4
Subterranean Morphology
and Mycorrhizal Structures
Stephan Imhof, Hugues B. Massicotte,
Lewis H. Melville, and R. Larry Peterson
4.1
Introduction
For most, if not all plants, subterranean parts are
less known than their aerial counterparts, due in
part to the difficulty in extracting a complete root
system (see Kutschera and Lichtenegger 1982,
1992; Kutschera et al. 2009) and the lack of morphological information in floras and taxonomic
descriptions because many herbarium specimens
do not include underground parts such as roots
and rhizomes. Likewise, information from the
fossil record is biased towards aerial structures
(Peterson 1992) although there have been discoveries of fossils showing fungal associations with
underground organs (e.g., Kidston and Lang
1921, Remy et al. 1994; Taylor et al. 1995;
LePage et al. 1997; Stockey et al. 2001). To date,
fossils of root-fungal associations of mycoheterotrophic plants are unknown.
S. Imhof (*)
Spezielle Botanik und Mykologie, Fachbereich Biologie,
Philipps-Universität, 35032 Marburg, Germany
e-mail: imhof@uni-marburg.de
H.B. Massicotte
Ecosystem Science and Management Program,
University of Northern British Columbia,
3333 University Way, Prince George,
BC, Canada
L.H. Melville • R.L. Peterson
Department of Molecular and Cellular Biology,
University of Guelph, Guelph, ON, Canada
In autotrophic plants, many scientific questions can be dealt with using generalized concepts of root structure and function (e.g.,
Kutschera and Lichtenegger 1992; Polomski and
Kuhn 1998; Gregory 2006). However, this certainly does not hold for mycoheterotrophic (MH)
plants. The structure of roots, rhizomes, or subterranean scale leaves of MH plants intimately
linked to the association with soil fungi is of critical ecological relevance because these plants
essentially depend upon fungi for their carbon
and perhaps other nutrient needs. Hence, the subterranean organs of MH plants often show
remarkable morphological and anatomical adaptations to meet their specific requirements. This
chapter, therefore, addresses the importance of
morphology and anatomy to complement modern
methods for understanding the fungal colonization patterns in MH plants and their relationships
to function.
In the following, we summarize the current
knowledge of structural aspects of the underground parts (for a peculiar exception, see
Afrothismia) of MH plants ranging from bryophytes to angiosperms, the latter in systematical
order following the Angiosperm Phylogeny
Group (APG 2009), which has been regularly
updated by Stevens (2001 onwards). We are
aware of the gradual differences between species
in terms of mycorrhizal dependence, however,
due to space limitations, we focus on the visibly
achlorophyllous species, and only include the
partially mycoheterotrophs where they add to the
common picture.
V.S.F.T. Merckx (ed.), Mycoheterotrophy: The Biology of Plants Living on Fungi,
DOI 10.1007/978-1-4614-5209-6_4, © Springer Science+Business Media New York 2013
157
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S. Imhof et al.
The final section interprets the available information in terms of detecting phylogenetic trends
of MH plants, in order to understand their evolutionary history, a subject that is receiving considerable attention in the mycorrhizal literature (see
Brundrett 2002).
4.2
Nonvascular Plants
4.2.1
Aneura
Aneura mirabilis (Aneuraceae/Hepaticae) was
described as Cryptothallus mirabilis by von
Malmborg (1933, 1934), although it was noted
earlier around 1914 (Schiffner 1934) and suggested to be either an Aneura or Riccardia species (e.g., Denis 1919; Schiffner 1934). Recently,
Cryptothallus was formally transferred to Aneura
by Wickett and Goffinet (2008) based on molecular and morphological characteristics. This decision is supported by the observation that the
endophyte in Aneura mirabilis belongs to the
same genus (Tulasnella) as that in Aneura pinguis (Bidartondo et al. 2003), and the mycorrhizal pattern in both species is very similar
(Ligrone et al. 1993).
Aneura mirabilis mostly occurs in maritime
climates (e.g., Sjörs 1949; Williams 1950;
Petersen 1972; Wiehle et al. 1989; Sergio and
Seneca 1997; Sergio and Garcia 1999; Boudier
et al. 1999) in cool, humid, mostly peaty environments with large mats of bryophytes (Wiehle
et al. 1989). Only a part of the seta and the sporangium is elevated above the surrounding mats
consisting of several moss species (von Malmborg
1933; Wiehle et al. 1989; Sergio and Garcia 1999;
Boudier et al. 1999). The whitish, vermiform to
lobular-coralloid, brittle gametophytes are only a
few centimeters in length and remain embedded
within the mosses or litter. Male and female
gametophytes differ in lobe shape (Williams
1950; Benson-Evans 1952; Wiehle et al. 1989).
The first structural work on the mycorrhiza in
A. mirabilis by Denis (1919) revealed intracellular fungal colonization with hyphal coils in the
ventral (lower) part of the thallus, although he
considered the specimen as an albino of another
chlorophyllous Aneura species. Von Malmborg
(1933) observed hyphae growing through the seta
into the sporangium and assumed that the fungus
is distributed together with the spores. Williams
(1950), unable to confirm this statement of von
Malmborg (1933), published the first detailed
investigations and provided drawings of the full
life cycle, including the pattern of mycorrhizal
colonization. The thallus lobes bearing antheridia
or archegonia are devoid of hyphae; starch is
deposited in the upper part of the thallus and
around the gametangia. In an ultrastructural comparison of green hepatics and Aneura mirabilis
(still called Cryptothallus), Pocock and Duckett
(1984) confirmed the concentration of fungal
colonization in the lower half of the thallus, but
more recently, Ligrone et al. (1993) showed that
the upper parts of the thallus can also become
colonized in later stages. Rhizoids are also colonized, albeit in an uncoiled manner (Duckett et al.
1990). Most likely, these straight hyphae within
rhizoids represent the connection to the external
substrate. The carbon of this liverwort probably
comes from surrounding beech (Fagus sylvatica)
trees (Read et al. 2000; Bidartondo et al. 2003),
with which it is connected via the mutual
Tulasnella mycorrhizal fungus, although Ligrone
et al. (1993) found dissimilar dolipore structures
in the endophytes of birch (Betula spp.) and
Cryptothallus. The fungal coils within the thallus
cells eventually degenerate to dark masses (von
Malmborg 1933; Williams 1950; Pocock and
Duckett 1984), interpreted as digestion, and the
cells can be reinfected (Ligrone et al. 1993).
Williams (1950) and Pocock and Duckett (1984)
stressed the difference in fungal identity between
Aneuraceae hosting basidiomycetes and resembling orchid mycorrhiza, in contrast to other liverworts hosting “phycomycetous” (today
considered as Glomeromycota, Schüßler et al.
2001) endophytes forming arbuscular mycorrhiza
(AM) in higher plants. This fact, together with
the identification of the fungi in A. mirabilis as
Tulasnella spp. (Read et al. 2000; Bidartondo
et al. 2003), has led to the hypothesis of a novel
acquisition of Tulasnella spp. as associates in
Aneuraceae. By attaining an epiphytic habit during
phylogeny, liverworts may have lost the original
4
Subterranean Morphology and Mycorrhizal Structures
symbiotic relationship with Glomeromycota.
Secondarily terrestrial Aneuraceae then could
have associated with new fungal partners (Kottke
and Nebel 2005; Bidartondo and Duckett 2009).
4.3
Seedless Vascular Plants
Mycoheterotrophy in the seedless vascular plants
is restricted to their gametophytic phase (Read
et al. 2000; Smith and Read 2008). Genera possessing acholorophyllous gametophytes (and
photosynthetic
sporophytes)
belong
to
Lycopodiaceae (e.g., Lycopodium, Huperzia,
Fig. 4.1a, b), Ophioglossaceae (Ophioglosssum,
Botrychium,
Helminthostachys,
Mankyua
Fig. 4.1d–f), Psilotaceae (Psilotum, Tmesipteris,
Fig. 4.1g, h), some species of Schizaea and
Actinostachys in the Schizaeaceae, and the monotypic species Stromatopteris moniliformis in the
Gleicheniaceae.
4.3.1
Lycopodiaceae (Fig. 4.1a–c)
It was recognized very early that subterranean
gametophytes of several Lycopodium species are
associated with endophytic fungi (Treub 1885,
1890; Bruchmann 1885, 1910; Lang 1899;
Burgeff 1938). Illustrations in Burgeff (1938)
and Boullard (1979) clearly show that fungi colonize the basal region of gametophytes shortly
after spore germination. Mature subterranean
gametophytes show variations in form from discshaped with convoluted margins (L. clavatum,
Fig. 4.1b, L. obscurum) to elongated, cylindrical
structures (L. complanatum = Diphasiastrum
complanatum, Bierhorst 1971; Gifford and Foster
1996). Gametophytes of all species have fungal
colonization restricted to a zone underlying more
surficial cells that give rise to antheridia and
archegonia (Bierhorst 1971, Fig. 4.1c).
Although the identity of the fungus was
unknown in these early studies, it was described
as being aseptate and forming intracellular hyphal
coils. An ultrastructural investigation of the fungal endophyte in association with achlorophyllous gametophytes of L. clavatum showed that
159
complex hyphal coils and vesicles formed but
arbuscules were absent (Schmid and Oberwinkler
1993a). Entrance of the fungus occurred either
through rhizoids, degenerated epidermal cells, or
between epidermal cells. Once within parenchyma cells of the gametophyte, host-derived
plasma membrane and wall material was deposited around invading hyphae. Hyphae were multinucleate and contained bacterium-like organelles
(BLOs). Hyphae became progressively more vacuolated and ultimately degenerated. The authors
came to the conclusion, based on a number of
unusual structural features, that this fungus-gametophyte interaction was unlike anything described
in the literature and could not be attributed to a
known mycorrhizal association. They therefore
proposed a new term “lycopodioid mycothallus
interaction” to describe the association.
More recently, based on structural features of
the fungi within cells, the fungal symbionts in the
gametophytes of all seedless vascular plants were
suspected to be members of the Glomeromycota
and to have the Paris-type arbuscular mycorrhiza
association (Read et al. 2000). Molecular studies
have confirmed this for the fungus associated
with two subterranean gametophytes of Huperzia
hypogaea collected in Ecuador: the fungus was
identified as belonging to a specific clade of
Glomus-Group A (Winther and Friedman 2008).
Observations of these sectioned gametophytes
confirmed earlier reports that hyphal coils are
restricted to the basal region and that arbuscules
are not formed.
4.3.2
Ophioglossaceae (Fig. 4.1d–f)
Gametophytes of Ophioglossum may be cylindrical (O. nudicaule, O. vulgatum, Boullard 1957;
Gifford and Foster 1996), globose (O. crotalophoroides, Mesler 1976), or highly branched (O.
palmatum, Mesler 1975). Fungal hyphae may be
evenly distributed but avoiding the meristematic
area and gametangia (Bierhorst 1971). Fungal
colonization occurs immediately after spore germination (Campbell 1908) and gametophytes do
not develop unless they are associated with the
appropriate fungus. Hyphal coils, some of which
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Fig. 4.1 (a–c) Lycopodiaceae, (d–f) Ophioglossaceae, (g–j)
Psilotaceae. (a) Lycopodium obscurum sporophyte showing strobili. (b) Lycopodium clavatum mycoheterotrophic
S. Imhof et al.
gametophytes with shoots (arrowheads). (c) Section of
L. obscurum gametophyte showing zone of arbuscular
mycorrhizal fungi (arrowheads) and antheridia (arrows).
4
Subterranean Morphology and Mycorrhizal Structures
161
have undergone degeneration, are illustrated in
gametophyte cells of O. pendulum (Burgeff
1938). Mesler (1975) described the endophytic
hyphae in gametophytes of O. palmatum as being
non-septate and multi-nucleate. He also showed
what he interpreted as vesicles in some gametophyte cells. Mesler (1976) gave a similar description of the fungal endophyte in O. crotalophoroides.
Details at the ultrastructural level are lacking for
gametophytes of Ophioglossum spp. and the
identity of the fungus remains unknown.
Gametophytes of Botrychium also vary in their
morphology from being tuber-like to disc-shaped
(Bruchmann 1906; Burgeff 1943; Gifford and
Foster 1996; Winther and Friedman 2007); endophytic fungi are restricted to a basal zone of parenchymatous cells (Bruchmann 1906; Bierhorst
1971). The fungus in B. lunaria has been
described as forming aseptate intracellular coils
and irregular vesicles (Bruchmann 1906). An
ultrastructural study of the fungus-gametophyte
interaction of this species (Schmid and
Oberwinkler 1993b) has provided additional
details. The intracellular hyphae contain vacuoles, endoplasmic reticulum, mitochondria, and
lipid-like bodies. They are enclosed by hostderived plasma membrane and wall material that
shows irregular outgrowths. Vesicles, some very
irregular in shape, contain BLOs, and lipids; they
can become very enlarged and then undergo
degeneration. The identity of the fungal endophyte has been determined for the subterranean
gametophytes of B. crenulatum (Fig. 4.1f) and B.
lanceolatum based on DNA sequence data
(Winther and Friedman 2007). The endophytes in
both species belong to a major clade of glomalean
fungi, Glomus-group A.
A third genus in the Ophioglossaceae,
Helminthostachys, is monotypic (H. zeylanica)
and native to the Indo-Malayan region (Gifford
and Foster 1996). It also forms achlorophyllous
mycoheterotrophic gametophytes (Lang 1902)
but little is known of the fungal association.
A new genus and species (Mankyua chejuense)
has been described from Cheju Island, Korea
(Sun et al. 2001) based on differences in sporophyte morphological characters from the other
genera in the family. Gametophytes have not been
described but are presumed to be subterranean.
Fig. 4.1 (continued) (d) Shoot of Botrychium virginianum
with fertile segment of leaf with sporangia (arrowhead).
(e) Mycoheterotrophic gametophyte (arrow) of B. virginianum with a root (arrowhead) and base of a shoot (double
arrowhead). (f) Intracellular hyphal coils of GlomusGroup A in a Botrychium crenulatum mycoheterotrophic
gametophyte. Photo courtesy of Jennifer Winther. (g)
Shoots of Psilotum nudum with synangia (arrowheads).
(h) Branched mycoheterotrophic gametophyte of P. nudum.
(i) Intracellular hyphal coils of an arbuscular mycorrhizal
fungus in a sectioned P. nudum mycoheterotrophic
gametophyte stained with Toluidine blue O. (j)
Transmission electron micrograph of hyphae within a
mycoheterotrophic gametophyte of P. nudum showing the
interface consisting of host plasma membrane (perifungal
membrane) (arrowheads) and host-derived intracellular
matrix (arrows). (k) Buxbaumia aphylla sporophyte,
1.5 cm high
4.3.3
Psilotaceae (Fig. 4.1g–j)
The two genera, Psilotum, with two species and
Tmesipteris, with ten species, have historically
been of considerable interest because of the belief
that they represented some of the most primitive
extant seedless vascular plants (Bierhorst 1971;
Gifford and Foster 1996). The lack of roots and
the presence of much reduced leaf-like structures
of the sporophyte strengthened this view.
However, based on molecular evidence, Smith
et al. (2006) include this family within the
Psilotales, an order belonging to the extant ferns.
Subterranean gametophytes of Psilotum are
highly variable cylindrical structures (Fig. 4.1h)
sometimes showing repeated branching (Bierhorst
1971). Asexual reproductive propagules (gemmae) are frequently developed (Bierhorst 1971).
Darnell-Smith (1917) was the first to succeed in
achieving spore germination and to monitor early
stages in gametophyte development. He reported
that endophytic fungi appeared as dense “skeins”
within interior cells of gametophytes and that
hyphae entered rhizoids. Other authors have
described an aseptate intracellular fungus thought
to be a phycomycete in either field-collected
gametophytes (Burgeff 1938; Boullard 1957) or
162
S. Imhof et al.
gametophytes growing in greenhouse pots containing various angiosperm species (Bierhorst
1953). Aspects of the ultrastructure of the gametophyte-fungus interaction have been described
from gametophytes collected from greenhouse
pots (Davis 1976; Peterson et al. 1981). The fungus in these gametophytes is aseptate and forms
complex coils (Fig. 4.1i) that undergo degeneration; arbuscules are not formed. Intracellular
hyphae are separated from the gametophyte cell
cytoplasm by host-derived plasma membrane
(perifungal membrane) and interfacial matrix
material (Peterson et al. 1981, Fig. 4.1j), characteristics of arbuscular mycorrhizal associations
(Bonfante and Perotto 1995). To date, the fungus
has not been identified but the structural characteristics are typical of a Paris-type arbuscular
mycorrhiza.
The fungal endophyte in subterranean gametophytes of Tmesipteris tannensis was described
by Lawson (1917) and Holloay (1921) as consisting of intracellular fungal coils (pelotons).
As with Psilotum gametophytes, the identity of
the fungus has not been determined.
4.3.4
Schizaeaceae
The gametophytes of the genus (Schizaea) in this
leptosporangiate fern family may either be
surficial and green, subterranean and achlorophyllous, or a combination of both, depending on
species and habitat (Bierhorst 1968, 1971).
Gametophytes of all species are associated with
endophytic fungi that have been described as
aseptate and frequently associated with rhizoids
(Bierhorst 1971; Swatzell et al. 1996).
Gametophytes of all species in the genus
Actinostachys are axial structures that are subterranean with fungal hyphae confined to a distinctive zone (Bierhorst 1968). The identity of the
fungi associated with achlorophyllous gametophytes in these two genera is unknown.
4.3.5
Gleicheniaceae
The monotypic genus Stromatopteris moniliformis (subfamily Stromatopteridaeae), has axial
subterranean gametophytes with coiled fungal
hyphae (Bierhorst 1971), reminiscent of Paristype arbuscular mycorrhizas. Although Bierhorst
(1971) concluded that the fungus present in the
gametophyte is the same as that in the photosynthetic sporophyte, this needs to be confirmed with
molecular methods.
Experimental evidence confirming transfer of
nutrients from fungi to the subterranean gametophytes of all seedless vascular plants is lacking.
4.4
Gymnosperms
4.4.1
Podocarpaceae
The New Caledonian endemic Parasitaxus usta
(not P. ustus, as many authors repeated the linguistically incorrect transfer from Podocarpus to
the feminine genus Parasitaxus by de Laubenfels
1972) is a succulent shrub or small tree (up to
2 m high) with wine-red scale leaves (Cherrier
et al. 1992; Schneckenburger 1999), unable to
photosynthesize (Feild and Brodribb 2005) and
only occurring closely associated with
Falcatifolium taxoides (also Podocarpaceae,
Sinclair et al. 2002). Root graft-like subterranean
connections between the two species have led to
the notion of parasitism in P. usta (de Laubenfels
1959; Köpke et al. 1981). However, Cherrier
et al. (1992) and an English version of that paper
adding a SEM micrograph (Woltz et al. 1994)
found an endophytic mycelium (called “ectendomycelium”) in both species, together with
haustorial-like connections apparent at the cellular level developing in tissues up to the cambium
of F. taxoides. The authors assume a symbiotic
association of the three partners but, based on
their anatomical observations, are convinced of
parasitism in this case. The latest investigation on
P. usta confirms the intimate vascular association
of both species, but results from stable carbon
isotope investigations suggest that most carbon is
provided by the fungus (Feild and Brodribb
2005). With respect to water physiology however,
P. usta has higher stomatal conductance and
lower water potential values relative to its host,
which is typical for parasitic angiosperms (Feild
and Brodribb 2005). Hence, apart from being a
4
Subterranean Morphology and Mycorrhizal Structures
gymnosperm, woody, and relatively large,
Parasitaxus is even more unique among heterotrophic organisms in possibly being a parasitic
and mycoheterotrophic plant at the same time.
4.5
Monocots
4.5.1
Petrosaviaceae (Petrosavia)
The three species of Petrosavia are distributed
from Japan to Java. The external morphology of
the underground structures does not differ much
among the species. Their subterranean rhizomes
can be branched and thus, may bear several
10–15 cm high scapes with terminal racemes or
corymbs of white flowers. Rhizomes measure up
to 1.5 mm in diameter and are densely covered by
sheathing scale leaves (Groom 1895a; Makino
1903; Stant 1970; Jessop 1979; Chen and Tamura
2000; Cameron et al. 2003). The filiform, hairless, approximately 0.5 mm thick and sparsely
branched adventitious roots, are initiated from the
rhizome, especially close to the base of the scape.
They most likely originate from the axils of the
scale leaves, as do the rhizome branches (Groom
1895a). In Petrosavia sakuraii, the roots predominantly grow horizontally through the substrate
and can be up to 20 cm long (Watanabe 1944).
This author also reports hyphae penetrating into
the roots 2–5 mm proximal from the root tip.
The epidermis is either ephemeral (Groom
1895a) or partly persistent (Stant 1970). The cortex consists of a suberized exodermis, 4–6 layers
of parenchyma cells, and an endodermis with
particularly strong u-shaped tertiary thickenings
surrounding the tetrarch central cylinder (Groom
1895a; Watanabe 1944; Stant 1970). This is similar to many mycoheterotrophic Burmanniaceae
(Johow 1889; Uphof 1929) with Dictyostega
orobanchoides as an extreme example (Imhof
2001, Fig. 4.4f). Watanabe (1944) mentions that
segments of older roots lose the cortex parenchyma but remain connected to the rhizome by
the central cylinder that is surrounded by the
fortified endodermis. The maintenance of connectivity between roots and rhizomes bearing
inflorescences is particularly important for MH
163
plants having filiform roots, since not only water
and nutrients but also carbohydrates must be
transported through these comparatively long
structures. A tertiary endodermis, a synapomorphy of monocots (Esau 1965), seems to be less
costly than the production of layers of lignified
tissue, which is the equivalent option for nonmonocots in order to protect the connectivity.
This economical advantage of monocots may be
part of the explanation as to why monocots
include disproportionately so many MH plants
(Imhof 2010).
Previous investigations on Petrosavia (Groom
1895a; Watanabe 1944; Stant 1970) report coiled
mycorrhizal hyphae within the cortex parenchyma cells. The figures and descriptions of
Watanabe (1944) resemble a Paris-type AM but
without the typical lateral arbuscules, which is
similar to the mycorrhiza found in Voyria truncata (Gentianaceae, Imhof and Weber 1997). The
advantage of the frequent feature of MH plants of
having a specialized mycorrhizal colonization
pattern allowing a selective digestion of hyphae
while keeping the fungus alive (see further), is
not apparent. Petrosaviaceae are a rather basal
clade of the monocots (Cameron et al. 2003; APG
2009), which might explain its plesiomorphic,
i.e., basic mycorrhizal pattern. Most recently,
Yamato et al. (2011a) confirmed the structural
descriptions of Watanabe (1944), and revealed
this mycorrhiza as an association with a highly
specific clade of Glomus-group A.
4.5.2
Thismiaceae
(Figs. 4.2–4.4 and 4.10)
Thismiaceae are either considered to be a tribe,
Thismieae, in the Burmanniaceae (e.g., Jonker
1938, Cronquist 1988) or a separate family (e.g.,
Agardh 1858; Thorne 1992; Takhtajan 1997;
Stevens 2001 onwards). APG (2009) is still reluctant to separate them from Burmanniaceae but
acknowledge the arguments for separation given
by Merckx et al. (2006). We regard them as a
family based on floral morphology (e.g., Maas
et al. 1986; Caddick et al. 2000) and molecular
evidence (Merckx et al. 2006).
164
Fig. 4.2 (a–h) Afrothismia hydra, (i, j) Afrothismia winkleri (Thismiaceae). (a) Seed (left, 0.6 mm long) and an
early germination stage (right) of A. hydra with disrupted
S. Imhof et al.
seed coat (sc), giving rise to a first root tubercle. (b–d)
More tubercles develop successively at the base of the initial one and the root extensions elongate. The seed coat (sc)
4
Subterranean Morphology and Mycorrhizal Structures
165
This genus is by far the largest of the family, with
a worldwide, although mostly tropical, distribution. The underground structures are quite variable. Most species have horizontal runner-like,
vermiform roots of 1–2 mm in diameter which
bear root-borne shoots (e.g., Groom 1895b;
Warming 1901; Pfeiffer 1914; Chantanaorrapint
2008; Chiang and Hsieh 2011) and give rise to
additional similar roots where the shoots emerge,
thus forming star-like clusters (e.g., Groom
1895b; Bernard and Ernst 1910; Pfeiffer 1914;
Larsen 1965; Saunders 1996; Yang et al. 2002;
Wapstra et al. 2005). This indicates the trend
towards a star-like radiating root system, typical
for MH plants. The runner-like parts of the roots
can be short (e.g., Thismia appendiculata,
Schlechter 1919), and in this case, the shoots
emerge in nest-like tufts above the soil surface. In
other species, the root system is reduced to a coralloid structure, e.g., Thismia yorkensis (Cribb
1995), T. goodii (Kiew 1999), or T. clandestina
and T. versteegii (Bernard and Ernst 1911).
Thismia versteegii shows similarities to the
unique fan-shaped roots of Thismia clavigera
(Stone 1980), which probably develop through
short, dichotomously branched and congenitally
merged roots. Thismia annamensis and T. tentaculata have short rhizomes bearing a dense
covering of vermiform roots (Larsen and
Averyanov 2007), also resulting in a star-like root
system. This is morphologically similar but ontogenetically quite different from the other species
mentioned above. The decision whether a condensed root system is developed by root-borne
shoots or shoot-borne roots is often difficult to
make and sometimes requires anatomical investigations (see Imhof 2004). Finally, some neotropical species have globose tubers (see Fig. 4.10g),
from which a shoot as well as numerous filiform
roots arise (e.g., Thismia hyalina, Miers 1866,
T. glaziovii, Poulsen 1890a, T. janeirensis,
Warming 1901, T. panamensis, Maas et al. 1986,
Fig. 4.8j, T. saülensis, Maas and Maas 1987).
Inferred from T. luetzelburgii, these tubers are
roots, giving rise to up to four endogenous flowering
shoots. The filiform roots can develop new tubers
at their apices (Goebel and Süssenguth 1924).
The fungal colonization of Thismia spp. has
been investigated quite early and in great detail.
Like many other MH plants, Thismia also shows
different fungal morphologies in distinct tissue
Fig. 4.2 (continued) is still attached. The whitish content
is the fungal colonization. (e) The rhizome has developed
into a shoot terminated by a 1 cm long flower. (f) A. hydra
in its natural habitat with the filiform root elongations
superficially clinging to the substrate. (g) A. hydra showing the strictly sympodial flowering mode with clusters of
root tubercles at the base of each pedicel. Three basal
flowers (A, B, C) have already detached, the following are
in dissemination stage with placentophore developed (D),
in fruit (E) and in bud (F). Note early tubercle (tb) development at the base of the flower bud. (h) Close up of the
placentophore (pl). (i) Flower of A. winkleri, measuring
1.5 cm from the subtending scale leaf to the bending of the
tube. (j) Root/rhizome system of A. winkleri
4.5.2.1 Haplothismia, Oxygyne, Tiputinia
The extremely rare Haplothismia exannulata from
India has vermiform to tuberous, up to 3.5 cm
long, roots radiating from the shoot base (Airy
Shaw 1952; Sasidharan and Sujanapal 2000). For
Oxygyne triandra from Cameroon (probably
extinct, Yokoyama et al. 2008), the subterranean
organs are unknown. The Japanese species,
O . shinzatoi and O. yamashitae, only known from
their type localities, have vermiform roots
(Yokoyama et al. 2008; Yahara and Tsukaya 2008),
and the original description of O. hyodoi, also
from Japan, states “rhizoma repens” (Abe and
Akasawa 1989). Tiputinia foetida is represented
by a single specimen from Ecuador (Woodward
et al. 2007), measuring about 9 cm in length. The
largest part of it is an orthotropous, vermiform,
4 mm thick root, giving rise to two subterranean
shoots, with only the terminal flower being epiterrestrial. The root cortex contains “intracellular,
looped, septate” hyphae (Woodward et al. 2007).
4.5.2.2 Thismia (Fig. 4.10g, h)
166
Fig. 4.3 Afrothismia saingei (Thismiaceae). (a) Rhizome
tip with many root tubercles; some tubercles have been
S. Imhof et al.
detached. The characteristic hyphal loops (green rectangle), developed in spiral lines within the tubercles, are
4
Subterranean Morphology and Mycorrhizal Structures
167
compartments, which sometimes are anatomically
different. In T. clandestina (coralloid root system) and T. aseroe (vermiform roots), the outer
cortex parenchyma layers contain straight hyphae
with only a few coils; in the middle cortex layers,
the hyphae are coiled but not digested; the inner
layers show amorphous fungal material (Groom
1895b; Janse 1896; Meyer 1909; Bernard and
Ernst 1911). In T. americana, T. rodwayi, and
T. javanica (vermiform roots), straight hyphae
are missing, instead, the outer cortex layer is
occupied by coiled hyphae which do not degenerate. Digestion takes place in the inner cortex
(Bernard and Ernst 1910; Pfeiffer 1914; Coleman
1936; McLennan 1958; Campbell 1968). Of the
species having root tubers, T. luetzelburgii
(Goebel and Süssenguth 1924) and T. glaziovii
(Poulsen 1890a) have been investigated. They
also show compartmentation of digested and
undigested fungal material, whereas the digestion is more prominent in the proximal and central part of the tuber. The filiform roots connecting
the mother tuber with smaller daughter tubers
bear straight undigested hyphae linking the two
tubers (Goebel and Süssenguth 1924). This is
partly reminiscent of structures found in
Afrothismia spp. (see next paragraph).
Due to the structural characteristics typical of
a Paris-type AM, the fungus colonizing Thismia
4.5.2.3 Afrothismia (Figs. 4.2–4.4)
The genus Afrothismia from tropical Africa is
characterized by its dense aggregates of small
tuberous roots elongated by a filiform extension
of various lengths between the species (Figs.
4.2g + j, 4.3a + c, and 4.4b). Although our chapter
deals with subterranean organs, this is not entirely
correct for some Afrothismia spp. In fact, the
peculiar root/rhizome/shoot systems often grow
entirely epiterrestrially (Fig. 4.2f), the filiform
part of the roots clinging to rotten wood, leaf litter, or bare soil (e.g., A. foertheriana, Franke
et al. 2004, A. hydra, Sainge and Franke 2005,
A. winkleri, Imhof pers. observ., Fig. 4.2i–j). Only
A. baerae (Cheek 2003a) and A. gesnerioides
(Imhof pers. observ., Fig. 4.4a) are known to be
rooted in the soil. The latter two species also differ by their conspicuously short filiform parts of
the roots (Fig. 4.4b, Cheek 2003a; Maas-van de
Kamer 2003). Afrothismia zambesiaca, described
from a herbarium specimen collected in 1955, is
only inferred to have an underground stem with
bulbils (Cheek 2009). The ontogeny of Afrothismia
hydra from seed to the open fruit has been
Fig. 4.3 (continued) visible from the outside. (b) Close
up of the green rectangle in (a). Tangential section through
two hypodermal cells colonized by looped hyphae that
are connected to each other. (c) Specimen (Wilks No.
1179) of A. saingei under investigation from the
Herbarium in Utrecht, labeled as Afrothismia winkleri.
(d) Cleared preparation of a filiform root extension showing straight growing hyphae and vesicles. (e) Transverse
section through a rhizome of A. saingei with coils of
enlarged hyphae (vh) in the cortex. Mostly only once per
tubercle these hyphae transit (green circle) over an inconspicuous separating layer (sl) into the hypodermis of a
tubercle to start the spiral line of hyphal loops (see (a, b)).
(f) Transverse section through a shoot/pedicel of A.
saingei. The enlarged hyphae in the rhizome cortex (e) are
continuous with the straight growing, also quite large
hyphae (sh) in the outer shoot cortex, thus connecting the
spatially separated tubercle clusters along the plant (c).
The inner cortex (ic) is free of hyphae. (g) Longitudinal
section through a young root tubercle showing the looped
coils in an alternating pattern in the hypodermis as to be
expected from its spiral arrangement (1–5) whereas all
other cortex cells contain degenerated hyphal coils (hc−).
(h) Longitudinal section through an old root tubercle
where the digestion of hyphal coils has advanced but the
epidermis now contains straight growing, nondegenerated hyphae (sh) linking those in the filiform root extension (she, see (d)) with the enlarged hyphae in the rhizome
cortex (see (e)). (i) Schematic view of the mycorrhizal
colonization pattern in A. saingei: Straight hyphae grow
through root extension and tubercle epidermis, enter the
rhizome cortex becoming enlarged and coiled, transit
once per tubercle into the hypodermis of the tubercle
starting a spiral line of looped interconnected hyphae
around it (hl), and from there send hyphal branches into
the rest of the cortex parenchyma for digestion (dh). The
red line signifies an impenetrable barrier to the fungus.
The spatially separated clusters of tubercles share the
fungus via straight hyphae growing along the shoot axis
in its outer cortex parenchyma
spp. almost certainly belongs to the
Glomeromycota. This has recently been confirmed
for T. rodwayi using molecular identification
methods (Merckx et al. 2012).
168
Fig. 4.4 Afrothismia gesnerioides (Thismiaceae). (a) A.
gesnerioides emerging about 1.5 cm above the soil.
S. Imhof et al.
In contrast to many other Afrothismia spp., roots and
rhizome are subterranean. (b) Root/rhizome system of
4
Subterranean Morphology and Mycorrhizal Structures
described (Imhof and Sainge 2008), and since
structurally the genus is quite consistent, this
example will be detailed here to represent the
whole genus. With germination, a root tubercle
without a filiform extension is generated (Fig.
4.2a). Successively, more tubercles develop on a
rhizome that is gradually increasing in size and the
root extensions elongate (Fig. 4.2b–d). This creates a globose to ovate, coarsely echinate structure
due to the characteristic roots. At some point, the
rhizome proceeds to grow without root development. This axis, now more accurately called a
shoot, will terminate with a flower (Fig. 4.2e), and
a side shoot appears in the uppermost scale leaf of
the shoot. The base of this side shoot also bears a
cluster of tubercles with extensions, and this shoot
will also end in a flower. This sympodial pattern is
repeated several times (Fig. 4.2g). The fruit is a
pyxidium, opening by means of a placentophore
(Fig. 4.2h, see details in Imhof and Sainge 2008).
The fungal colonization of Afrothismia saingei
is an extreme example of mycorrhizal complexity
(Imhof 1999a, treated as A. winkleri1). Briefly,
the pattern of colonization is as follows (sche-
169
According to Maas-van de Kamer and Maas (2010), the
material under investigation in Imhof 1999a (=Wilks no.
1179, received from the herbarium of Utrecht, labeled as
A. winkleri) turned out to be A. saingei (Franke 2004),
synonymous to A. gabonensis (Dauby et al. 2008).
matic view on Fig. 4.3i). The filiform root extension bears straight hyphae, continuous with those
in the epidermis of the tubercle (Fig. 4.3d + h).
These hyphae never pass from the epidermis into
the cortex of the tubercle but proceed around it
towards the rhizome. As soon as the fungus
reaches the rhizome at the tubercle base, it colonizes the rhizome cortex tissue with coiled, swollen, vesicle-like structures, but still does not show
signs of degeneration (Fig. 4.3e). From there, few
hyphae re-enter the tubercle from the rhizome
cortex, and grow towards the subepidermal layer
of the tubercle (Fig. 4.3e). Characteristic loops of
hyphae are developed in the subepidermal cells
(Fig. 4.3b + g), and an upward spiral line of cells
containing such looped hyphae proceed around
the tubercle (Fig. 4.3a). No digestion of hyphae
occurs to this stage. Side branches from these
hyphal loops enter the other cells of the tubercle
cortex, where they degenerate to amorphous
clumps (Fig. 4.3g, h). Connections to more distant tubercle clusters along the plant are provided
by straight growing hyphae in the outer cortex of
the shoot internodes (Fig. 4.3f, see details in
Imhof 1999a). This complicated plant structure
and colonization pattern represent a sophisticated
and ecologically functional system. The filiform
root extensions increase the surface for contact
with and invasion by hyphae, the root tubercle
increases the number of cells for colonization by
Fig. 4.4 (continued) A. gesnerioides, about 1 cm wide. (c)
Transverse section through a rhizome (rh) and longitudinal sections of root tubercles (tb) showing starch grains
(st) in the inner rhizome cortex and uncolonized root cortex, straight hyphae (sh) in the outer rhizome cortex, nondegenerated dense hyphal coils in the third cell layer of the
root tubercle (hc+) and degenerated hyphal coils in the
tubercle parenchyma (hc−). The hypodermis (hd) is largely
collapsed. (d) Longitudinal section through a young tubercle of A. gesnerioides where the hypodermis (hd) is still
visible. Fungal colonization has just started from the tubercle base in the third cell layer (hc+) and starch depositions
(st) are still present in the parenchyma, which will disappear when fungal colonization proceeds. First degenerated
hyphal coils (hc−) are also present in the inner parenchyma.
The root extension has not yet developed. (e) Longitudinal
section through a root tip of an older tubercle showing the
root extension (ex) partly in transverse view, colonized by
straight hyphae (sh) which proceed into the root epidermis
(ep, see (f)). (f) Root tubercle epidermis (ep) only contains
straight growing hyphae (sh) which never penetrate the
hypodermis (hd, collapsed) but are continuous with those
in the outer rhizome cortex. (g) Four neighboring tissues of
the root tubercle hosting distinct morphotypes of fungal
colonization: epidermis (ep) with straight hyphae (sh),
hypodermis (hd) as a barrier to the fungus, the third root
layer with nondegenerated dense hyphal coils (hc+) and
the multilayered root parenchyma containing degenerated
hyphal coils (hc−). (h) Transition of colonization (green
oval) between the straight hyphae (sh) in the outer rhizome
cortex and the nondegenerating hyphal coils (hc+) in the
third root layer of the tubercle across a layer continuous
with the otherwise impenetrable hypodermis (hd).
Uncolonized parenchyma cells and the inner rhizome cortex contain starch grains (st). (i) Schematic view of the
mycorrhizal colonization pattern in A. gesnerioides:
straight hyphae grow through root extension, tubercle epidermis and outer rhizome cortex, transit at the base of the
tubercle into its third layer to form dense coils (dc, green
texture), and branches from there colonize the inner tubercle parenchyma to become digested (dh). The red marked
hypodermis is impenetrable for the fungus
1
170
S. Imhof et al.
hyphae and eventual digestion, representing the
locations of the beginning and end of the mycorrhizal colonization pattern. Between these events,
the different hyphal forms serve three fundamental functions: (1) transportation and distribution
of carbohydrates and nutrients within the rootrhizome-complex, (2) storage, and finally (3) as a
carbon source for the plant following digestion.
The straight hyphae in the filiform root extension
and the epidermis allow for rapid transport of
nutrients and carbohydrates towards the rhizome.
The swollen hyphae in the rhizome cortex store
these substances, eventually for the benefit of the
plant. The spiral line of hyphal loops is the geometrically and economically optimal distribution
mode around the parenchyma of the tubercle.
With a minimum of living hyphae, this provides
short distances and limits the number of cell passages for side branches to penetrate into all parenchyma cells, necessary due to the quick
degeneration process therein. The fungus in
Afrothismia gesnerioides shows a similar colonization pattern with straight hyphae in the short
root extension (Fig. 4.4e) and the root epidermis
(Fig. 4.4f, g), as well as digestive tissue in the
inner root parenchyma (Fig. 4.4c, see details in
Imhof 2006). However, it does not develop a spiral line of hyphae around the tubercle parenchyma. Instead, dense coils of living irregular
hyphae develop in the third root layer, encompassing the parenchyma in a collar-like pattern
(Fig. 4.4c + f–h). Economically speaking, this
pattern is less efficient than that in A. saingei,
considering the amount of living fungal biomass
necessary to supply the digesting cells with
hyphal branches. Moreover, the rhizome of A.
Of the ten genera in this family, only Burmannia
contains green representatives. Burmannia
tenella is the only entirely achlorophyllous
neotropical species, others occur in Africa
(e.g., B. hexapterella) and Asia (e.g., B. championii, B. candida). However, many species with
intermediate mycoheterotrophic status, between
Fig. 4.5 (continued) structures (a), and vesicles (v). (f)
Transverse section through a root of D. orobanchoides
showing the central cylinder (cc) consisting of a central
tracheary element surrounded by one ring of smaller tracheary elements and the pericycle, a thickened endodermis
(en), two layers of small parenchyma layers, and epidermal cells (ep). The epidermal cells contain hyphal coils
(hc+) and arbuscules (a), the latter partly degraded. (g)
SEM micrograph of a rhizome of D. orobanchoides imbricately covered by peltate scale leaves with fringed margins. The leaf interstitials contain fungal hyphae (hy).
(h) Transverse section through a rhizome covered with
imbricate scale leaves (le) showing fungal colonization
including vesicles (v) within (ihy) and in between (ohy)
the scale leaves. The rhizome axis (rh) is not colonized
but contains starch grains (st). (i) Tangential section
through a rhizome (rh) including the imbricate scale
leaves (le) showing dense hyphal masses in the leaf interstitials (li). (j) Schematic view of the mycorrhizal colonization pattern of D. orobanchoides: the peltate scale
leaves and their interstitials are colonized by hyphal coils
and vesicles. The root is colonized only in the epidermis
by hyphal coils, arbuscules, and vesicles; the arbuscules
are the first to degenerate
gesnerioides contains straight growing hyphae
in its outer cortex, whereas the inner cortex
cells contain starch deposits (Fig. 4.4c), as does
the uncolonized tubercle cortex (Fig. 4.4d + h,
schematic view on Fig. 4.4i). This means that
A. gesnerioides, in contrast to A. saingei, converts the carbon delivered by the fungus into
starch grains. In the case of Afrothismia spp.
however, this appears as a unnecessary metabolic
step, since the carbon source is permanently present. Therefore, although the mycorrhizal patterns
in Afrothismia spp. are highly complex, they still
show signs for an ongoing evolutionary progression of mycorrhizal structures within the genus,
whereas A. gesnerioides can be considered to be
less advanced than A. saingei. More of the 12
Afrothismia species described so far should be
investigated to determine if intermediate structures exist (see 4.8 Trends, Conclusions, and
Future Directions).
The fungal species associated with Afrothismia
spp., as identified by molecular methods, all
belong to Glomus-group A (Franke et al. 2006),
and are species-specific (Merckx and Bidartondo
2008).
4.5.3
Burmanniaceae (Figs. 4.5–4.7)
4
Subterranean Morphology and Mycorrhizal Structures
Fig. 4.5 Dictyostega orobanchoides (Burmanniaceae).
(a) Inflorescence of D. orobanchoides, composed of a
bifurcate cincinnus. (b) Single preserved flower of
D . orobanchoides, 2.5 mm long. (c) Rhizome (rh) of
D. orobanchoides about 1.5 mm thick with a tuft of filiform
roots (r). Apically the rhizome turns into a shoot (s). (d)
171
Root of D. orobanchoides, cortex (rc) partly detached,
exposing the thickened endodermis (en) which encloses
the central cylinder (cc). The whitish cell contents are
fungal coils. (e) Longitudinal section through a root epidermis of D. orobanchoides. Fungal colonization consists
of coiled hyphae (hc+), partly decomposed arbusculate
172
Fig. 4.6 (a–c) Apteria aphylla, (d–e) Gymnosiphon divaricatus, (f–h) Hexapterella gentianoides, (i–k) Campylosiphon
congestus (Burmanniaceae). (a) Preserved flower (9 mm
long) and fruit of A. aphylla. (b) Top view of a flower of A.
aphylla (courtesy of H and PJM Maas). (c) Subterranean
S. Imhof et al.
system of A. aphylla. The shoot (s) is continuous with the
short (3 mm) orthotropous rhizome (rh) bearing numerous
filiform roots. The root cortex parenchyma is often disrupted, leaving the thickened endodermis (en) with central
cylinder enclosed as the only connection with the rhizome.
4
Subterranean Morphology and Mycorrhizal Structures
173
Fig. 4.7 (a–f) Burmannia tenella, (g–i) Burmannia
hexaptera (Burmanniaceae). (a) Preserved flower (6 mm
long) of B. tenella. (b) Root system of B. tenella with
several star-like radiating vermiform roots (r), about
1.2 mm thick, at the base of the shoot (s). (c) Inflorescence
of B. tenella, the bifurcate cincinnus usually consists of
a few flowers. (d) Transverse section through a root of
B. tenella with extensive fungal colonization of the multilayered cortex parenchyma. The central cylinder is
reduced and surrounded by a tertiary endodermis (en).
(e, f) The fungus in the root parenchyma cells of
B. tenella forms heteromorphic coils of hyphae of various
width (hc+), arbusculate structures (a) as well as vesicles
(v), often within one cortex cell. Degeneration begins
with the arbusculate structures (a−); the thicker hyphae
tend to persist longer. (g) Flowers of B. hexaptera emerging only a few centimeters above the soil surface. (h)
Preserved flower (1 cm long) of B. hexaptera. (i) Root
system of B. hexaptera comprised of vermiform roots
(r), about 1.2 mm thick, also with the tendency to radiate
at the base of the shoot (s), resulting in a coralloid
appearance
Fig. 4.6 (continued) (d) Subterranean system of G. divaricatus, very similar to that of A. aphylla (see (c)) with short
rhizome (rh) and exposed endodermis (en). (e) Top view of
a flower of G. divaricatus (courtesy of H and PJM Maas).
(f) Flower of H. gentianoides (courtesy of H and PJM
Maas). (g) Subterranean system of H. gentianoides, also
with a short rhizome (rh, 4 mm long) continuous with
the shoot (s). The rhizome bears filiform roots (r); light
coloration indicates fungal colonization. (h) Top view of a
flower of H. gentianoides (courtesy of H and PJM Maas).
(i) Preserved inflorescence of C. congestus. (j) Preserved
single flower (9 mm long) of C. congestus. (k) Subterranean
system of C. congestus with the shoot (s) continuous with
a slightly tuberous rhizome (rh), 9 mm long and 2.5 mm
thick, bearing filiform roots (r)
174
leafy Burmannias and achlorophyllous, scaleleaved species exist (Jonker 1938; Maas et al.
1986; Leake 1994), suggesting an evolutionary
trend towards mycoheterotrophy. All other genera
are fully mycoheterotrophic. The monotypic
genus Desmogymnosiphon (Guinea Lopez 1946)
is most probably a Gymnosiphon species (compare to Maas et al. 1986).
4.5.3.1 Apteria, Campylosiphon,
Dictyostega, Gymnosiphon,
Hexapterella, Marthella, Miersiella
(Figs. 4.5 and 4.6)
Except for Campylosiphon congestus and the
pantropical Gymnosiphon, all these genera are
exclusively neotropical (Jonker 1938; Maas et al.
1986). All species have the same basic architecture for their underground parts. The aerial shoots
are continuous with rhizomes, densely covered
by scale leaves. These scale leaves are conspicuously fringed in Dictyostega (Imhof 2001, Fig.
4.5g), which has led to the hypothesis they might
ecologically replace the missing root hairs
(Goebel and Süssenguth 1924; Maas et al. 1986).
The rhizomes can be longer (up to 7.5 cm in e.g.,
Miersiella umbellata, Maas et al. 1986, up to
4 cm in Dictyostega orobanchoides, Imhof 2001,
Fig. 4.5c) or rather short (e.g., Apteria aphylla
(Fig. 4.6c), Uphof 1929, Gymnosiphon longistylus, Hepper 1968, G. divaricatus (Fig. 4.6d),
Maas et al. 1986, Hexapterella gentianoides (Fig.
4.6g)), and can be slightly tuberous (e.g.,
Campylosiphon purpurascens, Maas et al. 1986,
Campylosiphon congestus, Fig. 4.6i–k). Many
filiform, less than 0.5 mm thick, sparsely branched
roots arise from the axils of the scales. Species
with short rhizomes, therefore, have a star-like
root system (Fig. 4.6c, d + g), but roots also
emerge as tufts on longer rhizomes (Imhof 2001,
Fig. 4.5c). As a peculiar exception in this group
of species, Gymnosiphon afro-orientalis develops little tubers of unknown origin beside scale
leaves and filiform roots at the short rhizome
(Cheek 2009), superficially reminiscent of those
found in Afrothismia (e.g., Fig. 4.4b), but fundamentally differing in being distinct from the
filiform roots.
S. Imhof et al.
Anatomically, these roots are characterized by
a much reduced central cylinder with one central
enlarged tracheary element surrounded by a ring
of much smaller tracheary elements, and a pericycle (e.g., Fig. 4.5f). The tertiary endodermis is
conspicuously reinforced (e.g., Marthella trinitatis, erroneously called Burmannia capitata by
Johow 1885, Gymnosiphon refractus (formerly
Cymbocarpa refracta, Merckx 2008), treated
under two different synonyms by Johow 1889 and
Goebel and Süssenguth 1924, Apteria aphylla,
Uphof 1929). In transverse sections of a
Dictyostega orobanchoides root, the fortification of
a single endodermal cell may even be wider than
the entire central cylinder (Imhof 2001, Fig. 4.5f).
This reinforcement protects the essential connection to the shoot. In fact, the thin-walled cortex tissue is often found to be disrupted (Figs. 4.5d and
4.6c, d + g) whereas the central strand is even hard
to disconnect using forceps (Imhof 2001, see section on Petrosavia for interpretation).
The two to three parenchyma layers and, in
particular, the often large-celled persistent epidermis (Johow 1889; Imhof 2001 and unpublished observations) are colonized by coils of
hyphae (Uphof 1929), vesicles, as well as arbuscular-like structures, often all together within a
single cell (Imhof 2001). The fungal material
often appears amorphous, suggesting a digestion
process (Fig. 4.5e, f).
Dictyostega orobanchoides also has fungal
colonization in the scale leaves (Fig. 4.5h) as well
as in the interstitials of their imbricate arangement
along the rhizome (Fig. 4.5g–i), but not in the rhizome axis. These hyphae and vesicles do not
show signs of degeneration, and it has been
hypothesized that they serve as a refugium for the
fungus, which in turn enhances the rhizomosphere
with the appropriate mycobiont (Imhof 2001, see
Fig. 4.5j for a schematic view). It can be interpreted as a strategy for a sustained use from the
fungus, analogous to the often complex colonization pattern in other MH plants (e.g., Voyria,
Afrothismia, Triuris, Sciaphila). More investigations might clarify the possible general relevance
of rhizomes and their scale leaves for the mycorrhiza in other Burmanniaceae.
4
Subterranean Morphology and Mycorrhizal Structures
Franke et al. (2006) found several Glomusgroup A species and an Acaulosporaceae in
Campylosiphon congestus (treated as Burmannia
congesta). Also, Dictyostega orobanchoides is
associated with Glomus-group A species (Merckx
et al. 2010), as are Apteria (Courty et al. 2011)
and Gymnosiphon spp. (Dirk Redecker, pers.
comm. cited in Leake 2005; Courty et al. 2011).
4.5.3.2 Burmannia (Fig. 4.7)
Burmannia species are more diverse with respect
to their subterranean structures than their sister
genera. Although they are sometimes similar to
the latter (e.g., B. championii, Ernst and Bernard
1911), they also can have thicker roots also arising from rhizomes (e.g., B. larseniana, Zhang
and Saunders 1999) or even vermiform, up to
2.6 mm thick roots and no (visible) rhizomes
(e.g., Burmannia candida, Smith 1911, B. liukiuensis, Terashita and Kawakami 1991, B. tenella,
Imhof 1999b, Fig. 4.7b, B. hexaptera, Imhof
unpublished, Fig. 4.7i). Others have tuberous
organs of uncertain nature (Burmannia hunanensis, Liu et al. 2001), with filiform roots. However,
more taxonomic investigation may result in new
classifications resolving some of this subterranean diversity, as in fact, Burmannia congesta,
having a tuberous rhizome, only recently was
attributed to Campylosiphon (Fig. 4.6i–k) by
molecular and morphological data (Merckx 2008;
Maas-van de Kamer and Maas 2010).
Root anatomy is also diverse. Epidermal cells
may be conspicuously enlarged (Johow 1889;
Ernst and Bernard 1911, 1912; Bernard and
Ernst 1914) or not (Colozza 1910; Ernst and
Bernard 1911; Imhof 1999b, Fig. 4.7d).
Depending on the variability of root thickness,
the cortex parenchyma layers can be from three
to many (Janse 1896; Ernst and Bernard 1911;
Larsen 1963; Terashita and Kawakami 1991;
Imhof 1999b, Fig. 4.7d), and can be uniform
(Imhof 1999b) or heteromorphic (Ernst and
Bernard 1911) or with lacunae (Johow 1889;
Malme 1896a; Colozza 1910). Similar to the
other genera of the family, the endodermis has
obvious tertiary reinforcements and the central
cylinder is much reduced (e.g., Malme 1896a;
175
Ernst and Bernard 1911; Terashita and Kawakami
1991; Imhof 1999b, Fig. 4.7d).
In species with filiform roots, the mycorrhizal
fungus colonizes epidermal cells (Johow 1889;
Ernst and Bernard 1911), whereas in the species
with thick roots, the cortex parenchyma cells are
colonized (Meyer 1909; Ernst and Bernard 1911;
Terashita and Kawakami 1991; Imhof 1999b,
Fig. 4.7d). In the thick roots of Burmannia
tenella, hyphal coils, vesicles and arbuscular-like
structures may occur together in a single cell
(Fig. 4.7e, f ). A colonization pattern with compartmentation of root tissue similar to other MH
plants is not obvious (Fig. 4.7d). However, a selective digestion of the thinner, arbusculate hyphae
but not the thicker hyphae within cells (Imhof
1999b, Fig. 4.7e, f ), seems to allow a sufficient
spread of the colonization within the cortex parenchyma by the latter, while carbon and nutrients are
obtained through digestion of the former.
The only Burmannia species which has been
investigated for the identity of its mycorrhizal
fungus is B. hexaptera (Fig. 4.7g–i). It is mycorrhizal with Glomus-group A species (Franke et al.
2006; Merckx and Bidartondo 2008).
4.5.4
Triuridaceae (Figs. 4.8–4.10)
Fossil specimens of this exclusively achlorophyllous family from the Upper Cretaceous (ca. 90
mya) are the oldest unequivocal monocotyledonous remnants known (Gandolfo et al. 2002).
Eleven genera are grouped in three tribes, the
Sciaphilae are pantropical, Triurideae neotropical (Maas-van de Kamer and Weustenfeld 1998),
and Kupeaeae only occur in tropical Africa
(Cheek 2003b). All genera except for Sciaphila
and Andruris (included in Sciaphila by van de
Meerendonk 1984) contain only one to three species. The affiliation of the family was long uncertain (Rübsamen-Weustenfeld 1991; Maas-van de
Kamer 1995; Maas-van de Kamer and
Weustenfeld 1998). Today, molecular methods
have assigned them to the Pandanales, which is
supported by structural features (Furness et al.
2002; Rudall and Bateman 2006).
176
Fig. 4.8 (a–i) Triuris hyalina, (j–k) Sciaphila ledermannii (Triuridaceae). (a) Subterranean shoot (s) of T. hyalina
with two nodes (n) bearing paired roots (r) in the axils of
S. Imhof et al.
the scale leaves. The female inflorescence has one
mature flower and one bud (b). (b) Female flower of T.
hyalina with the characteristic tail-like tepal appendages.
4
Subterranean Morphology and Mycorrhizal Structures
177
With only few exceptions, the subterranean
organs of Triuridaceae are rather uniform. The
epiterrestrial shoots are continuous plagiotropically to orthotropically with subterranean shoot
segments with various internode lengths without
increasing in diameter (Fig. 4.8a + c, d). In addition to occasional side shoots, the axils of the
nodal scale leaves bear pairs (sometimes solitary)
of long filiform roots about as thick as the shoots
(e.g., van de Meerendonk 1984; Maas and
Rübsamen 1986; Maas and Maas-van de Kamer
1989), which can be glabrous (e.g., Triuris hyalina,
Imhof 1998, Triuridopsis intermedia, Franke et al.
2000, Fig. 4.8a + c–e), sparsely hairy (e.g., several
Sciaphila spp., Schlechter 1913, Lacandonia
schismatica, Martinez and Ramos 1989) to conspicuously pilose (e.g., Soridium spruceanum,
Miers 1852. several Sciaphila spp., Johow 1889;
Hemsley 1907; Larsen 1972, Andruris spp.,
Schlechter 1913, Seychellaria madagascariensis,
Fig. 4.10c). Usually the scale leaves and, consequently, the root pairs are spaced along the subterranean shoot (Fig. 4.8a + c), but there can also be
dense clumps of filiform roots seemingly radiating from a single origin (e.g., several Sciaphila,
Triuris, and Peltophyllum spp., Larsen 1972;
Maas and Rübsamen 1986, Fig. 4.8d, e + j,
Seychellaria madagascariensis, Fig. 4.10c),
sometimes occurring in two or three tiers along
the subterranean shoot (Fig. 4.8d). There are also
a few species with more stout roots but also showing a star-like arrangement at the base of the shoot,
namely the three species of the Kupeaeae (Cheek
et al. 2003; Cheek 2003b, Fig. 4.10d–f), but also
Sciaphila polygyna (Imhof 2004, Fig. 4.9a–d).
Sciaphila ledermannii (Fig. 4.8i) has an intermediate root thickness (Fig. 4.8j). The star-like root
aggregations by filiform or stout roots, even if
they appear superficially very different, all follow
the same developmental pattern, that is maximally
one pair of shoot-borne roots per node, but are
formed by the initiation of a side shoot from the
scale leaf axil directly bearing a next node with
scale leaf, giving rise to another pair of roots and
a side shoot and so forth. The side shoots often do
not elongate, which explains the abundance of
roots (see details in Imhof 1998, 2004).
The tendency towards aggregations of thick
and short roots seems to be characteristic for
mycoheterotrophic plant families (Leake 1994,
Imhof 2010, this chapter). Hence, the quite recent
discovery of this feature in the Triuridaceae
(Cheek et al. 2003; Imhof 2003, 2004, see
Figs. 4.9d and 4.10f) was not too surprising.
The root anatomy of Triuridaceae is quite uniform also. Internal to the epidermis, there is a
Fig. 4.8 (continued) The apocarpous gynoecium is about
1.5 mm wide. (c) Subterranean shoots of T. hyalina with
spaced nodes (n) where paired roots (r) arise from each
scale leaf axil giving it a ladder-like appearance. The roots
are uniformly 0.4 mm thick. (d) Each node seen in (c)
may develop aggregations of paired roots (agr) as
explained in text. (e) An aggregation of roots seen in (d)
results in a star-like root system. At this stage, it may have
already borne several flowering shoots (detached). A new
shoot (s), 2 cm long, bearing a flower bud has developed.
(f) Transverse section through a T. hyalina root measuring
0.4 mm in diameter. The epidermis (ep) is mostly free of
hyphae and the exodermis (ex) is a barrier to the fungus
except for the short cells (shc) with thickend outer tangential walls serving as passage cells. The outer cortex parenchyma layer bears dense hyphal coils (hc+) which do not
become digested but may collapse when older. The middle
parenchyma layer consists of enlarged cells containing
mostly amorphous clumps of hyphal masses (hc−).
The inner cortex layer of much smaller cells is free of
hyphae. The endodermis (en) is only slightly suberized.
(g) Longitudinal section through a T. hyalina root showing epidermis (ep), exodermis (ex), the dense hyphal coils
(hc+) in the outer and the degenerated ones (hc−) in the
middle cortex parenchyma layer. Occasionally vesicles
(v) may occur in both layers. (h) Schematic view of the
colonization pattern in T. hyalina: after penetration of epidermis and a short cell of the exodermis the hyphae start
to coil and decrease their diameter while spread longitudinally and tangentially within the outer cortex parenchyma.
The dense coils of narrow hyphae (see (g)) send branches
into the middle parenchyma layer where they degenerate
to amorphous clumps. Vesicles may occur in both layers.
The red marked cells are impenetrable to the fungus. (i)
Inflorescence of S. ledermannii showing female flowers.
(j) Subterranean system of S. ledermannii consisting of a
short rhizome (rh) continuous with the epiterrestrial shoot
(s). The rhizome bears filiform roots (r) in this specimen
up to 9 cm long and 0.8 mm thick
178
Fig. 4.9 Sciaphila polygyna (Triuridaceae). (a) Top view
of a female flower of S. polygyna with its numerous carpels in fruiting stage (about 3 mm wide). (b) Same flower
S. Imhof et al.
from the lower side showing the tepals (tp) with hair tufts.
(c) Apex of an inflorescence of S. polygyna with numerous
flower buds. (d) Subterranean system of S. polygyna with
4
Subterranean Morphology and Mycorrhizal Structures
179
suberized exodermis (Fiebrig 1921; Imhof 1998,
2003) and two (Johow 1889; Tomlinson 1982),
three (Fiebrig 1921; Imhof 1998), to several
(Imhof 2003) cortical parenchyma layers. The
endodermis and/or pericycle may be reinforced
(Poulsen 1886, 1890b; Johow 1889, Milanez and
Meira 1943; Larsen 1963; Tomlinson 1982) or
not (Malme 1896b; Imhof 1998, 2003). The central cylinder is much reduced. Very characteristic
is the second cortex parenchyma layer, which
mostly consists of conspicuously enlarged cells
(Poulsen 1886, 1890b; Johow 1889; Fiebrig
1921; Tomlinson 1982; Imhof 1998, 2003), with
the exception of Sciaphila thaidanica according
to Larsen (1963, Figs. 4.8f, g and 4.9e).
Mycorrhizal colonization was recognized very
early (e.g., Poulsen 1886, 1890b; Johow 1889;
Janse 1896), with additional information added
later (Fiebrig 1921; Ohga and Sinoto 1932;
Milanez and Meira 1943; Palacios-Mayorga and
Pérez-Silva 1993), but details of the colonization
pattern were described rather recently (Imhof
1998, 2003; Franke 1999). Triuris hyalina attains
a sustained benefit from the endophytic fungus
by maintaining the hyphae in a functional state in
the first cortical parenchyma layer and digesting
them only in the enlarged cells of the second
parenchyma layer (Fig. 4.8f–h, see details in
Imhof 1998). An unpublished diploma thesis on
Sciaphila purpurea (Franke 1999) not only
yielded detailed information on morphology,
anatomy, and ecology of the reproductive parts,
but also confirmed the distinction of undigested
hyphae in the outer vs. the digestion of hyphae in
the inner enlarged cells of the root cortex. Beyond
that, the structural diversity of the mycorrhizal
colonization pattern in Sciaphila polygyna (Fig.
4.9a–d) is much more complicated and certainly
belongs to the most complex mycorrhizas known
(Fig. 4.9e–h). It includes four different morphologies of hyphae occurring in four distinct root tissue compartments. Moreover, it shows a disparate
colonization at the tip compared to the base as
well as the dorsal vs. the ventral side of the root,
creating a monosymmetrical (only one plane of
symmetry) root in transverse and longitudinal
sections (Fig. 4.9h, see details in Imhof 2003).
The purpose of these complex structures, except
for the strictly localized digestion in the “giant
cells” for a sustained carbon influx (Imhof 2003),
is not yet understood.
The fungus in Sciaphila secundiflora (Yamato
2001, treated as S. tosaensis, the two treated
as being synonymous by Ohashi 2000) was
determined by DNA sequencing to be a Glomus
species (Glomeromycota). More recently, S.
secundiflora (still called S. tosaensis) and
Andruris japonica (treated as Sciaphila japonica)
were described to associate with Glomus-group
A fungi (Yamato et al. 2011b), the phylotypes
extracted from each species being closely related
to another but quite distant when compared
between the two species. Sciaphila ledermannii
was also found to be colonized by a species from
Fig. 4.9 (continued) thick roots (r, several have detached)
radiating from the base of two shoots (s, one is detached).
For the architecture of this root system, see Imhof (2004).
(e) Transverse section through a central part of a 1.2 mm
thick root of S. polygyna surrounded by epidermis (ep) and
exodermis (ex) with short cells (shc) serving as the only
passage cells for fungal penetrations (p). Root anatomy and
mycorrhizal pattern are highly heteromorphic with cells of
the fourth root layer being much larger (“giant cells,” blue
border) than others dislocating the central cylinder (cc) out
of its central position to create a dorsiventral architecture of
the root. Fungal material degenerates (hc−) only in the
fourth layer. The third layer has loose hyphal coils with
swellings (yellow border), coils without swellings (not
marked) and very dense coils of thin hyphae (green border,
ventral side). Colonization by dense coils follows a
v-shaped pattern leaving a gap of colonization (g) in some
parts of the roots (see right hand side of (h)). (f) Tangential
section through the dorsal side of a S. polygyna root showing areas colonized by coils with many (yellow border) and
with less swellings. (g) Tangential section through the ventral side of the third root layer at the same magnification as
(f) indicating the differences of the three types of coils in
the third layer. (h) Schematic view of the mycorrhizal colonization pattern in S. polygyna in transverse (left) and longitudinal view (right). The coloration corresponds to those
in (e, f). Note that the different colonization morphotypes,
the appearance of giant cells, the digestion of hyphae
therein is also heteromorphic in the longitudinal view as it
is in transverse view (see details in Imhof 2003)
180
Fig. 4.10 (a–c) Seychellaria madagascariensis, (b–f)
Kupea martinetugei, (Triuridaceae) (g, h) Geosiris aphylla,
(Iridaceae) (i, j) Thismia panamensis (Thismiaceae). (a)
S. Imhof et al.
Female flowers of S. madagascariensis about 2 mm wide
with the basal filiform styles projecting above the carpels. (b) Young male flower of S. madagascariensis.
4
Subterranean Morphology and Mycorrhizal Structures
Glomus-group A as well as by an Acaulospora
sp. (Franke et al. 2006), whereas Merckx and
Bidartondo (2008) detected only a Glomus-group
A fungi in a specimen of S. ledermannii from
Mount Cameroon. Kupea martinetugei was associated with two closely related fungi of Glomusgroup A (Franke et al. 2006), confirmed by
Merckx and Bidartondo (2008).
4.5.5
Corsiaceae (Fig. 4.11)
181
and vesicles (Fig. 4.11g) rarely occur. The function of the branched structures is unknown but
they, along with the hyphal coils, may be involved
in the transfer of sugars from fungus to root cells
(Domínguez et al. 2009). Arachnitis uniflora also
develops unusual asexual propagules (Fig. 4.11b)
on its fleshy roots (Domínguez et al. 2006) that
are colonized by fungi from the parent root before
they detach. The propagules develop a shoot apical meristem and adventitious roots and ultimately new plants that presumably link to
neighboring photosynthetic plants for their source
of carbon (Domínguez et al. 2006, 2009)
4.5.5.1 Arachnitis
Roots of the inconspicuous plant Arachnitis
uniflora (Corsiaceae, Fig. 4.11a), one of two
unusual mycoheterotrophs in the genus confined
to a few locations in the southern hemisphere
(Dimitri 1972; Cribb et al. 1994; Ibisch et al.
1996; Domínguez and Sérsic 2004), are short and
fleshy, radiating from the shoot base, and lack
root hairs. Reiche (1907) described colonization
of peripheral parenchyma cells in roots by endotrophic mycorrhizal fungi whereas Colozza
(1910) called the plant “parassita,” adding
“fors’anche saprofita?” (= perhaps saprophytic?)
in brackets. More recently, Minoletti (1986)
referred to the colonization pattern as an ectendomycorrhiza because both intercellular and
intracellular hyphae were present in the outer
cortex of roots. Molecular methods have proven
that roots are colonized by an AM fungus belonging to Glomus-Group A (Bidartondo et al. 2002).
However, details of the structural characteristics
of the plant-fungus interaction are unlike other
plant associations with Glomus spp. (Domínguez
et al. 2006, 2009). Unusual branched structures
with inflated ends (Fig. 4.11c–g) form in addition
to hyphal coils (Fig. 4.11d) in the cortical cells of
the plant’s fleshy roots. Arbuscules do not form
4.5.5.2 Corsia
Only a general description of roots in Corsia species is given by van Royen (1972); no further
specific information can be found in the taxonomic section of his monograph. The roots are
filiform, unbranched, white to cream-colored,
growing horizontally through the humus layer.
Compared to the entire plant, they are “quite sizable” and “extend over considerable distance in
many directions” (van Royen 1972). They arise
from short, creeping rhizomes, with sheathing
scale leaves (Williams 1946; van Royen 1972).
Beccari’s (1877) and Schlechter’s (1905) drawings, however, show some root branches in Corsia
ornata and C. unguiculata, respectively. Similarly,
Cribb (1985), without discussing them, depicts
branching roots in C. pyramidata, also arising
from branching rhizomes a few centimeters
below the soil surface. Jones and Gray (2008),
describing the only Australian species C. dispar,
explained this discrepancy as an oversight by van
Royen, since many herbarium sheets of Corsia
spp. in the herbarium of Canberra have branched
roots as well. According to Jones and Gray
(2008), the rhizome in C. dispar is about 4 mm
Fig. 4.10 (continued) (c) Root aggregation of S. madagascariensis (compare Fig. 4.8e) showing four shoots (s)
and numerous pilose roots (r) up to 10 cm long and
0.6 mm thick. (d) Female inflorescence of K. martinetugei.
(e) Male inflorescence of K. martinetugei. (f) Root system
of K. martinetugei with several radiating, 1.5 mm thick
and up to 7 mm long, roots (r) at the base of the shoot (s).
(g) Subterranean parts of G. aphylla with a rhizomatous
tuber (t) and filiform roots (r) at its base. (h) Preserved
capitulum of G. aphylla. (i) Subterranean tuber (t) of T.
panamensis with filiform roots (r) radiating from it. (j)
Flower of T. panamensis (courtesy of H and PJM Maas)
182
Fig. 4.11 Arachnitis uniflora (Corsiaceae). (a) Flowering
stems of Arachnitis uniflora, each with a single flower.
Image courtesy of Laura Domínguez. (b) Propagules
(arrowheads) on a fleshy root (asterisk) of A. uniflora. (c)
Confocal microscopy of intracellular branched hyphal
structures of Glomus-Group A in a root of A. uniflora. (d)
Longitudinal section of resin-embedded root of A. uniflora
stained with toluidine blue O showing the apical meristem (asterisk), intracellular hyphae of Glomus-Group A
S. Imhof et al.
(arrowheads), and intracellular branched hyphal structures
(arrow). (e) Enlarged portion of a similar section of a A.
uniflora root showing intracellular branched hyphal structures of Glomus-Group A (arrows). (f) Clearings of root
cells of A. uniflora stained with acid fuchsin showing numerous intracellular branched hyphal structures of GlomusGroup A (arrows). (g) Clearings of root cells of A. uniflora
stained with acid fuchsin showing intracellular hyphae
(arrowheads) and a vesicle (arrow) of Glomus-Group A
4
Subterranean Morphology and Mycorrhizal Structures
thick and grows in annual increments. In contrast
to the information given by van Royen (1972),
Cribb (1985) and Jones and Gray (2008), a new
variety C. purpurata var. wiakabui (Takeuchi and
Pipoly 1998), later considered to be a separate
species (Jones and Gray 2008), has a conspicuously tuberous rhizome bearing the roots (interpreted from the drawing in Takeuchi and Pipoly
1998). This is partly reminiscent of the third
genus of the family, Corsiopsis, discussed below.
No anatomical studies exist on this genus which
could elucidate its mycorrhiza.
4.5.5.3 Corsiopsis
The monotypic Corsiopsis chinensis is only
known from a single herbarium specimen (Zhang
et al. 1999). The original description is of an
ellipsoid rhizome 12–15 mm long and 5 mm in
width, the drawing showing it in an orthotropous
orientation. Roots were not seen.
4.5.6
Orchidaceae (Fig. 4.12)
The family Orchidaceae has the largest number
of mycoheterotrophic genera of any plant family,
with approximately 35 % of more than 500 fully
mycoheterotrophic angiosperm species recognized (Leake 1994; Merckx et al. 2009; Imhof
2010). It is impossible to characterize the subterranean structures of all mycoheterotrophic orchid
species (see Rasmussen 1995 for a thorough discussion) but a few examples will demonstrate the
variability. Some species (e.g., Cyrtosia javanica)
have rhizomes bearing fleshy adventitious roots,
others (e.g., Epipogium aphyllum; Corallorhiza
spp., Rhizanthella garderi) with rhizomes only,
and others (e.g., Wullschlaegelia calcarata) with
roots, some of which are modified as tubers.
Regardless of the nature of the underground
structures, the majority of achlorophyllous orchid
species are associated with fungi that form intracellular hyphal coils (pelotons) similar to those in
photosynthetic orchids. These can develop within
the majority of root or rhizome cortical cells and
sometimes even in scale leaf tissue (Groom
1895c) and have an ephemeral existence since
they undergo digestion by host cells (Smith and
183
Read 2008). This process, termed tolypophagy
(Burgeff 1932), can be repeated with recolonization by pelotons of cells containing hyphal remnants and subsequent digestion of these. Often,
the cortex parenchyma is divided into an outer
“Pilzwirtsschicht” (fungus host layer), where the
coils do not degenerate, and an inner
“Pilzverdauungsschicht” (fungus digestion
layer), where digestion takes place (Magnus
1900; Burgeff 1932). Moreover, in some mycoheterotrophic species (e.g., Gastrodia spp.), a
process called ptyophagy occurs (Burgeff 1932;
Wang et al. 1997; Rasmussen 2002). While keeping the fungus host cell layers, this is characterized by only short hyphae penetrating the
single-layered and particularly voluminous digestion cells and releasing their contents into it without coiling (Janse 1896; Burgeff 1932; Campbell
1962, 1963, 1964). As such, it very much resembles the “hyphal pegs” in monotropoid mycorrhizas (Lutz and Sjolund 1973; Duddridge and
Read 1982). It is open to speculation if this can
be interpreted as an evolutionary progression
within orchid mycorrhiza, from non-differentiated colonization pattern (see e.g., Peterson et al.
2004), over the tissue compartmentation in host
and digestion layers, to the ptyophagy as a special type of the latter in few MH orchids. More
structural work is needed to elucidate this, but
since arbuscular mycorrhizas and ectomycorrhizas seem to have undergone evolutionary progression (Imhof 2009), it would be surprising if
this is not the case in orchid mycorrhizas.
Because of their “dust seeds,” consisting of a
rudimentary embryo and limited storage reserves,
all orchid species (Fig. 4.12a) growing in native
habitats require a suitable fungal partner to germinate and for the subsequent development of
the protocorm (Peterson et al. 1998, 2004). The
intracellular fungal hyphal coils (pelotons) are
essential features for metabolite transfer into
developing protocorms (Fig. 4.12b) and roots
(Fig. 4.12c). All orchid species can, therefore, be
considered to be mycoheterotrophic during this
early stage of their life cycle (Leake 2004). The
fungi involved are basidiomycete anamorphs
such as Ceratorhiza, Epulorhiza and Moniliopsis
which are capable of enzymatically reducing
Fig. 4.12 Orchidaceae. (a) Flowering stems of
Corallorhiza trifida. (b) Diagram of a developing orchid
protocorm with intracellular fungal hyphal coils (pelotons)
(arrowheads). (c) Section of an orchid root showing intact
pelotons (arrowheads) and degraded hyphae (arrows).
Image courtesy of Carla Zelmer. (d) C. trifida root cells
(arrow) showing peloton stained with chlorazol black E
(arrowheads). Scale bar = 10 mm. Image courtesy of Carla
4
Subterranean Morphology and Mycorrhizal Structures
185
complex carbohydrates to simple sugars that are
not only used for fungal growth but are also
transferred to developing protocorms to enable
seedling establishment to occur.
Although the majority of orchid species
develop photosynthetic adult plants, a considerable number of genera remain dependent on mycorrhizal fungi for carbon compounds throughout
their life cycle and therefore continue to be mycoheterotrophs. In these situations, developing seedlings link to photosynthetic plant species via
fungal mycelium, mostly belonging to members
of the Basidiomycota (Taylor and Bruns 1997).
Zelmer and Currah (1995) demonstrated that the
fungus isolated from roots of Corallorhiza trifida,
although not identified, formed pelotons in
Corallorhiza trifida root cells (Fig. 4.12d) and
typical ectomycorrhizas with lodgepole pine
(Pinus contorta, Fig. 4.12e). It was recently demonstrated by Zimmer et al. (2008) that the fungal
symbiont associated with C. trifida is a Tomentella
sp. (Thelephoraceae). Another example in which
seeds of Neottia nidus-avis (also a non-photosynthetic orchid) were enclosed in seed packets and
placed either near adult plants or at some distance
from them in a beech (Fagus sylvatica) woodland,
McKendrick et al. (2002) were able to show that
the genus Sebacina (anamorph, Epulorhiza) was
the symbiont involved in the stimulation of seed
germination. Adult plants of the mycoheterotroph
Neottia nidus-avis remain associated primarily
with the basidiomycete family Sebacinaceae
(Selosse et al. 2002) whereas Cephalanthera austinae (another mycoheterotroph) associates with
members of the Thelephoraceae (Taylor and Bruns
1997). Recently, Ogura-Tsujita and Yukawa
(2008) reported the extreme specificity of the
mycoheterotrophic orchid Eulophia zollingeri with
the fungal symbiont, Psathyrella candolleana in
the Agaricales (Basidiomycetes). In contrast,
mycoheterotrophic species within the genus
Epipactis have been reported to associate not only
with members of the Basidiomycota but also with
members of the Ascomycota, including Tuber
(truffle) species (Selosse et al. 2004).
Fig. 4.12 (continued) Zelmer and Randy Currah. (e) The
same fungus isolated from C. trifida root cells and inoculated on Pinus contorta roots formed typical ectomycorrhizas with a mantle (arrow) and Hartig net (arrowheads).
Scale bar = 25 mm. Image courtesy of Carla Zelmer and
Randy Currah. (f) Subterranean system of Neottia nidus-
avis, consisting of numerous roots, 1–2.5 cm long and 2 mm
thick, emerging from a short orthotropous rhizome. (g)
Subterranenean system of Wullschlaegelia calcarata, with
spindle-shaped root tubers (max. 2 cm long and 2 mm thick)
at a short rhizome
4.5.7
Iridaceae (Geosiris, Fig. 4.10g, h)
Unlike the other larger families comprising both
autotrophic and mycoheterotrophic species
(Orchidaceae, Burmanniaceae, Gentianaceae,
Polygalaceae, Ericaceae), Iridaceae do not comprise morphologically intermediate species with
reduced photosynthetic surface or amount of
chlorophyll. Geosiris aphylla (Fig. 4.10g, h) and
the recently described G. albiflora (Goldblatt and
Mannings 2010) are the only mycoheterotrophic
exceptions in the entire family. Systematically,
Geosiris has been treated as a member of
Iridaceae, Burmanniaceae or a family of its own
(see Rübsamen-Weustenfeld et al. 1994). Within
the Iridaceae, it has been considered as
Nivenioideae (Goldblatt 1990; Goldblatt et al.
1987, 1998), but recently a position in its own
subfamily Geosiridoideae, as suggested earlier
(e.g., Thorne 1983), has been confirmed
(Goldblatt et al. 2008).
Geosiris aphylla has an orthotropous, cormlike, oval to elongate rhizome with numerous
scale leaves. The flowering shoots arise from the
apical tip of this tuber-like organ, whereas at its
base numerous filiform roots develop (Fig.
4.10g), similar to the base of onion bulbs.
Anatomically, the rhizome consists of a wide cortex parenchyma surrounding a vascular cylinder
with occasional gaps due to leaf and bud traces. A
thin-walled epidermis and a fortified endodermis
186
S. Imhof et al.
around the vascular cylinder are present and some
isolated amphivasal (xylem around phloem) bundles are found in the ground tissue of the central
pith. The parenchyma cells, particularly in the
pith, contain starch grains (Goldblatt et al. 1987).
The roots also have a thin-walled epidermis, the
cortex has four layers of parenchyma cells and a
strongly fortified tertiary endodermis (Goldblatt
et al. 1987). As pointed out previously (see
Petrosavia), this again corroborates the view of
Imhof (2010), who considers a strong tertiary
endodermis as one of the common adaptations of
monocotyledonous MH plants in order to secure
the essential linkage of roots and shoots.
The only anatomical work on G. aphylla
(Goldblatt et al. 1987), aside from RübsamenWeustenfeld et al. (1994) studying embryology,
does not mention any fungal colonization of roots
or rhizomes. This is rather curious, since a carbon
source for this non-photosynthetic plant is
mandatory. A parasitic mode of life (sensu Weber
1993) is highly unlikely because, in contrast to
the roughly 4,500 eudicotyledonous parasitic
plants, monocots have never been found to be
parasitic (Raynal-Roques and Paré 1998; HeideJørgensen 2008). Possibly, Goldblatt et al. (1987)
may have overlooked the mycorrhizal structures.
Therefore, further anatomical investigations
focusing on the putative mycorrhiza of this species are necessary.
Epirixanthes from Southeast Asia is the only
genus in the Polygalaceae entirely devoid of
chlorophyll, although there are other species in
Polygala and Salomonia (e.g., Polygala setacea
(southeast USA) and Salomonia ciliata (Southeast
Asia and northern Australia) that also show
reductions in photosynthetic surface). The taxonomic accounts (e.g., Smith 1912; Ridley 1922;
Backer and van den Brink 1963; van der Meijden
1988; Hsieh et al. 1995; Pendry 2010) of the six
species of Epirixanthes do not yield information
on the subterranean organs. However, there are
two older and one contemporary study on the
mycorrhizal roots of E. papuana, E. elongata,
and E. cylindrica (Penzig 1901; van der Pijl 1934;
Imhof 2007).
The rhizome of E. papuana and E. elongata is
only a few millimeters long and continuous with
the aerial shoot. The scale leaf axils give rise to
sparsely branched filiform roots that are up to
12 cm long and have a maximum diameter of
0.65 mm (Imhof 2007, Fig. 4.13a + d). A primary
root was never found. Since the rhizome is short,
the roots seem to be radiating from the shoot
Fig. 4.13 (continued) (c) Inflorescence of E. papuana.
(d) Subterranean system of E. elongata similar to that of
E. papuana seen in (a). This specimen has basal shoot
ramifications. Labels as in (a). (e) Longitudinal section
through the cortex of an E. papuana root showing a part of
the straight, cascading hyphae (ch) in the outer parenchyma, coiled hyphae (hc+) in layer 2 (l2) and degenerated coils (hc−) in layer 1 (layers counted from the
endodermis). (f) Longitudinal section through parenchyma layers 1 (l1) and 2 (l2) of a E. elongata root. Layer
2 contains initially straight hyphae sending hyphal
branches (hb1) into layer 1 where they immediately
degenerate to amorphous clumps (hc−). (g) Tangential
section external to the central cylinder through an E. papuana root showing 2 cell rows of each layer 1 (l1), 2 (l2)
and 3 (l3). Hyphae in layer 2 remain functional (hc+) and
send branches centripetally into layer 1 (hb1) as well as
centrifugally into layer 3 (hb3), both of which digest the
fungal material (hc−). A part of the cascading hyphae
coming from the outer cortex layers is also visible (ch).
(h) Transverse section through an E. papuana root. The
epidermis (ep) as well as the outer three cortex parenchyma layers are not colonized by hyphal coils, layer 1
(l1) and 3 (l3) contain degenerated coils whereas layer 2
(l2 and dotted line) contains functional hyphae. The central cylinder inside the endodermis (en) is largely composed of lignified fibers. (i) Schematic view of the
mycorrhizal colonization pattern in Epirixanthes spp..
After penetration, the hyphae grow straight in a cascading
manner through the outer cortex (1), retain the straight
growth when reaching layer 2 but send branches into layer
1 for digestion (2), start to coil hyphae in layer 2 when the
mycorrhization proceeds (3) and then also send branches
in layer 3 for digestion (4)
4.6
Eudicots
4.6.1
Polygalaceae
(Epirixanthes, Fig. 4.13)
Fig. 4.13 Epirixanthes spp. (Polygalaceae). (a)
Subterranean system of E. papuana with approximately
0.6 mm thick roots (r) arising from a short rhizome (rh)
continuous with the epiterrestrial shoot (s). Additional
roots (ar) may develop along the shoot where it is connected to the soil. (b) Isolated flower of E. papuana
(spirit material), little more than 2 mm long, with three
tepals (te). Not all of the five sepals (se) are visible.
188
S. Imhof et al.
base, but additional roots may develop along the
shoot when it is still covered by soil or litter substrate (Imhof 2007, Fig. 4.13a). Epirixanthes
cylindrica has thicker roots (up to 0.75 mm
diameter) and the rhizome bearing the roots is
longer (Penzig 1901). Roots of Epirixanthes have
a triarch central cylinder with many lignified
fibers, a pericycle, a suberized endodermis, up to
seven cell layers of cortex parenchyma, and an
epidermis (Fig. 4.13h). The cells of the innermost
cortex parenchyma layer are larger in radial but
shorter in longitudinal direction than the other
cells (Penzig 1901; van der Pijl 1934; Imhof
2007, Fig. 4.13e, f + h).
Penzig (1901) recognized the coiled intracellular hyphae, their degeneration stages, especially
in parenchyma layers 1 and 3 when counted outwards from the endodermis, and the nearly fungus-free outer cortex layers. Van der Pijl (1934)
added the observation that the hyphae in layer
two grow in longitudinal direction and send
hyphal branches into the inner layer for digestion. The whole colonization pattern, however, is
more complicated and only perceivable when
considering sequential sections. After penetration, the hyphae grow straight through the cells of
the outer cortex, branch repeatedly and spread
coarsely in this root segment until they reach
layer 2 (Fig. 4.13e, layer 1 being the innermost
cortex parenchyma layer). There the hyphae keep
growing straight (Fig. 4.13f), but later develop
hyphal branches that coil within the cells (Fig.
4.13f, g). Directly after having reached layer 2,
lateral hyphae enter the anatomically distinct
layer 1 where they immediately swell and degenerate (Fig. 4.13e, f). In a later stage, layer 3 is also
colonized from coiled hyphae in layer 2, again
Eleven genera of mycoheterotrophic species are
now recognized in the Monotropoideae: Allotropa,
Cheilotheca, Hemitomes, Hypopitys, Monotropa,
Monotropastrum,
Monotropsis,
Pityopus,
Pleuricospora, Pterospora and Sarcodes, with
several species endemic to a particular continent
(Wallace 1975). Molecular phylogeny work has
revealed that Monotropa uniflora is more closely
related to Monotropastrum humile, whereas
Monotropa hypopitys seem to be sister of Pityopus
californicus (Bidartondo and Bruns 2001;
Tsukaya et al. 2008). Therefore, some standard
taxonomies (Stevens et al. 2004; Seybold 2011)
have erected Hypopitys as a separate genus from
Monotropa, and now use Hypopitys monotropa
coined by Crantz (1766).
The minute seeds of members of the
Monotropoideae have underdeveloped embryos
and minimal nutritive tissue and therefore depend
Fig. 4.14 (continued) mycorrhiza with mantle (asterisk),
fungal peg (arrow) and flask-shaped cystidia (arrowheads). (g) Scanning electron micrograph of large calcium
oxalate crystals (arrows) among flask-shaped cystidia. (h)
Freehand transverse section of root showing mantle
(asterisk) and labyrinthine branching of Hartig net (arrowheads). (i) Longitudinal section of resin-embedded root
stained with Toluidine blue O showing the apical meristem (asterisk), and the mantle covering the root apex. (j)
Paradermal section of resin-embedded root stained with
Toluidine blue O showing labyrinthine branching of
Hartig net hyphae and fungal pegs in transverse section
(arrows). (k) Higher magnification of a longitudinal section of resin-embedded root stained with Toluidine blue O
showing mantle (asterisk), Hartig net (arrowheads) and
fungal peg (arrow). (l) Transmission electron micrograph
showing detail of the fungal peg with finger-like wall
depositions (arrows)
with immediate degeneration in layer 3 (Fig.
4.13g, h). In contrast, the hyphae in layer 2 as
well as the straight hyphae in the outer cortex,
remain alive for nearly the lifetime of the root
(Fig. 4.13g, h). This rather complicated colonization pattern is interpreted as a reasonable strategy
in order to have maximum as well as sustained
benefit from the few fungal penetration events. It
includes a coarse (outer cortex colonization) as
well as a fine scale distribution mode (colonization in layer 2), specialized cells for digestion,
and tissue to keep the fungus alive (schematic
view on Fig. 4.13i, details see Imhof 2007).
4.6.2
Ericaceae (Monotropoideae,
Figs. 4.14–4.17)
Fig. 4.14 Monotropa uniflora (Ericaceae/Monotropoideae). (a) Cluster of flowering stems in a hardwood
forest in southern Ontario, Canada. (b) Young shoots and
associated root ball. (c) Mycorrhizal root tip showing
compact mantle with cystidia (arrowheads). Photo cour-
tesy of Brent Young. (d) Scanning electron micrograph of
a root tip showing cystidia. (e) Higher magnification scanning electron micrograph of portion of a mycorrhiza
with a calcium-oxalate crystal (arrowhead) among cystidia. (f) Scanning electron micrograph of a fracture of a
190
Fig. 4.15 Pterospora andromedea (Ericaceae/Monotro
poideae). (a) Two flowering stems in the boreal forest in
British Columbia, Canada. (b) Root ball showing mycorrhizal root tips. (c) Branched root tip showing colored
mantle characteristic of a Rhizopogon sp. (d) Scanning
electron micrograph of a portion of mantle showing
compact hyphae. (e) Higher magnification scanning electron micrograph showing irregular hyphae and abundant
S. Imhof et al.
small crystals (arrowheads). (f) Higher magnification
scanning electron micrograph showing details of various
crystals (arrowheads). (g) Longitudinal section of a
root showing the thick mantle (asterisk) and Hartig net
(arrows). (h) Longitudinal section of a root showing
the inner mantle (asterisk), Hartig net (arrowhead)
and fungal peg (arrow) penetrating the radial epidermal
cell wall
Fig. 4.16 (a, b) Allotropa virgata, (e–h) Pleuricospora
fimbriolata, (Ericaceae/Monotropoideae). (a) Flowering
shoots of Allotropa virgata. (b) Longitudinal section of a
root showing a small apical meristem (asterisk) and some
fungal colonization (arrowheads). (c) Higher magnification
showing Hartig net (arrowhead) and fungal peg (arrow).
(d) Transmission electron micrograph showing Hartig net
(arrowhead) and detail of the fungal peg with finger-like
wall depositions (arrows). (e) Emerged shoot with flowers
of Pleuricospora fimbriolata. Photo courtesy of Dan
Luoma. (f) Longitudinal section of a root showing the apical meristem (asterisk) and well-developed mantle (arrowheads). (g) Mantle (asterisk) and fungal peg (arrow)
penetrating the outer tangential wall of an epidermal cell.
(h) Transmission electron micrograph showing a fungal
peg with finger-like wall depositions (arrowheads)
192
Fig. 4.17 Pityopus californicus (Ericaceae/Monotropoideae). (a) Flowering stems of Pityopus californicus.
Photo courtesy of Barry Rice. (b) Developing embryo
with multilayered mantle (arrows) on developing root.
Remnants of seed coat are obvious (arrowheads). (c)
Longitudinal section of an older root with mantle covering
the apex. An emerging lateral root (arrow) is evident.
S. Imhof et al.
(d) Higher magnification showing mantle (arrows) and
Hartig net (arrowheads). (e) Detail of mantle (asterisk),
Hartig net (arrowheads), and fungal peg (arrow) penetrating the tangential wall of an epidermal cell. (f) Scanning
electron micrograph of fractured root showing a thick mantle (asterisk). (g) Transmission electron micrograph showing a fungal peg with finger-like wall depositions (arrows)
4
Subterranean Morphology and Mycorrhizal Structures
on the presence of a suitable fungus to provide
sugars and perhaps other nutrients needed for
germination and seedling establishment (Bruns
and Read 2000; Leake et al. 2004; Smith and
Read 2008). Massicotte et al. (2007) have shown
that, in Pityopus californicus, fungal hyphae
become associated with germinating seeds and
form a mantle as the embryo begins to elongate.
Later, a mantle, Hartig net, and fungal pegs form
in the developing root. Mycoheterotrophy is
therefore established very early in the life cycle
of these plant species. Their root systems vary
among species, ranging from large root balls
comprised of numerous mycorrhizal roots (e.g.,
Monotropa, Pterospora), to more diffuse root
systems with mycorrhizal roots distributed more
randomly (e.g., Pleuricospora, Monotropsis,
Allotropa). Hirce and Finocchio (1972) described
in detail the remarkably compact root system
and anatomy of M. uniflora and concluded that it
represents a variation of the normal dicotyledonous condition. They documented a decrease in
anatomical complexity of first order (hexarch
stelar configuration of vascular tissue, several
centimeters long and up to 1.4 mm thick, linking
adjacent plants) over second order (up to 8 mm
long and 0.85 mm thick) to the third order roots
(protostelic arrangement, max. 4 mm long and
0.5 mm thick), even if all roots are densely covered with a mantle (and are presumably active).
At the other extreme, Allotropa exhibits a more
loose system of elongated rhizomes with first
and second order adventitious roots (Massicotte
et al. 2010), and likewise in Monotropsis odorata
(treated as Cryptophila pudica) “the root system
resembles a slender, mostly repeated manybranched, creeping rhizome” (Wolf 1922).
Compared to Allotropa, Pleuricospora seems to
have a slightly more condensed subterranean
system (Massicotte et al. 2010).
In the Monotropoideae (formerly Pyrolaceae),
a progressive compaction of the root system,
from fibrous roots (e.g., Allotropa) to coralloid
roots (e.g., Pleuricospora) to tight rootballs (e.g.,
Monotropa) has been hypothesized as reflecting a
progressive dependence on epiparasitic mycotrophy (Furman and Trappe 1971), although this
remains to be tested physiologically.
193
Structurally, monotropoid mycorrhizas resemble ectomycorrhizas in that a mantle and a Hartig
net in this case confined to the epidermis, form
(Peterson et al. 2004). However, they possess a
unique feature, the invasion of epidermal cells by
short hyphae originating from the Hartig net or
inner mantle. These structures, referred to as fungal pegs (Lutz and Sjolund 1973; Duddridge and
Read 1982; Robertson and Robertson 1982;
Peterson and Massicotte 2004; Peterson et al.
2004) form either along the outer tangential wall
of epidermal cells, or at the base of the radial wall
of epidermal cells. Host cells respond by depositing additional cell wall material, in finger-like
projections, around each peg. It has been hypothesized (Lutz and Sjolund 1973; Duddridge and
Read 1982; Massicotte et al. 2005) that these
structures, resembling “transfer cells” in other
plant species, may be involved in nutrient transfer between the fungus and root cells although
there is no experimental evidence to support this.
In these systems, the Hartig net likely also plays
a role in nutrient transfer but this needs to be
confirmed. Kuga-Uetake et al. (2004) have shown
the close association of microtubules with the
fungal pegs in M. uniflora.
All of these species form monotropoid mycorrhizas with various fungal genera. The vast
majority of fungi colonizing monotropoid roots
are basidiomycetes and most of them have been
identified using molecular approaches (Cullings
et al. 1996; Lefevre 2002; Kretzer et al. 2000;
Bidartondo and Bruns 2001, 2002). In the following paragraphs, we explore these critical features
for five genera of Monotropoideae.
4.6.2.1 Monotropa uniflora (Fig. 4.14)
Monotropa uniflora, a northern hemisphere species (Fig. 4.14a), along with the Asian
Monotropastrum humile (Yokoyama et al. 2005;
Yamada et al. 2008; Matsuda et al. 2011) have a
strong affinity for fungi in the family Russulaceae,
including many species of Russula such as
R. brevipes, R. decolorans, R. nitida, as well as
Lactarius spp. (Young et al. 2002, Bidartondo
2005; Bidartondo and Bruns 2005; Yang and
Pfister 2006). Hypopitys monotropa (=Monotropa
hypopitys), in contrast, forms mycorrhizas mostly
194
with fungal species in the Tricholomataceae.
Excavated plants of M. uniflora reveal welldeveloped root balls (Fig. 4.14b), packed with
mycorrhizal tips of Russulaceae (Fig. 4.14c),
most forming numerous cystidia in the outer
mantle (Fig. 4.14d). Scanning electron microscopy of the mantle surface has shown that cystidia can take various forms including, awl-shaped
(Fig. 4.14e) and flask-shaped (Fig. 4.14f). As
well, frequent calcium-oxalate crystals may be
present (Fig. 4.14g). The mantle (Fig. 4.14h, i)
and Hartig net (Fig. 4.14h) are easily observed
but sectioning of mycorrhizal roots is required to
show the presence and structure of fungal pegs
(Fig. 4.14j–l).
4.6.2.2 Pterospora andromedea (Fig. 4.15)
Pterospora andromedea (Fig. 4.15a) and Sarcodes
sanguinea (not shown), are confined to western
North America and appear to associate almost
exclusively with the section of Rhizopogon
(Rhizopogonaceae) encompassing R. ellenae,
R. salebrosus and R. arctostaphyli (Kretzer et al.
2000; Bidartondo and Bruns 2001, 2002; Taylor
et al. 2002; Dowie et al., 2011). Large root balls
(Fig. 4.15b) dominated with Rhizopogon mycorrhizas (Fig. 4.15c) are evident on excavated
plants. A compact mantle with crystal inclusions
of variable dimensions and shapes (Fig. 4.15d–f)
is characteristic of the mycorrhizas of this species when viewed by scanning electron microscopy. Light microscopy shows a thick mantle, a
well-developed Hartig net (Fig. 4.15g) and a fungal peg apparatus penetrating the radial epidermal cell wall (Fig. 4.15h).
4.6.2.3 Allotropa virgata and
Pleuricospora fimbriolata
(Fig. 4.16)
Allotropa virgata (Fig. 4.16a) forms mycorrhizas
exclusively with Tricholoma magnivelare
(Lefevre 2002; Taylor et al. 2002), and is one of
the most specific host-fungal monotropoid symbiosis documented so far. Light microscopy
reveals sporadic colonization at the root surface
with a thin mantle (Fig. 4.16b) and a fungal peg
penetrating radial walls of epidermal cells (Fig.
4.16c, d). Pleuricospora fimbriolata (Fig. 4.16e)
S. Imhof et al.
parasitizes the fungal species Gautieria monticola (Bidartondo and Bruns 2001, 2002), a truffle
forming species belonging to the Gomphaceae
(Humpert et al. 2001). Typically, a well-developed mantle envelops the root (Fig. 4.16f) and
fungal pegs, penetrating the outer tangential walls
of the epidermis (Fig. 4.16g), are obvious.
Characteristic finger-like wall depositions are
found on the fungal peg (Fig. 4.16h).
4.6.2.4 Pityopus californicus (Fig. 4.17)
Pityopus californicus (Fig. 4.17a) also forms
mycorrhizas mostly with fungal species in the
Tricholomataceae, in this case Tricholoma myomyces (Bidartondo and Bruns 2005). However, a
developmental study on young mycorrhizal
embryos of P. californicus suggests other fungi
are present in earlier stages (Fig. 4.17b) that are
presumably replaced at later stages by T. myomyces (Fig. 4.17c, Massicotte et al. 2007). Mature
mycorrhizal roots typically show a thick mantle
(Fig. 4.17d + f), a well-developed Hartig net and
a fungal peg, penetrating the outer tangential
wall of epidermal cells (Fig. 4.17e). Small fingerlike projections can be seen on the fungal peg
(Fig. 4.17g).
4.6.3
Gentianaceae (Figs. 4.18–4.20)
In the Gentianaceae, 25 species in four genera are
mycoheterotrophic. Additional to the genera covered here, two others are also considered to be at
least partially “saprophytic” (Johow 1889;
Knoblauch 1894; Gilg 1895; Holm 1897, 1906;
Perrot 1898; Wood and Weaver 1982). The monotypic Obolaria virginica has scale-like leaves
along the lower stem with larger spatulateobdeltoid leaves towards the inflorescence. The
fleshy stem and leaves are purplish-green. The
roots are coralloid and mycorrhizal (Holm 1897;
Gillett 1959; Wood and Weaver 1982), like many
of the Voyria species described below. Bartonia
comprises four species, B. virginica, B. verna,
B. paniculata, the latter of which has two subspecies (Gillett 1959), and B. texana (Correll 1966).
All species have only scale leaves but an overall
greenish appearance. Compared to Obolaria
4
Subterranean Morphology and Mycorrhizal Structures
Fig. 4.18 (a–c) Voyria tenuiflora, (d, e) Voyria obconica,
(f–h) Voyria spruceana, (i–k) Exochaenium oliganthum
(Gentianaceae). (a) V. tenuiflora in its natural habitat. (b)
Coralloid shaped root system of V. tenuiflora with branched
roots (r) clumped at the base of a shoot (s). (c) Subterranean
organs of V. tenuiflora showing the tendency to radiating
roots (r) at the base of a shoot (s). The roots can be up to
1 mm thick and several centimeters long. (d) V. obconica
in its natural habitat (courtesy of H and PJM Maas).
(e) Subterranean system of V. obconica with stout, up to
1.5 mm thick and 1 cm long roots (r) at the base of a shoot
195
(s). (f) Characteristic fringed, tail-like thecae appendages
(ap) of V. spruceana. (g) Coralloid shaped root system of V.
spruceana, also having the tendency for a star-like structure
(this specimen measuring 14 mm in maximal extension). (h)
Preserved flower (1.2 cm long) of V. spruceana. (i)
Vermiform to filiform root of E. oliganthum with thickenings up to 0.8 mm where a light brown coloration indicates
fungal colonization (fc). (j) E. oliganthum tends to develop
radiating roots (r) at the base of a shoot (s). (k) Two preserved flowers (7 mm long) of E. oliganthum
196
Fig. 4.19 (a–g) Voyria truncata, (h–n) Voyria aphylla
(Gentianaceae). (a) Epiterrestrial part of V. truncata. (b)
Subterranean shoot (s) of V. truncata, spirally bent due to
S. Imhof et al.
soil obstructions. The shoot arises from the axil between a
main root (r) and a side root (sr). (c) Complete specimen
of V. truncata extracted from the soil, basally arising from
4
Subterranean Morphology and Mycorrhizal Structures
virginica, the Bartonia species are more delicate,
but also have fleshy, sparsely branched mycorrhizal root systems (Holm 1906). Recent physiological investigations using a stable isotope
distribution approach found strong indications
for a partial mycoheterotrophy in Obolaria virginica and Bartonia virginica (Cameron and Bolin
2010).
197
4.6.3.1 Exacum
Cotylanthera was originally a genus comprising
four achlorophyllous species, which was suspected to be closely related to the large genus
Exacum (e.g., Raynal 1967a, Klackenberg 1985,
2002, Yuan et al. 2003); the genus has now been
formally transferred into Exacum by Klackenberg
(2006). The taxonomic accounts (e.g., Miquel
1856; Gray 1871; Clarke 1885; Gilg 1895; Lace
1914; Hara 1975) mostly do not refer to the subterranean parts, but there are two rather old but
quite detailed morphological descriptions of the
roots for Exacum tenue (Janse 1896; Figdor 1897,
treated as Cotylanthera tenuis). Janse (1896)
described the roots as tufted around the stem
base. Such star-like root systems are a common
feature of most MH plants and interpreted as a
strategy to decentralize carbohydrate and nutrient
transport: a root system with many but hierarchically
equivalent roots can better compensate for the
failure of some of its elements than few but highcapacity roots, as is the case in most allorhizic
root systems (Imhof 2010). Combining the
information given by Figdor (1897) and Imhof
et al. (1994), the former showing a seedling of E.
tenue, the latter describing the ontogeny of
another gentian (Voyria tenella), the star-like root
system of E. tenue is probably generated by a primary root, developing ray-like lateral roots, and
only then does this structure give rise to a rootborne shoot. Later, further root-borne shoots are
a means of vegetative propagation. Root hairs, if
present, are much reduced (Figdor 1897). A considerable portion of the stem, up to half its length,
may also be subterranean, often bent due to
Fig. 4.19 (continued) a plagiotropous root (detached).
Only the upper, reddish branches were epiterrestrial and,
hence, appeared superficially as clustered but distinct
individuals. (d) Subterranean shoot (s) of V. truncata arising from a runner-like, plagiotropic root (r), intermingled
with roots of neighboring plants (rnp). (e) Runner-like
root (7 cm long and up to 2 mm thick) of V. truncata
extracted from the soil with several side roots (sr) in the
axils of which one or at most two root-borne shoots (s)
develop. (f) Longitudinal section through a V. truncata
root showing the epidermis (ep) and multilayered cortex
with intracellular hyphal coils in various stages of degradation. The green dotted line indicates a course of colonization from penetration to the inner cortex. The passage
through a short cell (shc) of the exodermis (ex) is not visible on this section but present on the subsequent one (not
shown). The pattern of newly inserted cell walls (arrows)
indicate an ongoing primary thickening. (g) Schematic
view of the mycorrhizal colonization pattern in V. truncata. After penetration of the epidermis and a short cell
(shc) of the exodermis as the only passage cells, the
hyphae grow in a coiling manner from cell to cell deeper
into the cortex. Extent of hyphal degradation increases
with cortex depth. (h) Flower of V. aphylla. (i) Shoot (s)
of V. aphylla arising from a net of runner-like, up to
0.5 mm thick roots (r), much smaller than in V. truncata.
(j) Radiating roots (r) at the base of a shoot (s) of
V. aphylla. (k) Tangential section through the cortex of a
V. aphylla root showing the epidermis (ep) with straight,
nondegenerated hyphae (sh), the exodermis (ex) with only
the short cells (shc, anatomically not particular distinct)
being used as passage cells for the hyphae, and the cortex
parenchyma with often degenerated hyphal coils (hc−).
Root hairs (h) only occur where organic material is
attached to the root. Fungal penetrations (p) mostly happen via the root hairs. (l) Tangential section just external
to the central cylinder through a V. aphylla root, showing
the epidermis (ep) with straight hyphae (sh) and mostly
degenerated hyphal coils (hc−) in the cortex parenchyma.
However, straight hyphae (sh) also occur in the innermost
parenchyma layers (ic), linked to the hyphae in the epidermis by nondegenerated coiled hyphae (not shown). (m)
Root of V. aphylla (r) attached to a root of a neighboring
plant (rnp). Root hairs (h) develop only at such root to root
connections. (n) Schematic view of the mycorrhizal colonization pattern in V. aphylla. After penetration of root
hairs, the hyphae grow straight in the epidermis, cross the
exodermis via passage cells (miss the red coloration),
build coils in the outer cortex parenchyma which partly
degenerate but also reach the innermost cortex parenchyma layers, where they again grow in a straight manner
along the root axis. From these inner straight hyphae
which do not become digested, branches grow back into
the outer cortex to build coils for digestion
198
Fig. 4.20 Voyria tenella (Gentianaceae). (a) Three specimens of V. tenella in various stages of development. The
younger specimens still show the nodding flower bud. (b)
Youngest specimen of V. tenella found, measuring 2 mm
S. Imhof et al.
in length. The primary root formed during germination
(the arched part on the left hand side) has initiated three
root primordia (on the right hand side). A shoot bud has
not formed at this stage. (c) The first shoot primordium (s)
4
Subterranean Morphology and Mycorrhizal Structures
199
obstacles in the soil (Figdor 1897). Similar observations were made in Voyria truncata (Imhof and
Weber 1997) and Triuris hyalina (Imhof 1998).
The roots are several centimeters long and irregularly thickened, with the thicker parts three
(Figdor 1897) to four times (Janse 1896) wider
than the thinner ones. Tangential cell divisions in
the outer cortex increase the number of parenchyma cell layers in those parts where fungal
hyphae are found within the inner cortex cells.
Uncolonized root segments, thus, have only four
parenchyma layers whereas the mycorrhizal parts
can have up to eight (Janse 1896). Cells in the
course of division do not become colonized, neither does the epidermis, exodermis nor the first
cortical parenchyma layer. The hyphae within the
cells are coiled (Figdor 1897) and show local
swellings. They form “sporangioles,” a term used
by Janse (1896) for degenerating hyphae. Figdor
(1897) also speaks of clumped masses of dead
hyphae. A prolonged primary thickening of
the root as described by Janse (1896) has also
been seen in Voyria truncata, although there the
cell divisions happen throughout the cortex, irrespective of being colonized or not (Imhof and
Weber 1997).
No information is given as to whether the subterranean part of the stem is colonized by the fungus. For comparison, more recent studies in Voyria
species did not find hyphae in shoot tissues (Imhof
and Weber 1997, 2000; Imhof 1997, 1999c),
although Svedelius (1902) reports hyphae in aerial
stems of V. tenella (treated as Leiphaimos azurea).
4.6.3.2 Exochaenium (Fig. 4.18i–k)
Exochaenium oliganthum (previously Sebaea
oligantha Kissling et al. 2009; Kissling 2012)
from Central Africa is the only species in this
genus that is achlorophyllous (Raynal 1967a,
Kissling et al. 2009). However, representatives
with little photosynthetic surface such as E. debilis, E. rara and E. pulsilla (Marais and Verdoorn
1963) suggest that partial mycoheterotrophy may
also be present in other species of the genus. As
is often the case, the subterranean organs are
neglected in taxonomic accounts (Gilg 1899;
Robyns 1962). Raynal (1967a), recognizing this
lack, described the roots as radiating from the
stem base, being few (her drawing shows five at
the shoot base), sparsely branched, terete, carnose, and up to 0.5 mm thick (Fig. 4.18i, j). This
description is very similar to that for achlorophyllous Exacum species discussed above. More
exciting is Raynal’s (1967a) finding of additional,
almost subterranean cleistogamous flowers covered by the leaf litter of the soil surface. In contrast to the straight epiterrestrial shoots, the stems
and pedicels of these “subterranean” flowers are
mostly positively geotropic, coiled, and intermingled with each other, but basically develop the
same flowers and fruits as the aerial counterparts,
except for some reductions in floral structures.
The fruits, due to positive geotropism, are geocarpic (Raynal 1967a).
Information on the roots additional to that
mentioned above is very scarce. Professor
Mangenot did investigate the mycorrhiza of
Fig. 4.20 (continued) appears only after the development
of a characteristically radiating root system (this specimen
is 4 mm wide in its maximal extension). (d) Young specimen of V. tenella, the shoot is 1 mm thick. (e) Flower of
V. tenella. (f) Longitudinal section through a young root
of V. tenella showing the penetration and the subsequent
direct growth of the straight hypha (sh) towards the inner
root cortex where it proceeds along the central cylinder.
(g) Longitudinal section through a mature root of V.
tenella showing the straight hyphae (sh) in the inner cortex parenchyma around the central cylinder (cc) and the
degenerated coils of hyphae (hc−) in the outer cortex. (h)
Transverse section through a root of V. tenella (0.8 mm in
diameter). The straight hyphae (sh) in the inner cortex
parenchyma are visible as small circles. The epidermal
tissue (et) consist of 2–3 layers of smaller cells never colonized by the fungus except for penetration points. Their
outermost cells slough off (so) and are replaced by derivates of the layers underneath. The obvious digestion of
hyphal coils (hc−) takes place in the majority of the cortex
parenchyma. (i) Schematic view of the mycorrhizal colonization pattern in V. tenella. After penetration, the hyphae
grows straighly towards the inner cortex layers which are
longitudinally elongated and proceed therein along the
central cylinder. Branches of these inner hyphae grow
back into the outer cortex and degenerate (dh)
200
Exochaenium oliganthum, but never published it.
Raynal (1967a) reports his findings communicated to her, which revealed fungal coils within
the cells very similar to the conditions in Neottia
nidus-avis (Orchidaceae). However, we know
today that orchid mycorrhizas (e.g., Smith and
Read 2008) and the AMs in gentians (e.g., Imhof
1999c) are only superficially alike. Recently,
molecular methods have detected a Glomusgroup A endophyte in Exochaenium oliganthum,
which seems to be highly specific for this species
(Franke et al. 2006).
4.6.3.3 Voyriella
There are roughly 30 publications mentioning the
monotypic Voyriella, many of them only as part
of an enumeration of gentianaceous genera. The
approximately dozen of taxonomic or geographic
accounts mostly lack information on subterranean organs. In fact, there are only five statements
on the roots of Voyriella parviflora: “radice
fibrosa” (Miquel 1851), “roots on the transition
from filiform to coralloid shape” (Johow 1889,
translated from German), “filiform” (Jonker
1936), and “30 mm long and 0.3 mm thick”
(Maas and Ruyters 1986). The latter statement is
simply restated by Pires O’Brien (1997) in a plant
checklist of the Jari river in Brazil. There is no
figure showing the roots of V. parviflora, but its
mycorrhizal fungus has been identified by 18S
rDNA sequencing. V. parviflora seem to be highly
specific to a basal clade of Glomus-group A of the
Glomeromycota (Bidartondo et al. 2002).
4.6.3.4 Voyria (Figs. 4.18–4.20)
There is considerable information available for
the roots of several of the 19 Voyria species. The
earliest observations by Aublet (1775) on Voyria
rosea indicated that it has irregularly tuberous
roots of the size of a fist and become eaten by the
indigenous people (Garipons of the Guianas)
after being cooked in a coal fire, tasting similar to
potatoes. However, although V. rosea has roots
up to 40 mm long and 15 mm (!) thick, it appears
more as a loosely coralloid root system rather
than a tuberculous one (Maas and Ruyters 1986).
S. Imhof et al.
Either the specimen seen by Aublet had densely
intermingled roots only appearing like a tuber of
that size, or Aublet confused it with another plant.
Aublet (1775) called the achlorophyllous genus
after the Garipon name of that edible plant,
Voyria. There is still some confusion on the nature
of the subterranean parts of Voyria spp. as they
are sometimes erroneously called rhizomes (e.g.,
Süssenguth 1937; Jonker 1936; Fukarek et al.
1994; Pringle 1995). Possibly, since the rootborne shoots (Imhof et al. 1994; Imhof and Weber
1997; Imhof 1997) often have to grow through a
considerable layer of soil before they reach the
surface (Fig. 4.19b–d, Imhof and Weber 1997,
see before under Exacum), they might have been
misinterpreted as orthotropous rhizomes.
Considerable differences, most probably representing evolutionary steps, occur within this
genus. Paralleled by the reduction of floral
(Oehler 1927; Maas and Ruyters 1986) and shoot
anatomical features (Johow 1885; Solereder
1908; Oehler 1927; ter Welle 1986), the root systems can also be arranged according to reduction
particularly in root length. Roots of Voyria truncata (a primitive member of the genus), can presumably be several meters long, growing
horizontally and runner-like (Fig. 4.19e) as deep
as 20 cm beneath the soil surface (Fig. 4.19c, d),
and up to 2 mm thick, frequently branched, and
give rise to two root-borne shoots in the axils of
side roots (Imhof et al. 1994; Imhof and Weber
1997, Fig. 4.19e). Hence, seemingly distinct
specimens above soil can well belong to the same
individual, either by shoot ramifications already
in the soil (Fig. 4.19c), or because of different
root sprouts originating from the same root (Fig.
4.19e). Field observations recognized V. truncata
shoots (Fig. 4.19a–d) emerging like a chain of
beads for several meters, denoting the root course
in the soil (Imhof et al. 1994). Aublet (1775) also
reports on roots of V. rosea being a foot deep in
the soil.
In contrast, the most advanced representative
of the genus, Voyria tenella, has a small, star-like
root system, shallowly rooted or only loosely
connected to the litter substrate (Imhof et al.
4
Subterranean Morphology and Mycorrhizal Structures
1994, Fig. 4.20a–e). Ontogenetic studies revealed
a root to appear first after germination (Fig.
4.19b) and only after a small star-like root system
form does a first shoot arise (Imhof et al. 1994,
Fig. 4.20c). In spite of the ray-like appearance,
the roots do not grow evenly in all directions. In
fact, there is a single pole of growth representing
the primary root which develops side roots at
very short distances creating the globose structure (Imhof 1997).
In conclusion, although the two root systems
of V. truncata and V. tenella seem to be very different, the root system of V. tenella is easily
interpreted as an extremely abbreviated root system of V. truncata. This notion is supported by
intermediate root systems linking V. truncata and
V. tenella, e.g., V. aphylla (Imhof 1999c, Fig.
4.19h–j), V. rosea (Maas and Ruyters 1986),
V. chionea (Progel 1865), and V. obconica (Imhof
and Weber 2000, Fig. 4.18d, e). The only African
representative, V. primuloides, which is considered to be sister to V. chionea (Albert and Struwe
1997), is the only species in the genus with prominent root hairs (Raynal 1967b).
The root anatomy and mycorrhizal colonization patterns in Voyria also support the progression proposed above. Voyria truncata has an
almost identical root anatomy to a young Gentiana
lutea (Perrot 1898), including a distinct dimorphic exodermis with short and long cells (Fig.
4.19f, g) and a substantial central cylinder with
lignified tracheary elements, but lacking secondary thickening. Instead, a prolonged primary
thickening is established (Fig. 4.19f), which constantly enhances the essential tissue for fungal
colonization and is interpreted as an important
adaptation to its mycoheterotrophy (Imhof and
Weber 1997). The type of arbuscular mycorrhiza
is also very similar to other gentians (Neumann
1934; Jacquelinet-Jeanmougin and GianinazziPearson 1983), except for the lack of lateral
arbuscules formed from the coiled intracellular
hyphae, typical for a Paris-type AM. The degradation of hyphal coils in the cells, best explained
as a digesting process of the plant to absorb carbon and nutrients from the hyphae, happens after
201
about 15 cell passages (Imhof and Weber 1997,
Fig. 4.19f).
The roots of V. aphylla (Fig. 4.19h, i) have all
anatomical elements of V. truncata but are
reduced in their size and there is a tendency for a
radiating formation of side roots at the shoot
bases (Fig. 4.19i, j). The mycorrhizal associations are also similar. However, some new features have been acquired: (1) a longitudinal
spread of straight hyphae within the epidermis as
well as the innermost cortex layers (Fig. 4.19k, l)
and (2) the development of root hairs only where
roots of neighboring plants are attached (Imhof
1999c, Fig. 4.19m). Whereas the hyphae in roots
of V. truncata are largely restricted regarding the
cortical spread, the nondegenerating hyphae in
the epidermis of V. aphylla are able to reach more
distant segments of the root as well. Direct hyphal
bridges from attached roots of neighboring plants
are frequent sources of fungal root penetrations
in several Voyria species. The locally developed
root hairs of V. aphylla increase this contact zone
and, in fact, receive most of the external fungal
penetrations (Imhof 1999c; Fig. 4.19k + m).
Hyphal passage through the exodermis still is
exclusively via the short cells, although those are
not as anatomically distinct as they are in V. truncata (Imhof and Weber 1997). Digestion of fungal material takes place in the cortex parenchyma
except for its innermost layers, where a still
imperfect internal spread along the central cylinder can be seen. The latter feature foreshadows
the highly efficient colonization pattern of the
further derived Voyria tenella, V. obconica and
V. flavescens (Fig. 4.20i).
The root anatomy of the most advanced
V. tenella had been investigated by Johow (1885)
and Vigodsky-de Philippis (1938, under the synonym Leiphaimos brachyloba). Both stress the
reduced character of the vascular system, the lack
of suberization of the endodermis in V. tenella,
and the voluminous root cortices (more details in
Imhof 1997, Fig. 4.20a–e). Johow (1885) also
recognized the coiled fungal mycelium within the
cortex cells, but called it “parasitic” and, in
accepting Drude’s hypothesis (1873), assigned its
202
presence to the attraction through a “particularly
rich flow of organic nutrients” (translated from
German) he assumed to be a compulsory attribute
of the roots of this “saprophytic” plant. He was
aware of Kamienski’s (1882) new notion of a
symbiotic association between fungus and plant,
yet with cautious criticism. Vigodsky-de Philippis
(1938) called the hyphae a “micelio micorrhizico.” However, these classical papers did not recognize the specialized colonization pattern. Root
penetrating hyphae initially grow straight towards
the innermost cortex layers and proceed in longitudinal direction along the reduced central cylinder. From there, hyphal branches grow back into
the outer cortex parenchyma where they begin to
coil, quickly inflate, and finally collapse into
amorphous clumps (see details in Imhof 1997;
Imhof and Weber 2000; Franke 2002, Fig. 4.20f–
h). By this means, a sustained use of only few
external penetrations of the fungus is attained,
maintaining the hyphae alive in the inner cortex
and only digesting branches of them in the outer
cortex: an intraradical fungus garden (Imhof
1997, Fig. 4.19i). In summary, within Voyria, we
can retrace not only morphological and anatomical reductions but also the evolutionary progression of a mycorrhiza (compare Figs. 4.19g, n and
4.20i), resulting in a highly efficient system to
benefit from a fungus.
The mycorrhizal fungi of several Voyria species have been determined by molecular
identification methods. Almost all of the endophytes belong to Glomus-group A of the
Glomeromycota. However, Voyria spp. seem to
not be as specific regarding their mycorrhizal
associates as other mycoheterotrophic species
(Bidartondo et al. 2002; Merckx et al. 2010).
Since mycoheterotrophic plants are very
difficult to cultivate, the unexpected emergence
of a Voyria species in the Botanical Garden in
Hamburg (Germany) shall be briefly reported
here. As an epiphytic stowaway on the trunk of a
tree fern (Alsophila salvinii) imported in the mid
seventies, a yellow Voyria (perhaps V. aphylla,
which is known to grow also epiphytically,
Groenendijk et al. 1997) was discovered around
1980. Unfortunately, it died in 1983, when its
host fern tree was placed outside during summer
(Poppendieck 1997).
S. Imhof et al.
4.7
Selected Species of
Questionable Trophic Status
4.7.1
Buxbaumia spp. (Bryophyta,
Fig. 4.1k)
The genus Buxbaumia is comprised of 12 species
in the northern hemisphere (Crosby et al. 2000;
Goffinet et al. 2008). In contrast to the majority
of mosses, the up to 3 cm high sporophyte is the
prominent phase of this genus and consists of a
bulky, oblique-oval capsule on top of the seta
(Eastwood 1939). This appearance has led to its
enchanting vernacular names, e.g., Elfcap Moss,
Humpbacked Elves, Bug-on-a-stick (Fig. 4.1k).
While most species have greenish sporophytes
(Udar et al. 1971; Ligrone et al. 1982; Stone
1983; Düll and Düll-Wunder 2008), the sporophytes of Buxbaumia aphylla and B. minakatae
(Okamura 1911; Iwatsuki and Sharp 1967) seem
to be largely devoid of chlorophyll. The gametophyte of Buxbaumia aphylla is minute and achlorophyllous (Goebel 1892; Dening 1928; Mueller
1972; Hancock and Brassard 1974). The possibly
perennial protonema (Steven and Long 1989),
consisting of single-lined threads of thick walled
cells (Mueller 1972), which may form velvety
mats (McClymont 1950), is green but of questionable trophic relevance (Haberlandt 1886;
Eastwood 1939). Early stages of the sporophyte
may show some green color (Goebel 1892;
Dening 1928; Eastwood 1936; Hancock and
Brassard 1974; Schoepe and Philippi 2000; van
Rompu and Stieperaere 2002), but many recent
as well as older publications consider it to have a
heterotrophic mode of life (e.g., Haberlandt 1886;
Eastwood 1936; Mueller 1972; Watson and
Dallwitz 2005 onwards; Düll and Düll-Wunder
2008). It is clear therefore that the relation of
auto- vs. heterotrophy in the whole genus is completely unknown and should be studied.
In contrast to Eastwood (1939), who believed
in direct absorption by Buxbaumia of organic
substances from humus or neighboring green
mosses, we know today that plants are unable to
do that directly, but are either parasitic (Kuijt
1969; Weber 1993) or mycoheterotrophic when
lacking chlorophyll (Leake 1994, 2005). However,
4
Subterranean Morphology and Mycorrhizal Structures
no information is yet available on mycorrhizal,
endophytic, or parasitic associations, which could
possibly explain the strange habit of these mosses.
The ultrastructural investigation on the green B.
piperi did not find any association with fungi
(Ligrone et al. 1982). Neither did the detailed
description (Stone 1983) on the foot and vaginula
of B. novae-zelandiae mention fungal hyphae,
but explained the dense indumentum with anastomosing elements as “rhizoidal outgrowth from
the epidermal cells of the fertile axis.” Udar et al.
(1971) also show drawings of longitudinal and
transverse sections of the tomentose and slightly
tuberous basis of the sporophyte of B. himalayensis, which superficially resembles an ectomycorrhiza. Unfortunately, the authors did not comment
on this feature at all. However, Haberlandt (1886)
has shown drawings of what he interpreted to be
rhizoids. These “rhizoids” could well be fungal
hyphae, and Haberlandt (1886) even explicitly
remarked on their striking similarity to hyphae,
particularly because of their frequent anastomoses, which, as far as we know, is not characteristic
for moss rhizoids. Similarly, Goebel (1892)
reports of anastomosing as well as achlorophyllous protonemata, which even show clamp-like
connections (line drawing in Goebel 1892). In
any case, contemporary studies providing photographic micrographs instead of drawings on the
micromorphology and anatomy of Buxbaumia
aphylla as well as stable isotope investigations
(see Chap. 8) in order to elucidate the trophic
mode, are urgently needed.
4.7.2
Pyrola picta (Pyrola aphylla,
Ericaceae)
Pyrola aphylla was first described by James
Edward Smith 1814 in Abraham Rees’
Cyclopaedia (Vol. 29, No. 7), calling it “leafless.”
Other authors as well, such as de Candolle (1838)
and Hooker (1840), described this species as having no leaves. However, Nuttall (1843, cited by
Holm 1898) detected leaves of this species, and
Holm (1898) ascertained subterranean shoots
connecting apparently leafless specimens with
rosettes of green leaves, proving that they belong
to the same individual. Also Andres (1914) noted
203
that P. picta can have few to no leaves. The same
observation was made by Camp (1940), who
revisited herbarium sheets and argued for a close
relationship, if not identity, of Pyrola aphylla,
P. picta and P. dentata. More recently, Haber
(1987) eventually merged the three species within
the highly variable Pyrola picta Sm.
In addition to the subterranean shoots (stolons) already mentioned, P. picta also has horizontally growing, runner-like, branching roots.
Root-borne shoots as well as adventitious roots
from the stolons can develop (Holm 1898). The
only specific investigations on the mycorrhiza of
P. picta is by Largent et al. (1980), calling it arbutoid and ericoid (both seen in specimens of
P. picta var. picta) and ericoid (in P. picta var.
aphylla). Other authors have called the mycorrhizas of Pyrola spp. arbutoid (Robertson and
Robertson 1985; Massicotte et al. 2008; Vincenot
et al. 2008), ectendomycorrhizal (Wang and Qiu
2006), or pyroloid (Cullings 1996). It also has
been considered as a linking mycorrhizal type
between arbutoid and ericoid mycorrhiza in a
new classification of mycorrhizas (Imhof 2009).
Because P. picta has green leaves, and the
extreme, leafless variant of P. picta, P. aphylla,
still has chlorophyll in the shoot bark (Holm
1898), it actually should not be fully mycoheterotrophic. However, Hynson et al. (2009)
found characteristic stable isotope signatures
typical for mycoheterotrophic plants in Pyrola
aphylla specimen. Interestingly, Pyrola picta
with leaves, although being the same species taxonomically, did not show signs of mycoheterotrophy according to carbon stable isotope signatures
(Hynson et al. 2009). Hence, the trophic status of
this species is ambiguous. Possibly, dependency
on the fungal carbon is not determined by the
species in the taxonomical sense but on the actual
ability for assimilation in a particular specimen.
4.8
Trends, Conclusions,
and Future Directions
MH plants have distinctive structural necessities
in contrast to autotrophic species due to their mycorrhizal dependence for carbon supply. Secondary
growth of roots, for example, is deleterious since
204
it sheds the primary tissue, which alone can host
the indispensable mycobiont. Moreover, the primary tissue, less important in autotrophic plants,
must be present in sufficient quantity. Most importantly, intracellular (in contrast to intercellular)
mycorrhizal colonization is a major prerequisite.
In fact, there is no mycoheterotroph having
an Arum-type AM or an ectomycorrhiza, both
of which are characterized by predominantly
intercellular hyphal growth. Obviously, the transfer of nutrients and carbohydrates provided by
those mycorrhizal types is not sufficient to support
achlorophylly. There also must be a high probability to become colonized by an appropriate fungus,
keeping in mind that MH plants are often quite
specific with respect to their endophyte (e.g.,
Kretzer et al. 2000; Taylor et al. 2002; Bidartondo
et al. 2002; Franke et al. 2004; Ogura-Tsujita and
Yukawa 2008). A widely branched allorhizic root
system seems to be suitable for this, but in turn, is
susceptible to functional failure of large parts by
only a single blocking, collapsing or disconnection event in a proximal segment. This is particularly critical when secondary growth for securing
the connection is impossible. In any case, the
transfer of carbon to the reproductive parts must
be either short or reliably assured. These challenges are reasons for the following convergent
evolutionary trends concerning subterranean
organs of MH plants in unrelated plant families:
1. Star-like root systems consisting of many
roots radiating from the base of the shoot,
either created by root-borne shoots or shootborne roots, reduce the risk of becoming disconnected to a major part of the root system
(e.g., Figs. 4.2g, 4.4b, 4.6c + g + k, 4.7b,
4.8e + j, 4.10c + f + g + i, 4.12f + g, 4.13a + d,
4.14b, 4.15b, 4.18b + e + g + j, 4.19j, 4.20a).
2. Short and thick roots shorten the transport distance of carbon to the shoot while retaining
the tissue volume of long and thin roots (e.g.,
Figs. 4.3a, 4.4b, 4.7b, 4.9d, 4.10f, 4.12f + g,
4.14b, 4.15b, 4.17e + g, 4.19c).
3. Specialized colonization pattern that enables a
sustained use of a few fungal penetrations
counterbalance the reduced probability to
become colonized in short and thick roots
compared to filiform roots (e.g., Figs. 4.3i,
S. Imhof et al.
4.4i, 4.5j, 4.8h, 4.9h, 4.13h, 4.14k, 4.16d,
4.19n, 4.20i).
4. Strong reinforcement of thin roots, either by
tertiary endodermae (in monocots) or the
development of multicellular fibrous tissue,
protect the carbon supply of the shoot (e.g.,
Figs. 4.5d + f, 4.6c + d, 4.13h).
The contradicting needs for a large root surface for
high infection probability and short distances for
carbon transport, has been discussed as the “mycoheterotroph’s dilemma” (Imhof 2010) and supposedly has shaped much of the subterranean organs
in MH plants during evolution. As an effect,
advanced MH plants within a family have stout,
clumped roots and (in orchids) rhizomes mostly
with a specialized fungal colonization pattern.
This trend is best exemplified in Voyria
(Gentianaceae, Imhof 1999c) and in Ericaceae
(Furman and Trappe 1971). Gentianales and
Ericaceae especially, having two fundamentally
distinct groups of mycorrhiza (AM group vs. ECM
group, Imhof 2009) but both show evolutionary
reductions from trees to achlorophyllous herbs
(Henderson 1919; Imhof 1999c) including changes
in mycorrhizal pattern, turn out to be a textbook
example for convergent evolution. In Triuridaceae
(Imhof 2003), Burmanniaceae (Imhof 2001),
Thismia (this chapter) and Afrothismia (both
Thismiaceae, Imhof 2006), and Orchidaceae
(Furman and Trappe 1971), this trend is partly
detectable, but further investigations are necessary for more support. Research on taxa-like
Geosiris (Iridaceae), Corsia (Corsiaceae), Kupea
(Triuridaceae), Haplothismia (Thismiaceae) and
others for which nothing is known concerning the
fungal structures, will also help to understand the
evolution of mycoheterotrophy. Moreover, given
that 15 investigated vascular MH plants associated with AM fungi (i.e., Monotropoideae and
orchids excluded) revealed 13 different mycorrhizal colonization patterns, there is a considerable
chance for more fascinating novelties. In orchid
mycorrhizas, although belonging to the oldest
fields of mycorrhizal research (e.g., Schleiden
1845), comparatively little is known on the two
existing types: tolypophagy and ptyophagy
(Burgeff 1932; Wang et al. 1997; Rasmussen
2002; Imhof 2009). Since the latter type was found
4
Subterranean Morphology and Mycorrhizal Structures
exclusively in achlorophyllous orchids so far (e.g.,
Janse 1896; Campbell 1963, 1964; Wang et al.
1997), an examination of the mycorrhizal structures of other MH orchids is highly desired.
In conclusion, mycoheterotrophy is based on a
number of specializations with respect to morphology and anatomy of the underground parts,
and, most importantly, on the evolution of sophisticated mycorrhizal pattern.
Acknowledgements Thanks to Mori Thomann for his
help with the Japanese paper of K. Watanabe, as well as
Jesper Hansen for translating parts of the Danish articles
by V. A. Poulsen.
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