jurnal antum

COMPARATIVE ANATOMY OF ROOT MERISTEM AND ROOT CAP IN

SOME SPECIES OF PODOSTEMACEAE AND THE EVOLUTION OF

ROOT DORSIVENTRALITY1

SATOSHI KOI,2 RIEKO FUJINAMI,3 NAMIKO KUBO,3 IKUE TSUKAMOTO,3 RIE INAGAWA,3 RYOKO IMAICHI,3 AND

MASAHIRO KATO2,4

2Department of Botany, National Science Museum, Amakubo, Tsukuba 305-0005, Japan; 3Department of Chemical and

Biological Sciences, Faculty of Science, Japan Women’s University, Mejirodai, Tokyo 112-8681, Japan

In the unusual aquatic Podostemaceae, the root is the leading organ of the plant body and is variously compressed and

submerged as it adheres to rock surfaces in rapid water. In an anatomical comparison of the root apical meristems and root caps of

33 species that represent the major lineages of the family, the dorsiventrality of root meristems varied and was classified into four

patterns: (1) The root cap is produced outward from a nearly radially symmetrical meristem. (2) The meristem and root cap are

markedly dorsiventral; the outermost cells of the hood-shaped cap are acroscopic derivatives from bifacial initials on the ventral

side, while the pattern on the dorsal side is similar to pattern 1. (3) Bifacial initials are on both the dorsal and ventral sides. (4) No

root cap is present. An evolutionary polarity may be evident from pattern 1 to 2 and then to 3. Pattern 2 arose in the early

evolution of the subfamily Podostemoideae and subsequently, pattern 3 arose in species with crustose roots, while the least

specialized pattern 1 is retained in Tristichoideae and Weddellinoideae. Pattern 4 characterized by caplessness may have appeared

recurrently in Tristichoideae and Podostemoideae. These evolutionary changes in the meristem preceded the specialization of

external root morphologies.

Key words: dorsiventrality, evolution, Podostemaceae, root, root cap, root apical meristem.

Podostemaceae are a family of ecologically and morpholog-ically unusual aquatic angiosperms. They occur on rocks inrapids and waterfalls in the tropics and subtropics of the world.Vegetative plants grow submerged in swift-running waterduring the rainy season and subsequently emerge andeventually dry during the dry season when the water leveldrops. Generally flowers open and seeds are set shortly afteremergence. Except for a few rootless species, the roots creepon and adhere to rock surfaces (in some species floating exceptthe base) and are dorsiventral and variously flattened intoshapes ranging from subcylindrical to ribbon-like or crustose(Fig. 1) (Willis, 1902; Troll, 1941; Rutishauser, 1997). Theroots bear adventitious shoots, both vegetative and reproduc-tive, on the lateral flanks or dorsal side and root hairs (rhizoids)on the ventral surface. Thus, the root is the leading organ in thebody plan of most Podostemaceae and is a remarkableadaptation to its unique environment, i.e., the border betweenhard rock surfaces and fast-running water. Molecular analysesplace Podostemaceae in the eudicot rosid Malpighiales,particularly close to Clusiaceae (Hypericaceae) (Savolainen etal., 2000; Soltis et al., 2000; Gustafsson et al., 2002). Theresults of these analyses suggest that such unique roots ofPodostemaceae were derived from cylindrical, capped rootscommon in Clusiaceae and other angiosperms, and that theepilithic roots were derived from underground roots that pene-trate soil.

The morphology of the root and root cap is determinedprimarily by the root apical meristem, because typically theradially symmetric meristem forms a cylindrical root.Anatomical information for the meristem is useful to un-derstand the root morphology of Podostemaceae, but thecellular patterns in the meristem in relation to the root properand cap have been analyzed in detail in only a few species. Koiand Kato (2003) described that in three species of Cladopuswith either narrow or broad ribbon-like roots covered by hood-shaped dorsiventral root caps, the outermost layer of the rootcap is an acroscopic extension from the ventral dermal layer ofthe root meristem. The crustose roots of Hydrobryumjaponicum and Zeylanidium olivaceum have a moderatelylayered structure of the meristem along the margin of the rootlobe, which is framed by a root cap (protective tissue) (Ota etal., 2001; Hiyama et al., 2002). The root of Indotristicharamosissima appears to have a meristem of the closed type,which is demarcated from the root cap (Rutishauser and Huber,1991). Thus, the results of previous studies differ considerablyin the organization of the root meristem and cap. However,very little information is available to consider the evolution ofthe root in Podostemaceae. The goal of this study was to definethe dorsiventrality of the root meristem and its derivative cap inthe diverse Podostemaceae and infer the evolutionary scenariothat led to their unique roots.

MATERIALS AND METHODS

Plant materials—Plants examined are shown in Table 1. Species wereselected to represent major clades of Podostemaceae (Kita and Kato, 2001,2004; Kato, 2004). They include species that may be in need of systematic andnomenclatural revision or have very recently been revised (see footnote a,Table 1). Vouchers are deposited in the Forest Herbarium, Department ofNational Parks, Wildlife and Plant Conservation, Bangkok (BKF), theUniversity of Tokyo Herbarium (TI), and National Science Museum Herbarium(TNS).

1 Manuscript received 3 July 2005; revision accepted 24 February 2006.

The authors thank T. Santisuk, T. Wongprasert, A. K. Pradeep, P.Mathew, D. B. Sumithraarachchi, F. Montata, J.-Q. Liu, S.-J. Lin, D.Darnaedi, G. G. Hambali, R. Lilwah, T. Kajita, M. Hasebe, T. Yamadaand Y. Kita for help during field trips and collections and Y. Kita forunpublished phylogenetic data. This study was supported in part byGrants-in-Aid for Scientific Research from the Japan Society for thePromotion of Science.

4 Author for correspondence (e-mail: [email protected])

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American Journal of Botany 93(5): 682–692. 2006.

Anatomy—Plants were fixed in FAA (formalin : acetic acid : 50% ethylalcohol ¼ 5 : 5 : 90). For anatomical observations with the light microscope,apical pieces of roots collected from the fixed plants were dehydrated in anethyl alcohol series, embedded in Historesin (glycol methacrylate, Leica,Heidelberg), cut into 2-lm thick sections, and stained with safranin, toluidineblue, and orange G (Jernstedt et al., 1992). We cut sagittal-, frontal-longitudinal, and cross sections (Fig. 2). For scanning electron microscopic(SEM) observations, fixed roots of Podostemum rutifolium var. ricciforme andMarathrum schiedeanum were dehydrated in an ethyl alcohol series, criticalpoint dried, coated with platinum-palladium, and observed with a Hitachi S-800microscope at 10 kV. Roots of Polypleurum stylosum and Vanroyenellaplumosa were similarly prepared and observed with a KEYENCE VE-8800SEM at 5 kV. Roots of Weddellina squamulosa and Terniopsis malayana weredehydrated in an ethyl alcohol and t-butyl alcohol series, freeze-dried, coatedwith platinum-palladium, and observed with a JEOL JSM-820S SEM at 5 kV.

Reconstruction of character evolution—The evolution of the root apicalmeristem and root cap organization was reconstructed by mapping characterstates (patterns 1–4 in this study plus rootlessness [Table 1; Fig. 3]) ontoa molecular phylogenetic tree of Podostemaceae adapted from Kita and Kato(2001, 2004), Kato et al. (2003), Kato (2004), and Y. Kita (University ofTokyo, unpublished data), using MacClade version 4 (Maddison andMaddison, 2005). Trees with added taxa (Kato et al., 2003; Kato, 2004; Kitaand Kato, 2004; Y. Kita, unpublished results of Dalzellia gracilis, Diamantinalombardii, Jenmaniella ceratophylla, and Rhyncholacis palmettifolia var.palmettifolia) are in consistency with Kita and Kato’s (2001) tree of the wholePodostemaceae.

RESULTS

The root apical meristem and the root cap varied structurallyamong the species of Podostemaceae examined. Rootmorphologies were classified into four histological patternswith two subpatterns, based on the presence or absence and theplace of bifacial meristems and the presence or absence of theroot cap (Table 1, Fig. 3).

Pattern 1—The roots of Indotristicha ramosissima, Ter-niopsis australis, T. malayana, T. sessilis, Weddellinasquamulosa, and Vanroyenella plumosa are variously com-pressed, subcylindrical or narrow ribbon-like (Figs. 4–10). InW. squamulosa the distal part of the root is almost cylindrical(Fig. 4). The apical meristem is dome-shaped in sagittal andfrontal-longitudinal sections (Figs. 5, 6). As seen in an opentype of root meristem (Barlow, 2002), the central portion of themeristem is not well delimited from the root cap, due to theabsence of predominant periclinal cell walls, while the

periphery is demarcated by cell walls formed by anticlinaldivisions of the epidermal cells below the inner root cap.Vanroyenella plumosa has a similar pattern. In comparison, inTerniopsis malayana, T. australis, T. sessilis, and Indotristicharamosissima, the distal part of the root is compressed andconical (Figs. 7–9). In relation to this, the apical meristem inthe Terniopsis species is a somewhat compressed dome and isseparated from the root cap by periclinal cell walls, except inthe smaller central part of the root. Such cell walls on the dorsalside are close to or apparently connected with those on theventral side in I. ramosissima (Fig. 10; Figs. 7, 8, 11 inRutishauser and Huber, 1991). The meristem of all the speciesis located in or below the center of the root apex and suppliedby the procambial strand running in or below the center.

The root cap is nearly radially symmetrical in Terniopsissessilis and Indotristicha ramosissima (Fig. 10), while it isdorsiventral with the dorsal part larger than the ventral in theother species of Tristichoideae and in Vanroyenella plumosa.The root cap has the most cell layers in the center andincreasingly fewer layers toward the proximal end in mostspecies (Figs. 8, 9). In Indotristicha ramosissima the cap isuniformly thick and multilayered, suggesting that there may becap initials in each layer (Fig. 10). Central root cap cells, theinnermost of which are small and densely stained, are likelyacroscopic derivatives from initials in the central part of themeristem, and the peripheral cap cells are probably basiscopicderivatives from the periphery of the meristem (Fig. 5, 6, 8–10). In comparison, the outermost root cap cells aretangentially the longest and sometimes broken (Figs. 6, 9,10; see also cell wall residues on the root cap surface proximalto the root cap). The thickness of the root cap varies from a fewto several cells among the species examined.

Pattern 2A—The roots of Diamantina lombardii, Endocau-los mangorense, Marathrum schiedeanum, Oserya coulteriana,and Polypleurum elongatum are compressed and subcylindrical,and the apical meristem is located near the ventral side, as alsoindicated by the procambial strand close to the ventral side(Figs. 11–15). Unlike pattern 1, cell wall residues are eitherabsent or scant on the root cap surface in species with patterns2A and 2B, indicating that the outermost cells did not break(Figs. 13, 15, 21, 24, 25). Compared to the larger dorsal part ofthe dorsiventral root cap, the ventral part is very small and one ortwo cells thick (Figs. 13, 15). In Oserya coulteriana the ventralroot cap has a more or less layered structure with the outer layerof cells longer than the inner layer, indicating that the outer cellsare older than the inner (image not shown). The periphery of thedorsal side of the root cap separates from the root proper as theinnermost cell walls break in association with the differentialgrowth of the root cap and root epidermis (Figs. 13, 15); similarto the case in pattern-1 roots (images not shown).

Marathrum schiedeanum has a meristem composed of anouter cell layer and densely staining inner cells inside the twocell layers of the root cap on the ventral side of the root. Theinner layer of the root cap lies in the same row as the outermostmeristem layer, while the outer layer of the cap covers it (Fig.13). Oserya coulteriana has a similar organization, witha thicker (three cells) root cap on the ventral side (image notshown). This histology implies that the outermost meristemlayer on the ventral side produces the root cap and epidermis,and the layer sequentially differentiates into a root cap,concomitantly with the appearance of a new meristem layerunder the root cap. In Diamantina lombardii cells of the

Fig. 1. Polypleurum wallichii in the wild with ribbon-like roots (R)creeping on rock surface and bearing adventitious shoots (S) on the flanks.Scale bar ¼ 5 mm.

May 2006] KOI ET AL.—DORSIVENTRAL ROOT MERISTEM IN PODOSTEMACEAE 683

outermost cap layer on the ventral side are elongated andpartially torn where a new meristem layer and a root cap layerderived from it are exposed (Fig. 15). Based on the cellalignment in this and other species with pattern 2A, the rootcap on the ventral side is comprised of acroscopic derivativesfrom the bifacial meristem of the outermost layer, while the

ventral epidermis are basiscopic derivatives. Although thepatterns of cell division are not obvious, both dorsal cap cellsand dorsal parts of the root are basiscopic derivatives of thedorsal meristem.

In Marathrum schiedeanum the lateral root arises endoge-nously near the vascular strand of a parent root. The young root

TABLE 1. Species examined in this study, those anatomically described, their sources, root morphologies, cellular patterns of root meristem and cap, asdefined in Results, and figures or references.

Species Source Root morphology Pattern Figure/Reference

Subfamily WeddellinoideaeWeddellina squamulosa Tul. Potaro River, Tumatumari, Guyana; Kato et al. GU-12 Subcylindrical 1 Figs. 4–6

Subfamily TristichoideaeIndotristicha ramosissima (Wight) P. Royen Chaithravahin River at Beenmarak, Kasaragod, Kerala, India;

Kato & Imaichi KI-26Subcylindrical 1 Fig. 10

Dalzellia gracilis C.J. Mathew, Jager-Zurn& Nileena

Punavoorthode River at Urulanthanny, Kerala, India;Kato et al. KI-115

Subcylindrical 4 — b

Terniopsis australis (C. Cusset & G. Cusset)M. Kato a

Katherine Gorge, Northern Territory, Australia; Kato AU-301 Subcylindrical 1 Kato et al., 2003

Terniopsis malayana (J. Dransf. & Whitmore)M. Kato a

Sirindhorn waterfall, Narathiwat, Thailand; Kato et al. TL-106 Subcylindrical 1 Figs. 7–9

Terniopsis sessilis H.C. Chao Ting River, Fujian, China; Kato CH-3 Subcylindrical 1 — b

Tristicha trifaria (Bory ex Willd.) Spreng. San Gabriel, Mixtepec, Oaxaca, Mexico; Kato et al. MX-3 Subcylindrical 4 Fig. 26Subfamily Podostemoideae

Apinagia longifolia (Tul.) P. Royen Essequibo River, Kurupukari, Guyana; Kato et al. GU-21 Subcylindrical 2B Fig. 24Cladopus austro-osumiensis Kadono & N. Usui Ogawa River, Kagoshima, Japan; Koi et al. s.n. Subcylindrical 2B — b

Cladopus fukienensis (H.C. Chao) H.C. Chao Buyun, Shanghan, Fujian, China; Kato CH-1 Subcylindrical 2B Koi and Kato, 2003Cladopus javanicus M. Kato & Hambali Curug Luhur River, Cisagu, Java, Indonesia; Kato et al. ID-105 Ribbon-like 2B Koi and Kato, 2003Cladopus nymanii H. Moller Cisarua River, NW of Mt. Pangrango, Java, Indonesia;

Kato et al. ID-3Ribbon-like 2B Koi and Kato, 2003

Cladopus taiensis C. Cusset Wang Takrai waterfall, Nakhon Nayok, Thailand;Kato et al. TL-201

Ribbon-like 2B — b

Diamantina lombardii A. Novelo,C.T. Philbrick & Irgang

Castatinha waterfall, Caraca, Minas Gerais, Brazil;F. Montana 8006

Subcylindrical 2A Fig. 15

Endocaulos mangorense (H. Perrier) C. Cusset Mandraka, E of Antananarivo, Madagascar; Imaichi et al. 70 Subcylindrical 2A — b

Hanseniella heterphylla C. Cusset Kaeng Sopha waterfall, Tung Saleng Luang, Phitsanulok,Thailand; Kato et al. TL-311

Crustose 3 Kato et al., 2004

Hydrobryum japonicum Imamura Ogawa River, Kagoshima, Japan; Kato & Imaichi s.n. Crustose 3 Ota et al., 2001Jenmaniella ceratophylla Engl. var. ceratophylla Potaro River, Tumatumari, Guyana; Kato et al. GU-17 Subcylindrical 4 Fig. 27Marathrum schiedeanum (Cham.) Tul. San Gabriel, Mixtepec, Oaxaca, Mexico; Kato et al. MX-2 Subcylindrical 2A Figs. 11–14Oserya coulteriana Tul. Rio San Antonio, Guerrero, Mexico; Kato et al. MX-7 Subcylindrical 2A — b

Paracladopus chiangmaiensis M. Kato a Mae Wang stream, Chiang Mai, Thailand; Kato et al. TL-808 Subcylindrical 2B Fig. 25Podostemum rutifolium Warm. var. ricciforme

(Liebm.) A. Novelo & C.T. PhilbrickSan Marcos, Veracruz, Mexico; Kato et al. MX-8 Subcylindrical 2B Figs. 18–23

Polypleurum elongatum (Gardner) J.B. Hall Kelani Ganga at Kitulgala, Central District, Sri Lanka;Kato et al. SL-12

Subcylindrical 2A/1? — b

Polypleurum stylosum (Wight) J.B. Hall Kozhipara waterfall, Malappuram, Kerala, India;Kato & Imaichi KI-48

Ribbon-like 2A? Figs. 16, 17

Polypleurum wallichii (R. Br. ex Griff.) Warm. Nang Rong waterfall, Khao Yai, Thailand; Kato et al. TL-201 Ribbon-like 2A? — b

Rhyncholacis palmettifolia P. Royenvar. palmettifolia

Potaro River, Tumatumari, Guyana; Kato et al. GU-11 Subcylindrical 2B — b

Thawatchaia trilobata M. Kato, Koi & Y. Kita Mae Wang stream, Chiang Mai, Thailand; Kato et al. TL-809 Crustose 3 Kato et al., 2004Thelethylax minutiflora (Tul.) C. Cusset Amboavaha, near Antsirabe, Madagascar; Imaichi et al.

44, 45 Mandraka, E of Antananarivo, Madagascar;Imaichi et al. 70

Ribbon-like 2B — b

Vanroyenella plumosa A. Novelo &C.T. Philbrick

Vicinity of San Gabriel, Mixtepec, Oaxaca, Mexico;Kato et al. MX-5

Subcylindrical 1 — b

Zeylandium lichenoides (Kurz) Engl. Pathamkayam, Calicut, Kerala, India; Kato & Imaichi KI-35 Ribbon-like 4 Hiyama et al., 2002Zeylanidium maheshwarii

C.J. Mathew & SatheeshThusharagiri waterfall, Calicut, Kerala, India;

Kato & Imaichi KI-41Crustose 3 Hiyama et al., 2002

Zeylanidium olivaceum (Gardner) Engl. Adam’s Peak, Nuwara Eliya, Central prov., Sri Lanka;Kato et al. SL-9

Crustose 3 Hiyama et al., 2002

Zeylanidium subulatum (Gardner) C. Cusset Mahaweli Ganga at Ambagamuwa, Kandy, Central prov.,Sri Lanka; Kato et al. SL-18

Subcylindrical 4 Hiyama et al., 2002

a The species previously referred to as Malaccotristicha were nomenclaturally combined with the genus Terniopsis, and Paracladopus chiangmaiensisis a new genus and species (Kato, in press).

b Image is not shown.

684 AMERICAN JOURNAL OF BOTANY [Vol. 93

produces a root apical meristem and a dorsiventral root capwhile developing within the parent root (Fig. 14). The root capunevenly covers the root tip very much like that in pattern 2Ain the adult or pattern 1.

The broad ribbon-like roots of Polypleurum stylosum and P.wallichii may have unusual forms of pattern 2A. The root capis very small compared to the root width (Fig. 16). In P.stylosum there is no sharp histological difference between theroot apical meristem and cap, except in cell size andstainability. The root cap comprises 2–4 cell layers, theinnermost of which lies in roughly the same row as the dorsaland ventral epidermis (Fig. 17). The outermost meristem layeron the ventral side is located under a broken cap cell. In anothermaterial, the outermost meristem layer is placed undera unilayered cap and partly exposed because cap cells areruptured (data not shown). In P. wallichii, the outermostmeristem layers on both dorsal and ventral sides, as in pattern4, form a single, continuous layer enveloping the innermeristem cells. The root cap is one cell thick and vestigial. Itmay be intermittently differentiated from the outermostmeristem layer.

Pattern 2B—Apinagia longifolia, Cladopus austro-osumien-sis, C. taiensis, Paracladopus chiangmaiensis, Podostemumrutifolium var. ricciforme, and Thelethylax minutiflora, likemost other species of Podostemaceae, have subcylindrical orribbon-like roots (Figs. 18–21, 24, 25). Rhyncholacis is usuallydescribed as lacking roots, so that the plant is interpreted asbeing composed of a ‘‘stem’’ and leaves (Royen, 1951; see alsoEngler, 1930). However, in our field observations we noted thatRhyncholacis palmettifolia has adventitious roots at the base ofshoots. The roots of those species including such adventitiousroots have similar structures in the meristem and root cap, sothey are described together.

Their root meristem is dorsiventral and composed ofa convex layer on the dorsal side, usually a less convex layeron the ventral side, and the inner cells (Figs. 21, 24, 25). Themeristem is located near the ventral side of the root andsupplied by a procambial strand also close to the ventralsurface. The dorsiventrality is verified by cross sections of theroot tip (Figs. 19, 20). The ventral epidermis is a basiscopicderivative of the bifacial meristem, whereas particular initialsthat produce the dorsal epidermis are not obvious. The dorsalcortex is thicker than the ventral, comprising the outer cortex ofsmaller cells and the inner cortex of larger cells, while the innercortex is slightly developed in the ventral cortex (Figs. 21, 24,25), the same as in pattern 2A (Figs. 13, 15).

The root cap is hood-shaped with its apical part covering thehemispheric root tip and is remarkably dorsiventral with thedorsal part long, gutter-like and free (Fig. 18). Cladopustaiensis has a small root cap with the dorsal part barely free. Inmost species with pattern 2B examined, the root cap is a few toeight cells thick and composed of smaller cells near the rootapex and larger cells away from the apex. The root cap is thethickest (thicker than the root proper) in Thelethylaxminutiflora among the species examined, mainly due to largercells. The outermost cells of the root cap on the ventral side liein the same row as the outermost layer of the root meristem(Figs. 21, 25). In Apinagia longifolia cells of the second tofourth outer layers also are aligned this way (Fig. 24). Hence,those cap cells are most likely acroscopic derivatives from theinitials. These cap-producing initials are one to several celllayers thick; the thinnest is in Cladopus austro-osumiensis andthe thickest in Apinagia longifolia. The dorsal root cap, alongwith the dorsal epidermis and cortex, is a basiscopic derivativefrom the dorsal meristem. Proximal to the tip, the innermostroot cap cells on the dorsal side are distorted from the different

Fig. 3. Diagram of cross and sagittal sections through root tips ofPodostemaceae depicting three patterns of root apical meristem andhypothesized evolutionary changes (indicated by open arrows) in rootmeristem and cap organization. Cells of the root cap and root proper areproduced in directions (small solid arrows) from root initials (outermostcentral initials, ventral initials, or dorsal and ventral initials are shown bysquares). Asterisks indicate the center of root meristem. In the change frompattern 1 to pattern 2 (2A, 2B), reduction of the ventral portion of the rootmeristem (shown by thin and thick lines) was accompanied by theestablishment of a bifacial meristem. Further reduction of the dorsalportion was involved in the change to pattern 3. See Table 1 for specieswith those patterns. Figure abbreviations: C, cap cell; CS, cross section;D, dorsal side; FL, frontal longitudinal section; I, initial; PC, procambium;PR, parent root; R, root; RAM, root apical meristem; RC, root cap; S,shoot; SL, sagittal longitudinal section; V, ventral side.

Fig. 2. Diagram of the three sectioning planes of a model root ofPodostemaceae (e.g., Terniopsis) with ventral side facing rock substratum(not drawn). D, dorsal; V, ventral.


growth rates between the root cap and epidermis and areeventually broken so that the root cap is detached proximally(Figs. 19–21, 24, 25).

In Podostemum rutifolium var. ricciforme the lateral rootarises endogenously near the vascular strand of a parent root(Fig. 22). The root cap of the young root developing within theparent root unequally covers the root tip, a structure similar topattern 1 or 2A. Shortly after emerging from the parent root,the apical meristem and the root cap become similar to those ofthe mature root, but the meristem is still covered by the rootcap on the ventral side as well as on the dorsal (Fig. 23). Newventral initials seem to arise under the root cap and produceroot cap cells and epidermal cells bidirectionally, and in

a further elongating lateral root, the outermost root cap cells areacroscopic derivatives of the ventral bifacial meristem, thesame 2B pattern as in the adult (Fig. 21).

Pattern 3—The root is crustose and lobed in Hanseniellaheterophylla and Thawatchaia trilobata, which, together withthe similarly crustose-rooted Hydrobryum, form a clade in thesubfamily Podostemoideae (Kita and Kato, 2001, 2004; Kato etal., 2004). The uniform marginal root meristem occurs alongthe root lobe margin. Re-examination of materials used by Otaet al. (2001) and Kato et al. (2004) shows that the meristem iscomposed of dorsal and ventral layers that sandwich the moreor less layered inner cells. The band-like root cap (protective

Figs. 4–10. Root tips of Weddellinoideae and Tristichoideae of pattern 1. Dorsal side is on top in SLs. Arrowheads indicate periclinal cell walls alongthe boundary of the root cap and root proper. 4–6. Weddellina squamulosa. 4. SEM of dorsal side. Note root cap cell residues proximal to root cap. 5. FL.6. SL. 7–9. Terniopsis malayana. 7. SEM of dorsal side. 8. FL. 9. SL. Inset, magnification of area with asterisk, showing young epidermal and root capcells (C) under old root cap on the ventral side. 10. Indotristicha ramosissima, SL. Scale bars ¼ 100 lm, 25 lm in Fig. 9 inset.


tissue) of collenchymatous cells also has a similar structurewith less obvious layering because of cell enlargement. Cellalignment suggests that the dorsal and ventral layers of the rootcap are acroscopic derivatives from the dorsal and ventralinitials of the root meristem via anticlinal cell divisions (Fig. 3).

Pattern 4—Although Dalzellia gracilis, Tristicha trifaria,and Jenmaniella ceratophylla are assigned to different

subfamilies (Tristichoideae and Podostemoideae), their rootmorphology is simple and quite similar. The root is compressedsubcylindrically and does not have a root cap (Figs. 26, 27).The naked apical meristem is located near the center of the rootand is composed of inner cells coated by a dermal layer ofrectangular cells. The dermal cells divide anticlinally toproliferate initials and epidermal cells and less frequentlypericlinally to produce inner cells.

Figs. 11–17. Root tips of Podostemoideae of pattern 2A. Dorsal side is on top in SLs. 11–14. Marathrum schiedeanum. SEM of 11. obliquely lateraland 12. ventral sides. Arrowhead indicates the ventral margin of root cap. 13. SL. Inset, magnification of area with asterisk showing a row of young capcells (C) and initials (I). 14. Oblique SL of young lateral root embedded in cross-sectioned parent root. Arrowheads indicate the border of root cap and rootproper. 15. Diamantina lombardii, SL. Inset, magnification of area with asterisk showing a row of new root cap cells (C) and ventral initials (I) undera broken cap. 16–17. Polypleurum stylosum. 16. SEM of dorsal side. 17. SL. Scale bars¼ 100 lm, 10 lm in Fig. 13 inset, 25 lm in Fig. 15 inset.


Root meristem evolution—In addition to the four patternsdescribed, there is a fifth rootless state in Dalzellia zeylanica(Willis, 1902; Imaichi et al., 2004; Kato, in press), Mourerafluviatilis Aublet (Rutishauser and Grubert, 1999), andRhyncholacis (Royen, 1951). Adventitious roots may arisefrom the shoot in M. fluviatilis (Jager-Zurn, 2005) and R.palmettifolia. The reconstructed evolution of the root apicalmeristem and its derived root cap in the species examined hereand reported (Table 1, Fig. 3) shows that in the family, pattern1 of Tristichoideae and Weddellinoideae is plesiomorphic,while pattern 2 is apomorphic (Fig. 28). Tristichoideae alsoinclude the apomorphic pattern 4 and rootless pattern. WithinPodostemoideae, pattern 2 is plesiomorphic, and patterns 1, 3,4 and the rootless pattern are apomorphic. Patterns 3 and 4 arelikely to have arisen recurrently. Subpatterns 2A and 2B aredistributed in phylogenetically unrelated clades and eachsubpattern occurs in African, American, and Asian species.

DISCUSSION

In our comparison among species representing most majorclades of Podostemaceae, we found four cellular patterns, withtwo subpatterns, of the root meristem and cap (Table 1, Fig. 3).Pattern 1 occurs in Tristichoideae and Weddellinoideae, while

pattern 2 is widely distributed in Podostemoideae, with theexception of Vanroyenella plumosa (Table 1, Fig. 28). Pattern3 is exclusive to Asian Podostemoideae with crustose roots.Differences among the patterns are obvious in development ofthe root cap, which is a product of the root meristem. Inpattern-1 roots, the root cap develops outward and toward theproximal end, like in most angiosperms (Barlow, 2002). Incontrast, cells of root caps with pattern 2 have an age gradientalong the length of the root cap with older cells toward the freeproximal end on the dorsal side of the root, while pattern-3roots have a proximodistal gradient. The diversification in rootmeristem organization is likely to have preceded changes ofexternal morphology except for the root cap, because thesubcylindrical or ribbon-like roots do not appear very differentin the subfamilies. Nonetheless, pattern 2, which is plesiomor-phic in Podostemoideae, is possibly partly involved in thediversification of the subfamily with ca. 260 or more of in totalca. 270 species or more.

Among the four patterns, pattern 1 is the most similar to thepattern in ordinary angiosperm roots with radially symmetricalapical meristems (Esau, 1965). Pattern 1 of Indotristicha ra-mosissima and species of Terniopsis may be called inter-mediate-open type (Groot et al., 2004) and that of Weddellinasquamulosa and Vanroyenella plumosa with the largest region

Figs. 18–23. Root tips of Podostemum rutifolium var. ricciiforme of pattern 2B. Dorsal side is on top in SLs. 18–21. Mature roots. 18. SEM of lateralside. 19–20. Selected serial CSs. 21. SL. Inset, magnification of area with asterisk showing a row of young ventral cap cells (C) and initials (I). Numbers 19and 20 indicate locations equivalent to those in Figs. 19 and 20, respectively. 22–23. SLs of developing lateral roots at different stages. 22. Young rootembedded in cross-sectioned parent root. 23. Root extruding from parent root. Arrowhead indicates root cap cells beginning to break. Scale bars¼ 300 lmin Fig. 18, 100 lm in Figs. 19–23.


of cap-producing initials, open type (Barlow, 2002) or basic-open type (Groot et al., 2004). Most other Podostemoideae alsohave similar types with respect to the dorsal half of themeristem, although the ventral half differs from that of pattern 1.Groot et al. (2004) proposed that the root apical meristem of theclosed type evolved in the Malpighiales. Podostemaceae aresister to Hypericaceae assigned to the Malpighiales (Savolainenet al., 2000; Soltis et al., 2000; Gustafsson et al., 2002). BecauseHypericaceae are likely to have a closed-type meristem, thepatterns of Podostemaceae may have been derived from thetype, as a result of a change in the orientation of root capproduction.

Although the patterns differ in the presence or absence andthe distribution of the unique bifacial meristem as well as thepresence or absence of the root cap, there are developmentaltransitions. In Podostemum rutifolium var. ricciforme withpattern 2B, a young lateral root has a poorly dorsiventralmeristem similar to that of pattern 1 or 2A. In Marathrumschiedeanum with pattern 2A, a young root has either pattern 1or 2A. These changes may imply that pattern 2 is derived frompattern 1, or pattern 2B is derived from pattern 2A, althoughthe polarity is only partially consistent with the phylogeneticrelationship. Furthermore, transitions between patterns 2 and 3

are seen in some Asian species. Both patterns exist inHydrobryum japonicum in which, compared with the matureroot of pattern 3, a young regenerating root has pattern 2B (Fig.29 in Ota et al., 2001).

There is another similarity in root development betweenCladopus of pattern 2 and Hydrobryum of pattern 3. InCladopus the lateral root forms exogenously from the marginalmeristem of a parent root where there is no root cap, involvingits young dorsal and ventral epidermises (Koi and Kato, 2003).Then the dorsal epidermal cells of the developing root apexdifferentiate into the root cap, while the hypodermal cellsbecome the dorsal meristem. In contrast, the ventralmeristematic dermal cells become a bifacial meristem ina developmental mode similar to that of Hydrobryum withcrustose roots. In H. japonicum a new meristem initiatesexogenously in a certain part of the nearly differentiatedmeristem of a parent lobe, and then gives rise to bifacialmeristem layers on both dorsal and ventral sides (Ota et al.,2001).

Along with the comparative morphology among the threepatterns and the general angiosperm root meristem organiza-tion, the inferred evolution of the root meristem and capsuggests that pattern 1 is the least specialized and preserves

Figs. 24–27. SLs of root tips of Podostemoideae of pattern 2B (24, 25) and pattern 4 (26, 27). Dorsal side is on top. 24. Apinagia longifolia. Inset,magnification of area with asterisk showing rows of initials (I) and root cap cells (C) on the ventral side. 25. Paracladopus chiangmaiensis. Inset,magnification of area with asterisk showing a row of cap cells (C) and initials (I). 26. Tristicha trifaria. Inset, magnification of area with asterisk showingpericlinal divisions of dermal initials. 27. Jenmaniella ceratophylla. Scale bars¼ 100 lm, 20 lm in Figs. 24 and 26 insets, 50 lm in Fig. 25 inset.


a radially symmetrical apical meristem typical of cylindricalroots, that pattern 2 is derived from it, and that the mostspecialized pattern 3 is derived from pattern 2 (Figs. 3, 28).Pattern 2, seen only in Podostemaceae, appeared early in theevolution of the subfamily Podostemoideae. The suggestedspecialization is consistent with that of other morphologies. Forexample, in contrast to the seedling of Terniopsis malayana ofTristichoideae that forms both a primary shoot and a radicle,seedlings in Podostemoideae lack the radicle and havea reduced primary shoot, and sometimes lack both (Suzuki etal., 2002; Kita and Kato, 2004). The shoot apical meristem ispresent in the adult of Tristichoideae and Weddellinoideae, butabsent from Podostemoideae (Imaichi et al., 2005; Koi et al.,2005; references cited therein).

In regard to the unique bidirectional histogenesis of the rootmeristem, we speculate that the evolution from pattern 1 topattern 2 involved the appearance of the bifacial meristem onthe ventral side of the root, and the subsequent evolution frompattern 2 to pattern 3 involved the addition of such a meristemon the dorsal side. The pattern-2 meristem is one-sided; it islocated much closer to the ventral side of the root than themeristem of pattern 1. Along with the establishment of

a bifacial meristem, a reduction of the ventral portion of theancestral dome-shaped meristem may have been involved inthe evolution of pattern 2, with a subsequent reduction of thedorsal portion during the evolution of pattern 3 (Fig. 3).

The evolution from pattern 2 to pattern 3 took place in closeassociation with the transformation from the subcylindrical orribbon-like root to the crustose root, which is caused bymodification of the organization of the meristem. The ratherbroad, ribbon-like root of Cladopus javanicus with pattern 2B(Koi and Kato, 2003) is formed by the prominent marginalmeristem located proximolateral to, and derived from, theapical meristem. The marginal meristem is similar to that ofpattern 3 in being long, uniform, and more differentiated thanthe apical meristem, although it is capless. An increase in themarginal meristem and a reduction in the apical meristem arelikely to have contributed to the appearance of the crustoseroot.

Pattern 4, characterized by the absence of the root cap, isseen in Dalzellia gracilis, Tristicha trifaria (Rutishauser, 1997;this study), Zeylanidium lichenoides, Z. subulatum (Hiyama etal., 2002), and Jenmaniella ceratophylla. Ameka et al. (2003)described the absence of the root cap in Ledermanniella

Fig. 28. Reconstructed evolution of root apical meristem and cap organization (patterns 1–4) with rootless state in Podostemaceae (for method seeMaterials and Methods). Character states of taxa are shown in Table 1.


browlingii (J. B. Hall) C. Cusset. Organography (Rutishauser,1997) and phylogeny (Kita and Kato, 2001) indicate that, eventhough caplessness is unusual, the organ observed iscomparable with a root. Their root meristems, coated witha single dermal meristem layer, are very similar due to theirstructural simplicity, although they belong to differentsubfamilies or clades of the subfamily Podostemoideae. Rareoccurrences of pattern 4 in a few remote, nonbasal clades ofPodostemaceae and also other angiosperms (Guttenberg, 1968)suggest that pattern 4 is derived recurrently from the cappedroots of pattern 1 or 2 (Fig. 28). Although the character state ofthe common ancestor to the species of Polypleurum andZeylandium is equivocal, the derivation from pattern 2 is morelikely than from pattern 3, because the crustose root, which istightly linked with pattern 3, is a derivative relative to thesubcylindrical or ribbon-like roots (Suzuki et al., 2002).Dalzellia gracilis is sister to a clade of D. zeylanica andIndotristicha ramosissima, and all are sister to Tristichatrifaria. Therefore, the root cap may have either been regainedlater in the evolution of Indotristicha ramosissima orrecurrently lost in Tristicha trifaria and Dalzellia gracilis.Similarly, a regain (Hiyama et al., 2002) or a loss may haveoccurred in the Polypleurum-Zeylandium lineage.

Temporary absence of the root cap is often visible duringcapped-root development in Podostemaceae. In seedlingculture experiments on nine capped species, the secondaryroot developing from the hypocotyl is capless early indevelopment and has an apical meristem quite similar to thatof these capless species (Suzuki et al., 2002). In the crustose-rooted Hydrobryum japonicum, the young root lobe is capless,then the root cap (protective tissue) is derived from thedifferentiated distal portion of the marginal root meristem (Otaet al., 2001). The same case may hold for Zeylanidiummaheshwarii (Hiyama et al., 2002). Our observations suggestthat in Polypleurum wallichii cap formation may temporarilycease in pattern 2, much like that in pattern 4. Taking intoaccount all these results (Ota et al., 2001; Suzuki et al., 2002)and the aforementioned development of lateral roots froma capless portion of a parent root in Cladopus species (Koi andKato, 2003), a likely scenario for root cap disappearance isthat, compared to the delayed cap formation in capped species,cap formation is suppressed or lost at maturity in some speciesthat are remotely related phylogenetically. The root cap playsa central role in the perception of environmental signals such asgravity, water gradients, mechanical impedance, and pathogens(Sievers et al., 2002). This significant role in perception isprobably not played or transferred to an unidentified tissue inthe capless species of Podostemaceae.

In conclusion, there is variation in the root apical meristemand cap organization in Podostemaceae. The least specializedorganization (pattern 1) is present in Tristichoideae andWeddellinoideae. In the remarkably dorsiventral, one-sidedroot apical meristem (pattern 2) of Podostemoideae, the ventrallayer is bifacial to yield the root cap acroscopically. Thecrustose root of some Podostemoideae has bifacial meristemlayers on both dorsal and ventral sides (pattern 3) so that theroot cap is an acroscopic derivative from the meristem. A rootmeristem with pattern 1 may have become specialized topattern 2 and then to pattern 3 and may have causeda specialization of the root morphology in the evolution ofPodostemaceae. A few species are devoid of the root cap,representing another extreme specialization (pattern 4).

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