Annals of Botany 109: 873 886, 2012

doi:10.1093/aob/mcs018, available online at www.aob.oxfordjournals.org

RESEARCH IN CONTEXT

Evolution of mixed-linkage (1  3, 1  4)-b-D-glucan (MLG) and xyloglucan

in Equisetum (horsetails) and other monilophytes
Xinxin Xue and Stephen C. Fry*
The Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, School of Biological Sciences, The University of
Edinburgh, Daniel Rutherford Building, The Kings Buildings, Mayfield Road, Edinburgh EH9 3JH, UK

Present address: The Biodiversity Research Centre, Department of Botany, Botanical Garden and Centre for Plant Research,
The University of British Columbia, 2212 Main Mall, Vancouver V6T 1Z4, Canada.
* For correspondence. E-mail S.Fry@ed.ac.uk

Received: 24 November 2011 Returned for revision: 20 December 2011 Accepted: 13 January 2012 Published electronically: 28 February 2012

Background and Aims Horsetails (Equisetopsida) diverged from other extant eusporangiate monilophytes in
the Upper Palaeozoic. They are the only monilophytes known to contain the hemicellulose mixed-linkage
(1  3, 1  4)-b-D-glucan (MLG), whereas all land plants possess xyloglucan. It has been reported that
changes in cell-wall chemistry often accompanied major evolutionary steps. We explored changes in hemicellu-
loses occurring during Equisetum evolution.
Methods Hemicellulose from numerous monilophytes was treated with lichenase and xyloglucan endogluca-
nase. Lichenase digests MLG to di-, tri- and tetrasaccharide repeat-units, resolvable by thin-layer
chromatography.
Key Results Among monilophytes, MLG was confined to horsetails. Our analyses support a basal trichotomy of
extant horsetails: MLG was more abundant in subgenus Equisetum than in subgenus Hippochaete, and uniquely
the sister group E. bogotense yielded almost solely the tetrasaccharide repeat-unit (G4G4G3G). Other species
also gave the disaccharide, whereas the trisaccharide was consistently very scarce. Tetrasaccharide : disaccharide
ratios varied interspecifically, but with no consistent difference between subgenera. Xyloglucan was scarce in
Psilotum and subgenus Equisetum, but abundant in subgenus Hippochaete and in the eusporangiate ferns
Marattia and Angiopteris; leptosporangiate ferns varied widely. All monilophytes shared a core pattern of xylo-
glucan repeat-units, major XEG products co-chromatographing on thin-layer chromatography with non-fucosy-
lated hepta-, octa- and nonasaccharides and fucose-containing nona- and decasaccharides.
Conclusions G4G4G3G is the ancestral repeat-unit of horsetail MLG. Horsetail evolution was accompanied by
quantitative and qualitative modification of MLG; variation within subgenus Hippochaete suggests that the struc-
ture and biosynthesis of MLG is evolutionarily plastic. Xyloglucan quantity correlates negatively with abundance
of other hemicelluloses; but qualitatively, all monilophyte xyloglucans conform to a core pattern of repeat-unit
sizes.

Key words: Equisetum, Hippochaete, Equisetum bogotense, ferns, eusporangiate monilophytes, leptosporangiate
monilophytes, evolution, cell wall ( primary), hemicellulose, mixed-linkage beta-glucan, xyloglucan.

IN T RO DU C T IO N et al., 2001). To date, differences in cell-wall composition

Major changes in primary cell wall composition often accom-
panied landmark steps of plant evolution, especially during
colonization of the land and vascularization (Stace, 1981;
Kenrick and Crane, 1997a; Popper and Fry, 2003).
Accordingly, a potential use of primary cell-wall composition
in understanding phylogenetic relationships has been consid-
ered, e.g. in algae (Popper and Fry, 2003), bryophytes
(Ligrone et al., 2002; Popper and Fry, 2003, 2004), lycophytes
and monilophytes (Buckeridge et al., 1999; Matsunaga et al.,
2004; Popper and Fry, 2004) and angiosperms (Nothnagel and
Nothnagel, 2007). Cell-wall components differ between plant
taxa in parallel with their evolution and diversification
(Popper, 2008) at both the monosaccharide and polysaccharide
levels (Fig. 1). For instance, the earliest diverging extant vas-
cular plants, lycopodiophytes, are unique among land plants in
containing high levels of 3-O-methyl-D-galactose (Popper
are commonly used as taxonomic markers in algal classifica-
tion but have not been applied to land-plant classification
(Stebbins, 1992; Buckeridge et al., 1999; Graham and
Wilcox, 1999).
The plant primary cell wall is a strong and cohesive network
of cellulose microfibrils, probably tethered by hemicelluloses.
Usually the principal tethers are xyloglucan (in dicots and
non-commelinid monocots) or glucuronoarabinoxylan [in
commelinid monocots, e.g. the Poaceae (grasses and
cereals); Smith and Harris (1999)]. Structures and proportions
of the hemicelluloses are variable between species and organs,
and even within tissues (Harris, 2005; Fry, 2011). A third
major hemicellulose, mixed-linkage (1  3, 1
4)-b-D-glucan (MLG), occurs in certain algae (Ford and
Percival, 1965; Nevo and Sharon, 1969) including at least
one charophytic species (Micrasterias denticulata; Eder
et al., 2008), and in a narrow range of land plants (Stone

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874 Xue & Fry Monilophyte hemicelluloses

Streptophytes
Embryophytes

Tracheophytes
Euphyllophytes

Monilophytes Seed plants

Angiosperms

Charophytes

Bryophytes

Lycophytes

Eusporangiate
monilophytes

Leptosporangiate
ferns

Gymnosperms

Commelinid
monocots

Non-commelinid
monocots

Dicots
abundance
Increasing

Monosaccharide residues
MeRha
MeGal
Polysaccharides
Xyloglucan
Mannans
Xylan
RG-II
MLG
Homo-GalA

F I G . 1. Scheme of streptophyte phylogeny, shown together with the relative abundance of selected primary cell wall components across major plant
groups. Wall composition differs at both the monosaccharide and the polysaccharide levels. MeGal, 3-O-methyl-D-galactose; MeRha, 3-O-methylrhamnose;
MLG, (1  3, 1  4)-b-D-glucan; RG-II, rhamnogalacturonan-II; Homo-GalA, homogalacturonan. Intensity of the shades indicates relative abundance.
MLG (dotted shading) is present in only one genus (Equisetum) of eusporangiate ferns and one order (Poales) of the commelinid monocots.

and Clarke, 1992; Buckeridge et al., 2004; Trethewey et al., cereals, MLG is synthesized in the Golgi apparatus (Carpita
2005; Fry et al., 2008a; Srensen et al., 2008). Within land and McCann, 2010) and typically has a structure of the type
plants, MLG has been found in only two, distantly related, . . . G4G4G3G4G4G3G4G4G4G3G4G4G3G4G4G3 . . .
groups: the angiosperm order Poales (Smith and Harris, where G represents a b-D-glucose residue, and 3 and 4 repre-
1999; Buckeridge et al., 2004; Trethewey et al., 2005) and sent (1  3) and (1  4) bonds, respectively. The underlined
the fern ally genus Equisetum (Fry et al., 2008a; Srensen segments are effectively cello-oligosaccharides interconnected
et al., 2008). Since MLG occurs in two evolutionarily very by hinges. The (1  4) linkages give rigidity, whereas (1 
distant land-plant taxa, it appears that its biosynthesis is 3) linkages confer flexibility and water solubility (Woodward
either deeply conserved or has arisen twice through convergent et al., 1983). Luttenegger and Nevins (1985) found a marked
evolution (Srensen et al., 2010). variation in the MLG content (1 14 %) of Zea mays coleoptile
Although the polysaccharide MLG is found in both the primary cell walls during changes in the rate of cell expansion,
Poales and the Equisetales, only the latter possess MLG : xylo- suggesting a role of MLG in rapid growth in the Poales.
glucan endotransglucosylase (MXE), an enzyme capable of MLG can be structurally characterized by analysis of
grafting MLG to xyloglucan chains by transglycosylation the oligosaccharides produced on digestion with Bacillus
(Fry et al., 2008b). This observation may indicate that MLG subtilis lichenase. This commercial endo-glucanase cleaves
serves different biological roles in the cell walls of these two the (1  4) bond following a (1  3) bond (Meikle et al.,
plant orders. A difference in role is also suggested by the 1994). From poalean MLG, lichenase thus generates the tri-
fact that poalean MLG is principally a feature of young, saccharide G4G3G plus a minority of the tetrasaccharide
rapidly expanding tissues, whereas the MLG of Equisetum per- G4G4G3G (Stone and Clarke, 1992) (these sequences are
sists and may even increase during ageing (Fry et al., 2008a). always quoted from non-reducing to reducing end).
MXE activity reaches its peak in old, tough Equisetum stems, While poalean MLG is composed of G4G3G . G4G4G3G,
suggesting a role in tissue strengthening (Fry et al., 2008a). Equisetum MLG when digested with lichenase releases
MLG is a long, unbranched, coiling b-D-glucan chain with G4G4G3G and the disaccharide G3G (laminaribiose) as the
(1  4) linkages plus a minority of (1  3) linkages. In predominant products (Fry et al., 2008a; Srensen et al.,
 Xue & Fry Monilophyte hemicelluloses
2008). The trisaccharide : tetrasaccharide ratio in poalean
MLG ranges between 1.5 in Zea mays and 4.5 in wheat
(Triticum aestivum) flour (Buckeridge et al., 1999; Li et al.,
2006). In addition to G4G3G and G4G4G3G, oligosaccharides
with degree of polymerization (DP) up to 14 have been
reported in barley MLG (Lazaridou and Biliaderis, 2007);
and Equisetum MLG yielded a lichenase product tentatively
identified as DP9 (Fry et al., 2008a).
Functionally, the trisaccharide : tetrasaccharide ratio affects
the strength of an MLG gel, as demonstrated by studies
using the large-deformation mechanical test (Lazaridou
et al., 2004), suggesting a significance of this ratio in
governing the strength of the primary cell wall.
In the primary cell walls of many land plants, except the
commelinid monocots, xyloglucan is the major hemicellulose.
Composed of long and slightly flexible chains, xyloglucans
can hydrogen-bond strongly to cellulose, and probably tether
adjacent microfibrils, contributing to wall architecture.
Xyloglucans have a backbone of (1  4)- b-D-glucan (qualita-
tively identical to cellulose), many of the glucose residues
(typically approx. 75 %) bearing an a-D-xylopyranose (Xylp)
residue at position 6. A (1  4)-linked glucose is abbreviated
G; one with xylose attached (forming a disaccharide called
isoprimeverose) is abbreviated X (Fry et al., 1993).
Additional sugar residues are attached to some of the
isoprimeverose groups, generating structures such as b-D-
Galp-(1  2)-a-D-Xylp-(1  6)-b-D-Glc (abbreviated L)
and a-L-Fucp-(1  2)-b-D-Galp-(1  2)-a-D-Xylp-(1  6)-
b-D-Glc (abbreviated F).
Xyloglucan can be broken down for analysis by digestion
with xyloglucan endoglucanase (XEG; Pauly et al., 1999),
which yields oligosaccharides such as the heptasaccharide
XXXG, the two octasaccharides XXLG and XLXG, the two
nonasaccharides XXFG and XLLG, and the decasaccharide
XLFG (Hoffman et al., 2005).
The monilophyte Equisetum hyemale and the lycophyte
Selaginella kraussiana have been reported to possess addition-
al xyloglucan repeat-units not yet detected in other plants.
These are D and E, which are identical to L and F, respective-
ly, except that the b-D-Galp residue is replaced by a-L-Arap
(Pena et al., 2008). This is a highly conservative replacement:
despite the a/b and D/L differences in the nomenclature,
b-D-Galp and a-L-Arap differ from each other only in the
fact that the former has a -CH2OH group in place of an -H.
Equisetum hyemale xyloglucan includes the sequences
XLEG and XLDG, which very closely resemble XLFG and
XLLG, respectively (Fig. 2).
Extant vascular plants are divided into two major clades:
lycophytes and euphyllophytes, the latter ( plants with true
leaves) being divided into spermatophytes and monilophytes
(Kenrick and Crane, 1997b; Pryer et al., 2001). The horsetails
(genus Equisetum), the psilophytes, and the eusporangiate and
leptosporangiate ferns, are all monilophytes (Pryer et al., 2001,
2004). The class Equisetopsida (syn. Sphenopsida) emerged in
the Upper Devonian, and became diverse and abundant in the
Palaeozoic swamp forest (Delevoryas, 1962). It includes both
the extant genus Equisetum and extinct herbaceous and arbor-
escent horsetails such as the Calamitaceae (Bateman, 1991).
The extant herbaceous horsetails are considered living
fossils as Equisetum is the only surviving genus, forming a
875
crown group (i.e. a clade including all the extant members
from their most recent common ancestor) of the
Equisetopsida total group (a clade containing all descendants
of a common ancestor, whether extant or extinct). Equisetum
might even be the oldest surviving genus of all vascular
plants owing to the ancient history of this lineage (Hauke,
1978).
Both molecular- and fossil-based estimates of age suggest
that extant Equisetum had diverged from the fossil genus
Equisetites in the Tertiary of the Cenozoic (approx. 49 Ma)
(Des Marais et al., 2003; Pryer et al., 2004). However,
recent comparisons of several anatomically preserved
Equisetum fossils from the Jurassic and Cretaceous period in-
dicate a much earlier Mesozoic origin of the crown group
(approx. 100 200 Ma) (Stanich et al., 2009; Channing et al.,
2011). This argument is based on the distinct morphological
features found in Mesozoic Equisetum (from sinter and other
volcanic deposits), which closely resemble but differ from
both the Triassic Equisetites fossil compressions and the
extant horsetails (Baron, 1889; Channing et al., 2011).
Leaves of Equisetum have the combined characteristics of
apparent microphylls (leaves with a single vascular bundle)
plus the megaphyll feature of having a gap in the stem vascu-
lature where the leaf bundle diverges. The fossil evidence is
clear that the apparent microphylls of Equisetum actually rep-
resent reduced megaphylls, based on observations from the
genus Sphenophyllum in the sister group Sphenophyllales,
which have more complex megaphylls from which type the
leaves of Equisetum are apparently reduced (Kenrick and
Crane, 1997b).
Recent phylogenetic analysis of the 15 recognized species
of the genus Equisetum shows a basal trichotomy: there are
two monophyletic subgenera [Hippochaete (including the neo-
tropical E. giganteum and E. myriochaetum) and Equisetum
(all of which are of temperate distribution in the northern
hemisphere)] plus the diminutive neotropical species
E. bogotense as a sister group (Hauke, 1978; Des Marais
et al., 2003). [Note: Equisetum in this manuscript implies
the genus unless subgenus is specified.] Species in the sub-
genus Equisetum typically have superficial stomata with
highly branched vegetative shoots, whereas most species in
Hippochaete have sunken stomata with generally unbranched
vegetative shoots (Hauke, 1959).
Phylogenetic study of Equisetum is difficult owing to the
long history of isolation and frequent inter-specific hybridiza-
tion within but not between each subgenus. In addition, horse-
tails are capable of reproducing vegetatively, further
complicating phylogenies as sterile hybrids can persist.
While the phylogenetic relationships among species of
Hippochaete have shown a fair degree of consistency across
studies (Des Marais et al., 2003; Guillon, 2004, 2007), the
phylogeny of subgenus Equisetum is still disputable, despite
there being more distinguishing morphological features
within the group than in Hippochaete. One suggested reason
for the lack of reconciliation between molecular and morpho-
logical classification is the homoplasic characters (such as
stem dimorphism), arising through convergent evolution as
analogous features, not being differentiated in classical taxo-
nomic treatments (Guillon, 2004, 2007). The systematic diffi-
culty of Equisetum is not unique in the early-divergent
876 Xue & Fry Monilophyte hemicelluloses

OH HO
HO HO OH
DP10 OH OH O

Xy
CH2 CH3 OH
O

Xy

l
HO O

Ar
OH O

Fu
CH3 CH2OH

a
HO O O O

c
Ga
CH2OH HO O O
O O
Fu

HO

l
HO O O O GlcRT
c

GlcRT CH2 OH OH
HO
CH2 OH OH
HO O O OH
HO O O OH
CH2 OH Glc
OH OH Glc OH O
CH2 OH

Xy
O OH OH OH
Xy

l
OH OH O O
l

OH

Ga
O O O
Ga

OH

l
O HO O
l

HO O CH2
CH2
O O
O O Xyl
Xyl OH Glc
Glc HO OH
OH OH O
HO
O HO O
CH2 OH
HO O OH
CH2 O
O Glc
Glc XLFG OH XLEG
OH HO
HO
OH
OH

HO HO HO
DP8 OH OH
OH
OH
CH2 O O
Xy

Xy
OH
OH
l

l
Ar O
O O O CH2OH
Ga

CH2OH
a
O O
l

O HO O
HO
GlcRT GlcRT
CH2 OH OH CH2 OH OH
O O
Xyl Xyl
OH O O OH O O
OH OH
HO Glc HO Glc
O OH O OH
HO HO
O O
Xyl CH2 O OH Xyl CH2 O OH
OH OH
HO O HO O
O Glc O Glc
HO OH HO OH
CH2 CH2
O O OH O O OH
Glc Glc
OH OH
HO XXLG HO XXDG
OH OH

F I G . 2. Representative xyloglucan oligosaccharides. Top, decasaccharides; bottom, octasaccharides; left, structures found in most vascular plants; right, struc-
tures detected by Pena et al. (2008) in Equisetum hyemale and Selaginella kraussiana. Note the minor chemical difference (grey ovals) between the left-hand and
right-hand structures. The TLC system shown in Fig. 7 would be expected to group the two similar decasaccharides together and the two similar octasaccharides
together. Sugar residues are labelled in grey: Ara, a-L-arabinopyranose; Fuc, a-L-fucopyranose; Gal, b-D-galactopyranose; Glc, b-D-glucopyranose; Xyl,
a-D-xylopyranose. GlcRT indicates the reducing terminal glucose group.

lineages of euphyllophytes: there is a significant morphologic- M AT E R I A L S A N D M E T H O D S

al gap between ferns and seed plants as a result of age, extinc-
tion and lack of fossil record (Pryer et al., 2001, 2004).
The objectives of the present work were to explore the oc-
currence and abundance of MLG in diverse monilophytes,
and to define MLG structures from both horsetail subgenera
and E. bogotense to test whether differences arose during the
course of evolution in the genus Equisetum. We also aimed
to develop a simple visual method for surveying the quantita-
tive and qualitative variation occurring in xyloglucan through-
out the monilophytes.
Plant materials
Plant sources are listed in Table 1. Most fresh plants were
obtained from the Royal Botanic Garden Edinburgh (RBGE)
living collection with accession numbers indicated, with the
exception of E. arvense and E. fluviatile which were collected
from the wild in Edinburgh. Vouchers of E. bogotense were
courtesy of New York Botanic Garden; four independent herb-
arium specimens were combined to make an adequate sample.
In the genus Equisetum (except for the herbarium specimen),
 Xue & Fry Monilophyte hemicelluloses 877

TA B L E 1. Classification, sources, AIR yields, and relative abundance of MLG subunits and xyloglucan of all species analysed

Relative oligosaccharide yields

Source/accession Polymer content
Classification Species number* Age (mg g21) MLG4 MLG3 MLG2 XGOs

HETEROSPOROUS LYCOPHYTE
Selaginellaceae Selaginella willdenowii (Desv. ex Poir.) 19762969(E) Y 117 +
Baker
EUSPORANGIATE MONILOPHYTES
Ophioglossaceae Ophioglossum vulgatum L. 19695522(E) Y 67 +
Psilotaceae Psilotum nudum (L.) P. Beauv. 20040835(E) Y 45 +
M 109 +
Equisetaceae} Equisetum bogotense Kunth. NYBG herbarium (H) nd ++ + []
subgenus Equisetum arvense L. RBGE Y 91 ++ + + +
Equisetum M 74 ++ + + ++ +
Equisetum fluviatile L. KB pond Y 32 ++ + + +
M nd ++ + + +
Equisetum pratense Ehrh. 19821716(E) Y 95 ++ + + +
M 129 ++ + + + +
Equisetum sylvaticum L. 19821736(E) Y 90 ++ + +
M 164 ++ + + + []
Equisetum bowmanii C.N. Page 20060765A(E) Y 129 ++ + ++
(E. telmateia E. sylvaticum) M 98 ++ + + + []
Equisetum litorale Kuhlewein ex Rupr. 19821740(E) Y 58 ++ + + + +
(E. arvense E. fluviatile) M 97 ++ + + + +
Equisetum robertsii Dines 20060766A(E) Y 81 ++ + + ++ ++
(E. arvense E. telmateia) M 74 ++ + + ++
subgenus Equisetum hyemale var. affine Engelm. 19734597B(E) Y 35 + ++ +
Hippochaete M 104 + + +
Equisetum myriochaetum Schltdl. & Cham. 19734598(E) Y 35 + + + ++
M 100 ++ + + +
Equisetum ramosissimum ssp. debile Hauke 19731694(E) M nd + ++ + []
Marattiaceae Marattia fraxinea Sm. 19697184(E) Y 68 ++ +
M 65 ++ +
Angiopteris evecta Hoffm. 20060955(E) Y 82 ++ +
M 58 ++
Angiopteris lygodiifolia Rosenstock 19763705A(E) Y 29 ++ +
M 57 ++ +
LEPTOSPORANGIATE MONILOPHYTES
Osmundaceae Osmunda regalis L. 19913878A(E) Y 108 ++ +
M 203 +
Todea barbara (L.) T. Moore 19652792(E) Y 98 ++ +
M 134 +
Hymenophyllaceae Trichomanes speciosum Willd. 19992144(E) Y 191 ++ +
M 224 ++
Dipteridaceae Dipteris conjugata Reinw. 20021886A(E) Y 145 +
M 249 +
Lygodiaceae Lygodium japonicum (Thunb.) Sw. 19734355(E) Y nd ++
M nd +
Anemiaceae Anemia sp. 19933657(E) Y 116 ++ +
M 154 ++
Marsileaceae Marsilea drummondii A. Braun 19933710(E) Y 100 +
M 111 +
Thyrsopteridaceae Thyrsopteris elegans Kunze 20031267A(E) Y 108 ++ +
M 70 +
Cyatheaceae Cyathea spinulosa Wall. 19941397A(E) Y 87 ++ +
M 54 ++
Woodsiaceae Woodsia obtusa (Spr.) Torrey 20061094A(E) Y 99 ++ +
M 73 ++
SPERMATOPHYTES
Gymnosperm Chamaecyparis lawsoniana Parl. KB Y 216 ++
M 255 ++
Angiosperm Nymphaea lotus L. 20040381(E) Y 26 ++ +
M 278 ++ +

* Sources/accession numbers: (E), Royal Botanic Garden, Edinburgh (RBGE) accession no.; KB, Kings Buildings campus, Edinburgh; NYBG, New York
Botanic Garden.

Age of tissues tested: Y, young tender tissues; M, mature tough tissues; H, herbarium tissue.

Polymer contents: yield of alcohol-insoluble residue (mg AIR g21 fresh weight); nd, not determined.

Oligosaccharides indicative of MLG and xyloglucan: MLG4, MLG3, MLG2, tetra-, tri- and disaccharide of MLG released by lichenase; XGOs,
xyloglucan oligosaccharides released by XEG. Yields of the oligosaccharides are scored as: , undetectable; +, inconsistently or barely detectable; +, ++ ,
+ ++ , low, moderate and high abundance; [ ], not determined.
}
E. bogotense cannot be placed clearly in either subgenus.
878 Xue & Fry Monilophyte hemicelluloses
young tender tissues were taken from the base of each inter-
node wrapped inside the leaf sheath; mature tissues were the
tougher parts of internodes. In other genera, young (tender)
and mature (tough) tissues were selected empirically.
Preparation of alcohol-insoluble residue (AIR) and
hemicellulose B
Methods followed previous protocol (Fry, 2000). AIR was
obtained by homogenizing plant materials in 96 % ethanol, fil-
tering on Miracloth (Calbiochem, EMD, USA), then repeated-
ly rinsing with 96 % ethanol until all the chlorophyll had been
removed and the filtrate was colourless. After drying at room
temperature, AIR (100 mg) was shaken at 37 8C for 17 h
with 5 mL 6 M NaOH containing 0.1 % NaBH4. The super-
natant was neutralized with acetic acid and the suspension
was dialysed in 12 14-kDa cut-off tubing (Medicell Int.
Ltd, London, UK) against running tap-water for 3 d. The sus-
pension from inside the sac was centrifuged at 6000 g for
20 min. Both the pellet (hemicellulose A) and the supernatant
(hemicellulose B) were retained for analysis.
Lichenase digestion
Hemicellulose was digested with lichenase (from Bacillus
subtilis; Megazyme), yielding MLG repeat-units, as described
by Popper and Fry (2003). Hemicellulose solution [200 mL,
0.5 % w/v in pyridine/acetic acid/chlorobutanol/water (1 : 1 :
0.5 : 98, v/v/w/v; PyAW)] was mixed with 200 mL lichenase
solution (1.43 unit mL21) and incubated at 20 8C with gentle
shaking for 16 h. Digestion was then stopped with 200 mL
formic acid. The products were dried, re-dissolved in 150 mL
PyAW, and analysed by thin-layer chromatography (TLC).
Xyloglucan endoglucanase (XEG) digestion
XEG (a gift from Novo Nordisk, Bagsvrd) from
Aspergillus aculeatus contained traces of pectinase and
b-galactosidase. Conditions for XEG digestion, with minimal
side reactions, were devised in preliminary studies, with
citrus pectin and tamarind seed xyloglucan as substrate,
monitoring galacturonic acid and galactose production,
respectively.
For the XEG digestion, 200 mL of sample (0.5 % w/v; sus-
pension of hemicellulose A or solution of hemicellulose B) in
PyAW was mixed with 200 mL of 0.05 % (w/v) XEG in PyAW
and incubated at 25 8C for 64 min. The reaction was stopped
with 200 mL formic acid. Products were dried and re-dissolved
in 150 mL water; 3 mL was analysed by TLC.
Thin-layer chromatography
Samples were loaded on to a TLC plate (Merck, silica
gel 60, 20 20 cm; Sigma-Aldrich, UK) and developed in
either butanol/acetic acid/water (2 : 1 : 1, v/v/v) or butanol/
ethanol/water (2 : 1 : 1, v/v/v), as specified in the figure
legends. Sugars were stained with thymol (Frankova and Fry,
2011).
R E S U LT S
Preparation of alcohol-insoluble residue
Samples of young and mature tissues from 27 species, mainly
monilophytes, were converted to alcohol-insoluble residue
(AIR; i.e. total polymers, a high proportion of which will be
cell wall material). The species investigated are listed in
Table 1, and most are illustrated in Fig. 3. There was consid-
erable variability between species in total polymer content
per gram fresh weight (Table 1), reflecting wide variation in
the species adaptations to diverse environments (wet and
dry, exposed and sheltered, insolated and shaded, etc.).
However, data for all 21 monilophyte species in which both
mature and young tissues were quantified show a mature :
young ratio (of mg AIR obtained per g fresh weight) of
1.42 + 0.15 (mean + s.e., N 21), supporting our subjective
description of these samples as tough and tender, respect-
ively. The mature tissues are likely to be richer in secondary
cell wall material, deposited after the cells had lost the
ability to expand.
Mixed-linkage (1  3, 1  4)-b-D-glucan (MLG)
Hemicelluloses from AIR of numerous species of monilo-
phyte plus a representative lycophyte and two spermatophytes
were tested for MLG by TLC analysis of lichenase digests.
Hemicellulose A (i.e. the alkali-extracted material that precipi-
tates when neutralized) gave almost no MLG oligosaccharides
(chromatograms not shown). On the other hand, the hemicellu-
lose B (i.e. that which remains in solution after neutralization)
from certain species gave a series of oligosaccharides diagnos-
tic of MLG. Production of these oligosaccharides was depend-
ent on the presence of lichenase (Fig. 4), confirming that they
were not due to artifactual degradation of polysaccharides. A
summary of all the following MLG oligosaccharide data is
given in Table 1.
All species of the genus Equisetum gave MLG oligosacchar-
ides, but in varying yields (Fig. 4). The major product in most
Equisetum species was the tetrasaccharide G4G4G3G, in con-
trast to the situation with poalean MLG (e.g. fully lichenase-
digested commercial barley MLG; Fig. 4), which gave
mainly the trisaccharide G4G3G. There was little consistent
difference, either quantitative or qualitative, between young
and mature tissues. No non-Equisetum monilophytes, either
eusporangiate or leptosporangiate, gave detectable MLG oligo-
saccharides (Fig. 4 and all other species listed in Table 1; chro-
matograms not shown). Likewise, no MLG was detected in the
lycophyte Selaginella willdenowii or either of the non-
commelinid spermatophytes examined (results not shown).
Samples rich in starch may give traces of malto-oligosacchar-
ides on lichenase digestion owing to slight contamination of the
enzyme with amylases. Therefore, digests were analysed by
TLC in two different solvent systems, with a
malto-oligosaccharide marker ladder. MLG oligosaccharides
were confirmed to be present in all Equisetum samples
(Fig. 5). Somewhat different patterns were noted among the pro-
ducts generated from the nine surveyed species of horsetail. In
general, there was a higher concentration of MLG in the hemi-
cellulose of subgenus Equisetum and E. bogotense than in
 Xue & Fry Monilophyte hemicelluloses 879

A B C D

E E F G H I

J K L M M

M N O P Q

R S S T U V

V W X Y Z

F I G . 3. Plant specimens analysed in the present work. (A) Selaginella willdenowii; (B) Equisetum bowmanii; (C) Equisetum robertsii; (D) Equisetum
arvense; (E, E ) Equisetum fluviatile; (F) Equisetum sylvaticum; (G) Equisetum pratense; (H) Equisetum hyemale var. affine; (I) Equisetum myriochaetum;
(J) Ophioglossum vulgatum; (K) Psilotum nudum; (L) Marattia fraxinea; (M, M , M ) Angiopteris evecta; (N) Angiopteris lygodiifolia; (O) Osmunda
regalis; (P) Todea barbara; (Q) Dipteris conjugata; (R) Trichomanes speciosum; (S, S ) Lygodium japonicum; (T) Anemia sp.; (U) Marsilea drummondii;
(V, V ) Thyrsopteris elegans; (W) Cyathea spinulosa; (X) Woodsia obtusa; (Y) Chamaecyparis lawsoniana; (Z) Nymphaea lotus. Extraneous species, e.g.
the pink-flowering Geranium seen in (D), were removed prior to analysis of the plants of interest. Letters A Z are colour-coded to agree taxonomically with
the labelling on Figs 4 7.
880 Xue & Fry Monilophyte hemicelluloses

Glc
Glc
M2
Lam2 M2
M4
MLG3 M4
MLG4
M7
M7
MLG6
MLG7
Markers a

Markers b
E. arvense Y

E. arvense Y+
E. arvense M
E. arvense M+
E. fluviatile Y
E. fluviatile Y +
E. hyemale Y
E. hyemale Y +

E. myriochaetum Y
E. myriochaetum Y +
E. myriochaetum M
E. myriochaetum M+
E. pratense Y
E. pratense Y +
E. pratense M
E. pratense M+
Markers c
Markers a

Markers b
E. litorale Y
E. litorale Y+
E. robertsii Y
E. robertsii Y+
E. robertsii M
E. robertsii M+
Angiopteris lygodiifolia Y
Angiopteris lygodiifolia Y +
Angiopteris lygodiifolia M
Angiopteris lygodiifolia M+
Osmunda regalis M
Osmunda regalis M+
Todea barbara Y
Todea barbara Y +
T odea barbara M
Todea barbara M+
Markers c
subgenus subgenus subgenus Eusporangiate Leptoporangiate
Equisetum Hippochaete Equisetum fern fern

F I G . 4. Lichenase digestion products of hemicellulose B from several monilophytes, with enzyme-free controls. Digests of: Y, young tender tissues; M mature
tough tissues; + lichenase-treated; , enzyme-free. The TLCs were developed in butanol/acetic acid/water, and stained with thymol. Markers a and b: oli-
gosaccharides of DP 3 7 obtained by partial (a) and complete (b) lichenase digestion of commercial barley MLG (MLG3, G4G3G; MLG4, G4G4G3G; MLG6,
G4G3G4G4G3G; MLG7, G4G4G3G4G4G3G and/or G4G3G4G4G4G3G). Markers c: M2, maltose; M4, maltotetraose; M7, maltoheptaose. Laminaribiose
(Lam2) was detectable in some digests. Spots marked * are contamination, not seen on replicate plates.

subgenus Hippochaete. In some samples of E. hyemale MLG similar across the eusporangiate and leptosporangiate monilo-
was almost undetectable. phytes, resembling that in the seed-plants Chamaecyparus and
The trisaccharide G4G3G (the major repeat-unit in poalean Nymphaea. The major repeat-unit classes co-migrated with
MLG) was consistently a very minor component of Equisetum XLFG, XLLG, XXFG, XXLG (and/or XLXG), and XXXG
MLG. The tetrasaccharide G4G4G3G and disaccharide G3G (DP 10, 9NF, 9F, 8 and 7, respectively, where NF and F indicate
( laminaribiose) were the major repeat-units. There was no non-fucosylated and fucosylated), giving a characteristic ---|
consistent difference between the subgenera in tetrasacchar- pattern, where | represents a band.
ide : disaccharide ratio. In some samples from both subgenera There was taxonomic variation in the intensity of the five
(e.g. some samples of mature E. arvense and mature bands in the core ---| pattern of xyloglucan oligosacchar-
E. ramosissimum; Fig. 5), the disaccharide predominated ides. For example, Marattia, Angiopteris, E. hyemale and
over the tetrasaccharide; but in other samples the tetrasacchar- Nymphaea exhibited relatively high intensities of the DP9NF
ide was predominant (Fig. 6). The very isolated ( probably fragment. In addition, many species also exhibited a probable
earliest-diverging) species E. bogotense uniquely had MLG DP6 band running slightly faster than XXXG.
composed almost solely of the tetrasaccharide. A discordant selection of species, upon XEG digestion, also
gave two major, unidentified oligosaccharides that appeared to
be a di- and trisaccharide of glucose. After purification by gel-
Xyloglucan
permeation chromatography, they gave no isoprimeverose on
Xyloglucan-derived oligosaccharides were almost undetect- Driselase digestion, and only glucose on acid hydrolysis. On
able in digests of hemicellulose A (chromatograms not shown). TLC, the disaccharide and trisaccharide migrated slightly
Therefore, only hemicellulose B was analysed in detail. A slower than cellobiose and cellotriose, respectively (marker
summary of the yields of hemicellulose B xyloglucan-derived chromatograms, not shown). The disaccharide was also distin-
oligosaccharides is given in Table 1. As expected, xyloglucan guished chromatographically from authentic maltose, isomal-
tended to be more abundant in the hemicellulose B of young tose and gentiobiose, and remains an unidentified product of
tissues than of mature (Fig. 7), the former being richer in XEG action. The lack of xylose, and non-identity with
primary cell walls. Among the eusporangiate monilophytes, cello-oligosaccharides, suggests that the two small oligosac-
total xyloglucan was scarce in the horsetail subgenus Equisetum charides were not derived from any known xyloglucan struc-
and in Psilotum nudum, but abundant in the horsetail subgenus ture. They were particularly abundant in XEG digests of the
Hippochaete and in the ferns Marattia and Angiopteris. The lep- eusporangiate fern Angiopteris, two leptosporangiate ferns
tosporangiate ferns varied widely in xyloglucan content: in some (Osmunda and Lygodium), and the gymnosperm
(Osmunda, Todea, Trichomanes, Anemia, Thyrsopteris, Cyathea Chamaecyparis, but were not abundant in any Equisetum
and Woodsia) it was abundant, whereas in others (Dipteris, species nor in Nymphaea, an early-diverging dicot nested in
Lygodium and Marsilea) it was scarce (Fig. 7). the basal ANITA (Amborella, Nymphaea, Illicium,
In all cases where the yield of xyloglucan oligosaccharides was Trimenia and Austrobaileya) grade according to APGII (2003).
sufficient for a TLC profile to be clearly discerned, a core Hemicellulose B preparations from the tested species gave
pattern of five classes of oligosaccharide repeat-unit was widely varying yields of glucose on XEG digestion, and
 Xue & Fry Monilophyte hemicelluloses 881

A
BEW

Glc

Lam2

Mlt2

MLG3
Mlt3

MLG4
Mlt4

Mlt5
Mlt6
Mlt7
Mlt8
Mlt9
Mlt10
1

10

11

12
13

14

15

16

17

18

19
B
BAW
Glc

Lam2
Mlt2

Mlt3
MLG3
Mlt4
MLG4

Mlt5

Mlt6
Mlt7
Mlt8
Mlt9
Mlt10
1 Glucose

2 Malto-ladder

4 E. arvense Y
3 Festuca arundinacea

5 E. arvense M

6 E. fluviatile M

7 E. sylvaticum Y

8 E. x bowmanii Y

9 E. robertsii Y

10 E. hyemale Y

11 E. hyemale M

12 E. myriochaetum Y

13 E. myriochaetum M

14 E. ramosissimum M

15 E. bogotense H

16 Laminaribiose

17 Cellobiose

18 Maltose

19 Gentiobiose

Markers Markers
subgenus subgenus
Poales Equisetum Hippochaete

F I G . 5. Lichenase digestion products of hemicellulose B from nine Equisetum species. The TLCs were developed either in butanol/ethanol/water (A) or in
butanol/acetic acid/water (B), and stained with thymol. A lichenase digest of the hemicellulose B from an MLG-rich grass, Festuca arundinacea, was run in
parallel. Abbreviations: H, herbarium specimen; M, mature tissue; Y, young tissue; Glc, glucose; Lam2, laminaribiose; MLG3-4, tri- and tetrasaccharide
repeat-unit of MLG; Mlt2-10, maltose to maltodecaose (malto-ladder obtained by partial hydrolysis of amylose on long-term storage of a sterile aqueous so-
lution). Spots marked * are contamination, not seen on replicate plates.

there was a tendency for glucose to correlate with the yield of lichenan and yeast glucan) gave small amounts of glucose
the small oligosaccharides mentioned in the previous para- on digestion with XEG under the conditions used. Therefore,
graph. Several authentic polysaccharides (including starch, the glucose detected was not necessarily of xyloglucan origin.
882 Xue & Fry Monilophyte hemicelluloses

A
BEW

Glc

Lam2
Mlt2

MLG3
Mlt3

MLG4
Mlt4

Mlt5
Mlt6
Mlt7
Mlt8
Mlt9
1

3
4

5
6

7
8
9

10

11

12

13

14
15

15
16
17
18

19

20
21
22
B
BAW
Glc

Lam2
Mlt2

Mlt3
MLG3
Mlt4
MLG4

Mlt5

Mlt6
Mlt7
Mlt8
Mlt9
Mlt10
1 Glucose

2 Malto-ladder

4 E. arvense Y

5 E. arvense M
6 E. fluviatile Y

7 E. fluviatile M

8 E. sylvaticum M
9 E. x bowmanii Y

10 E. x bowmanii M
11 E. litorale Y

12 E. litorale M

13 E. robertsii Y

13 E. robertsii M
15 E. pratense Y

15 E. hyemale Y
16 E. hyemale M
17 E. myriochaetum Y
18 E. myriochaetum M

19 Laminaribiose

20 Cellobiose
21 Maltose
22 Gentiobiose
3 Festuca arundinacea

Markers Markers
subgenus subgenus
Poales Equisetum Hippochaete

F I G . 6. Lichenase digestion products of hemicellulose B from additional Equisetum species. Other details as in Fig. 5.

DISCUSSION suggests fluctuations in MLG biosynthesis during the evolu-

tion of the horsetails. Likewise, in the Poales, levels of MLG
Evolution of MLG in Equisetum
can vary dramatically between species, subspecies and var-
The fact that MLG is consistently abundant in the subgenus ieties and even between genetically identical plants grown
Equisetum, and scarce or very scarce in E. hyemale and under different environmental conditions (Stone and Clarke,
E. myriochaetum but relatively high in E. ramosissimum, 1992).
 Glc
DP2
Gal DP3

GalA

Xue & Fry Monilophyte hemicelluloses

DP6?
DP7
XXXG DP8
XXLG DP9F
DP9NF
XLLG DP10
Markers
Selaginella willdenowii Y
Psilotum nudum Y
Psilotum nudum M
E. arvense Y
E. arvense M
E. fluviatile Y
E. hyemale Y
E. hyemale M
E. hyemale M
E. myriochaetum Y
E. myriochaetum M
E. pratense Y
E. pratense M
E. x litorale Y
E. x litorale M
E. x robertsii Y
Markers
E. x robertsii M
Marattia fraxinea Y
Marattia fraxinea M
Angiopteris evecta Y
Angiopteris evecta M
Angiopteris ligodiifolia Y
Angiopteris ligodiifolia M
Osmunda regalis Y
Osmunda regalis M
Todea barbara Y
Todea barbara M
Trichomanes speciosum M
Trichomanes speciosum Y
Dipteris conjugata Y
Dipteris conjugata M
Markers
Lygodium japonicum Y
Lygodium japonicum M
Lygodium japonicum M
Anemia sp. Y
Anemia sp. M
Marsilea drummondii Y
Marsilea drummondii M
Thyrsopteris elegans Y
Thyrsopteris elegans M
Cyathea spinulosa Y
Cyathea spinulosa M
Woodsia obtusa Y
Woodsia obtusa M
Chamaecyp. lawsoniana Y
Chamaecyp. lawsoniana M
Markers
Nymphaea lotus lamina Y
Nymphaea lotus petiole Y
Nymphaea lotus petiole M
Red: subgenus Equisetum
Blue: subgenus Hippochaete
Euspor- Equisetum Eusporangiate Leptosporangiate Gymno- Basal
angiate ferns ferns sperm dicot

F I G . 7. Xyloglucan endoglucanase (XEG) digestion products of hemicellulose B from numerous monilophytes and three other vascular plants. The TLCs were developed in butanol/acetic acid/water, and
stained with thymol. Markers (labelled at left): Gal, galactose; GalA, galacturonic acid; XXXG, XXLG and XLLG, oligosaccharides of DP 7, 8 and 9, respectively, from tamarind xyloglucan. Size-classes of
products (labelled at right) are indicated by their degree of polymerization (DP); e.g. DP7 is a heptasaccharide. DP9F, Nonasaccharides containing a fucose residue; DP9NF, lacking fucose. One residue of
fucose (6-deoxy-L-galactose) considerably increases chromatographic mobility compared with an oligosaccharide of similar size lacking a deoxy-sugar. The DP9F band is highlighted with a yellow stripe.
Abbreviations: M, mature tissue; Y, young tissue.

883
884 Xue & Fry Monilophyte hemicelluloses

The phylogenetic position of E. bogotense is suggested to be Evolution of xyloglucan in the monilophytes

either sister to Hippochaete or basal to the entire genus (Des
Although small amounts of xyloglucan may be present in
Marais et al., 2003; Guillon, 2004, 2007). Thus the abundance
mature xylem walls (Mellerowicz et al., 2008), this hemicellu-
in E. bogotense of an MLG built almost only of the tetrasac-
lose is mainly a component of primary cell walls. As therefore
charide repeat-unit, G4G4G3G, suggests that this structure is
expected, young tissues were richer in xyloglucan than mature
a primordial feature of the genus Equisetum. In all other
tissues, which possess a higher proportion of secondary walls.
Equisetum species, the disaccharide repeat-unit G3G is also
Xyloglucan-derived oligosaccharides were detectable in all
an appreciable component. MLG has been retained abundantly
monilophytes tested, though it was a very minor component
in the subgenus Equisetum, but almost lost in some members
in some. In fact, xyloglucan is thought to be present in all land-
of the subgenus Hippochaete. Its reappearance in one
plants; however, it appears not to be essential for viability in
Hippochaete species (E. ramosissimum) was in a form with
arabidopsis (Cavalier et al., 2008), suggesting that other
a highly unusual repeat-unit composition, predominantly
cell-wall components can replace it functionally when
based on the disaccharide G3G. This may indicate that MLG
xyloglucan biosynthesis is compromised.
acquired a subtly different role in this species. However,
Among the eusporangiate monilophytes, the total xyloglu-
E. ramosissimum is closely related to E. hyemale (Des
can content showed interesting taxonomic trends. It was
Marais et al., 2003; Guillon, 2004, 2007), implying a very
scarce throughout the MLG-rich horsetail subgenus
rapid evolution of MLG structure.
Equisetum, suggesting that MLG and xyloglucan may be
It has been suggested that the Equisetopsida total group
capable of serving equivalent roles in primary cell-wall archi-
diverged from the Marattiales in the period from
tecture. It was also very low in abundance in Psilotum nudum,
mid-Triassic to Upper Palaeozoic (238 295 million years
which is extremely rich in a different hemicellulose, gluco-
ago) (Des Marais et al., 2003; Pryer et al., 2004). The com-
mannan (Popper and Fry, 2004), suggesting that the latter
plete absence of MLG in the extant Marattiales and all other
may be able to replace xyloglucan functionally. Indeed,
non-Equisetum monilophytes, and its presence in all
mannose-rich polysaccharides are consistently abundant in
Equisetum species, would suggest that MLG was acquired
the cell walls of eusporangiate monilophytes (Popper and
later than the Equisetopsida Marattiales split and at least by
Fry, 2004; Nothnagel and Nothnagel, 2007). On the other
the crown group diversification in the Tertiary or Mesozoic.
hand, xyloglucan was relatively abundant in the MLG-poor
Nevertheless, it cannot be excluded that MLG was originally
horsetail subgenus Hippochaete and in the (MLG-free) euspor-
present in all eusporangiate monilophytes but then lost
angiate ferns Marattia and Angiopteris. Most of the
except in Equisetum. The invention of MLG in the seed
(MLG-free) leptosporangiate ferns also had high xyloglucan
plant lineage appears to be an entirely separate event. The
contents in their young tissues, although Dipteris, Lygodium
Poales date back to the Cretaceous (.65 million years ago)
and Marsilea had less. It will be interesting to determine
(Bremer, 2002) and poalean MLG is therefore assumed to
whether these last three fern genera functionally replace
have been acquired around this time.
xyloglucan with a different hemicellulose.
The significance of the different tetrasaccharide : trisacchar-
A core pattern of five xyloglucan repeat-units,
ide : disaccharide ratios in the MLGs of different species is un-
co-migrating on TLC with XLFG, XLLG, XXFG, XXLG
certain. An MLG composed mainly of one specific repeat-unit
and XXXG, was recognizable in all monilophytes studied, re-
(either trisaccharide or tetrasaccharide) will tend to self-
sembling that in the two representative seed-plants tested (a
aggregate, thus readily coming out of aqueous solution or
gymnosperm and a basal dicot). This core pattern even
forming a stiff gel (Lazaridou et al., 2004). Such MLGs are
applies to E. hyemale, which has recently been reported to
those of the lichen Cetraria islandica and many Equisetum
possess several unusual xyloglucan repeat-units not found
species (with predominantly the trisaccharide or the tetrasac-
in most other plants (Pena et al., 2008). It is likely that TLC
charide repeat-unit, respectively). We have indeed noted that
in a single solvent system does not separate oligosaccharides
pure dried MLG from Equisetum is difficult to redissolve in
containing the L repeat-unit from those in which L is replaced
water. Correspondingly, wheat MLG, which is mainly com-
by the chemically similar D, nor those containing F from those
posed of the trisaccharide repeat-unit (trisaccharide : tetrasac-
in which F is replaced by the chemically similar E (Fig. 2).
charide ratio 4.5 : 1), is less water-soluble than oat MLG,
Thus, the DP10 band in E. hyemale (and possibly other
which has a less extreme ratio (trisaccharide :
Equisetum spp.; Fig. 7) is expected to include all three of
tetrasaccharide 2 : 1) (Lazaridou et al., 2004; Li et al., 2006).
the very similar fucose-containing decasaccharides reported
It may be predicted that MLGs possessing a high proportion
by Pena et al. (2008): XLEG, XLFG and XDEG. Likewise,
of disaccharide repeat-unit (e.g. that of E. ramosissimum) will
the DP9F band will include both XXFG and XXEG; the
be highly flexible molecular chains, since the (1  3) bond
DP9NF band XLDG, XLLG and XDDG; and the DP8 band
allows free rotation. Where a disaccharide is flanked by two
XXLG, XLXG and XXDG. If the E. hyemale oligosaccharides
tetrasaccharides, there is a six-glucosyl run with alternating
reported by Pena et al. (2008) are grouped into the five classes
(1  3) and (1  4) bonds ( . . . G4G3G4G3G4G . . . ); such
that are resolvable by TLC, their data show a DP10 : DP9NF :
domains would be expected neither to self-aggregate readily
DP9F : DP8 : DP7 ratio of 23 : 46 : 5 : 22 : 4. In contrast, our
nor to hydrogen-bond to the surface of cellulose microfibrils.
TLC patterns (which are similar for young E. hyemale and
The self-aggregation and solubility of MLGs and their
E. myriochaetum) show a ratio of roughly 24 : 7 : 17 : 23 : 29
ability to hydrogen-bond to cellulose are all features that
(estimated by intensity of thymol staining; Fig. 7). Although
may be expected to influence the biological characteristics of
our data are only semi-quantitative, they nevertheless show
this cell-wall polysaccharide.
 Xue & Fry Monilophyte hemicelluloses
appreciable differences from the ratios found by Pena et al.
(2008), especially in exhibiting a higher abundance of the hep-
tasaccharide and the fucose-containing nonasaccharides and
much less of the non-fucosylated nonasaccharides such as
XLLG.
Interspersed with the five core pattern bands are additional
bands that show greater taxonomic diversity. For example,
many of the species tested also possess a probable hexasac-
charide. This may be XXGG, a major repeat-unit of many
members of the Poales and Solanales (Hoffman et al., 2005;
Fry, 2011), although it was not definitively identified. The
DP6 component was found in varying abundance across the
monilophytes and in the basal early-diverging dicot
Nymphaea, but was least abundant in the genus Equisetum,
supporting the view that hemicellulose structures show phylo-
genetically based variation.
CON C L U S IO NS
After confirming that MLG is confined, among all monilo-
phytes tested, to Equisetum, we also show varied MLG struc-
tures among the subgenera Equisetum and Hippochaete. The
sister group E. bogotense possesses its own potentially ances-
tral MLG structure, very predominantly composed of the tetra-
saccharide repeat-unit, G4G4G3G. Other members of the
genus seem to have refined this simple structure, e.g. varying
in the tetrasaccharide : disaccharide ratio. Furthermore, MLG
was lost as a predominant hemicellulose of the primary cell
wall (by E. hyemale and E. ramosissimum) at least once
during the adaptive radiation of the genus.
Xyloglucan, in contrast to MLG, is found in all land plants
tested, and a core pattern of repeat-units (co-migrating with
XLFG, XXFG, XXLG/XLXG, XXXG) appears consistently
in the TLC profiles of all species tested. However, xyloglucan
has become a very minor hemicellulose in some monilophytes
especially Psilotum and the horsetail subgenus Equisetum,
which possess abundant alternative hemicelluloses (mannans
in both; also MLG in Equisetum).
It is clear that land plants have experimented extensively
with the structures and proportions of their hemicelluloses
during evolution.
ACK N OW L E DG E M E N T S
We thank RBGE horticultural staff Fiona Inch and Andrew
Ensoll for the collection of plant materials, and New York
Botanic Garden for herbarium material of E. bogotense. We
thank Christopher Jeffree and Quentin Cronk for constructive
comments on the manuscript. This work was supported by
the BBSRC.
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