Ontogeny of the Mycoheterotrophic Species Afrothismia Hydra (Burmanniaceae)

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Ontogeny of the mycoheterotrophic species Afrothismia hydra (Burmanniaceae)

Article in Botanical Journal of the Linnean Society · April 2008 DOI: 10.1111/j.1095-8339.2008.00787.x

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Stephan Imhof Sainge Nsanyi Moses Philipps University of Marburg Tropical Plant Exploration Group (TroPEG)

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Ontogeny of the mycoheterotrophic species Afrothismia hydra (Burmanniaceae)

STEPHAN IMHOF1* and MOSES N. SAINGE2

1Spezielle Botanik und Mykologie, Fachbereich Biologie, Philipps-Universität, D-35032 Marburg, Germany 2Centre for Tropical Forest Science (CTFS), University of Buea, Department of Plant and Animal Sciences, Buea, Southwest Cameroon

Received 10 April 2007; accepted for publication 4 October 2007

The complete ontogeny of the mycoheterotrophic Afrothismia hydra (Burmanniaceae) from seed to seed dispersal is presented. The oblong–ovoidal seeds are up to 0.7 mm long. They germinate with root tissue only, disrupting the seed coat and developing a primary ovoid root tubercle. At the proximal end of the tubercle, a second tubercle arises and further root initials indicate the sequential growth of more root tubercles with filiform extensions resulting in a small root aggregate. The seed coat often remains attached to this structure. When the root aggregate enlarges, a central axis to which all roots are connected becomes visible. This axis has a growth pole where new root tubercles arise. The same growth pole will later develop into a stem with scale leaves finally terminating in a flower. Flowers develop sympodially when the mature plant is only several centimetres long. After anthesis, the corolla tube disintegrates, leaving a pyxidium which opens by means of a peculiar elongating placenta, here called ‘placentophore’. The placentophore elevates the placenta with attached seeds above the flowering level and is interpreted as an adaptation to ombrohydrochory. The reduction of hypocotyl, cotyledon and primary shoot is discussed with regard to the classical germination concepts of monocotyledons and with mycoheterotrophic dicotyledons. © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society, 2008, 157, 31–36.

ADDITIONAL KEYWORDS: germination – root morphology – seed morphology – fructiﬁcation.

INTRODUCTION Sainge & Franke, 2005; Sainge et al., 2005; Cheek, 2006), adding to three species and one variety that The exclusively mycoheterotrophic Afrothismia spp. have been described in the past century (Engler, (Burmanniaceae) are peculiar flowering plants, not 1905; Schlechter, 1906; Cowley, 1988). Because of the only for their minute habit and achlorophylly, but also extensive reduction of morphological characters, the with respect to flower (e.g. Franke, 2004; Franke, systematic position of the Burmanniaceae has been Sainge & Agerer, 2004; Sainge, Franke & Agerer, uncertain for decades (for an overview, see Maas 2005), root structures (e.g. Imhof, 1999a; Maas-van de et al., 1986). Today, combined analysis of molecular Kamer, 2003) and mycorrhizal colonization pattern and morphological data have affirmed their affiliation (Imhof, 1999a, 2006). These rare species mostly grow to the Dioscoreales (Caddick, Rudall & Wilkin, 2000; superficially attached to leaf litter or soil surface and Caddick et al., 2002a,b; APG, 2003), but the data on only few species are shallowly rooted in the soil. They the infraordinal relationship of the tribes Burman- occur in undisturbed lowland or submontaneous rain nieae and Thismieae (sisters according to Caddick forest of central Africa and are highly endangered by et al. (2002b), in contrast to Thismieae + Tacca as habitat destruction (e.g. Cheek, 2003; Franke et al., sister to Burmannieae + other Dioscoreaceae accord- 2004). Recently, eight species have been discovered ing to Merckx et al. (2006)) are still ambiguous. (Cheek, 2003; Maas-van de Kamer, 2003; Franke, Afrothismia spp., like most other mycoheterotrophic 2004; Franke et al., 2004; Cheek & Jannerup, 2005; plants (MHP), are difficult to cultivate, probably because of the specificity regarding their mycorrhizal *Corresponding author. E-mail: [email protected] fungi (Franke et al., 2006), hence, young stages have

© 2008 The Linnean Society of London, Botanical Journal of the Linnean Society, 2008, 157, 31–36 31 32 S. IMHOF and M. N. SAINGE never been depicted. The germination of MHP (Leake, which can now be interpreted as the mycorrhizal 1994, 2004) other than orchids (e.g. Irmisch, 1853; fungus (see arrow in Fig. 3). A second tubercle has Bernard, 1899; Weber, 1981; Smreciu & Currah, 1989; developed from the proximal part of the first tubercle Rasmussen & Whigham, 1993; Umata, 1998; Mc- – the part that was initially hidden in the disrupted Kendrick et al., 2002; Yagame et al., 2007) and seed coat. The new tubercle is not completely filled Ericaceae (e.g. Velenovsky, 1907; Christoph, 1921; with fungal material; the distal part appears hyaline. Francke, 1935; Leake et al., 2004; Bidartondo & Bruns, Its tail-like extension has grown to 1 mm in length. 2005) remains unknown. This article elucidates the on- Close to the proximal part of both tubercles, where the togeny of Afrothismia hydra Sainge & Franke, includ- seed coat is still attached, the beginning of a third ing its germination. As all species of Afrothismia are tubercle arises (Fig. 3). This pattern of root tubercle similar in their root–rhizome system, the development development repeats several times, resulting in a presented here most likely holds for the whole genus. star-like structure, the developmental sequence of which is still detectable (Fig. 4). At this stage, the MATERIALS AND METHODS longest filiform root extension was 9 mm long. In the course of further tubercle development, the proximal During an expedition to Cameroon, following an invi- parts of the roots build a common axis. This axis is the tation from Dr George B. Chuyong (University of first visible shoot organ (rhizome), having a distal Buea, Cameroon), and under the guidance of Vincent growth pole where new tubercles arise and a proximal Merckx (Leuven, Belgium), we were able to collect end sometimes indicated by the still attached seed coat flowering material of Afrothismia hydra from Korup (Fig. 5). The distal pole also rarely develops scale National Park near Chimpanzee Camp (5°3′50,20′′N/ leaves (Fig. 6). Intracellular raphides are visible exter- 8°51′25,23′′E, c. 136 m above sea level, 9 September nally because of their distinctive light reflection and 2006, no. 188 Imhof & Sainge) and Rengo Rock Camp the root extensions have grown to a length of 13 mm in (5°2′11,16′′N/8°49′50,58′′E, c. 120 m above sea level, the root–rhizome system in Figure 6. 10.09. 2006, no. 189 Imhof & Sainge). After 2 weeks of Eventually, the rhizome elongates without develop- fixation in FPA (37% formalin : propionic acid : 50% ing root tubercles, but bears scale leaves and finally ethanol at a ratio of 0.5 : 0.5 : 9), specimens were terminates in a flower (Fig. 7). Flower buds show the transferred into 70% ethanol for permanent storage tepal lobes and the urceolate inferior ovary early, but at the Philipps-University Marburg. Unexpectedly, the corolla tube is still short (Fig. 8). At this stage the dissection under the microsope revealed very young pre-anthetic flower is still encompassed by the sub- plant stages within the few earth clumps that accom- tending bract. The axils of the scale leaves bear the panied the larger specimen and that could be ordered beginnings of side shoots (Figs 8, 10), the uppermost according to the time passed after germination. Mor- of which will continue the inflorescence for each phological investigation and photography was carried branch of the monochasial cyme. These side shoots out using a Leica SD6 microscope in combination with always develop additional root tubercle aggregates at a digital photo device (Leica DFC280). their bases (Fig. 9). After anthesis the corolla tube and lobes disinte- RESULTS grate and only the capsule crowned with the style Seeds of Afrothismia hydra are shaped like a grain of remains. The ovary wall is translucent and the devel- rice, 600–700 mm long and 250–300 mm wide. The two oping seeds are visible, attached to the column-like ends of the seeds are slightly dissimilar; one is rather placenta connected to the basal (and initially also the rounded, the other more truncated. The truncated upper) part of the capsule (Fig. 10). As a result of elon- side is attached to the placenta. The outer seed coat gation of the basal part of the placenta, the connection is hyaline, encompassing a light-brown inner struc- to the upper part of the capsule is disrupted; the oper- ture with dark tips (Fig. 1). culum separates from the proximal part of the fruit The youngest stage found after germination is barely (thus characterized as a pyxidium, Fig. 10) and the longer than the seed itself and shows disruption of the seeds, arranged like the feathers in a feather duster, seed coat through the emergence of a first tubercle, yet are exposed (see inset of Fig. 10). Placental elongation missing a tail-like extension. The seed coat opened at elevates the seeds above flowering level (Fig. 10). the rounded tip of the seed, whereas the truncated end shows a hyphae firmly connected to the seed. The DISCUSSION young tubercle, with the exception of the outer one or two cell layers, contains whitish material (Fig. 2). The This article presents a process never previously next stage (Fig. 3) shows that the initial tubercle has depicted. Among MHP, only orchids (reviewed by grown a short tail-like extension (only about 150 mm Arditti et al., 1990; Peterson, Uetake & Zermer, 1998) long) and is fully packed with the whitish material, and Monotropoideae (for literature, see Introduction)

Figure 1–6. Fig. 1. Seed of Afrothismia hydra. A, truncated side; B, rounded side. Scale bar, 0.2 mm. Fig. 2. Germination of Afrothismia hydra, the seed coat disrupted as a result of primary root tubercle development. A hypha is firmly attached to one end of the seed (arrow). Scale bar, 0.2 mm. Fig. 3. A second root tubercle (2) has developed, not yet fully colonized by the root fungus. The first tubercle (1) shows distinct hyphal coils (arrow) in its tissue. First filiform root extensions (re) occur. The seed coat (sc) is still attached and close to the attachment point a third root initial (3) is visible. Scale bar, 0.2 mm. Fig. 4. More root tubercles and initials have developed, the sequence of which (1–5) is still detectable. Scale bar, 0.2 mm. Fig. 5. A primary shoot axis (s) becomes visible; at the growth pole (gp) new tubercles arise. The seed coat (sc) is still attached. Scale bar, 0.5 mm. Fig. 6. The shoot axis (s) with further tubercles has grown; a scale leaf (sl) is visible at the growth pole. The filiform root extensions (re) in this specimen reach 13 mm in length. Scale bar, 0.5 mm.

Figure 7–10. Fig. 7. Young flowering plant of Afrothismia hydra. Scale bar, 5 mm. Fig. 8. Flower bud of Afrothismia hydra enveloped in a bract (b). Corolla lobes (cl) and inferior ovary (o) are already differentiated. A scale leaf below (sl) bears an initial (i) for a side shoot. Scale bar, 1 mm. Fig. 9. Sympodium of Afrothismia hydra with six successive sympodial branches (1–7); the terminal parts of the older are already disintegrated (1*-4*). The bases of the side shoots possess a dense mass of root tubercles. Scale bar, 5 mm. Fig. 10. Close-up of Figure 9 showing an open (op) and a closed pyxidium (cp) and a developing flowering shoot (s). The seeds of the open pyxidium are exposed by the elongating basal part of the placenta (pp, placentophore). The young flowering shoot bears a flower bud (fb) as well as the first additional root tubercles (r) at the basal part. Inset: the elongation of the placentophore pushes the lid from the pyxidum. Scale bars, 1 mm. have been examined for their early ontogeny. Caddick primary shoot becomes visible during germination; et al. (2000) described floral ontogeny in Burmanni- instead, Afrothismia hydra seems to germinate with a aceae and, only once, young stages (but not the germi- root only. The root character of the first-emerging nation) of Voyria tenella Hook. (Gentianaceae) have structure becomes obvious when it develops into a been shown (Imhof, Weber & Gómez, 1994). In the light tubercle with filiform extension; this structure has of the results presented here, the seedling concepts for been proved to be a root in other studies (Imhof, 1999a, monocotyledons (Tillich, 1992, 1995) are difficult to 2006). Clearly, because of the difficulties in applying apply in Afrothismia. No cotyledon, hypocotyl or classical germination concepts, the comparable initial

© 2008 The Linnean Society of London, Botanical Journal of the Linnean Society, 2008, 157, 31–36 ONTOGENY OF AFROTHISMIA HYDRA 35 structures of orchids are termed ‘protocorms’ (Treub, of the pyxidium (see picture in Sainge & Franke, 1890; Bernard, 1909) and Velenovsky (1907) coined the 2005), it seems to be the result of meristem growth term ‘procaulom’ for the corresponding organ in the rather than cell elongation. Future anatomical inves- Ericaceae. ‘Protocorm’ is still in use as it represents an tigations will help to clarify this hitherto unknown organ distinct from the rest of the corm (Leroux, phenomenon. Ecologically, uplift of the seeds helps to Barabé & Vieth, 1997). In contrast, contemporary pub- widen seed dispersal when the seed puff is hit by lications on the germination of Monotropoideae avoid raindrops. Hence, in accordance with Stone (1980), the expression ‘procaulom’, but also hesitate to call the who assumed Thismia clavigera (Becc.) F.v.M. to be germinating organ a ‘root’, despite its identical appear- dispersed by rain splash, we believe Afrothismia ance to mature roots and the immediate development hydra to be ombrohydrochorous and, in analogy to of the hyphal mantle that is characteristic of roots gynophore and carpophore, we suggest the expression (Leake et al., 2004; Bidartondo & Bruns, 2005). How- ‘placentophore’ for the unique structure lifting the ever, in Voyria tenella, the youngest stage found (2 mm seeds from the pyxidium. in length) consisted entirely of root tissue, including a central vascular cylinder and the specific mycorrhizal ACKNOWLEDGEMENTS colonization pattern, without any trace of shoot tissue. We are very grateful to Dr George B. Chuyong (Buea) Only when a little star-like root clump has developed for his invitation, as well as to Vincent Merckx does the first shoot bud emerge (Imhof et al., 1994; (Leuven) for giving us the opportunity, to take part in Imhof, 1997). The results of the present study repre- his expedition. Many thanks also to Paula Rudall sent an analogous germination strategy. Considering (Kew) and an anonymous referee for their valuable the tiny seeds with few storage compounds and the fact remarks on the manuscript. that, in both Afrothismia spp. (Imhof, 1999a, 2006) and Voyria spp. (Imhof, 1997, 1999b), only the root tubercle REFERENCES parenchyma is able to break down hyphae for carbon Angiosperm Phylogeny Group (APG). 2003. An update of supply, immediate development of roots during germi- the angiosperm phylogeny group classification for the orders nation is essential in combination with an early colo- and families of flowering plants. Botanical Journal of the nization by the appropriate fungus (see Fig. 2). These Linnean Society 141: 399–436. constraints similarly hold for Monotropoideae and, Arditti J, Ernst R, Yam WT, Glabe C. 1990. 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