeneous, in which the vacuoles are filled with translucent, safranin-staining tannins (Fig. 35), or somewhat fibrillar in appearance (Fig. 36). However, these types, when more than one are present, often appear to intergrade. Thus, tannin cell type was not reconstructed on the cladogram.

Tannin Cell Presence and Distribution
The distribution of tannin cells among various floral tissues is variable in the family and somewhat difficult to quantify (Table 2). Only in two species, Heteranthera mexicana and H. rotundifolia, were tannin cells not observed at all. However, tannin cells were essentially absent in eight other taxa, being very sparsely present only in the receptacular, perianth, and stylar region of Eichhornia meyeri and very scantily present in the receptacular region only of Eichhornia paniculata, Heteranthera multiflora, H. reniformis, H. seubertiana, H. zosterifolia, Monochoria hastata (also scantily present in the perianth), and M. korsikovii. The distribution of tannin cells in the other investigated species varies somewhat. Tannin cells are abundantly found in the majority (four to six) of floral tissue regions in Eichhornia crassipes, E. diversifolia, Heteranthera dubia, Monochoria vaginalis, Pontederia cordata var. cordata, and P. sagittata. Tannin cells are moderately distributed (found in any significant quantities in only one or two floral tissue regions) in Eich-hornia heterosperma, Hydrothrix gardneri, and Reussia subovata. (Scholleropsis lutea could not be observed for this feature because of the poor quality of material available.) Tannin cells were observed in the floral tissues of some Haemodoraceae and in all Philydraceae. The presence or absence of tannin cells is phylogenetically significant in Haemodoraceae, as all members of the subfamily Conostylidoideae possess them and all examined members of subfamily Haemodoroideae lack them (Simpson 1990; erroneously termed "sclereids"). The phylogenetic significance of floral tannin cells in monocots as a whole has not yet been investigated. Although very few taxa have been examined to date, the utility of this feature shows promise as a systematic character.
The adaptive significance of floral tannin cells is not known. As with vegetative tannin cells, they may function to deter herbivory. It is also suspected that tannin cells may function to add hardness to floral tissues, e.g., in the anther connective regions of members of Haemodoraceae and Philydraceae.  All examined members of Pontederiaceae have raphide crystals (Fig. 37) that are birefringent in polarized light and presumably composed of an oxalate salt. Prismatic (Fig. 38) or styloid crystals (Fig. 39), which differ from raphides in being composed of a single crystal (either rectanguloid or spindle-shaped, respectively), are found within the family only in the two investigated members of Pontederia and the single investigated species of Reussia. Prismatic and styloid crystals appear to intergrade and are treated as a single "type" here. (This character was not plotted, but for Pontederiaceae is presumed to be similar to Fig. 51.) Raphide crystals were observed in all outgroups. Prismatic/styloid crystals, however, were observed only in all investigated members of Philydraceae.

Crystal
6. Placentation  Placentation has long been used as a descriptive character in plant systematics. However, traditional placentation types encompass a number of discrete features and are better subdivided into separate characters that more accurately assess homology. Part of the difficulty with the term "placentation" is in being correlated with the presence, absence, or fusion of septa. In addition, the term can refer to either of two things: (1) the point of attachment of one or more ovules, in which case it refers to a region, without allusion to the morphology of tissue; or (2) the morphology of the placental tissue to which one or more ovules are attached.
We propose a recoding of the character "placentation" to take into account three separate developmental processes: (1) the fusion or lack thereof of septa during carpel development; (2) the positioning of placental tissue relative to the locule(s) of the ovary; and (3) the relative outgrowth (evagination) of placental tissue.
6A. Septal fusion (Fig. 55).-This refers to whether or not the septa (if present) are centrally fused and, if so, the vertical extent of that fusion. Three character state terms are proposed to categorize septal fusion, although additional terms may be needed in other groups: (i) synseptalous ( = septa "connate" or "fused"), if the septa are fused the full length of the ovary locules (Fig. 46, 50); (ii) aposeptalous ( = septa "distinct" or "unfused"), if the septa are not fused at all or only very little, generally at the extreme base of the ovary (Fig. 48, 49); and (iii) hemiseptalous ( = septa "halfconnate" or "half-fused"), if the septa are fused only near the base and not fused at the apex of the ovary (Fig. 47).
In Pontederiaceae, nine of the investigated species are synseptalous (Eichhornia crassipes being almost hemiseptalous), seven are aposeptalous (although in two species, Heteranthera mexicana and H. reniformis, the septa often barely touch but have a commissure between them), and four are clearly hemiseptalous (except Eichhornia meyeri, in which the septa are fused only at the lower % of the locules; Table 2; Fig. 55, 42). Septal fusion corresponds with the traditional placentation types of axile, parietal, and axileparietal. However, it is more precise as to what is being described and allows for the description of additional aspects of features typically included in placentation.
All members of Haemodoraceae are synseptalous. Philydraceae are polymorphic with respect to this character. (See Fig.55; Table 2).
6B. Placenta position (Fig. 56).-This feature refers to the placement of placentae or ovules relative to the ovary and/ or septa, regardless of the morphology of the placental tissue (below). We propose, for the group studied, two character states that take into account a number of ovule attachment positions: (i) continuous, if the ovules are attached in the middle region of the locule(s), typically extending almost the entire length of the locule; and (ii) apical, if ovules are attached at the apex of the locule(s). Almost all taxa examined have a continuous placenta position. Only the species of Pontederia and Reussia have an apical placental position (Graham et al. 1998; see also Table 2; Fig. 55). The species of Eichhornia examined have a somewhat subapical placenta position, the tissue being in the upper 3 ;.4 to 16 of the locule; however, this is probably correlated with the presence of basal septal nectaries; see Fig. 42, below. All of the outgroups have a continuous (to medial) ovule attachment.
6C. Placenta evagination (Fig. 57).-This feature describes the morphology of any differentiated outgrowth of tissue, arising from the ovary wall or septa, to which one or more ovules are attached. This character describes the outgrowth of, for example, ridges or expanded pads of tissues. Placenta morphology would likely have states customized for a particular group of study.
For Pontederiaceae, we propose the following character states for this character: (i) unevaginated, if the placental tissue does not form expanded ridges of tissue (e.g., From our observations, all members of Pontederiaceae have evaginated placentae to some degree except for the investigated species of Pontederia and Reussia. The lack of expanded placentae in these two genera is certainly corre-lated with their reduced number of ovules (see below). All investigated Philydraceae have a 2-ftanged placental evagination; however, Haemodoraceae lack placental evaginations.
In summary, the inadequacy of traditionally termed placentation types is evident. For example, using traditional placentation terms, both Eichhornia crassipes (Fig. 46) and Pontederia cordata var. cordata (Fig. 50) are typically described as "axile" (e.g., as in Cook 1998), and Eichhornia meyeri (Fig. 47) might be termed "axile-parietal." However, Eichhornia crassipes (Fig. 46) and Pontederia cordata var. cordata (Fig. 50) are similar in only one of our revised characters, each having fused septa (termed synseptalous in our system), and differ in two characters: ovule attachment position (medial vs. apical) and placental morphology (evaginated vs. unevaginated). On the other hand, these two Eichhornia species are identical in these latter two features and differ only in septal fusion (aposeptalous in E. meyeri). Note also that, even though these two Eichhornia species differ in septal fusion, their overall septal morphology is rather similar. In fact, the septa of E. meyeri almost touch at the center of the ovary (Fig. 11, 12, 4 7). This similarity in the two Eichhornia species may allude to their being functionally similar with regard to fruit dehiscence and seed dispersal. Pontederia spp. and Eichhornia spp. also differ in ovule number and fertile carpel number, features that may be correlated with our subdivision of placentation as septal fusion, ovule attachment position, and placenta morphology; see below.
7. Ovule Number (Table 2) All examined family members have numerous (more than ten) ovules per carpel, except for the same two investigated members of Pontederia and the single investigated species of Reussia, which have a single ovule in one functional carpel. This reduced ovule number is obviously correlated with functional carpel number and with fruit type; Pontederia and Reussia have single-seeded nutlets/achenes in an anthocarp, whereas all other Pontederiaceae have capsules. Of Pontederiaceae with multiple ovules per carpel (and therefore a 2ftanged or slightly 2-flanged placenta) all have at least two rows of ovules, one on each of the flanges (e.g., "1 + 1" in Table 2). Some taxa have two, three, or even four rows of ovules on each flange. In the latter, the number of rows of ovules typically decreases to 1 + 1 apically, probably due to spatial constraints at the apex of the ovary. The number of ovule rows per carpel varied somewhat within a taxon and is correlated (as might be expected) with the degree of placental evagination (whether prominently or slightly 2flanged). Because of this variation, we chose not to plot this feature on the cladogram (but see Table 2).
Only Barberetta of Haemodoraceae has a single ovule, correlated with a similar nutlet/achene fruit type. Several other taxa of Haemodoraceae (Pyrrorhiza neblinae Maguire & Wurdack and all species of Dilatris P. J. Bergius, Phlebocarya R. Br., and Wachendorfia Loefl. have one ovule per carpel, but these all occur within three functional carpels. All Philydraceae have numerous ovules per carpel, these with several rows of ovules per flange. Haemodorum has two rows of ovules per placenta and per carpel. Tribonanthes has multiple rows of ovules, but no placental flanges. (Fig. 58, Table 2) Pontederiaceae are variable with respect to the presence or absence of septal nectaries. Of twenty species investigated to date, seven species have septal nectaries: Eichhornia crassipes, E. heterosperma, E. meyeri, E. paniculata, Pontederia cordata var. cordata, P. sagittata, and Reussia subovata (see Fig. 1, 32; Table 2). Interestingly, Eichhornia diversifolia lacks septal nectaries, as do all investigated species of Monochoria, Heteranthera and the single species of Hydrothrix and Scholleropsis. These results corroborate those of the eight species observed by Strange et al. (2004); only the observation of lack of septal nectaries in Eichhornia diversifolia (which they did not observe) add to their general conclusions, indicating polymorphism in this likely polymorphic genus.

Septal Nectaries
Of the seven taxa in Pontederiaceae that have septal nectaries, no significant variation was noted in structure. All occur within the tissues of the three septa and consist of one to two layers of densely staining epithelial cells (e.g., see Fig. 1, 5, 32). The position of the septal nectaries varies somewhat. All occur at or near the base of the superior ovary and extend from about 114 to over 3/4 the length of the ovary (Fig. 42). The species of Pontederia and Reussia examined have the longest relative septal nectary length, probably correlated with the fact that these taxa also have a single ovule with an apical placenta position.
Septal nectaries are found in the great majority of Haemodoraceae (see Simpson 1993) and are absent in Philydraceae (Table 2). A reconstruction of septal nectary gain/loss infers that presence of septal nectaries is an ancestral feature for Pontederiaceae, having been lost three times within the family: in the Heteranthera!Hydrothrix clade, in the Monochoria clade, and in Eichhornia diversifolia (Fig. 58). These independent losses may well be correlated with a shift in pollination mechanism, whereby a nectar reward is no longer adaptive.

CONCLUSIONS
These studies of the floral anatomy of the family Pontederiaceae reveal several characters that are generally well correlated with recent hypotheses of phylogenetic relationships (Graham et al. 1998). For example, tribe Pontederieae of Cook (1998), containing Pontederia and Reussia, seems well supported not only by a reduced functional carpel number (or "pseudomonomery," Fig. 51), reduced ovule number, and apical placental position (Fig. 56), which are features previously known from morphological studies and correlated with the anthocarp fruit type, but also in having a derived floral aerenchyma distribution (Fig. 52), lacking placental evagination (Fig. 57), and being the only Pontederiaceae with styloid or prismatic crystals. The clade containing Monochoria, Pontederia, and Reussia, and three Eichhornia species may be united by the presence of unique epithelial cells, although this would require a loss of such bodies in three Eichhornia species, which together form a distinct clade (Fig. 53). A clade containing the above taxa plus Eichhornia crassipes appears united in being the only taxa with com-pletely synseptalous ovaries (Fig. 55). Heteranthereae of Cook (1998), equivalent to the Heteranthera!Hydrothrix clade appears united by the loss of septal nectaries, although septal nectaries were also lost in other members of the family, specifically in all Monochoria species and in E. diversifolia (Fig. 58). Some characters show a gradation of states, e.g., septal fusion.
This study also points out the need to further dissect some standard morphological characters, such as placentation. Our division of what is usually treated as a single character into three characters: septal fusion, ovule/placenta position, and placenta evagination, gives greater insight into the direction and significance of these floral features.
The adaptive significance of some observed floral anatomical features, such as crystal type and epithelial cell occurrence, is unknown, and others, such as placenta position and ovule number, are undoubtedly correlated with fruit type. However, the presence or absence of septal nectaries may be significant with respect to pollination mechanisms. Tannin cell occurrence and distribution might possibly be related to herbivory. The interesting occurrence of floral aerenchyma may be adaptive with respect to fruit floatation, a hypothesis that needs testing. An increased number of ovules and ovule rows may be an adaptation for increasing total seed output; the decreased number of ovules in Pontederia and Reussia is correlated with a specialized, single-seeded fruit type.
In conclusion, we feel that these floral anatomical data may be valuable in further elucidating the phylogenie relationships in Pontederiaceae. In particular, problems with rooting the tree (see Graham et al. 1998) may be solved with the addition of these non-molecular data to a molecular data set.