Seedling development in Hanseniella, Hydrobryum and Thawatchaia ...

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Jul 11, 2012 - Abstract In order to understand the evolution of the body plan characteristic of the unique aquatic Podostemaceae, seedlings of crustose-rooted ...
Plant Syst Evol (2012) 298:1755–1766 DOI 10.1007/s00606-012-0676-7

ORIGINAL ARTICLE

Seedling development in Hanseniella, Hydrobryum and Thawatchaia (Podostemaceae), and implications on body plan evolution in the Hydrobryum clade Satoshi Koi • Petcharat Werukamkul La-aw Ampornpan • Masahiro Kato



Received: 30 January 2012 / Accepted: 12 June 2012 / Published online: 11 July 2012 Ó Springer-Verlag 2012

Abstract In order to understand the evolution of the body plan characteristic of the unique aquatic Podostemaceae, seedlings of crustose-rooted species of the Hydrobryum clade, i.e., Hanseniella heterophylla, Hydrobryum loeicum, Hy. tardhuangense, Hy. vientianense and Thawatchaia trilobata, were studied by sectioning and scanning electron microscopy. In all the species the plumule/primary shoot and the radicle/primary root did not form in the seedling. The adventitious root formed exogenously from the lateral side of the hypocotyl and developed initially into a semicircular root, which became crustose during early seedling development. Tufted-leaves arose endogenously from the root. Within the context of the phylogeny of the Hydrobryum clade, we postulate an evolutionary scenario in which the plumule was lost at the divergence of the Hydrobryum clade from the Cladopus clade, much later than the radicle was lost at the divergence of the clade of Podostemoideae and Weddellinoideae from Tristichoideae. S. Koi (&) Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan e-mail: [email protected] P. Werukamkul Faculty of Science and Technology Rajamamgala, University of Technology Phra Nakorn, 1381 Pibul Songkhram Road, Bangsue, Bangkok 10800, Thailand L. Ampornpan Department of Biology, Srinakharinwirot University, Sukumwit 23 Road, Bangkok 10110, Thailand M. Kato Department of Botany, National Museum of Nature and Science, 4-1-1 Amakubo, Tsukuba, Ibaraki 305-0005, Japan

The seedling body plan, with the exogenous crustose root playing the role of a leading organ, common in the clade of Hanseniella, Hydrobryum and Thawatchaia, appeared after the divergence of Hydrodiscus with a branched shoot as a leading organ. Keywords Evolution  Hydrobryum  Podostemaceae  Root  Seedling development

Introduction The body plan of terricolous angiosperm plants is characterized by the vertical shoot–root axis, allowing adaptation to land environments layered with upper aerial and lower soil zones. Within the framework of the modular construction, the adult plant construction primarily follows the seedling one established during embryogenesis. In most angiosperms, a plumule and a radicle occur at both ends of the hypocotyl, and develop into a primary shoot and a primary root, respectively (Steeves and Sussex 1989). The seedling morphology of angiosperms has been relatively stable, except for the number of cotyledons, through evolution and diversification (Esau 1977). In contrast, the seedling and older plants of Podostemaceae are submerged under water in rapids and waterfalls during the rainy season, and usually the roots (rarely the shoots) are creeping on and adhering to rock surfaces, and produce adventitious shoots (root-borne shoots) on their flanks or dorsal surfaces. Podostemaceae has great variation in seedling morphology among species with different adult-plant morphologies (Mohan Ram and Sehgal 1997; Suzuki et al. 2002; Kita and Kato 2005; see also Table 2). Differences are distinct among the three subfamilies. In the subfamily Tristichoideae, the seedling of Terniopsis brevis

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M. Kato is most similar to other angiosperms in having a primary shoot and a primary root that develop from a plumule and a radicle, respectively, at both ends of hypocotyl, and also forming adventitious roots endogenously from the hypocotyl (hypocotyl-borne roots) (Kita and Kato 2005; Katayama et al. 2011). Either root subsequently develops into a leading creeping organ with adventitious shoots (Kita and Kato 2005). The seedling of Indotristicha ramosissima (Wight) P. Royen forms a plumule, developing into a primary shoot, but no radicle, and forms exogenously adventitious roots from the base of the hypocotyl (Mukkada and Chopra 1973; Nagendran et al. 1981; Mohan Ram and Sehgal 1997; Rutishauser 1997). The seedling of the rootless Dalzellia zeylanica Wight has a rudimentary plumule that terminates after two leaves are formed, and neither a radicle nor adventitious root occurs (Mukkada 1969; Ja¨gerZu¨rn 1995; Imaichi et al. 2004). The crustose adhering shoot originates from one of the axillary shoot meristems in the cotyledons (Ja¨ger-Zu¨rn 1995; Imaichi et al. 2004). In the subfamily Weddellinoideae, the seedling of Weddellina squamulosa Tul. forms a primary shoot, but is devoid of a primary root (Koi and Kato 2007). The seedling morphologies of the subfamily Podostemoideae have been studied mainly in Asian species. Most species of Asian Podostemoideae, such as Cladopus (including Torrenticola), Griffithella, Hydrobryopsis, Polypleurum and Zeylanidium, have an unrecognizable plumule that is buried between the cotyledons and develops rudimentarily into only a few leaves. They are devoid of radicles. An adventitious root arises endogenously in the hypocotyl and grows into a ribbon-like or crustose creeping root (Vidyashankari and Mohan Ram 1987; Sehgal et al. 1993, 2002, 2007; Suzuki et al. 2002; Katayama et al. 2011). The seedling morphology of African and American Podostemoideae species is still poorly known except a few species, e.g., Mourera fluviatilis Aubl. and Marathrum schiedeanum Cham., both of which form a primary shoot (perhaps determinate), but no primary root (Oropeza et al. 1998; Rutishauser and Grubert 1999; Rutishauser et al. 1999), and Podostemum ceratophyllum Michx., which has a primary shoot axis with a few leaves but no primary root (Philbrick 1984). Podostemum ceratophyllum forms a secondary root on a lateral side of the hypocotyl (Philbrick 1984). The body plan of the seedling of Hydrobryum, Asian Podostemoideae, is extremely derived. Suzuki et al. (2002) and Katayama et al. (2011) showed that the plumule/primary shoot and the radicle/primary root are lacking, and the hypocotyl-borne root is exogenous from the lateral side of the hypocotyl in two Hydrobryum species [including Synstylis micranthera (P. Royen) C. Cusset]. The root develops into a broadly flattened crustose root with tiny filiform leaves on the dorsal surface (Ota et al. 2001; Kato 2004). Hydrobryum forms a monophyletic clade (here called the

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Hydrobryum clade) with Hanseniella, Hydrodiscus and Thawatchaia, in which Hydrodiscus exhibits distinct seedling and adult body plans (Koi and Kato 2010a). Hydrodiscus koyamae (M. Kato & Fukuoka) Koi & M. Kato is monocotyledonous, devoid of the plumule/primary shoot and the primary root/radicle, and has an adventitious shoot (hypocotyl-borne shoot) endogenous from the lateral side of the hypocotyl (Koi and Kato 2010a). The adult plant comprises only a huge branched shoot (Koi and Kato 2010a). These available data indicate that drastic changes occurred in seedling morphology at the divergence of the Hydrobryum clade. However, seedling morphology was poorly known in most other species, so the evolutionary process in seedling morphology in the clade remained uncertain. This article describes the seedling morphology of Hanseniella heterophylla C. Cusset, Thawatchaia trilobata M. Kato, Koi & Y. Kita, and three species of Hydrobryum in order to contribute to better understanding of the processes in body-plan evolution in the Hydrobryum clade. The term ‘root’ is used here for the dorsiventrally flattened body of Hydrobryum and other podostemads, although there are possible alternatives (see ‘‘Discussion’’).

Materials and methods Seedlings and seeds were collected in the field (Table 1). Voucher specimens are deposited in the Herbarium, Department of Botany, National Museum of Nature and Science, Tsukuba (TNS), and the Forest Herbarium, Department of National Parks, Wildlife and Plant Conservation, Bangkok (BKF). For seedling culture, Kita and Kato’s (2005) method was followed. Seeds were sterilized in 1 % sodium hypochlorite (Wako, Osaka, Japan) with 0.1 % polyoxyethylene sorbitan monolaurate (Tween 20) (Nacalai Tesque, Kyoto, Japan) with vortex for 5 min and washed in sterile distilled water. The rinsed seeds were placed on 1.5 % agar with 0.05 % HYPONeX (Hyponex Japan, Tokyo, Japan), and cultivated aquatically in 0.05 % HYPONeX at 23 °C in 14 h light:10 h darkness. Morphological observations followed the method of Koi et al. (2009). Seedlings from the field and cultivated ones were fixed with FAA (formaldehyde:acetic acid:50 % ethyl alcohol = 5:5:90) and 4 % formaldehyde (w/v), respectively. For scanning electron microscopic (SEM) observation, we used a critical point dryer HCP-2 (Hitachi, Tokyo, Japan) and an ion sputter E-1010 (Hitachi), and made observations with an S-4700 (Hitachi) at 2 kV. For anatomical observations, we used Technovit 7100 (Heraeus Kulzur, Wehrheim, Germany), cut embedded material into 2-lm-thick sections, and stained them with safranin, toluidine blue and Orange G (Jernstedt et al. 1992).

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Table 1 Materials used in this study Species

Source and voucher

Seedling and mature embryo collected in the field Hanseniella heterophylla C. Cusset

Gaeng Gliang, Phu Ruea District, Thailand; TWA-119

Hydrobryum loeicum M. Kato

Wangtad waterfall, Na Haeo District, Thailand; TWA-118

Hy. tardhuangense M. Kato

Tat Hueang waterfall, Na Haeo District, Thailand; TL-314, TWA-115

Hy. vientianense (M. Kato & Fukuoka) Koi & M. Kato

Phu Luang wildlife sanctuary, Wangsapung District, Thailand; TWA-127

Seedling cultured from seed Ha. heterophylla

Gaeng Gliang, Phu Ruea District, Thailand; TPK-15

Thawatchaia trilobata M. Kato, Koi & Y. Kita

Nam Tha river, Nam Ha NPA, Luang Namtha, Laos; LK-312

The evolution of seedling characters was reconstructed onto a phylogenetic tree of Asian Podostemoideae adapted from Kita and Kato (2001, 2004), Koi and Kato (2010a, 2012) and Koi et al. (2012), using the program MacClade 4.0 (Maddison and Maddison 2000). Unavailable characters were treated as missing data.

(Fig. 1d). The root primordium developed into a crustose semicircular root, where subsequently a tuft of leaves arose endogenously (Fig. 1e). As no morphogenesis was visible in the seedling, it did not form the primary shoot between the cotyledons (Fig. 1f). Further, the developing root became lobed and produced additional tufts of leaves in the more distal part of the root (Fig. 1g). The leaf was filiform with thin dense hairs on the ventral side (Fig. 1g).

Results Thawatchaia trilobata Seedlings of Ha. heterophylla, T. trilobata, Hydrobryum tardhuangense M. Kato and Hy. loeicum M. Kato were observed by both sectioning anatomy and scanning electronic microscopy (SEM), and those of Hy. vientianense (M. Kato & Fukuoka) Koi & M. Kato were observed by SEM. The seedlings examined showed the same general body plan, although the developmental anatomy of the hypocotyl-borne root was not observed in Hy. loeicum or Hy. vientianense because their seedlings, with the initiation of the hypocotyl-borne root, were not available. The mature embryo/post-embryonic young seedling comprised two cotyledons and a short hypocotyl in all the species examined. The seedling development is described separately for Ha. heterophylla, T. trilobata and three species of Hydrobryum. Hanseniella heterophylla There was no recognizable plumule between the cotyledons in seedlings just after germination [10 days after sowing (DAS)] (Fig. 1a, b). There were lightly stained cells at the base of the hypocotyl, indicating that the primary root did not form at the bottom of the hypocotyl (Fig. 1a, b). The adventitious root (hypocotyl-borne root) formed exogenously from one side of the basal half of the hypocotyl at right angles to the plane of the cotyledons and became as thick as the length of the hypocotyl (Fig. 1c–e). A procambial strand of elongate cells was differentiated proximal to the root meristem (Fig. 1d, arrowhead). Rhizoidal hairs were produced on the bottom of the hypocotyl

Shortly after germination (8 DAS), the seedling was devoid of a recognizable plumule between the cotyledons (Fig. 2a, b). A procambial strand of elongate cells ran from the hypocotyl through the cotyledons (Fig. 2a, b). Cells at the base of the hypocotyl were relatively small and densely stained (Fig. 2a, b). In the developed seedling (14 DAS), the dermal and inner cells of the hypocotyl were meristematic at the early stage (Fig. 2c, d). Subsequently, the dermal cells of the ventral surface of the hypocotyl became vacuolated and bore rhizoids, while the tissue on the dorsal surface remained meristematic (Fig. 2e–h). The dorsal meristematic tissue of the hypocotyl continuously developed into the flattened root primordium, resulting in an indistinctive boundary between the hypocotyl and the root (Fig. 2i–k). The primary shoot did not occur between the cotyledons (Fig. 2l). Subsequently, the root primordium grew into a crustose semicircular root (Fig. 2m, n). Three Hydrobryum species In the mature embryo of Hy. tardhuangense, the cotyledons and the hypocotyl were entirely meristematic, and a recognizable plumule did not form between the cotyledons (Fig. 3a, b). The primary shoot did not occur between the cotyledons in the germinated seedling (Fig. 3c, e). The primary root also did not form at the basal end of the hypocotyl, where the rhizoids were formed (Fig. 3d, g). An adventitious root formed exogenously on the side of the basal half of the hypocotyl at right angles to the cotyledons

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Fig. 1 Seedlings and young plants of Hanseniella heterophylla. Longitudinal sections of seedling parallel to cotyledons shortly after germination (a, b). Cells at distal part of cotyledons and the base of the hypocotyl are lightly stained. a Seedling with two cotyledons and short hypocotyl enveloped with ruptured seed coat. b Enlargement of a, showing absence of a plumule between cotyledons. SEM image (c) and longitudinal section at right angles to cotyledons (d) of seedlings with initiating hypocotyl-borne root. c Hypocotyl-borne root in semicircle arises on the side at right angles to cotyledons. d Exogenously developing root at basal half of the hypocotyl.

Arrowhead indicates elongate procambium cells. SEM image (e) and longitudinal section parallel to cotyledons (f) of seedlings with further developing hypocotyl-borne root. e Unidirectionally expanding root of older seedling and endogenous adventitious leaves on dorsal surface. f Absence of primary shoot axis between cotyledons. g SEM image of young plant with lobed root bearing tufts of leaves. Arrowheads indicate hairs on leaves. C, cotyledon; H, hypocotyl; L, leaf; R, root; Rh, rhizoid. Scale bars 100 lm in a, d, f; 50 lm in b; 200 lm in c, e, g

and gradually expanded into a crustose root parallel to the surface (Fig. 3c, d, f, g). A leaf formed endogenously in a very young root (Fig. 3h). In Hy. loeicum and Hy. vientianense, the primary shoot did not form between the cotyledons (Fig. 4a–e). The rhizoidal hairs were produced at the basal end of the hypocotyl instead of the primary root in Hy. loeicum (Fig. 4c). An adventitious root formed from the hypocotyl at right angles to the cotyledons (Fig. 4a, d, e). In Hy. loeicum, the hypocotyl-borne root was as thick as the length of the hypocotyl, which therefore was apparently indistinguishable (Fig. 4a, b, d), whereas the hypocotyl was visible in Hy. vientianense. The root primordium grew into a crustose semicircular root, from

the dorsal surface of which tufted leaves arose endogenously (Fig. 4d, e).

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Discussion Homology of hypocotyl-borne root The nature of the Podostemaceae root, including the foliose roots of the Hydrobryum clade, has been controversial. The root of Terniopsis, which is among the most basal genera in Podostemaceae, exhibits typical root features, i.e., an almost radially symmetric root cap, endogenous branching, endogenous production of an adventitious shoot, and

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Fig. 2 Seedlings of Thawatchaia trilobata. a Longitudinal section parallel to cotyledons of seedling shortly after germination [8 days after sowing (DAS)]. b Enlargement of a, showing absence of plumule between cotyledons. Note that the base of the hypocotyl is meristematic. Light micrograph (c) and longitudinal section at right angles to cotyledons (d) of seedlings (14 DAS) with meristematic hypocotyl. c Front view of seedling. d Entirely meristematic hypocotyl. Note a dermal cell (arrowhead) differentiating into a rhizoid on the ventral surface (left side). SEM images (e–g) of seedlings (18 DAS) older than c. e Front view of seedling. f Enlargement of e, showing smooth dorsal surface of hypocotyl. g Back view (ventral side) of seedling, showing ventral surface of hypocotyl forming rhizoids. h Longitudinal section at right angles to cotyledons of seedling (14 DAS) as old as e–g, showing initiating

hypocotyl-borne root. Ventral dermal tissue of the hypocotyl is vacuolated and differentiating to rhizoids. i, j Light micrographs of seedling with developing hypocotyl-borne root at 14 DAS. i Front view of seedling. j Lateral view of seedling of i, showing developing hypocotyl-borne root with rhizoids on ventral side. k SEM image of seedling (14 DAS) as old as i, showing hypocotyl-borne root continuously developed from hypocotyl with rhizoids on ventral side. l Section parallel to cotyledons of seedling (14 DAS) as old as k, showing absence of primary shoot between cotyledons. m Light micrograph of seedling with semicircular hypocotyl-borne root at 14 DAS. n SEM image of further developing hypocotyl-borne root (53 DAS). C, cotyledon; H, hypocotyl; R, root; Rh, rhizoid; Sc, seed coat. Scale bars 100 lm in a, c, e, i–n; 50 lm in b, d, f–h

development from the radicle and from the hypocotylborne root (Imaichi et al. 1999; Kita and Kato 2005; Koi et al. 2006). Therefore, the root of Terniopsis is

homologous to the ordinary root, although it is dorsiventrally subcylindrical and has chloroplasts for photosynthesis. The root of the other species of Tristichoideae,

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Fig. 3 Seedlings of Hydrobryum tardhuangense. Longitudinal section of mature embryo parallel to cotyledons (a, b). a Mature embryo with two cotyledons and hypocotyl. b Enlargement of a, showing absence of plumule between cotyledons. SEM images of seedlings (c, d). c Front view of young seedling with cotyledons, hypocotyl and hypocotyl-borne root. d Lateral view of seedling as old as c, showing developing hypocotyl-borne root with rhizoids on ventral side. e Longitudinal section of hypocotyl parallel to cotyledons of seedling

at a stage similar to c and d, showing absence of primary shoot axis. f SEM image of hypocotyl-borne root at a stage older than c and d. Longitudinal sections of hypocotyl-borne roots (g, h). g Hypocotylborne root initiating from base of the hypocotyl. h Developing hypocotyl-borne root with endogenous leaf primordium. C, cotyledon; H, hypocotyl; L, leaf; R, root; Rh, rhizoid. Scale bars 50 lm in a, b; 100 lm in c–h

Weddellinoideae, and American, Madagascan and a part of African Podostemoideae share endogenous branching and endogenous production of adventitious shoots with the root of Terniopsis (e.g., Rutishauser 1997), although the origin of the roots in the seedlings remains uncertain, except in a few species (Table 2). Asian and a part of African Podostemoideae have specialized, exogenously branching roots. Sehgal et al. (2002, 2007) studied the development of seedlings of Indian Hydrobryopsis sessilis (Willis) Engl., Griffithella hookeriana (Tul.) Warm. and Polypleurum stylosum (Wight) J.B. Hall, and described that the ‘roots’ are capless, show a tunica-corpus organization like a shoot

apical meristem (SAM) and originate from the meristem of the primary shoot, reaching the conclusion that the root is homologous with the shoot. In fact, the root in the adult plant of P. stylosum produces a root cap, which arises in a post-seedling stage (Koi et al. 2006). Such a delayed cap formation is likely in Cladopus species, Asian Podostemoideae, in which the root primordium originates near the base of the meristematic leaf primordium (Suzuki et al. 2002; Koi and Kato 2003) in a pattern similar to the species examined by Sehgal et al. (2002, 2007). The thallus of Hydrobryum has a marginal meristem instead of an apical meristem (Ota et al. 2001). However, the thallus has a

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Seedling development in Hanseniella, Hydrobryum and Thawatchaia (Podostemaceae)

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Fig. 4 Seedlings of Hydrobryum loeicum (a–d) and young plant of Hy. vientianense (e). a SEM image of semicircular hypocotyl-borne root below cotyledons. Hypocotyl is invisible. b Enlargement of a showing absence of plumule between cotyledons. c Longitudinal section of seedling as old as a, showing absence of plumule. d SEM

image of developing hypocotyl-borne root with cotyledons at proximal base and leaf on dorsal side. e SEM image of subcircular hypocotyl-borne root with cotyledons at proximal base and leaves in tufts. C, cotyledon; H, hypocotyl; L, leaf; R, root; Rh, rhizoid. Scale bars 200 lm in a, e; 100 lm in b–d

protective tissue fringing the marginal meristem and branches exogenously, and leaves arise endogenously on its dorsal surface, suggesting that the thallus is comparable to the root of other Asian Podostemoideae (Ota et al. 2001; Koi and Kato 2003). Therefore, roots of Asian Podostemoideae would be appropriately interpreted as a specialized root (but see ‘‘Exogeny of hypocotyl-borne root’’ below).

site of secondary root in the seedling (Table 2). In type I, the plumule and radicle are present, and a hypocotyl-borne root arises endogenously near the procambium in the hypocotyl (e.g., Terniopsis brevis; Kita and Kato 2005; Katayama et al. 2011). In type II, the plumule/primary shoot is present but rudimentary, i.e., devoid of a distinct SAM, the radicle/primary root is absent, and a hypocotylborne root is endogenous (e.g., Cladopus javanicus M. Kato & Hambali; Suzuki et al. 2002). Type II can be divided into two subtypes: a procambium is present in the hypocotyl and a hypocotyl-borne root occurs near the procambium (type II-a), or a procambium is absent and a hypocotyl-borne root occurs near the base of the plumule/ primary shoot (type II-b) (Table 2). Type III is observed in Indotristicha ramosissima, in which the plumule/primary

Seedling morphology of the Hydrobryum clade The present and previous studies found various seedling morphologies of Podostemaceae, which can be assigned to six types based on the presence/absence of the plumule/ primary shoot and radicle/primary root and the initiation

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Table 2 Types of seedling body plan in Podostemaceae Type

Plumule/ primary shoot

Radicle/ primary root

Hypocotylborne root

Species (source)

Type I

?

?

Endogenous

Terniopsis brevis M. Kato (Kita and Kato 2005; Katayama et al. 2011)

Type II-a

?a

-

Endogenousc

Zeylanidium olivaceum (Gardner) Engl. (Suzuki et al. 2002)

Type II-b

?a

-

Endogenousd

Cladopus javanicus M. Kato & Hambali, C. (= Torrenticola) queenslandicus (Domin) C.D.K. Cook & Rutish., Polypleurum wallichii (R. Br. ex Griff.) Warm., Zeylanidium subulatum (Gardner) C. Cusset (Suzuki et al. 2002); Griffithella hookeriana (Tul.) Warm. (Vidyashankari and Mohan Ram1987; Sehgal et al. 2007); Hydrobryopsis sessilis (Willis) Engl. (Sehgal et al. 2002); P. stylosum (Wight) J.B. Hall (Sehgal et al. 1993, 2007; Suzuki et al. 2002); Z. lichenoides Engl. (Suzuki et al. 2002; Katayama et al. 2011)

Type III

?

-

Exogenous

Indotristicha ramosissima (Wight) P. Royen (Mukkada and Chopra 1973; Vidyashankari 1988; Mohan Ram and Sehgal 1997; Rutishauser 1997)

Type IV-a Type IV-b

? ?b

-

-

I. tirunelveliana B.D. Sharma, Karthik. & B.V. Shetty (Uniyal 1999) Dalzellia zeylanica Wight (Mukkada 1969; Ja¨ger-Zu¨rn 1995; Imaichi et al. 2004); Indodalzellia gracilis (Mathew, Ja¨ger-Zu¨rn, & Nileena) Koi & M. Katoe (Koi and Kato 2010b)

Type IV-c

?a

-

-

Type V

-

-

Exogenous

Castelnavia princeps Tul. et Wedd. (Warming 1882); Mourera fluviatilis Aubl. (Rutishauser and Grubert 1999) Hydrobryum griffithii (Wall. ex Griff.) Tul., Hy. micrantherum (P. Royen) C.D.K. Cook & Rutish. (=Synstylis micranthera) (Suzuki et al. 2002); Hy. japonicum Imamura (Katayama et al. 2011); Hy. loeicum, Hy. tardhuangense, Hy. vientianense, Ha. heterophylla, Thawatchaia trilobata (present study)

Type VI

-

-

-

Hydrodiscus koyamae (M. Kato & Fukuoka) Koi & M. Katof (Koi and Kato 2010a)

Type uncertain

?

?

NA

Tristicha trifaria (Bory ex Willd.) Spreng. (Schnell and Cusset 1963; Grubert 1976)

Type uncertain

?

-

NA

Marathrum schiedeanum (Cham.) Tul. (Oropeza et al. 1998; Rutishauser et al. 1999); Podostemum ceratophyllum Michx. (Philbrick 1984); Rhyncholacis applanata Goebel (Goebel 1893); Weddellina squamulosa Tul. (Koi and Kato 2007); Willisia selaginoides (Bedd.) Warm. ex Willis (Mohan Ram and Sehgal 1997; Uniyal and Mohan Ram 2001)

?, Present; –, absent NA, no available data a

The plumule is devoid of a distinct shoot apical meristem (Katayama et al. 2011 and references therein)

b

The plumule produces only two leaves, and a shoot developed in the axil of the cotyledon is a leading organ

c

The root arises near the procambium of the hypocotyl

d

The root arises near the base of the plumule A root arises adventitiously from the shoot

e f

The shoot arises endogenously from the hypocotyl

shoot is present, the radicle/primary root is absent, and a hypocotyl-borne root is exogenous (Mukkada and Chopra 1973; Vidyashankari 1988; Mohan Ram and Sehgal 1997; Rutishauser 1997). In type IV, the plumule/primary shoot is present, but the radicle/primary root and hypocotyl-borne root are absent (e.g., Dalzellia zeylanica; Mukkada 1969; Ja¨ger-Zu¨rn 1995; Imaichi et al. 2004). Type IV can be divided into three subtypes (Table 2): the plumule develops into a tunica-corpus organized dome-shaped SAM (type IV-a); the plumule is rudimentary and produces only two leaves, and an axillary shoot meristem of a cotyledon develops (type IV-b); the primary shoot is devoid of

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distinct SAM (type IV-c). In type V, the plumule/primary shoot and radicle/primary root are absent, and a hypocotylborne root is exogenous on the lateral side of the base of the hypocotyl [e.g., Hydrobryum micrantherum (P. Royen) C.D.K. Cook & Rutish. (syn. Synstylis micranthera); Suzuki et al. 2002]. Type VI is observed in Hydrodiscus koyamae, in which the plumule/primary shoot, radicle/ primary root and hypocotyl-borne root are absent (Koi and Kato 2010a). The present study shows that Ha. heterophylla, T. trilobata and the three species of Hydrobryum have the same seedling body plan, in which the primary shoot and the

Seedling development in Hanseniella, Hydrobryum and Thawatchaia (Podostemaceae)

primary root are absent and a hypocotyl-borne root arises exogenously near the base of the hypocotyl. The seedling morphologies of these species are the same as those of Hydrobryum griffithii (Wall. ex Griff.) Tul., Hy. micrantherum and Hy. japonicum Imamura (Suzuki et al. 2002; Katayama et al. 2011). Therefore, all genera of the Hydrobryum clade except Hydrodiscus have the type V seedling body plan (Table 2).

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(Fig. 5a). Therefore, it is most likely that the loss of the plumule occurred at the base of the Hydrobryum clade (Fig. 6). The present study suggests that data of the developmental patterns of Hanseniella and Thawatchaia Zeylanidium subulatum

a

Griffithella hookeriana Polypleurum stylosum Polypleurum wallichii

Loss of plumule

Willisia selaginoides Zeylanidium olivaceum

Based on comparative developmental studies, Katayama et al. (2011) suggested that a morphologically unrecognizable embryonic shoot meristem, i.e., a cryptic plumule, is formed during embryogenesis in Zeylanidium lichenoides Engl. Cell division patterns until the 16-cell stage of the embryo are common between Terniopsis brevis with a distinct plumule in the seedling and Z. lichenoides, and, consequently, a putative organizing center (OC) of the SAM is established in both species. After the 16-cell stage, a dermal cell layer above the OC undergoes anticlinal cell division to form a distinct plumule in T. brevis, whereas no anticlinal cell division occurs in the dermal layer, and the OC is buried at the base of the cotyledons in Z. lichenoides. Katayama et al. (2011) noted that a leaf arises from the cells of the putative OC in Z. lichenoides, implying that a primary shoot axis between the cotyledons is developed from a cryptic plumule in Podostemoideae. Katayama et al. (2011) also revealed that cell division patterns at the 8- to the 16-cell stage of embryos are different between the T. brevis and Z. lichenoides seedlings and the primaryshoot-less Hydrobryum japonicum Imamura, and suggested that such a developmental change caused the loss of the cryptic plumule in Hydrobryum. In the Hydrobryum clade, Hydrodiscus is basal and sister to the rest of the genera, of which Hanseniella is close to Thawatchaia and together sister to Hydrobryum, although these relationships are supported weakly (Koi and Kato 2010a). The Hydrobryum clade is sister to the Cladopus clade comprising Cladopus and Paracladopus, and this Hydrobryum–Cladopus clade is sister to the clade including Polypleurum and Zeylanidium, together forming a monophyletic Asian group (see Fig. 6; Kita and Kato 2001; Koi et al. 2012). The examined species of the Cladopus, Polypleurum and Zeylanidium clades have type II seedling morphologies, i.e., the plumule is devoid of distinct SAM, produces several leaves and then terminates in the seedling. The radicle is absent, and a hypocotylborne root arises endogenously (Table 2). Based on the molecular phylogenetic relationships, we suggest that the plumule-less seedling of the Hydrobryum clade is autoapomorphic, compared with the plesiomorphic seedling with the plumule, a type common in other angiosperms

Zeylanidium lichenoides Hydrobryopsis sessilis Cladopus javanicus Cladopus queenslandicus Paracladopus chiangmaiensis Hydrodiscus koyamae Hanseniella heterophylla Thawatchaia trilobata Hydrobryum tardhuangense Plumule Present Absent Unknown Equivocal

Hydrobryum loeicum Hydrobryum vientianense Hydrobryum griffithii Hydrobryum micrantherum

Zeylanidium subulatum

b

Griffithella hookeriana Polypleurum stylosum Polypleurum wallichii Willisia selaginoides Zeylanidium olivaceum Zeylanidium lichenoides Hydrobryopsis sessilis Cladopus javanicus Cladopus queenslandicus Paracladopus chiangmaiensis Hydrodiscus koyamae Hanseniella heterophylla Thawatchaia trilobata Hypocotyl-borne root Endogenous Exogenous Absent Unknown Equivocal

Hydrobryum tardhuangense Hydrobryum loeicum Hydrobryum vientianense Hydrobryum griffithii Hydrobryum micrantherum

Fig. 5 Reconstructed character phylogeny of plumule (a) and hypocotyl-borne root (b) in Asian Podostemoideae. Trees are based on the phylogenetic tree of Koi and Kato (2010a). Character states after Suzuki et al. (2002), and Koi and Kato (2010a). Loss of plumule and gain of exogenous origin of hypocotyl-borne root occurred in the Hydrobryum clade

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1764 Podostemaceae Podostemoideae Asian clade

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Fig. 6 Scenario for seedling evolution in Podostemaceae. Phylogenetic tree is based on the trees of Kita and Kato (2001), Koi and Kato (2010a), Ruhfel et al. (2011), and Koi et al. (2012). Character states after Table 2. Major character evolutions are shown and others, e.g., loss of radicle in Tristichoideae and loss of hypocotyl-borne root in Podostemoideae, are not shown. Gain of endogenous hypocotylborne root appeared at the origin of Podostemaceae or earlier than divergence of Podostemaceae and Hypericaceae. Gain of exogenous hypocotyl-borne root appeared at the Hydrobryum clade or after divergence of Hydrodiscus

S. Koi et al.

Loss or degeneration of root Gain of exogenous hypocotyl-borne root Loss of plumule

Reduction of plumule Loss of radicle Gain of endogenous hypocotyl-borne root

embryos would be useful to better understand the loss of the plumule in the Hydrobryum clade. The seedling of Hydrodiscus is atypical in having a single cotyledon (Koi and Kato 2010a). Some Hydrobryum species also have a single cotyledon, and the reduction of the number of cotyledons occurred at least twice independently in the Hydrobryum clade (S. Koi, unpublished result). The parallel reduction of the cotyledon number seen in the Hydrobryum clade may be related to a developmental defect of the plumule during embryogenesis. Exogeny of hypocotyl-borne root The present and previous studies (Suzuki et al. 2002; Koi and Kato 2010a) show that all genera of the Hydrobryum clade except Hydrodiscus form hypocotyl-borne roots exogenously. This is in contrast to other Podostemoideae and Tristichoideae species (except I. ramosissima), in which hypocotyl-borne roots are formed endogenously (Table 2). The exogenous root is apomorphic compared with the plesiomorphic endogenous root (Fig. 5b; Kita and Kato 2005).

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In Hydrobryum embryos, the procambium is not well differentiated, and the inner cells of the hypocotyl are arranged in a random array (Katayama et al. 2011). Katayama et al. (2011) speculated that the random arrangement of the inner cells disturbs basipetal auxin flow in the species. In Arabidopsis thaliana, polar auxin transport through the vascular tissue controls the formation of a lateral root usually from the pericycle of vascular tissue (Fukaki and Tasaka 2009). If the speculation by Katayama et al. (2011) is correct, then the change in auxin flux pattern, along with lacking the plumule, which is a source of auxin (Zhao 2010), may be involved directly or indirectly in the shift of the initiation site of the hypocotyl-borne root at the divergence of the Hydrobryum clade. Generally, exogeny is a developmental trait of shoot and leaf (and also flower), and endogeny is the one of root (Steeves and Sussex 1989). Therefore, root exogeny in the seedling of the Hydrobryum clade makes the boundary of its root from the shoot fuzzy. Root branching in the adult is exogenous in Asian Podostemoideae (Koi and Kato 2003), whereas the adventitious root in the seedling forms endogenously or exogenously from the hypocotyl

Seedling development in Hanseniella, Hydrobryum and Thawatchaia (Podostemaceae)

(Table 2). The crustose root morphology of the Hydrobryum clade does not necessarily involve the exogenous origin of the hypocotyl-borne root, because the root is endogenous in the crustose-rooted Zeylanidium olivaceum (Gardner) Engl. (Suzuki et al. 2002). It may be possible that the root of the Hydrobryum clade contains the nature of another organ like the shoot or hypocotyl. This possibility of fuzzy morphology can be tested by genetic analysis. Katayama et al. (2010) offered genetic evidence for the fuzzy morphology of shoot and leaf in Cladopus and Hydrobryum. Our results allow re-investigation of the seedling morphology of Hydrodiscus, the most basal genus in the Hydrobryum clade. It is distinct in body plan from the rest of the clade (Koi and Kato 2010a). The seedling is devoid of a plumule/primary shoot and a radicle/primary root, and consists of a hypocotyl and a single cotyledon, giving rise to the rootless adult plant consisting of only a branched leafy and floriferous shoot. The shoot develops endogenously from the inner meristematic cells of the bulged hypocotyl. Koi and Kato (2010a) interpreted that the bulge is an outgrowth of the hypocotyl and, therefore, a hypocotyl-borne root is not formed. The present results, however, lead us to another interpretation. In the Thawatchaia seedling, the whole hypocotyl is meristematic at the early stage and appears to differentiate continuously into a root. Furthermore, the seedlings of Hanseniella and the three Hydrobryum species examined form the first adventitious shoot close to the hypocotyl in the hypocotyl-borne root. Here we can interpret that the bulge of the Hydrodiscus seedling is an extremely vestigial root or a transitional organ to the root that arises exogenously from the hypocotyl and forms only one adventitious shoot endogenously before its growth terminates. If this interpretation is acceptable, the exogenous origin of the hypocotyl-borne root was acquired in the Hydrobryum clade, while such a root degraded in Hydrodiscus koyamae (Fig. 6). The diversification of body plan in the present interpretation seems to have proceeded more smoothly or step by step than Koi and Kato’s (2010a) scenario that Hydrodiscus koyamae lost the hypocotyl-borne root and acquired the endogenous hypocotyl-borne shoot. Further analysis is needed to reveal the homology of the bulge of the Hydrodiscus seedling and eventually the evolution of body plan of the Hydrobryum clade. In summary, our reconstructed character phylogeny (Fig. 6) shows that an endogenous hypocotyl-borne root forming adventitious shoots was acquired at the origin of Podostemaceae [or earlier (S. Koi, unpublished result)], and subsequently the radicle was lost in the subfamilies Podostemoideae and Weddellinoideae, and the plumule was reduced and comprises, e.g., a tuft of leaves in Podostemoideae (Kita and Kato 2005; Koi and Kato 2007;

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Katayama et al. 2011). Later, the plumule was completely lost, and the exogeny of the hypocotyl-borne root was gained in the Hydrobryum clade (Suzuki et al. 2002; Katayama et al. 2011). The adventitious root is vestigial or lost, and the adventitious shoot was enlarged in Hydrodiscus (Koi and Kato 2010a). The seedling body plan is perhaps basically common across the Hydrobryum clade, although a saltational evolution happened at the divergence of Hydrodiscus and the rest of the genera. Acknowledgments We thank Natsu Katayama and Thawatchai Wongprasert for their help in the field studies, and two anonymous reviewers for their helpful suggestions for the manuscript. This study was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science.

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