Desiccation Tolerance in Bryophytes: A Reflection of the Primitive Strategy for Plant Survival in Dehydrating Habitats?

Abstract

Bryophytes are a non-monophyletic group of three major lineages (liverworts, hornworts, and mosses) that descend from the earliest branching events in the phylogeny of land plants. We postulate that desiccation tolerance is a primitive trait, thus mechanisms by which the first land plants achieved tolerance may be reflected in how extant desiccation-tolerant bryophytes survive drying. Evidence is consistent with extant bryophytes employing a tolerance strategy of constitutive cellular protection coupled with induction of a recovery/repair mechanism upon rehydration. Cellular structures appear intact in the desiccated state but are disrupted by rapid uptake of water upon rehydration, but cellular integrity is rapidly regained. The photosynthetic machinery appears to be protected such that photosynthetic activity recovers quickly. Gene expression responds following rehydration and not during drying. Gene expression is translationally controlled and results in the synthesis of a number of proteins, collectively called rehydrins. Some prominent rehydrins are similar to Late Embryogenesis Abundant (LEA) proteins, classically ascribed a protection function during desiccation. The role of LEA proteins in a rehydrating system is unknown but data indicates a function in stabilization and reconstitution of membranes. Phylogenetic studies using a Tortula ruralis LEA-like rehydrin led to a re-examination of the evolution of desiccation tolerance. A new phylogenetic analysis suggests that: (i) the basic mechanisms of tolerance seen in modern day bryophytes have changed little from the earliest manifestations of desiccation tolerance in land plants, and (ii) vegetative desiccation tolerance in the early land plants may have evolved from a mechanism present first in spores.

Full text

788
I
NTEGR
.C
OMP
.B
IOL
., 45:788–799 (2005)
Desiccation Tolerance in Bryophytes: A Reflection of the Primitive Strategy for Plant Survival
in Dehydrating Habitats?
1
M
ELVIN
J. O
LIVER
,
2,
*J
EFF
V
ELTEN
,
3,
*
AND
B
RENT
D. M
ISHLER
,
4,
†
*USDA-ARS, Cropping Systems Research Laboratory, 3810 4th St, Lubbock, Texas 79415
†University Herbarium, Jepson Herbarium, and Department of Integrative Biology, University of California, 1001 Valley Life
Sciences Bldg # 2465, Berkeley, California 94720
S
YNOPSIS
. Bryophytes are a non-monophyletic group of three major lineages (liverworts, hornworts, and
mosses) that descend from the earliest branching events in the phylogeny of land plants. We postulate that
desiccation tolerance is a primitive trait, thus mechanisms by which the first land plants achieved tolerance
may be reflected in how extant desiccation-tolerant bryophytes survive drying. Evidence is consistent with
extant bryophytes employing a tolerance strategy of constitutive cellular protection coupled with induction
of a recovery/repair mechanism upon rehydration. Cellular structures appear intact in the desiccated state
but are disrupted by rapid uptake of water upon rehydration, but cellular integrity is rapidly regained.The
photosynthetic machinery appears to be protected such that photosynthetic activity recovers quickly. Gene
expression responds following rehydration and not during drying. Gene expression is translationally con-
trolled and results in the synthesis of a number of proteins, collectively called rehydrins. Some prominent
rehydrins are similar to Late Embryogenesis Abundant (LEA) proteins, classically ascribed a protection
function during desiccation. The role of LEA proteins in a rehydrating system is unknown butdataindicates
a function in stabilization and reconstitution of membranes. Phylogenetic studies using a Tortula ruralis
LEA-like rehydrin led to a re-examination of the evolution of desiccation tolerance. A new phylogenetic
analysis suggests that: (i) the basic mechanisms of tolerance seen in modern day bryophytes have changed
little from the earliest manifestations of desiccation tolerance in land plants, and (ii) vegetative desiccation
tolerance in the early land plants may have evolved from a mechanism present first in spores.
I
NTRODUCTION
Green plants are believed to have colonized the land
from a fresh water origin (Mishler and Churchill,
1985), requiring adaptive mechanisms that permit
avoidance and/or survival of dehydration. In the initial
ventures into the dehydrating atmospheres of land hab-
itats, plants were of a very simple architecture and had
yet to evolve more complex morphological or physi-
ological strategies to prevent water loss. Water would
have been quickly lost from the cells of these plants
once it was no longer present in liquid form around
them. Thus primitive land plants would, in all likeli-
hood, spend a significant amount of time in equilibri-
um with the surrounding air, which in most cases
would mean that the plants would be desiccated. Even
at a relative humidity of 50% (at 28
8
C) a plant when
equilibrated would experience a water potential of ap-
proximately
2
100 MPa (Gaff, 1997), a water deficit
that is lethal to the majority of modern day flowering
plants. Early land plants would have had to evolve
mechanisms to survive such harsh drying treatments
in order to have successfully exploited habitats on
1
From the Symposium Drying Without Dying: The Comparative
Mechanisms and Evolution of Desiccation Tolerance in Animals,
Microbes, and Plants presented at the Annual Meeting of the Society
for Integrative and Comparative Biology, 4–8 January 2005, at San
Diego, California.
2
Author to whom editorial correspondence should be addressed.
Present address: USDA-ARS Plant Genetics Research Unit, 205
Curtis Hall, University of Missouri, Columbia, Columbia, MO
65211; e-mail: olivermj@missouri.edu
3
E-mail: jvelten@lbk.ars.usda.gov
4
E-mail: bmishler@calmail.berkeley.edu
land. Simply put, we hypothesize that primitive plants
must have been desiccation-tolerant in both vegetative
and reproductive stages in order to colonize the land.
If this is so, what can we learn about this earliest
of adaptive traits that shaped the evolution of land
plants? How has the ability of plants to survive des-
iccation evolved over time? These are not simply es-
oteric questions. The answers we might discover not
only have evolutionary importance, they have practical
application as well. The ability of plant cells to re-
spond to and cope with severe water deficits has eco-
nomic and agricultural implications that directly relate
to crop productivity in an ever challenging and chang-
ing environment. An understanding of these responses
and tolerance mechanisms could be vital if we are to
respond to the increasing need for a stable and suffi-
cient food supply.
Vegetative desiccation tolerance is broadly distrib-
uted among modern day plant taxa (Alpert, 2000). Tol-
erance is relatively common, but not universal, in the
bryophytes (Proctor, 1990; Proctor and Pence, 2002),
but it is much rarer in vascular plants (tracheophytes),
plants that developed the morphological adaptations
required to transport water, i.e., tracheids and vessel
elements. Porembski and Barthlott (2000) estimate that
there are only about 300 desiccation-tolerant species
of tracheophytes. As discussed in the final section of
this paper, phylogenetic analyses suggest that with the
evolution of the tracheophytes, vegetative desiccation
tolerance was lost (Oliver et al., 2000, see below) and
that its occurrence in a few clades of tracheophytes
represents independent evolutions (or re-evolutions),
presumably in response to selection pressures associ-

789B
RYOPHYTE
D
ESICCATION
T
OLERANCE
ated with arid niches. By investigating the underlying
mechanisms that allow these plants to survive the ex-
treme water deficits that characterize desiccation, we
can gain an understanding of how this trait has evolved
and what processes are important in its establishment
or acquisition. In keeping with the view that plants and
plant tissues achieve desiccation tolerance by virtue of
the inherent properties of their cellular components
(protoplasm), as discussed by Bewley (1979), this dis-
course will center on the cellular mechanisms that
characterize the various forms of vegetative tolerance
in land plants. Furthermore, the intention is to focus
on the mechanisms of tolerance exhibited by bryo-
phytes and to compare them to the tolerance mecha-
nisms of angiosperms to infer the possible adaptive
nature of the various aspects of the response of these
plants to desiccation. It is in the study of bryophytes
that we can come closest to learning how early plants
established themselves in the potentially lethal dehy-
drating terrestrial habitats as they colonized the land.
Comparisons with the mechanisms for tolerance that
have evolved in the morphologically more complex
tracheophytes allow for the generation of hypotheses
concerning the evolution and ecology of this important
trait and underlying genetic and physiological pro-
cesses.
M
ECHANISTIC
C
ONSIDERATIONS
Bewley (1979) outlined three protoplasmic proper-
ties of plant cells that have to be present for desicca-
tion tolerance to be established: 1. Limit damage from
desiccation and/or rehydration to a minimum, 2. Main-
tain cellular integrity in the desiccated state, and 3.
Activate or mobilize repair mechanisms upon rehydra-
tion. Basically these criteria translate into tolerance
mechanisms that protect and/or repair plant cells such
that desiccation or rehydration damage is ultimately
negated. Several possible strategies for desiccation tol-
erance can be proposed based on the two basic pro-
cesses of cellular protection and repair. Plant cells can
either constantly maintain the processes and compo-
nents necessary to protect cells, rendering desiccation
tolerance a constitutive trait, or these can be induced
when dehydrating conditions are encountered. Simi-
larly the processes and activities associated with repair
could be constitutive or induced following rehydration.
As both cellular protection and repair are likely to be
essential for any mechanism of desiccation tolerance,
all combinations of constitutive versus inducible for
these two elements are possible. In addition, as dis-
cussed above, since vegetative desiccation tolerance
within land plants has evolved (or re-evolved) multiple
times it is theoretically possible that we could encoun-
ter any one of the possible variations mentioned above
when investigating the individual mechanisms exhib-
ited in different species of tolerant plants. However, it
seems reasonable to assume that since tolerance mech-
anisms have evolved from a shared ancestral strategy,
later manifestations of this trait have evolved mecha-
nisms that are fundamentally similar. As will be dis-
cussed later, from the evidence available to us, this
does appear to be the case.
The actual components and processes involved in
both cellular protection and repair are in all probability
highly conserved (at least in function), constrained by
the underlying physics associated with the desiccation
of plant cells. Indeed, from what we do know this
appears to be true, at least for the components involved
in cellular protection (see below and Oliver et al.,
2000; Phillips et al., 2002; Buitink et al., 2002). How-
ever, much needs to be done in this area before we
can fully understand either of these processes.
Desiccation tolerance in bryophytes
Bryophytes today experience an environment, at
least with regards to water relations, that we postulate
ancient plants faced when the land was first colonized
(see above). Water transport occurs externally to the
plant, which is generally one cell layer thick and water
is freely lost to or gained from the surrounding habitat
across the cell membrane. This rapid and direct equil-
ibration of cell water content to that of the environ-
ment is called poikilohydry. When free water is de-
pleted from the surface of the plant the leaf cells im-
mediately moves towards equilibrium with the water
potential of the surrounding air; the plant desiccates.
Depending upon the relative humidity of the air this
can be a slow or rapid progression to equilibrium and
dryness; the higher the humidity the slower the drying
rate. The extent of water loss also depends upon the
relative water content of the air and its temperature
(see the example given earlier). As discussed in detail
by Proctor and Pence (2002), most bryophytes can sur-
vive moderate levels of desiccation (to
2
20 to
2
40
MPa) for short periods, certainly beyond the range that
most crop species can survive (
2
1.5 to
2
3 MPa).
Some bryophytes however, can tolerate severe desic-
cation and for extended periods, for example Tortula
caninervis, a desert species, can remain at around
2
540 MPa (equilibrated to the atmosphere above ac-
tivated silica gel; 2–4% RH) for up to six years and
still recover normal activity and growth (Oliver et al.,
1993; unpublished data). The drying rate, length of
desiccation, intensity of desiccation, prior dehydration
(hardening), and temperature, all have an effect on the
ability of desiccation-tolerant bryophytes to recover
from the drying event (extensively reviewed by Proc-
tor and Pence, 2002). Drying rates can be very rapid
in exposed habitats but clump architecture can slow
water loss such that in general mosses can exert a ru-
dimentary control over the drying rates (Proctor, 1980;
Rice et al., 2001). In the Organ Mountains of New
Mexico, clumps of Tortula ruralis would sustain hy-
dration for 3 to 4 hr when artificially moistened and
subsequently reach an equilibrated dryness at 6 hr (un-
published observations). Experimentally, many bryo-
phytes can survive extremely rapid desiccation, i.e., to
2
540 MPa in less than 30 min. Rehydration is almost
instantaneous (30 to 90 sec) and recovery rates are also
generally very rapid with most bryophytes reaching

790 M. J. O
LIVER
ET AL.
full recovery within a few hours. The recovery of
some processes, such as photosynthesis and respira-
tion, can be extremely rapid: in the case of photosys-
tem II, recovery occurs within a few minutes (Proctor,
2001). The rate of recovery does, however, depend
upon the rate at which the prior desiccation occurred
(Oliver and Bewley, 1997). In contrast, alldesiccation-
tolerant angiosperms only survive such drying events
if the rate of water loss is very slow (in days to weeks)
and take up to 24 hr or more to recover (Oliver and
Bewley, 1997; Alpert and Oliver, 2002; Proctor and
Pence, 2002).
With their strategic phylogenetic positioning, de-
scended from early divergence events in the history of
land plants, bryophytes are ideally suited as models
for understanding how primitive plants survived the
rigors of colonizing dehydrating and else wise stressful
habitats as they moved onto the land. That bryophytes
can endure such extremes of dehydration and recover
so quickly is a testament to the effectiveness of the
cellular strategy for desiccation tolerance these plants
evolved.
B
RYOPHYTE
M
ECHANISM FOR
D
ESICCATION
T
OLERANCE
Many desiccation-tolerant bryophytes do apparently
share a common mechanism for the tolerance of cel-
lular dehydration. Of the desiccation-tolerant bryo-
phytes that have been extensively studied, mainly the
highly tolerant species of the genus Tortula (in partic-
ular T. ruralis), all appear to utilize a mechanism that
directs the constitutive protection of cellular structures
coupled with a rehydration-induced repair/recovery
process (Oliver and Bewley, 1997; Oliver et al., 2000;
Alpert and Oliver, 2002). The evidence for this con-
clusion has been recently and extensively reviewed in
the literature (Oliver et al., 2000; Alpert and Oliver,
2002). In this report we will summarize this evidence
along with the addition of some new data that is di-
rected at both an understanding of the tolerance mech-
anism exhibited by bryophytes and its place in the
evolution of this trait among land plants.
C
ONSTITUTIVE
C
ELLULAR
P
ROTECTION
The presence of a constitutive cellular protection
component to the bryophyte mechanism for desicca-
tion tolerance can be inferred by their ability to tolerate
rapid desiccation events. Desiccation, and the ensuing
metabolic quiescence, can occur too quickly to initiate
and establish cellular protective measures. Protein syn-
thesis, a prerequisite for most stress response mecha-
nisms in plants, is extremely sensitive to cellular de-
hydration and is rapidly lost during drying of T. ruralis
gametophytes (reviewed by Bewley, 1979; Bewley and
Krochko, 1982). Oliver (1991) clearly demonstrates
that there are no novel transcripts recruited by the pro-
tein synthetic machinery during drying. In contrast,
desiccation-tolerant angiosperms cannot survive des-
iccation if water loss occurs rapidly (less than 12 hr)
and it is well established that the mechanism of tol-
erance in these plants utilizes a dehydration-induced
cellular protection strategy requiring both novel tran-
scription patterns and new protein synthesis (see be-
low, Ingram and Bartels, 1996; Oliver et al., 2000;
Alpert and Oliver, 2002; Phillips et al., 2002). Apart
from the metabolic considerations, ultrastructural ob-
servations strongly suggest that membranes of vege-
tative cells of desiccation-tolerant bryophytes do not
suffer observable damage during drying. Freeze-frac-
ture electron microscopy has enabled the ultrastructur-
al investigation of dried plant cells to progress with
little fear of artifact generation and was used success-
fully to demonstrate that membranes in seeds and pol-
len retain normal lipid bilayer organization at very low
water contents (Thompson and Platt-Aloia, 1982;
Platt-Aloia et al., 1986). Similar studies with T. rur-
alis, and the spike moss Selaginella lepidophylla, dem-
onstrated that this is also the case in the cells of the
leaf tissues of these two plants (Platt et al., 1994).
Finally there is also biochemical evidence to support
a constitutive cellular protection strategy in bryo-
phytes. In orthodox seeds and vegetative tissues of
desiccation-tolerant angiosperms two cellular compo-
nents, the Late Embryogenesis Abundant (LEA) pro-
teins and soluble sugars accumulate in response to des-
iccation (for review see Phillips et al., 2002; Buitink
et al., 2002; Kermode and Finch-Savage, 2002). These
two components are generally considered critical in the
acquisition of cellular desiccation tolerance, although
the actual function of the LEA proteins remains un-
clear (Cuming, 1999). The genes encoding the Group
II LEA proteins, termed dehydrins, are generally in-
duced in response to water deficits (Close, 1997) and
in the case of Craterostigma plantagineum, a desic-
cation-tolerant angiosperm, during desiccation (In-
grams and Bartels, 1996) resulting in the accumulation
of dehydrin proteins in the drying cells. In T. ruralis
dehydrins are apparently constitutively expressed, at
least at the protein level (Bewley et al., 1993). The
accumulation of soluble sugars has long been corre-
lated with the acquisition of desiccation tolerance in
plants and other organisms (Crowe et al., 1992; Ver-
tucci and Farrant, 1995). In seeds, pollen, and most
plants that accumulate soluble sugars in response to
desiccation utilize the disaccharide, sucrose. In Cra-
terostigma plantagineum, 2-octulose stored in the hy-
drated leaves is converted to sucrose during drying to
such an extent that in the dried state it comprises about
40% of the dry weight (Bianchi et al., 1991). Sugars
are a major contributing factor to vitrification (biolog-
ical glass formation) of the cytoplasm of dried cells
(Buitink et al., 2002). Sucrose makes up approximate-
ly 10% of the dry mass of T. ruralis gametophytes and
does not change in amount during desiccation or re-
hydration in the dark or light (Bewley et al., 1978).
Thus, it appears important to maintain a constant, and
presumably sufficient, amount of this sugar in this
moss. The lack of an increase in soluble sugars in re-
sponse to desiccation appears to be a common feature
of tolerant mosses (Smirnoff, 1992).

791B
RYOPHYTE
D
ESICCATION
T
OLERANCE
C
ELLULAR
D
AMAGE
:D
ESICCATION OR
R
EHYDRATION
?
If vegetative bryophyte cells are essentially intact in
the dried state as the evidence seems to indicate, what
damage is there that would require a rehydration-in-
duced (or activated) repair system as first suggested by
Bewley (1979)? Cellular damage could occur during
desiccation, as has been seen in sensitive tissues (Wal-
ters et al., 2002), which are associated with such ac-
tivities as lipid oxidation and free radical generation.
In the bryophyte studies we know that such activities
occur (Smirnoff, 1992) but, as is obvious from the
ultrastructural observations, visible and extensive
membrane damage is not evident. We do know that
membranes are altered in desiccation-tolerant tissues
during desiccation; e.g., Buitink et al. (2000), using
electron paramagnetic resonance (EPR) spectroscopy,
demonstrated that tolerant tissues differ from sensitive
tissues in the partitioning of amphiphilic substances
into membranes. Cellular damage can also occur dur-
ing extended periods in the dried state through oxi-
dative and other chemical activities, the extent of dam-
age depends on the degree of desiccation and time
(Walters et al., 2002; Buitink et al., 2002). Cellular
damage also occurs during the inrush of water as re-
hydration progresses. Since any damage caused by the
desiccation phase is only manifest following rehydra-
tion, it is difficult to say which of the two processes
of dehydration or rehydration is the root cause of the
damage. Rehydration is certainly damaging to dried
cells and can result in significant injury that desicca-
tion-tolerant systems strive to prevent (Osborne et al.,
2002). Orthodox seeds are capable of slowing the re-
hydration process, allowing for some order in an oth-
erwise chaotic event. Bryophytes however, have no
such capability and rehydrate almost instantaneously
when water is added. The first indication that there is
an alteration in the cellular structure during rehydra-
tion of dried plant cells is the leakage of solutes from
the protoplasm. In desiccation-tolerant tissues this
leakage is transient and the extent of leakage is de-
pendent upon the rate at which the prior desiccation
event occurred. The faster the drying rate the more
solutes are leaked during rehydration (Bewley and
Krochko, 1982; Oliver and Bewley, 1984; Oliver et
al., 1993). It is generally accepted that membrane
phase transitions are the cause of rehydration leakage
in vegetative tolerant tissues (Crowe et al., 1992).
As water enters the dried cells of T. ruralis the con-
densed cytoplasm rapidly expands to fill the empty cell
cavity formed by plasmolysis (Tucker et al., 1975)
Within five minutes chloroplasts are swollen and glob-
ular in shape and their outer membranes are folded and
separated from the thylakoids, which themselves are
no longer compacted (Tucker et al., 1975; Bewley and
Pacey, 1978). The extent of thylakoid disruption is de-
pendent upon the prior speed of desiccation; the more
rapid the drying rate the more disruption occurs. Mi-
tochondria also swell and exhibit disruption of the in-
ternal membrane structures (cristae), but the appear-
ance of this organelle upon rehydration is not affected
by the rate of desiccation. Similar results have been
reported for other desiccation-tolerant moss species
(reviewed by Oliver and Bewley, 1984). It has been
suggested that such alterations in cellular structure ob-
served following rehydration as simply artifacts of fix-
ation procedures used on rehydrating dried materials,
especially as many of these studies were reported in
the early 1980s. In the case of the ultrastructural stud-
ies concerning Tortula this seems unlikely as fixation
was delayed until 5 minutes following the re-addition
of water so that cells were fully hydrated, andsimilarly
treated hydrated control tissues were apparently unaf-
fected by the methods of fixation used in these studies.
In all cases organelles regain normal structure within
24 hr of the readdition of water. Rehydrated cells of
dried gametophytes of the desiccation-sensitive moss
Cratoneuron filicinum exhibit identical structural ab-
normalities as those seen in T. ruralis but in this case
the cells never regain a normal appearance and die
(Bewley and Pacey, 1978; Krochko et al., 1978). Des-
iccation and rehydration (imbibition) induced damage
has been extensively investigated in desiccation-tol-
erant seeds and pollen (reviewed by Osborne et al.,
2002; Walters et al., 2002) and indicates not only dam-
age and alterations in membranes but also cytoskele-
ton, nucleus, and DNA (chromatin). In bryophytes we
have little direct evidence for cellular damage other
that what has been observed ultrastructurally.
I
NDUCED OR
A
CTIVATED
C
ELLULAR
R
EPAIR
Much of what we can determine about the repair or
reconstitution requirement in bryophytes has been in-
ferred from physiological, biochemical, and genetic re-
sponses that have been monitored during the initial
phases of recovery following rehydration. As elo-
quently stated by Proctor and Pence (2002) ‘‘full re-
covery must involve a diversity of processes’’ and as
is becoming clear, the complexity of recovery and re-
pair will be difficult to detail and characterize.
Early work (see Bewley, 1979 for review) estab-
lished the ability of Tortula ruralis, a primary model
for these studies, and other mosses to rapidly recover
synthetic metabolism when rehydrated. This rapid re-
covery is in part possible because of the efficient se-
questration of the components of protein synthesis,
with the exception of some initiation factors, during
drying (Oliver and Bewley, 1997). During the first two
hours following rehydration of dried T. ruralis there
is an extensive alteration in gene expression as mea-
sured by changes in the patterns of proteins synthe-
sized during this time compared to hydrated controls.
By 2D gel analysis Oliver (1991) was able to detect
the termination or decrease in synthesis of 25 proteins
(termed hydrins) and the initiation or substantial in-
crease in the synthesis of 74 others (termed rehydrins).
Although this is, of course, an underestimate of the
rehydration-manifested alterations in protein synthesis
this study was able to demonstrate that the controls
over changes in synthesis of these two groups of pro-

792 M. J. O
LIVER
ET AL.
teins are not mechanistically linked. It takes a certain
amount of prior water loss to fully activate the syn-
thesis of rehydrins upon rehydration. Perhaps this is a
strategy that has evolved to link the amount of energy
expended in repair to the amount of damage potenti-
ated by differing extents of drying.
Oliver (1991) also demonstrated that the alterations
in the protein patterns synthesized following rehydra-
tion of Tortula gametophytes occur within a back-
ground of a qualitatively unaltered transcript popula-
tion indicating that the change in gene expression with
rehydration is brought about by a change in transla-
tional controls. Using cDNA clones corresponding to
T. ruralis rehydrins, Scott and Oliver, (1994) con-
firmed that the basis of the control of gene expression
centered upon translation and not transcription, and
this has since been further validated in genomic level
studies (see below). This is in direct contrast to the
case in desiccation-tolerant angiosperms that utilize a
desiccation induced change in transcriptional control
to direct the synthesis of genes required for desiccation
tolerance (Ingram and Bartels, 1996; Phillips et al.,
2002). However, the manner in which the control of
translation is achieved in Tortula does appear to de-
pend upon the previous rate of desiccation. Rehydrin
transcripts accumulate for the first hour following re-
hydration of rapid-dried gametophytes, apparently to
replenish a transcript pool that has been reduced by
rapid desiccation (Scott and Oliver, 1994; Wood and
Oliver, 1999; Velten and Oliver, 2001). In contrast, re-
hydrin transcripts accumulate during the drying phase
if desiccation is slow, i.e., 4 to 6 hr (Oliver and Wood,
1997; Wood and Oliver, 1999). Further studies clearly
demonstrate that these transcripts accumulate not as a
result of transcription but because they are selectively
sequestered in mRNP particles (Wood and Oliver,
1999). Upon rehydration it is postulated that these
transcripts are rapidly released and utilized for the rap-
id synthesis of rehydrins. This is supported by their
rapid inclusion in the polysomal fraction of rehydrated
gametophytes (Scott and Oliver, 1994; Wood and Ol-
iver, 1999). These findings suggest that the repair
mechanism in rehydrated slow-dried gametophytes,
perhaps reflecting the natural response, is activated
rather than induced (which implies a de novo assembly
of components). The repair mechanism in rehydrated
rapid-dried gametophytes would have to rely upon
what little has been assembled until new components
can be synthesized and assembled. This would be con-
sistent with the slower recovery time and more visible
damage associated with rehydrated rapid-dried game-
tophytes. There is a caveat to this postulate in that
because rapid desiccation is achieved in very dry at-
mospheres it may also reflect the depth of desiccation
achieved in rapid-dried gametophytes.
A number of recent physiological studies of the re-
covery of photosynthesis following rehydration of a
number of desiccation-tolerant mosses have ques-
tioned the need for protein synthesis and repair with
respect to the recovery of chloroplasts and chloroplast
functions (reviewed by Proctor and Pence, 2002; Proc-
tor and Tuba, 2002). Essentially, recovery of chloro-
plastic function, as measured by chlorophyll fluores-
cence measurement (Fv/Fm), is extremely rapid (10 to
20 min) and is unaffected by protein synthesis inhib-
itors in the dark for rehydrated dried gametophytes
(dried to
2
70 MPa) of several desiccation-tolerant
mosses (Proctor and Smirnoff, 2001; Proctor, personal
communication). Protein synthesis is required however
if rehydration occurs in the light, presumably to repair
photo-oxidative damage. CO
2
uptake and assimilation,
however, is not instantaneous and does require protein
synthesis to recover. Measurements of the rapid recov-
ery of photosynthesis upon rehydration for dried T.
ruralis also suggest that protein synthesis may not be
required for the recovery of chloroplast structure and
function (Tuba et al., 1996; Csintalin et al., 1999).
There are some caveats to these studies. The effec-
tiveness of protein synthesis inhibitors is difficult to
assess as they can also affect the rate of uptake of the
radiolabelled amino acids used to assess protein syn-
thetic rates. Protein synthesis inhibitors also rarely
completely prevent protein synthesis even when used
in combination. Proctor and Smirnoff (2001) report an
effective inhibition of 90% after 20 min in the pres-
ence of two inhibitors (to inhibit both cytoplasmic and
organellar synthesis), however, no uptake measure-
ments or amino acid pool size measurements were re-
ported. The level of desiccation obtained in the mosses
used in these investigations is generally relatively
moderate, approximately
2
70 MPa and as discussed
above the rate and depth of desiccation has a major
effect on the amount of cellular damage seen in bryo-
phytes. Even with these considerations it does appear
that the photosynthetic machinery (at least Photosys-
tem II) is well protected in the dried state and may
require little repair. It is interesting to speculate that,
because of the need to rapidly utilize the time that
water is available and the need for energy to repair
other cellular damage, part of the bryophyte mecha-
nism of tolerance lies in a focused and effective pro-
tection of the chloroplast.
Before progressing into some of the latest genomic-
level studies it is worth mentioning that desiccation
tolerance can be induced in some bryophytes, those
that are not constitutively tolerant and live in mesic
habitats. Beckett (1999) demonstrated that desiccation
tolerance (as measured by ion leakage) of a mesic Atri-
chum species could be increased by a previous drying
treatment and that addition of abscisic acid (ABA) to
the hydrated moss could produce the same increase in
tolerance. Several studies have demonstrated that ABA
treatment of Funaria hygrometrica not only increases
tolerance to desiccation, allowing for survival of rapid
desiccation that is normally lethal, but also results in
the induction of the synthesis of a number of proteins
that accumulate during drying (Werner et al., 1991;
Bopp and Werner, 1993). ABA has similar effects on
the tolerance of some liverworts to desiccation (Hell-
wege et al., 1994) and its precursor lunularic acid con-

793B
RYOPHYTE
D
ESICCATION
T
OLERANCE
trols the switch from a sensitive to a tolerant stage of
Lunularia cruciata (Schwabe and Nachomony-Bas-
comb, 1963).
The involvement of ABA and tolerance in these
bryophytes is of interest because it is reminiscent of
the strategy used by desiccation-tolerant angiosperms
that involves, at least in part, the induction of gene
expression associated with the acquisition of desicca-
tion tolerance by an elevation in endogenous ABA (re-
viewed by Phillips et al., 2002). It is ABA that is
thought to be a major hormonal component to the in-
duction of the cellular protection system during drying
in seeds and most, if not all, desiccation-tolerant tra-
cheophytes (Oliver et al., 2000). Interestingly ABA
was undetectable in T. ruralis gametophytes in either
the hydrated or drying stages of the wet-dry-wet cycle
and was not detected in dried tissues and ABA does
not induce dehydrin accumulation in hydrated game-
tophytes (Bewley et al., 1993; Oliver, unpublished ob-
servations). This again illustrates the complexity of the
desiccation tolerance phenotype, and raises some in-
teresting evolutionary questions such as: Did mesic
bryophytes forego the constitutive cellular protection
aspect of desiccation tolerance in favor of an inducible
system that allows them to better compete in a mesic
habitat? Or, did the constitutive cellular protection sys-
tem evolve from a primitive developmental system in
spores that allowed some mosses to move into pro-
gressively more extreme xeric habitats? Did the de-
velopmentally programmed acquisition of desiccation
tolerance in seeds, that apparently utilizes an ABA in-
duction pathway, evolve from an environmentally in-
duced mechanism similar to that seen in mesic bryo-
phytes? Only a greater understanding of the underlying
genetic aspects of this interesting trait will enable us
to answer such questions as these.
G
ENOMIC
A
PPROACH TO THE
R
EPAIR
A
SPECTS OF
D
ESICCATION
T
OLERANCE IN
B
RYOPHYTES
One approach to understanding the full nature of the
desiccation-rehydration response in bryophytes is to
determine what transcripts are available during the
wet-dry-wet continuum and which are targeted for
translation. As we have discussed, in desiccation-tol-
erant bryophytes the gene expression response to des-
iccation and rehydration is controlled at the level of
translation. To study this requires a genomics level ap-
proach combining EST based assessments of transcript
populations coupled with microarray profiling assays
to determine not only the available amount of any par-
ticular transcript but also its recruitment potential for
protein synthesis.
It is only recently that bryophytes have garnered
attention in regards to genomics in any form, and
much of this has centered on the development of Phys-
comitrella patens, a desiccation-sensitive species, as a
plant model for development and genetics (Kamisugi
and Cuming, 2004; Holtorf et al., 2004; Fujita et al.,
2004). For desiccation-tolerant species only Tortula
ruralis has served as a model for genomic level in-
vestigations (Wood and Oliver, 2004). In small-scale
studies we isolated 18 rehydrin cDNAs (Scott and Ol-
iver, 1994) followed by a further 152 ESTs from a
cDNA expression library made from sequestered
mRNAs extracted from slow-dried gametophytes
(Wood et al., 1999). Only 29% of these cDNAs dem-
onstrated any significant similarity to previously iden-
tified nucleotide and/or peptide sequences. Of those
that could be assigned a possible identity and putative
function several were ribosomal proteins (perhaps in-
dicative of the role of translation in the response), ear-
ly-light inducible proteins (ELIPs), and desiccation-re-
lated polypeptides, in particular LEA proteins. It is of
interest that 71% of the sequences could be classified
as novel, this may of course be a consequence of a
deficiency of plant genes, in particular bryophyte
genes, within the public databases but it may also in-
dicate that there are genes involved in desiccation re-
sponses or tolerance that have yet to be isolated. This
was underscored in a more extensive analysis of the
rehydration transcriptome of T. ruralis that determined
that 40.3% of the 5,563 clusters (contig groups rep-
resenting individual genes) derived from an EST col-
lection of 10,368 cDNA clones could not be assigned
identity by comparison with sequences in all available
public databases (Oliver et al., 2004). The EST col-
lection described by Oliver et al. (2004) was derived
from a non-normalized rehydration specific library,
non-normalized to allow for some assessment of tran-
script abundance during the initial two hours following
rehydration. Genome ontology (GO) mapping of the
Tortula clusters gave a broad look at what cellular ac-
tivities appear to be emphasized in the rehydrated ga-
metophytes and, as expected from our previous bio-
chemical investigations, the protein synthetic machin-
ery (both in structure and control), membrane structure
and metabolism, and the need to reestablish or main-
tain plastid integrity were central to the response. The
GO analysis also generated new directions and hy-
potheses to follow by indicating the prominence of
membrane transport, phosphorylation and signal trans-
duction, in the rehydration response to desiccation.
Signal transduction is especially intriguing with re-
gards to desiccation tolerance in T. ruralis given that
translational controls appear more important in the al-
teration of gene expression than do alterations in tran-
scriptional activities, although the rehydration cDNA
library was derived from rehydrated rapid-dried ga-
metophytes that do appear to rely on transcription to
replenish the supply of rehydrin transcripts.
Of the top 30 most abundant transcripts reported for
the rehydration transcriptome seven appear to encode
LEA or LEA-like proteins (Table 1 and Oliver et al.,
2004). This led us to suggest that LEA proteins in
Tortula may have had a role in protecting cellular con-
stituents during rehydration as well as, or instead of,
its generally assumed role of importance in protection
during the drying process. The significance of this was
amplified in an expression profile study using a cDNA
microarray constructed from the 5,563 individual clus-

794 M. J. O
LIVER
ET AL.
T
ABLE
1. Abundant LEA-like protein transcripts in the Tortula rehydration library.
#ESTs
cluster Description Bit score E-value
40 Q9XFD0 ABA-inducible protein WRAB1 (LEA/RAB-related COR) 63.93 2.88E-09
37 O16527 Caenorhabditis elegans CE-LEA 75.87 6.51E-13
32 Q9ZRF8 Hydrophobic LEA-like protein (Oryza sativa) 100.91 1.84E-20
28 P13934 Late embryogenesis abundant (LEA) protein 76 (B. napus) 51.60 1.42E-05
22 O16527 Caenorhabditis elegans CE-LEA 50.83 2.02E-05
21 Q9RV58 Protein DR1172-LEA type 1 family 55.45 1.01E-06
18 Q9LF88 Putative late embryogenesis abundant protein (Arabidopsis) 53.91 2.63E-06
Clusters are ordered by the number of individual ESTs that constitute a distinct cluster. Bit scores and E-values are derived from a BLASTx
search of a customized database derived from UNIPROT as described in Oliver et al., 2004.
T
ABLE
2. Expression profile of LEA-like transcripts in the Tortula ruralis EST collection.
Tortula ID Gene ID Total REH Poly REH
Cluster
p
1809 Q8S7U3 Putative LEA protein 7.5 3.4
Cluster
p
3490 Q9XES7 Seed maturation protein 7.2 6.6
Cluster
p
42 LEA3MAIZE LEA protein 6.3 6.0
Cluster
p
392 Q9ZQW6 Lea protein 5.7 2.1
Cluster
p
319 O16527 CE-LEA 5.3 3.0
Cluster
p
1646 Q9LJ97 Seed maturation protein 4.7 4.6
Cluster
p
136 LEA1HORVU (P14928) 4.6 4.5
Cluster
p
147 Q39801 51kDa seed maturation protein 4.5 2.8
Cluster
p
861 Q9LJ97 Seed maturation protein 4.4 3.7
Cluster
p
41 O16527 CE-LEA 4.4 2.9
Cluster
p
28 LEA1HORVU (P14928) 4.4 5.4
Cluster
p
41 O16527 CE-LEA 4.2 3.5
Cluster
p
596 O16527 CE-LEA 4.2 2.1
Cluster
p
139 Q9FKV7 LEA protein-like 4.2 4.7
Cluster
p
36 LEA3MAIZE LEA protein 3.8 3.9
Cluster
p
1647 Q8GWT7 Putative LEA 3.7 4.2
Cluster
p
1409 Q9LF88 Putative LEA protein 3.4 2.0
Cluster
p
2300 Q9XES7 Seed maturation protein 3.2 2.7
Cluster
p
40 LEA1HORVU (P14928) 3.2 2.7
Cluster
p
2020 O04371 Rehydrin Tr288 3.0 2.1
Cluster
p
935 Q39660 LEA protein homolog 2.9 2.1
Cluster
p
144 O16527 CE-LEA 2.8 2.4
Cluster
p
935 Q39660 LEA protein homolog 2.5 2.3
Cluster
p
578 Q8GV49 LEA1 protein 2.3 2.5
Total and polysomal RNA was isolated from rapid-dried rehydrated 1–2 hr (treated) and hydrated (control) gametophytes. Three biological
replicates from each condition were probed with the Tortula cDNA array in a triple dye-swap experiment for a total of six hybridizations per
condition. Data are expressed as mean ratios (rehydrated/hydrated). A total of 24 LEA-like transcripts were up-regulated more than two-fold
(P
#
0.05) in response to a dehydration-rehydration event in both the total and polysomal RNA fractions.
ters derived from our EST collection. Twenty-four of
the clusters that exhibited at least a two-fold increase
in the accumulation of its corresponding transcripts in
gametophytes that had been rehydrated for between 1
and 2 hr following rehydration have sequence similar-
ity to known LEA protein sequences (Table 2). These
transcripts are also elevated greater than two-fold in
the polysomal RNA fraction indicating their recruit-
ment into the translational mRNA pool. These data are
consistent with the classification of these transcripts as
rehydrins, and in fact one of the putative LEA like
proteins whose transcript is elevated in these two frac-
tions is the rehydrin Tr288, a rehydrin that we have
described as a LEA-like protein (Velten and Oliver,
2001). Each cluster represents an independent nucle-
otide sequence even though several match the same
LEA sequence from the public databases (indicative of
the conserved nature of these proteins) which implies
that there are several LEA proteins in T. ruralis that
are available to the gametophytes following rehydra-
tion. If LEA proteins are preferentially synthesized in
T. ruralis in response to rehydration, for which our
evidence indicates but does not directly demonstrate,
then it is reasonable to assume that LEA proteins play
a role in cellular recovery in bryophytes as well as, or
not, a role in cellular protection during dehydration.
What the actual role of LEA proteins could be during
the recovery from desiccation is yet to be determined
but some clues have recently been uncovered that al-
low us to postulate a testable hypothesis. Koag et al.
(2003) demonstrated that the maize DHN1, a group 2
LEA (Dehydrin), was capable of binding to lipid ves-
icles with some degree of specificity. The DHN1 pro-
tein preferred small vesicles to larger ones and vesicles
that contained a significant proportion of acidic phos-
pholipids. In addition, in binding to the vesicles, the
protein was determined to undergo a conformational
change resulting in an increase in alpha-helicity within

795B
RYOPHYTE
D
ESICCATION
T
OLERANCE
F
IG
. 1. Mapping of Tr288 orthologs to an unrooted phylogenetic
network of a basal portion of land plant phylogeny (synthesized
from several previous studies, published and unpublished). Those
genera whose names are in italics have been classified as desicca-
tion-tolerant according to reports in the literature, see Proctor and
Pence (2002), and from unpublished observation (Oliver and Mish-
ler).
the protein secondary structure. The authors suggest
dehydrins may undergo a function-related conforma-
tional change at the water-membrane interface that
may aid in the stabilization of small lipid vesicles or
other endomembrane structures within cells. In collab-
oration with Julia George, at the University of Illinois,
we have similar data using the Tr288 LEA-like protein
from T. ruralis. Tr288 has no sequence similarity, nu-
cleotide or amino-acid, to any other plant protein in-
cluding the LEA proteins with the exception of a sin-
gle putative ‘‘K-Box’’ motif, a signature motif of an-
giosperm dehydrins, near the carboxy-terminal of the
Tr288 protein (Velten and Oliver, 2001). However,
Tr288 is structurally similar to the LEA protein family
in all other respects. It is highly hydrophilic, glycine-
rich (19.6%), and contains 15 copies of a conserved
amino acid sequence motif that is predicted to form
amphipathic alpha helices. Purified protein, derived
from expression of a Tr288 construct in E. coli, pref-
erentially binds to lipid vesicles that are composed of
a 3:1 mixture of phosphotidylcholine and phosphatidic
acid. If the vesicles are constructed of phosphotidyl-
choline alone, Tr288 appears to partially solubilizethe
lipid (unpublished data). Although we have yet to
demonstrate any alteration in the conformation of the
protein as it interacts with the lipid vesicles or any in
vivo confirmation of this activity it does appear that at
least some LEA proteins may function as lipid stabi-
lizing proteins. This leads us to the hypothesis that in
rehydrating T. ruralis gametophytes LEA proteins may
serve a role in stabilizing membranes, or perhaps lipid
transport for reconstitution of damage membranes,
during the cellular upheaval that results from the un-
fettered inrush of water during rehydration.
P
HYLOGENETIC
R
ELATIONSHIPS OF
T
R
288
The apparent emphasis on the synthesis of LEA pro-
teins during the initial periods following rehydration
of dried gametophytes and the possibility that they
play a role in membrane stability or reconstitution sug-
gests that these genes may play a key adaptive role in
the evolution of desiccation tolerance in plants. In or-
der to gain some insight into this possibility, we uti-
lized the conserved nature of the nucleotide sequences
corresponding to the 15 amino-acid repeats within the
Tr288 gene to develop a PCR-based strategy to iden-
tify possible Tr288 orthologs in a wide range of land
plant species. The purpose of such an investigation
was to determine the evolutionary relationship of the
Tr288 gene to desiccation tolerance. This approach is
limited by the ability of the PCR primers to allow
amplification of a fragment from the ortholog and any
conclusions that are drawn are done so under the ca-
veat that the absence of a PCR fragment may simply
mean that the primer was incapable of allowing exten-
sion of a DNA product because of a mismatch at the
3
9
end. To alleviate this problem, at least in part, we
chose the most highly conserved sequence within the
repeats for the 3
9
portion of the primers and cloned
and sequenced each PCR product to be sure that it
contained sequence similar to that of the T. ruralis
Tr288. The location of the PCR primers within the
conserved portion of the repeats increased our poten-
tial for isolating an othologous sequences because the
primers, if there are more than two repeats, will gen-
erate several possible PCR fragments rather than just
one and if each is cloned and sequenced will generate
a better sequence comparison for the ortholog assess-
ment.
The results of this analysis were mapped onto a phy-
logenetic tree, synthesized from previous studies, to
gain an insight into the evolutionary history of this
gene (Fig. 1). On this tree, Tr288 orthologs appear to
correlate well with desiccation tolerance within the
bryophyte taxa, with the exception of the tropical des-
iccation-tolerant members of the Exostratum,Octoble-
pharum,Leucophanes, and Anthrocormus genera
(even though Mitthyridium, a related genus, does have
a Tr288 ortholog). This raises the interesting possibil-
ity that these tropical mosses that dry under elevated
temperatures may have a mechanism of tolerance that
differs from that seen in more temperate species and
one that has lost the need for a Tr288 homolog or has
evolved a more efficient form of this type of protein.
Of even more significance is that a Tr288 ortholog is
completely absent from the liverworts even though
many are desiccation-tolerant. This may be a conse-
quence of the fact that liverworts branched off earliest
in land plant evolution and that the ancestral form of
the Tr288 gene either had yet to evolve into a structure
that would allow detection by our PCR approach, or
that these primitive plants may actually have a differ-
ent strategy and mechanism of desiccation tolerance.

796 M. J. O
LIVER
ET AL.
F
IG
. 2. Simplified phylogeny of the land plants, showing the reconstruction of presence of vegetative desiccation tolerance (in at least some
members of indicated clade) in black, non-tolerance in white, and equivocal reconstruction by hatching. The arrow marks where the ancestral
condition can unequivocally be reconstructed as non-tolerant. Branching topology based on Shaw and Renzaglia, 2004; Pryer et al., 2004;
Kelch et al., 2004; distribution of desiccation-tolerance based on Wood and Peng (2005) and Proctor and Pence, 2002.
Tr288 orthologs are still present in the tracheophytes.
This could mean that the gene has been co-opted into
a different role, perhaps still involved in dehydration
tolerance mechanisms or possibly involved in propa-
gule desiccation tolerance, or has become simply an
inactive gene remnant. Although all of these possibil-
ities are simply speculative this analysis has opened
up some interesting hypotheses and research directions
to be tested and followed. This study has also led us
to re-evaluate what we understand of the evolution of
desiccation tolerance in the land plants.
P
HYLOGENETIC
I
NFERENCES
R
EVISITED
An evolutionary scenario for the evolution of veg-
etative desiccation tolerance was first given by Oliver
et al. (2000), and presented there as a testable hypoth-
esis. Because of the widespread occurrence of vege-
tative desiccation tolerance in the bryophytes (the ear-
liest diverging living clades of land plants), they ar-
gued that it is likely that it was primitively present in
the land plants. They further hypothesized that vege-
tative desiccation tolerance was subsequently lost in
the early evolution of tracheophytes and then re-
evolved multiple times in several separate tracheo-
phyte lineages. Their postulated mechanistic explana-
tion for this pattern can be summarized as follows: the
primitive mechanism of tolerance exhibited by the first
plants probably involved a constitutive level of cellular
protection coupled with an efficient and active repair
process, similar to what we have described above for
modern-day desiccation-tolerant bryophytes. This des-
iccation tolerance came at a cost, because metabolic
rates are low in tolerant plants as compared to plants
that do not maintain costly mechanisms for tolerance.
As plants evolved to fill the various niches available
to them on dry land, loss of tolerance was favored
because of the internalization of water relations as the
vascular plants became larger and more complex.
Genes that had originally evolved for vegetative cel-
lular protection and repair were possibly recruited for
different but related processes such as the response to
water stress and, more important, the desiccation tol-
erance of reproductive propagules (seeds). In turn, Ol-
iver et al. (2000) speculated that once established in
seeds, the developmentally induced cellular protection
system became available for induction in vegetative
tissues by environmental cues that are related to dry-
ing. This may have led to an evolutionarily more re-
cent vegetative desiccation tolerance mechanism,
evolved from that programmed into seed development,
as certain phylogenetically scattered lineages of angio-
sperms spread into very arid environments.
This series of hypotheses needs to be tested in light
of more recent phylogenetic knowledge, and greater
knowledge of the distribution of desiccation tolerance
in land plants. Figure 2 shows a simplified picture of

797B
RYOPHYTE
D
ESICCATION
T
OLERANCE
the overall phylogenetic distribution of desiccation tol-
erance in the current phylogeny of land plants. It is
evident from this figure that the basal condition for the
land plants has now become equivocal, given this
more detailed picture. It makes sense, given the ar-
guments in the earlier part of this paper, thatvegetative
desiccation tolerance is likely to have been present in
the earliest land plants. However, it is difficult to dem-
onstrate this formally with character reconstructions at
present. Part of the problem comes from the lack of
knowledge about desiccation tolerance in the out-
groups, the close ‘‘green algal’’ relatives of land plants
(Chara,Coleochaete, and relatives; Lewis and Mc-
Court, 2004). Also, there are key groups near the base
of liverwort and moss phylogenies that do not appear
to be desiccation-tolerant (although these need to be
surveyed in greater detail). It has been a long time
since the branches near the base of this tree diverged
from each other (450–500 MYA), extinction has taken
its toll, and many of the modern taxa (such as Sphag-
num) are highly modified in structure and physiology
from their ancestral conditions. To resolve this char-
acter reconstruction question, better sampling for des-
iccation tolerance is needed, especially among the bas-
al lineages. It is also likely that if highly derived taxa
such as Sphagnum were once desiccation-tolerant, but
lost the phenotype, telltale signs will remain in their
genomes such as the presence of rehydrins that can be
discovered by comparative genomics. So there is hope
of resolving this important ancestral reconstruction is-
sue someday.
The possibility that vegetative desiccation tolerance
may not have been present at the very bottom of the
land plant phylogenetic tree suggests that it may be
necessary to expand on the hypothesis given in Oliver
et al., 2000. Vegetative desiccation tolerance is cer-
tainly not the full story. It is quite possible that an
even more primitive condition in precursors to land
plants is desiccation-tolerant spores. Perhaps the ear-
liest manifestation of desiccation tolerance was in the
spores of land plants (and probably their charophyte
alga relatives), and then desiccation tolerance became
expressed in vegetative tissues in early land plants. As
previously postulated by Oliver et al. (2000), expres-
sion appears to have been lost in the vegetative tissues
of the early tracheophytes, yet regained in the some
widely separated lineages. We don’t know what hap-
pened in spores, however—it is quite possible that des-
iccation tolerance was widely retained throughout in
the spores of tracheophytes. Very little is known about
desiccation tolerance in spores, and it is imperative
that this be studied soon in a breadth of taxa to test
this expanded hypothesis.
It is clear from Figure 2, that vegetative desiccation
tolerance was ancestrally lacking in the tracheophytes
(starting from the branch shown with arrow). The
number of times it was regained is still not certain,
given the lack of complete surveys for the presence of
desiccation tolerance. However, recently increased res-
olution of the angiosperm phylogeny allows a clearer
picture of the distribution of the unusual phenotype of
desiccation tolerance in this most diverse group of land
plants. Using the online phylogenetic trees at the An-
giosperm Phylogeny Website (Stevens, 2001), and
mapping on the most recent survey of the occurrence
of desiccation tolerance (Proctor and Pence, 2002), al-
lows the following insights: (1) within the angio-
sperms, at least ten phylogenetically independent cases
of re-evolution of desiccation tolerance occurred; (2)
at least four of these cases arose in the monocots, and
probably more in the large families Poaceae and Cy-
peraceae; (3) no cases occur in basal dicots, but six
occur in the Eudicots; (4) Four cases occur in the As-
terids, but interestingly there are none known so far in
the Rosids. There will certainly be more cases found
as more angiosperms are studied, and it will be inter-
esting to see what habitat and phylogenetic correla-
tions are uncovered.
A
CKNOWLEDGMENTS
We thank Peter Alpert for putting in the lion’s share
of the work organizing the symposium, Michael Proc-
tor and one anonymous reviewer provided helpful
comments. We also thank Sarah Learmonth for her
expert technical help. Work presented here was sup-
ported in part by USDA-ARS CRIS project 6208-
21000-012-00 (MJO and JV) and by NSF grants DEB-
9712347 (the Deep Gene RCN) and DEB-0228729
(the Green Tree of Life project) to BDM. Mention of
a trademark or proprietary product does not constitute
a guarantee or warranty of the product by the United
States Department of Agriculture, and does not imply
its approval to the exclusion of other products that may
also be suitable.
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