New Phytol. (1998), 140, 9–23
Adaptations for an amphibious life :
changes in leaf morphology, growth rate,
carbon and nitrogen investment, and
reproduction during adjustment to
emersion by the freshwater macrophyte
Littorella uniflora
B  W. E. R O B E*    H. G R I F F I T H S
Department of Agricultural and Environmental Science, Ridley Building, The University,
Newcastle upon Tyne, NE1 7RU, UK
(Received 13 January 1998 ; accepted 4 June 1998)

Littorella uniflora (L.) Ascherson is a small, perennial, amphibious rhizophyte of rosette life-form which is
common along the margins of lakes, tarns and reservoirs where water-level fluctuations are often rapid and
unpredictable. The majority of plants are continuously submersed and reproduce vegetatively, but a small
proportion become completely emersed for variable lengths of time, when flowering and seed set occur. To find
out how L. uniflora adjusts to sudden emersion we studied the plants at a reservoir where water level falls each
spring and remains low throughout the summer ; L. uniflora adjusted very quickly showing a degree of phenotypic
plasticity not expected in a ‘ stress tolerator ’, including the production of a new set of terrestrial leaves with
reduced lacunal volume and increased stomatal density, a rapid increase in leaf growth rate, and flowering within
3–4 wk. Comparison of terrestrial L. uniflora with aquatic plants growing permanently submersed in lake and tarn
habitats showed that three to fourfold more carbon (C) and nitrogen (N) was incorporated into above-ground
biomass by emersed plants. However, ramet production in the aquatic environment appeared to be more costly,
in terms of C and N invested, than terrestrial flower and seed production. The combination of continuous,
submersed vegetative spread with the capacity for a high degree of phenotypic plasticity allowing some flower and
seed production to occur during brief periods of emersion seems to account for the success of this plant in the
amphibious niche.
Key words : Littorella uniflora (L.) Ascherson (Shoreweed), aquatic macrophyte, emersion, heterophylly, seasonal
growth.

There are many species of higher plant which lead an
amphibious existence. These plants have evolved
from terrestrial ancestors and show varying degrees
of specialization for aquatic life, from species which
tolerate only short periods of submersion to others
which complete their whole life cycle under water
(Arber, 1920 ; Sculthorpe, 1967 ; Raven, 1984).
Although there has long been keen interest in the
remarkable plasticity of leaf form shown by many of
* To whom correspondence should be addressed.
E-mail : w.e.robe!ncl.ac.uk
these plants, and more recently in their photosynthetic physiology and C and N metabolism
(Arber, 1920 ; Sculthorpe, 1967 ; Cook, 1969 ;
Bodkin, Spence & Weeks, 1980 ; Bowes & Salvucci,
1989 ; Goliber & Feldman, 1989 ; Maberly & Spence,
1989 ; Nielsen, Gacia & Sand-Jensen, 1991 ; Pedersen & Sand-Jensen, 1992 ; Nielsen, 1993 ; Madsen &
Breinholt, 1995 ; Keeley, 1996 ; Pedersen & SandJensen, 1997), the adaptations which allow amphibious plants to withstand sudden emersion and
submersion and to proliferate in both aquatic and
terrestrial environments are not well understood.
Climate change, by affecting both precipitation and
rates of water abstraction, may well subject such
Printed from the C JO service for personal use only by...
10
W. E. Robe and H. Griffiths
Early June
Aquatic
Early July
Flowering
Early September
Seed-bearing
(a)
(b)
(c)
(d)
(e)
(f )
Figure 1. The study site at Thirlmere Reservoir in the Cumbrian Lake District and the appearance of the
plants in early June (a, d ), early July (b, e) and early September (c, f ).
plants to more frequent and extreme fluctuations in
environment and long-term changes in habitat.
Amphibious plants play an important role in the
ecology of fresh waters and a better understanding of
their capacity to respond to water level fluctuations is
now timely.
Littorella uniflora L. Ascherson (Shoreweed) is a
common species at the margins of tarns, lakes and
reservoirs throughout the UK, northern Europe and
North America (see Fig. 1, and Clapham, Tutin &
Warburg, 1981 ; Raven et al., 1988 ; Robe & Griffiths,
1992). This small perennial plant (4–6 cm high) has
a rosette of cylindrical evergreen leaves, a short stem
and a large root system. In lake and tarn habitats, the
majority of plants are submersed throughout the
year in 0n3–2 m of water and reproduce vegetatively,
often covering large areas (e.g. Pearsall, 1920 ; Misra,
1938 ; Spence, 1964 ; Farmer & Spence, 1986).
Like many amphibious macrophytes, L. uniflora
only flowers when emersed. Plants in shallow water
at lake margins often become emersed during the
summer but emersion and re-submersion are unpredictable and, for this small plant, often rapid.
Sometimes the plants are exposed to the aerial
environment for only a few days, or water level does
not fall far below the sediment surface (Aulio, 1986 ;
Nielsen et al., 1991), but they can also be completely
emersed for weeks or months, and at reservoirs
survive and grow terrestrially on dry land for even
longer (Arber, 1920 ; Farmer & Spence, 1986 ;
Hostrup & Wiegleb, 1991 ; W. E. Robe ; unpublished
observations).
For amphibious plant species the transition from
aquatic to terrestrial environments involves an
adjustment to extremely different conditions. In the
aquatic environment, the slow rate of CO diffusion
#
potentially limits photosynthesis, leading to a range
of additional inorganic C uptake strategies (Raven,
Osborne & Johnston, 1985 ; Maberly & Spence,
1989 ; Madsen & Sand-Jensen, 1991). Low O
#
concentrations in anaerobic sediments may inhibit
root metabolism, nutrient supply and uptake (SandJensen, Prahl & Stokholm, 1982 ; Christensen,
Revsbech & Sand-Jensen, 1994), and nutrient concentrations are generally very low in oligotrophic
habitats such as those in which L. uniflora is most
common (see Robe & Griffiths, 1994). A wide range
of light intensities is encountered (Spence, 1981 ;
Raven, 1984), while epiphytes and pleustophytes
(Roelofs, 1983), wave action and silting (Pearsall,
1920 ; Spence, 1981) are additional hazards. In
contrast, in the terrestrial environment, atmospheric
CO is readily available, and nitrate (the form of
#
nitrogen mainly utilized by L. uniflora ; Schuurkes,
Kok & Den Hartog, 1986) may increase in concentration as nitrification increases in aerobic sediments.
However, water deficit becomes the major potential
growth-limiting factor.
Previous work on the ecophysiology of L. uniflora
has focused almost exclusively on how this plant is
adapted for submersed growth. Aquatic L. uniflora
have thick, stiff, nearly cylindrical leaves containing
an extensive system of intercellular gas channels, or
lacunae, which continue through the stem and into
the roots. CO supply to the leaves is predominantly
#
by diffusion within the lacunal system from the CO #
enriched sediments, where concentrations are in the
range 0n3–3n0 mol m−$ (Roelofs, 1983 ; Boston,
Adams & Pienkowski, 1987 ; Robe & Griffiths, 1990,
1992). Daytime CO supply within the leaves is
#
boosted by recycling of respiratory CO and Cras#
sulacean Acid Metabolism (CAM), and lacunal
concentrations are typically 0n45–2n70 mol m−$
(1–6 %) and saturating for photosynthesis in vitro
Printed from the C JO service for personal use only by...
Littorella uniflora : adjustment to emersion
(Madsen, 1987 a, b ; Raven et al., 1988 ; Robe &
Griffiths, 1988, 1990, 1992). Photosynthetically
generated O diffuses from the leaf surfaces and via
#
the lacunae into the roots and rhizosphere, facilitating nutrient supply and uptake. (Sand-Jensen et
al., 1982 ; Robe & Griffiths, 1990 ; Christensen et al.,
1994). Nitrogen requirements are reduced by N
storage, a small life form and a slow growth rate
(Robe & Griffiths, 1994). Plants growing permanently submersed produce approximately one
ramet per plant per year (Sand-Jensen & Sondergaard, 1978 ; Boston & Adams, 1987 ; Robe &
Griffiths, 1992).
Although separate studies had compared the
growth rate (Nielsen & Sand-Jensen, 1997), leaf
morphology (Hostrup & Wiegleb, 1991), CAM
activity (Aulio, 1986 ; Groenhof, Smirnoff & Bryant,
1988), gas exchange (Nielsen et al., 1991) and
photosynthetic enzyme activity (Beer et al., 1991) of
aquatic and terrestrial L. uniflora, the degree of
emersion and duration of exposure to the terrestrial
environment varied and there was no integrated
picture of how this species adjusts to emersion when
growing in situ at a single location. To try to remedy
this situation we have studied this species at a
reservoir where the plants are submersed during the
winter and emersed regularly in late spring, remaining on dry land until early winter. In this paper
we describe the changes which took place in leaf
morphology (including stomatal density and lacunal
volume), growth rate, dry-matter turnover and C
and N investment as aquatic plants became terrestrial, flowered and set seed. We also draw on an
earlier study of permanently submersed L. uniflora
in lake and tarn habitats (Robe & Griffiths, 1992), to
compare the costs and benefits of submersed vegetative proliferation with flower and seed production
in the terrestrial environment.
  
11
fine plastic netting supported 0n4 m above the plants
by wooden posts.
Measurement of environmental parameters
Photosynthetic photon flux density (PPFD) in the
400–700 nm wavelength range was measured using a
submersible PAR quantum sensor, and microvolt
integrator (Delta-T Devices Ltd., Cambridge, UK).
Relative humidity was determined with a dewpoint
meter (Protimeter, Marlow, Bucks, UK). Water
content of sediments taken from around the roots of
the plants was determined from their weight before
and after drying to constant weight at 60 mC.
Plant morphology, and carbon and nitrogen content
Shoots, individual leaves, roots, stems, stolons,
ramets, flowers and seeds were gently washed,
blotted dry, weighed, then dried to constant weight
at 60 mC. Total C and N content of the material was
determined using a Europa2 Roboprep\Tracermass
system (Europa Scientific, Crewe, UK). Whole-leaf
volume was determined by displacement of water.
Thin transverse sections of fresh leaf, stem and root
material were cut by hand, mounted in water and
viewed and photographed under a Leica2 DMRB
microscope. Lacunal volume as a percentage of leaf,
stem and root volume was estimated from a comparison of surface areas made using stereology (Steer,
1981), with a 5 mm or 10 mm point\line lattice
applied to enlarged photographs of magnified sections. Leaf surface impressions were made with
Xantopren2, a dental impression material (Bayer,
Germany) applied either immediately after removal
of the leaf from the plant (aquatic L. uniflora) or to
leaves growing in situ (terrestrial plants). Clear
acetate copies were made and examined under the
microscope ; stomatal numbers were counted before
photographs were taken as described above.
Field site and programme of measurements and
sampling
Leaf turnover and growth rate, flowering and seed
production
This study was carried out at Thirlmere Reservoir,
Cumbria, UK (National Grid Reference NY
323133 ; Fig. 1) where regular and extensive fluctuations of water level guaranteed rapid emersion of
L. uniflora in late spring and prolonged exposure of
the plants to the terrestrial environment during
summer and autumn. Plants were sampled on three
occasions ; in early June when still submersed, in
early July after three to four weeks out of water, and
in early September after three months on dry land.
Growth rate measurements ran concurrently from
late May until mid-October. Care was taken to
sample and make growth-rate measurements in areas
of similar plant size and density, and these areas were
protected from grazing by wildfowl with a layer of
Measurements were begun in late May, when the
upper part of the site was briefly emersed before
being re-submersed for two weeks following heavy
rain. Twenty-four plants without ramets (referred to
as ‘ parent plants ’ in this study) were chosen (six in
each of four locations approx. 0n5 m apart) in a
typical area of the site where plants were large and
fairly closely packed. No attempt was made to thin or
weed round them. All existing leaves were identified
using stainless steel washers attached with a loop of
nylon cord, and their length and width were
measured (Robe & Griffiths, 1992). The plants were
re-examined at approx. 4-wk intervals from midJune until mid-October, new leaves were labelled
and the lengths and widths of all leaves were
Printed from the C JO service for personal use only by...
12
W. E. Robe and H. Griffiths
measured ; male flower stems were counted and
measured in July. Length-to-dry-weight relationships were used to calculate growth rates. These
plants did not produce ramets during the course of
this study. As the seeds (for their location see Results
section) could not be obtained without damaging the
plants, the seed complements of adjacent L. uniflora
were determined in September.
Statistics
Statistical analysis of the data was carried out
following Parker (1979). Details of each analysis are
included in the Results sections, or in the captions to
tables and figures, as appropriate.

Study site and time-course of emersion
In the 100 yr since the establishment of Thirlmere
Reservoir, a very large population of L. uniflora had
built up on the gently sloping, southern shore in
deep silty\sandy sediments (Fig. 1). During the
winter of 1994 and spring of 1995 the plants were
submersed in up to 2 m of water. Spring was wet and
it was not until late May\early June that the plants
were accessible in still, shallow water a few cm above
the leaf tips (Fig. 1 a). Within a few days of the first
sampling (early June) water level fell rapidly and the
submersed plants were quickly emersed (within
24 h). By early July (Fig. 1 b) the plants had been out
of water for 3–4 wk, and by early September (Fig.
1 c) had been growing on dry land for 3 months. The
summer and early autumn of 1995 were amongst the
warmest and driest in the UK this century, with long
spells of sunny weather and only very light rain in
September and October. Environmental conditions
at the site on the three sampling dates are outlined in
Table 1.
Growth form, leaf morphology and reproduction
The appearance of the plants is illustrated in Figure
1 and Table 2. The rosettes of submersed L uniflora
sampled in early June (Fig. 1 d ; referred to in this
study as ‘ aquatic ’ plants) comprised mainly short,
thick cylindrical leaves growing from a short stem.
Many plants possessed ramets (daughter plants
attached to the stem by stolons) which had probably
developed under water during the previous winter
(Table 2). The emersed plants sampled in early July
(Fig. 1 e ; described in this study as ‘ flowering ’ L.
uniflora) were visibly taller, with a new set of longer,
narrower, terrestrial leaves and a greater shoot dry
weight (Table 2). Male flower buds were growing
from the stem at the base of the new leaves. Female
flowers (sessile ovaries with long stigmas) were
clustered at the base of the leaves (for details see
Arber, 1920 ; Sculthorpe, 1967). Flowering across
the entire site was condensed into a 1- to 2-wk period
in early-to-mid-July. By early September (Fig. 1 f ),
after a further 2 months on dry land, the rosettes
were noticeably shorter and flatter. Shoot fresh
weight was less, and while the long leaves produced
in June and July were still present on the outside of
the rosettes, the youngest mature leaves were shorter
and narrower than those in July. All leaves showed a
tendency to grow prostrate. Each plant possessed on
average 8n5 fruits (single-seeded nuts or drupes)
Table 1. Environmental conditions at the site at Thirlmere Reservoir,
Cumbria, on a typical day during sampling in early June, early July and
early September 1995
Early June
Aquatic
PPFD ( µmol m−# s−")
Leaf tips of aquatic plants
Mean integrated
Instantaneous range
Water surface\leaf tips of
terrestrial plants
Mean integrated
Instantaneous range
Temperature (mC)
Relative humidity (%)
Sediment water content (%)
Early July
Flowering
Early September
Seed-bearing
909
600–1800
23
61
36
497
350–500
16
89
18
718
590–1180
1132
960–1900
22
51
Mean integrated, and instantaneous, photosynthetic photon flux density
(PPFD) were measured over a 2–3-h period around midday. In early June the
leaf tips of aquatic Littorella uniflora were approx. 4 cm below the water surface,
and measurements of surface and leaf-tip PPFD were made simultaneously. The
relatively low PPFD and temperature, but higher relative humidity, in early
September reflect temporarily misty conditions caused by low cloud.
Printed from the C JO service for personal use only by...
Littorella uniflora : adjustment to emersion
13
Table 2. Characteristics of Littorella uniflora growing at Thirlmere
Reservoir, Cumbria in 1995
Early June
Aquatic
Shoot
f. wt (g)
d. wt (g)
Leaves per plant
Leaf length (cm)
All leaves
Youngest mature
leaves
Leaf width (cm)
All leaves
Youngest mature
leaves
Leaf volume (cm$)
Male flower
Stems per plant
f. wt per stem (g)
d. wt per stem (g)
Female flowers
f. wt per plant (g)
d. wt per plant (g)
Seeds per plant
Mean seed weight (g)
Stem
f. wt (g)
d. wt (g)
Root system
f. wt (g)
d. wt (g)
Ramet f. wt (g)
Stolon
f. wt (g)
d. wt (g)
Early July
Flowering
Early September
Seed-bearing
0n44p0n08 a
0n029p0n006 a
5n42p0n58 a
0n51p0n09 a
0n047p0n007 b
7n87p1n11 b
0n28p0n07 b
0n035p0n009 ab
5n83p1n40 a
4n80p1n28 a
4n76p0n52 a
7n68p1n87 b
9n28p0n99 b
7n28p3n40 b
4n86p2n18 a
0n272p0n068 a
0n346p0n035 a
0n167p0n043 b
0n133p0n017 b
0n177p0n021 c
0n105p0n006 c
0n312p0n073 a
0n250p0n097 b
0n106p0n038 c
2n54p0n88
0n020p0n005
0n0025p0n0007
0n003p0n001
0n0003p0n0001
8n50p3n38
0n001p0n0003
0n08p0n01 a
0n014p0n005 a
0n07p0n04 a
0n013p0n006 a
0n25p0n08
0n022p0n006
0n064p0n02
0n29p0n12
0n037p0n012
0n09p0n03 a
0n027p0n009 b
0n042p0n006
0n003p0n0005
Results are for 10–25 plants, except in the case of female flower f. wt (n l 5
plants), and root system f. wt where only 2–3 sets of nearly intact roots could be
obtained. Mean lengths and widths of all leaves are for the entire leaf
complement of the 24 plants used for growth rate measurements ; n l 117, 187
and 123 leaves in June, July and September, respectively. Leaf volume was
determined by displacement of water. Ramets possessed 2–4 leaves, total length
4n8 cm, and 1–5 short roots 1 cm long ; their f. wt includes leaves and roots.
Mean stolon length was 5 cm. Mean results are shownp. Since sample sizes
were not always equal, for each characteristic the significance of the difference
of the means at the three sampling dates was tested pairwise using t-tests
corrected where necessary for small sample sizes and significantly different
variances. Means followed by a different letter are significantly different at the
P 0n001 level, except for leaves per plant in June and July (P 0n002), leaf
volume in June and July (P 0n01) and shoot f. wt in June and September
(P 0n02).
lightly attached to the stem amongst the outer
brown, senescent leaves (removed in the figure) and
is referred to in this study as ‘ seed-bearing ’.
Aquatic L. uniflora possessed the large root system
(reaching a depth of 30 cm in the sediments and
comprising 30 % of fresh and dry weight ; Table 2)
typical of this species (see Raven et al., 1988). The
size of the root system appeared to remain relatively
constant following emersion, although it became
difficult to extract intact roots systems from the
drying sediments.
Leaf turnover
The changing appearance of the shoots after emersion was due to rapid leaf turnover as illustrated in
Figure 2, which shows the leaf complement of an
average plant of L. uniflora on six occasions between
late May and mid-October. In late May, just before
emersion, aquatic plants possessed 5–6 leaves (Table
2), numbered 0–5 in Figure 2. The plants were
examined again in mid-June, mid-July, mid-August,
mid-September and mid-October, and during this
Printed from the C JO service for personal use only by...
Leaves of each age category
per Plant
14
W. E. Robe and H. Griffiths
1·00
0·75
0·50
0·25
–2 –1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
Leaf complement
Figure 2. The changing leaf complement of an average
plant of Littorella uniflora growing in situ at Thirlmere
Reservoir between late May and mid-October. At the first
measuring date in late May ($), just before emersion, all
healthy leaves on each of 24 aquatic plants were numbered.
The eldest leaf was numbered 0 and the youngest leaf 5.
Leaf numbering provided an index of leaf composition
which could be updated as each new leaf was produced and
each senesced leaf was shed. Thus, leaves formed after late
May were numbered 6 onwards. In mid-June (#), a few
days after emersion, the leaf complement of the plants was
numbered 0–8, in mid-July (>), 4–5 wk after emersion,
3–13, in mid-August () 4–17, in mid-September (
)
6–19, and in mid-October (=) 9–21. The y axis shows the
leaves of each age category (0–21) on an average plant, p
where larger than the symbol.
time each produced, on average, 16 new leaves,
numbered 6–21, and shed 11–12 leaves, numbered
0–11. The first new leaf was present within a week
after emersion. Rates of leaf shedding and production, expressed as leaves per plant per day (p),
were 0n026p0n048 and 0n107p0n030, respectively,
between May and June samplings, 0n135p0n028 and
0n154p0n034 between June and July, 0n067p0n021
and 0n062p0n024 between July and August,
0n133p0n049 and 0n067p0n030 between August and
September, and 0n058p0n030 and 0n073p0n019 between September and October.
Each new leaf was slightly different in size and
shape, resulting in the gradation of leaf types on the
rosettes at each sampling date (Figs 1 d–f ). Thus, in
early July, one or two green and healthy aquatic
leaves (4n75 cm lengthi0n35 cm width ; Table 2)
remained on the outside of the rosettes of flowering
L uniflora but there was a gradation towards the fully
terrestrial type (mean dimensions ; 9n28i0n13 cm ;
Table 2) nearer the centre. The largest leaves
recorded were 12n0i0n12 cm in early July and
13n5i0n12 cm in August. In early September, seedbearing plants retained 1–3 of the long outer leaves
formed soon after emersion, but inner leaves were
progressively shorter until only, on average,
4n86i0n10 cm at maturity (Table 2), with some as
small as 2n7i0n1 cm at maturity.
Stomatal density and lacunal volume in leaves stems
and roots
Aquatic and terrestrial leaves differed dramatically
in stomatal density and lacunal volume, as shown in
Figures 3 and 4. In Figure 3 a, b impressions of the
adaxial (upper) surfaces of the distal halves of
youngest mature leaves of aquatic and flowering L.
uniflora are compared. Youngest mature aquatic
leaves possessed a few scattered stomata (Fig. 3 a) at
low frequency (9 mm−# ; Fig. 4 a) near the tips which
generally appeared to be closed. These appeared to
became functional after emersion, since open stomata
were found on aquatic leaves remaining on the
outside of flowering rosettes in July (Fig. 1 e). In
contrast, terrestrial leaves on flowering L. uniflora
(Fig. 3 b) showed a frequency of stomata
(90–100 mm−# ; Fig. 4 a) similar to that found on true
terrestrial species (Larcher, 1983 ; Willmer, 1983).
Stomata were present on both leaf surfaces, along the
whole length of the leaf (although most frequent on
the distal half of the flatter, adaxial surface) and were
functional, with varying degrees of aperture visible.
However, on many of the leaves examined in
September, up to 50 % of the stomates appeared to
be occluded by an unknown substance.
Figure 3 c and d shows transverse sections through
the middle portions of typical youngest mature
leaves of aquatic and flowering L. uniflora. The thick
aquatic leaves (Fig. 3 c) showed the well developed
lacunal system typical of this species, with gas
channels occupying 48 % of total leaf volume and
lined by green photosynthetic cells (Fig. 4 b, see also
Robe & Griffiths, 1988, 1990). In contrast, the
narrow terrestrial leaves of flowering and seedbearing plants contained a lacunal volume only
12–13 % of total leaf volume (Figs 3 d and 4 b).
Figure 3 e and f shows transverse sections through
a typical stem and root of flowering L. uniflora. Gas
channels occupied 6–12 % of total volume, a similar
percentage to that in the leaves (12–13 % ; Fig. 4 b).
The lacunal system in stems and roots seemed to be
unaffected by emersion, as sections from aquatic and
seed-bearing L. uniflora were of the same appearance.
Growth rate and dry matter turnover : terrestrial
and submersed L. uniflora
Since L. uniflora is a perennial plant with a rosette
life-form and simultaneous leaf-shedding and production (Fig. 2), shoot dry weight does not accurately
reflect changes in the rate of leaf growth. In Figure
5 a, therefore, relative rates of above-ground drymatter gain and loss of emersed L. uniflora at
Thirlmere are presented separately. In Figure 5 b
they are compared with those of plants growing
submersed throughout the summer and autumn at
Esthwaite Water, a lowland eutrophic lake, and Red
Tarn, an upland oligotrophic tarn. Data for Esthwaite Water and Red Tarn L. uniflora, including
environmental conditions at each site, were collected
in a study in the Cumbrian Lake District in 1986 (see
Robe & Griffiths, 1992). L. uniflora were planted in
openwork plastic baskets which were relocated in the
Printed from the C JO service for personal use only by...
Littorella uniflora : adjustment to emersion
15
(a)
(b)
(c)
(d )
(e)
(f )
Figure 3. Impression of the adaxial surface of the distal half of a typical youngest mature leaf. (a) Aquatic
Littorella uniflora sampled in early June. (b) Flowering L. uniflora sampled in early July. Magnificationi100,
scale bar 0n1 mm. Transverse sections through middle portions. (c) A typical youngest mature leaf of aquatic
L. uniflora showing only about one third of the whole section. (d ) A typical terrestrial leaf from flowering L.
uniflora. (e) A representative stem from flowering L. uniflora showing only about one sixth of the whole section.
( f ) A typical root from flowering L. uniflora. Magnificationi50, scale bar 0n1 mm.
sediments in 0n25–0n4 m of water and could be
removed for measurement. The data are now
presented on a dry-matter basis. In Figure 5 a, b,
each data point shows simply the gain or loss of dry
weight during the preceding 4-wk period as a
function of dry weight at the beginning of that
period. The data are plotted against time on an
arithmetic scale and for clarity are presented as
single points rather than in histogram form.
At Thirlmere, leaf dry-matter production increased sharply after emersion, from 20 mg g−" d−"
between late May and early June to 44 mg g−" d−"
between early June and early July (Fig. 5 a).
However, after flowering, production slowed greatly,
to 9 mg g−" d−" July–August and 3 mg g−" d−" in the
4 wk to September. Growth resumed slightly, but
not significantly, to 5 mg g−" d−" between September
and October, following light rain.
The rapid turnover of leaves at Thirlmere (Fig. 2)
was accompanied by substantial dry-matter losses
(Fig. 5 a). By September–October losses greatly
outweighed new growth as outer leaves were shed
and rosettes became smaller (Fig. 1). Thus, relative
growth rate, calculated as the change in shoot dry
weight over a 4-wk period as a function of the initial
dry weight of the shoots (p), increased from
13n2p1n9 mg g−" d−" in June to 22n8p2n9 mg g−" d−"
in July, but was negative in August (k0n81p0n5),
September (k18n5p0n7) and October (k5n8p1n3).
Leaf production by parent plants at Esthwaite and
Red Tarn also peaked between June and July, (Fig.
5 b), although dry-matter losses due to leaf senecence
Printed from the C JO service for personal use only by...
(a)
Early June
Aquatic
Early July Early September
Flowering Seed-bearing
Stomatal density
(stomata per mm2)
100
75
50
25
Lacunal volume
(% of total leaf volume)
0
(b)
50
40
30
Leaf dry matter turnover (mg g–1 d. wt day –1)
W. E. Robe and H. Griffiths
Gains
June
Flowers
Figure 4. (a) Stomatal density on the adaxial surface of the
distal half of youngest mature leaves of Littorella uniflora.
Results for 6–9 leavesp. (b) Lacunal volume as a
percentage of whole-leaf volume estimated from photographs of leaf transverse sections using stereology (Steer,
1981). Results for three or four leaves youngest mature
leavesp.
were even more severe, outweighing dry-matter
production for most of the season. For example, at
Red Tarn relative growth rate () (p) reached
3n1p1n5 mg g−" d−" in July but ranged from k0n57 to
k2n89 mg g−" d−" for the rest of season, while at
Esthwaite,  peaked at only 2n6p1n2 mg g−" d−" in
June, remaining between k0n5 and k7n4 thereafter.
Comparison of Figure 5 a, b shows that in June–
July the rate of production of leaf dry-matter by
emersed plants at Thirlmere reached a level three
and fourfold higher than that achieved by plants
continuously submersed at Red Tarn and Esthwaite
respectively. Student’s t-tests corrected for the small
sample sizes and significantly different variances
indicate that rates of leaf dry-matter production by
emersed and submersed L. uniflora are highly
significant (P 0n001) and this is confirmed by
Figure 5.
At Thirlmere, flowering was complete by midJuly (Fig. 5 a). As there are no data on the length of
time required for fruit\seed maturation by L.
uniflora, we have assumed the 2–3 wk suggested for
some other aquatic species (Arber, 1920 ; Sculthorpe,
1967). In contrast, vegetative propagation by submersed parent plants did not reach a peak until July
or August, as parent-plant leaf production declined
(Fig. 5 b). However ramet growth, with negligible
senescence, continued into the autumn and winter.
Whilst flower and seed production involved relatively low rates of dry matter production, ramet leaf
production proceeded at rates similar to those of
parent plants (the rate of ramet leaf production
August
September
Seeds
0
20 40 60 80 100 120 140
10 30 50 70 90 110 130
Days after emersion
(b)
45
40
35
30
25
20
15
10
5
0
–5
–10
–15
–20
October
Losses
10
0
(a)
July
45
40
35
30
25
20
15
10
5
0
–5
–10
–15
–20
20
Leaf dry matter turnover (mg g–1 d. wt day –1)
16
Gains
July
June
August
May
September
November
Losses
–20 0 20 40 60 80 100 120 140 160
–30 –10 10 30 50 70 90 110 130 150 170
Days
Figure 5. (a) Rates of leaf dry matter gain and loss for
Littorella uniflora growing terrestrially at Thirlmere
Reservoir following emersion in early June. Data are for 24
plants all of which flowered. Leaves of parent plants
(——), male and female flowers and seeds (– – –). Since the
plants were not harvested, changes in leaf length were
converted to dry weight using a length to dry weight ratio
of 1 cm l 1n01, 0n92, 0n84, 0n76 and 0n76 mg for June, July,
August, September and October data points respectively.
For male flower stems (mean length 6 cm), a length to dry
weight ratio of 1 cm l 0n58 mg was used. Dry weight data
for female flowers and seeds were taken from Table 2. (b)
Rates of leaf dry-matter gain and loss for L. uniflora
growing submersed at Esthwaite Water and Red Tarn,
also in the Cumbrian Lake District, during 1986 (Robe &
Griffiths, 1992). Data are for 28 plants. At Esthwaite, 21
parent plants (
) produced a total of 39 ramets (), and
at Red Tarn 21 plants (>) produced 33 ramets (=).
Leaves of parent plants (——), ramets (– – –). Leaf length
was converted to dry weight using a length to dry weight
ratio of 1 cm l 1n6 mg and 1 cm l 1n0 mg for Esthwaite
and Red Tarn plants respectively (Robe, 1989). In (a) and
(b) each data point shows the increase or decrease in dry
weight during the preceding period of approximately four
weeks as a function of the dry weight of parent plants at the
beginning of that period, expressed as a rate per day. We
assumed that ramets were independent of the parent when
they possessed a total leaf length of 12 cm or more, and
Printed from the C JO service for personal use only by...
Littorella uniflora : adjustment to emersion
17
Table 3. Total carbon and nitrogen content (mg g−" d. wt) of Thirlmere Littorella uniflora at the three sampling
dates in 1995
Early June
Aquatic
Thirlmere
Shoots
Senesced leaves
Stems
Roots
Ramets
Flower stems
and buds
Seeds
Esthwaite
Shoots
Roots
Ramets
Red Tarn
Shoots
Roots
Ramets
Early July
Flowering
Early September
Seed-bearing
C
N
C
N
C
N
433
36n8
465
438
424
31n1
38n3
39n5
443p4n5 a
425
464p23n0
450p4n8 c
28n7p2n4 a
11n1
23n7p2n1
21n1p3n6 c
457p31n0 a
420
455p11n0
441p19n0 c
19n2p1n7 a
10n9
15n1p1n1
7n2p0n9 c
473
32n7
499
10n8
405p2n1
396p4n2
25n4p1n7
25n0p1n5
400p3n4 b
421p4n3 d
24n8p1n1 b
22n8p2n7 c
396p9n8 b
409p13n2 d
382p0n7
24n0p2n2 b
18n2p1n6 d
24n1p0n3
397p7n6
423p26n3
23n6p4n1
26n0p1n4
400p6n4 b
437p11n5 cd
22n6p4n3 b
21n3p3n4 c
388p7n5 b
420p6n5 d
394p2n1
22n9p1n2 b
22n2p2n0 d
29n4p0n5
Data are for 7–10 plants. The material collected in early June at Thirlmere was combined. Data for ramets are for
leaves and stolons. The results for July and September are shownp. Senesced leaves were brown but still attached
to the plants ; data are for leaves from approx. 30 plants combined. Data for permanently submersed plants at Esthwaite
Water and Red Tarn, collected at the same time of year during an earlier study (see text and Robe & Griffiths, 1992),
are included for comparison (n l 4p). The significance of the differences in the mean C and N content of shoots or
roots of Thirlmere, Esthwaite and Red Tarn L. uniflora was tested pairwise using t-tests corrected for the small sample
sizes and, where necessary, for significantly different variances. Means followed by a different letter are significantly
different from corresponding values in the same column at the P 0n001 level, except for shoot N and root C at
Thirlmere and Esthwaite in July, and root C at Thirlmere and Esthwaite in September (P 0n002), and shoot C at
Thirlmere and Esthwaite, and Thirlmere and Red Tarn in September (P 0n01).
reaching 9n7 mg g−" d−" at Red Tarn in July and
9n2 mg g−" d−" at Esthwaite in August).
Carbon and nitrogen content : terrestrial and
submersed L. uniflora
The C content of shoots and roots of Thirlmere L.
uniflora, which undergo regular emersion, was higher
than that of permanently submersed Esthwaite and
Red Tarn plants throughout the season (Table 3).
This was most noticeable in the case of shoot C,
which was 11–18 % higher. At Thirlmere, the C
content of male flower stems and buds, as well as of
seeds was 8–12 % higher than that of leaves and
roots, and 22 and 29 % higher than ramets of
Esthwaite and Red Tarn plants, respectively.
thereafter calculated their rate of leaf dry-matter turnover
on the basis of their own dry weight. Means are shownp
where larger than the symbol.  followed by pairwise
comparisons with the Studentized range test, showed that
at Thirlmere rates of leaf dry matter gain differed
significantly (P 0n01) between June, July, August and
September. For the Red Tarn population, parent plant
leaf dry-matter gain differed significantly (P 0n01)
between June, July, August and November, but at
Esthwaite mid-season differences in parent-plant leaf
growth were not significant.
The N content of Thirlmere plants was also
initially higher than that of the permanently submersed lake and tarn L. uniflora (Table 3). Thus in
early June, the N content of Thirlmere shoots
(36n8 mg g−" d. wt) was 46 and 50 % higher than that
of Esthwaite and Red Tarn plants, respectively.
However, by early September, the N content of
Thirlmere plants (shoots, roots and stems) had been
drastically reduced, and t-tests indicated that these
reductions were significant (P 0n001). Thus in
September, shoot N (19n2 mg g−" d. wt) was 20 %
lower than that of Esthwaite leaves, and root N
(7n2 mg g−" d. wt) was 60 % lower than that of
Esthwaite roots.
Carbon and nitrogen investment in vegetative and
reproductive biomass : terrestrial and submersed L.
uniflora
Table 4 compares the season (above-ground) C and
N balance of L. uniflora growing terrestrially at
Thirlmere and submersed at Esthwaite and Red
Tarn. The gains in dry matter due to leaf growth,
and losses due to leaf senescence at each sampling
date between late May and September (Fig. 5) have
been combined, converted to C and N content using
Printed from the C JO service for personal use only by...
18
W. E. Robe and H. Griffiths
Table 4 a. Carbon investment in, and loss from, above-ground biomass of Littorella uniflora growing emersed at
Thirlmere Reservoir, and submersed at Esthwaite Water and Red Tarn, Cumbria, between late May and
September 1995 (mg C g−" d. wt d−")
Terrestrial plants
at Thirlmere
Aquatic plants at
Esthwaite Water
Aquatic plants at
Red Tarn
Investment in new
ramets
Stolons
Leaves
Loss from
abscissed
ramet leaves
4n31p1n23
0n46p0n12
2n29p2n63
0n29p0n45
3n54p0n97
0n67p0n19
1n57p1n69
0n42p0n74
Investment in
new leaves
Loss from
abscissed
leaves
Flowers
Seeds
11n29p2n41 a
8n79p2n19
1n11p0n39
1n54p0n37
3n73p1n59 b
4n39p2n43 b
Data are for the 24 plants whose growth was monitored at Thirlmere, all of which flowered. At Esthwaite and Red
Tarn the growth of 28 plants was measured. At Esthwaite 21 plants produced 39 ramets and at Red Tarn 21 plants
produced 33 ramets. For each plant increments in leaf d. wt gain or loss at each measuring date (calculated and converted
to C and N, using the data in Figure 5 and Table 3 for Thirlmere plants, and in Robe & Griffiths (1992) for Esthwaite
and Red Tarn plants) were combined and expressed as a function of the d. wt of the shoots in May and the number of
days between May and September measurements. Investment in stolons was based on a length to d. wt relationship of
1 cm l 0n69 mg and mean stolon lengths of 6n9 cm at Esthwaite and 4n8 cm at Red Tarn. D. wt of stolons was equivalent
to 63 % and 52 % of a mature leaf of average length on Esthwaite and Red Tarn plants respectively. Means are
shownp. The significance of the differences in mean C and N investment in new leaves at the three sites was tested
using t-tests corrected for the small sample sizes (variances were not significantly different). Means followed by different
letters are significantly different (P 0n001).
Table 4 b. Nitrogen investment in, and loss from, above ground biomass of Littorella uniflora growing emersed
at Thirlmere Reservoir and submersed at Esthwaite Water and Red Tarn between May and September 1995
calculated as described in the caption to Table 4 a above (mg N g−" d. wt d−")
Terrestrial
plants at
Thirlmere
Aquatic plants
at Esthwaite
Water
Aquatic plants
at Red Tarn
Investment
in new
leaves
Loss from
abscissed
leaves
Investment in new ramets
Stolons
Leaves
Loss from
abscissed
ramet leaves
Flowers
Seeds
0n77p0n20 a
0n48p0n09
0n088p0n058
0n035p0n005
0n26p0n16 b
0n30p0n15
0n036p0n039
0n133p0n128
0n018p0n027
0n30p0n22 b
0n23p0n14
0n039p0n011
0n109p0n167
0n039p0n085
the data in Table 3, and expressed as a function of
the dry weight of parent plants in late May. The
results are expressed as a rate per day to overcome
slight differences in the interval (a few days) between
May and September measurements at the three
locations. The C and N losses calculated here are the
maximum which could occur. Although L. uniflora,
like other perennial species usually found in oligotrophic habitats, is thought to store and recycle C
and N from senescing leaves, no study has quantified
these processes in this species. Incorporation of C
and N into flowers, seeds, and ramet leaves and
stolons, have been calculated in the same way using
data in Tables 2 and 3. Figure 6 compares the cost of
flowers, seeds and ramet production in terms of C
and N invested, relative to that incorporated into
parent–plant leaves.
Between late May and September 2n5- to 3n0-fold
more C and N was invested in leaves by parent plants
at Thirlmere than at Esthwaite Water and Red Tarn
(Table 4). However, losses of C and N due to
senescence were substantial in both environments.
At Thirlmere, 78 % of C and 62 % of N assimilated
into leaf biomass was subsequently lost, while at
Esthwaite 116 % of C and 115 % of N and at Red
Tarn 81 % of C and 77 % of N were lost.
Incorporation of C and N into reproductive tissue
as flowers plus seeds at Thirlmere (2n65 mg C g−" d−"
and 0n12 mg N g−" d−"), and ramets (stolons plus
leaves) at Esthwaite (2n75 mg C g−" d−" and 0n17 mg
N g−" d−") and Red Tarn (2n24 mg C g−" d−" and
0n15 mg N g−" d−"), were relatively similar. Thus,
despite heavy losses from adult plants through leaf
turnover and senescence, end-of-season C and N
balances were positive in both aquatic and terrestrial
environments.
Printed from the C JO service for personal use only by...
Carbon investment (mg C g–1 d. wt day –1)
Littorella uniflora : adjustment to emersion
(a)
14
Seeds
12
Flowers
10
8
6
Ramets
Ramets
4
2
Nitrogen investment (mg N g–1 d. wt day –1)
0
(b)
0·90
Seeds
Flowers
0·75
0·60
0·45
Ramets
Ramets
0·30
0·15
0
Thirlmere
Esthwaite
19
L. uniflora. If ramets were importing all the C and N
required until September, vegetative reproduction
by submersed plants required a proportionally very
much larger investment of C and N by parent plants
than did flowering and seed production (see Fig. 6).
Thus at Thirlmere, allocation of C to flowers and
seeds was 23 % of that invested in parent–plant leaf
biomass ; however, at Esthwaite, ramet production
required 74 % of the C invested in parent-plant
leaves and at Red Tarn 51 %.
In Table 5 we have calculated how much initial
investment of C by parent plants at Esthwaite and
Red Tarn would be required if ramets became
completely independent earlier. At Esthwaite, independence at the stage of four small leaves, which
18 % of the ramets had reached by July and 36 % by
August, would require an allocation (stolons plus
leaves) of 1n97 mg C g−" d−" by parent plants, equivalent to 53 % of their investment in parent-plant leaf
biomass. Independence at the two leaf stage, which
31 % of the plants had reached by July, and 59 % by
June, would reduce C allocation to 1n69 mg C g−"
d−". Although this is considerably less than the
absolute investment in flowers and seeds at Thirlmere (2n65 mg C g−" d−"), it still represents 45 % of
the C investment in parent-plant leaf biomass
compared with the 23 % allocated to flowers and
seeds by emersed plants at Thirlmere. The only way
to reduce the cost of ramets further would be to
reduce stolon length.
Red Tarn
Figure 6. Investment of (a) carbon and (b) nitrogen in
leaves (8), seeds, flowers and ramets by parent plants of
Littorella uniflora at Thirlmere, Esthwaite and Red Tarn.
The data are taken from Table 4.
For these calculations we have assumed that all
ramets (including secondary and tertiary) remained
connected via stolons to and fully dependent on,
parent plants, since there are no data on the length of
connection or physiological integration of ramets of

Changes in leaf morphology and growth form
following emersion : environmental triggers
Littorella uniflora responded to emersion with a
rapid change from aquatic to terrestrial growth
forms. Submersed and emersed plants of similar
contrasting appearance have been reported in the
older literature (Gluck, 1911 ; Arber, 1920) but the
time-scale of these changes and the underlying
Table 5. Carbon investment in ramets (stolons and leaves) by submersed
Littorella uniflora at Esthwaite Water and Red Tarn, Cumbria in 1995
(mg C g−" d. wt d−")
Esthwaite
Red Tarn
Investment in ramets with
four leaves
Investment in ramets
with two leaves
Stolons
Leaves
Stolons
Leaves
0n46p0n13
0n67p0n19
1n51p1n47
1n36p1n57
0n46p0n13
0n67p0n19
1n23p0n12
0n99p1n20
Calculations were carried out as described in the caption to Table 4 a,
assuming that dependence of primary ramets on the parent plant ended at the
stage of either (a) four small leaves (total length approx. 6 cm) or (b) two small
leaves (total length approx. 3 cm). Secondary and tertiary ramets joined to the
primary ramet were also assumed to be independent of the parent once the
primary ramet had ceased to be dependent. Results are shownp.
Printed from the C JO service for personal use only by...
20
W. E. Robe and H. Griffiths
transition in leaf morphology had not been described
in detail.
This study has clearly established that L. uniflora,
like many amphibious macrophytes (Arber, 1920 ;
Sculthorpe, 1967) is heterophyllous, producing distinct aquatic and terrestrial leaves differing in size,
shape, lacunal volume and stomatal density (Figs 1,
3 ; Table 2). In L. uniflora there is no ontogenetic
sequence of leaf forms irrespective of whether the
plant is growing submersed or on land, and the
observed morphological plasticity must therefore
have been triggered by some external and\or internal
stimuli. Experimental work with other species
suggests that leaf morphology can respond to
photoperiod, temperature, light regime, CO and O
#
#
concentration, moisture conditions and mechanical
pressure, and that the response might be mediated
by plant growth substances (see, for example,
Bradshaw, 1965 ; Sculthorpe, 1967 ; Bristow, 1969 ;
Anderson, 1978 ; Bodkin et al., 1980 ; Deschamp &
Cooke, 1983 ; Goliber & Feldman, 1989 ; Maberly &
Spence, 1989 ; He, Morgan & Drew, 1996 ; Hellwege,
Dietz & Hartung, 1996 ; Trewavas & Mulho! , 1997).
For L. uniflora at Thirlmere, emersion was
accompanied by negligible changes in photoperiod
and temperature (Table 1). PPFD reaching leaf
surfaces is likely to have increased (see Table 1) but,
for plants in shallow water, light intensity is so
variable because of weather conditions (e.g. Robe &
Griffiths, 1992) that short-term increases in PPFD,
and accumulation of carbohydrate resulting from
higher rates of photosynthesis, are unlikely to be
reliable signals of emersion. The changing ratio of
red to far-red light, which stimulates the development of aerial-type leaves in emergent species
inhabiting deep, clear water (e.g. Hippuris vulgaris in
2 m ; Bodkin et al., 1980), as they grow towards the
surface, seems less likely to be a good indicator of
emersion for L. uniflora. The relatively small
absorption of far-red in the few centimetres of water
which cover the plants before emersion is often offset
by absorption of red wavelengths by organic material, other vegetation and micro-organisms
(Spence, Bartley & Child, 1987 ; Smith, 1994).
However, it does seem possible that the gradations in
leaf form (e.g. Arber, 1920) and stomatal density
observed in submersed L. uniflora in habitats with a
wide range of water depths (W. E. Robe and
H. Griffiths ; unpublished data), might be a response
to light quality.
CO concentrations in the leaf lacunae of L.
#
uniflora will not have changed in the period immediately after emersion if, as suggested by Nielsen
et al. (1991), CO continues to be taken up from the
#
sediments. However, aquatic leaves have apical
openings (hydathodes) in direct contact with the
vascular system (Hostrup & Weigleb, 1991), stomata
at the leaf tips which become functional after
emersion, and, probably, a thinner cuticle than true
terrestrial species. Their sudden exposure to an
environment of low relative humidity seems likely to
result in rapid transpirational water losses (see
Nielsen & Sand-Jensen, 1997), possibly leading to a
progressive reduction in leaf turgor. In several other
amphibious species, emersion seems to have a direct
effect on leaf form via water status, and such
responses might involve ABA (for example, Deschamp & Cooke, 1983 ; Goliber & Feldman, 1989).
In L. uniflora, deteriorating water status may also be
linked with the tendency for prostrate growth seen at
the early September sampling.
Changes in growth rate following emersion :
underlying causes
It was not possible to compare the effect of emersion
on seasonal growth rate and C and N investment in
reproduction of L. uniflora at Thirlmere Reservoir,
since water abstraction was so rapid that no submersed plants remained after mid-June. We have
therefore used data for permanently submersed
plants growing at two submersed sites, oligotrophic
Red Tarn and eutrophic Esthwaite Water, collected
in an earlier study (it was not practical to study
emersion at these sites because of the unpredictability of fluctuations in water level). Such a comparison of plants at different sites must be interpreted
with caution since factors other than emersion may
be having an effect.
The increase in growth rate immediately following
emersion, to a level three to fourfold higher than that
in permanently submersed plants (Fig. 5 a), could
have several possible causes. Improved CO supply
#
could be a factor if photosynthesis of aquatic L.
uniflora is still limited by resistances to CO transport
#
in the unstirred boundary layer around the roots, in
the root wall, or within the lacunal system or
mesophyll (see Sand-Jensen et al., 1982 ; Richardson
et al., 1984 ; Raven et al., 1985, Farmer, Maberly &
Bowes, 1986 ; Raven et al., 1988 ; Beer et al., 1991 ;
Madsen & Sand-Jensen, 1991 ; Schuette, Klug &
Klomparens, 1994), and if atmospheric CO replaced
#
the sediment supply, as suggested by the reduced
lacunal volume and high stomatal density of terrestrial leaves. However, the lacunal system in roots and
stems, and a reduced lacunal system in leaves, was
retained by plants which had been growing emersed
for several months at Thirlmere (and for two years at
another drained reservoir ; W. E. Robe ; unpublished
observations). This could have enabled CO from
#
the sediments to continue to diffuse into leaves, not
only those of emergent plants close to the water’s
edge with roots still submersed (Nielsen et al., 1991),
but also those of L. uniflora growing for long periods
on dry land, if CO concentrations in terrestrial
#
sediments remained high (see Russell, 1973 ; Rowell,
1994). Perhaps continuity of gas exchange with the
sediments is important in enabling emersed L.
Printed from the C JO service for personal use only by...
Littorella uniflora : adjustment to emersion
uniflora to adjust rapidly to unpredictable resubmersion.
Alternatively, supplies of mineral nutrients might
have increased as sediments dried and became
aerobic, although the similarity in performance of
submersed plants at Esthwaite and Red Tarn, sites
of greatly contrasting nutrient status (Robe &
Griffiths, 1992), and the lack of response of L.
uniflora to nitrogen additions under controlled
conditions (Robe & Griffiths, 1994) suggest that this
may not have been a major factor. Another possibility
is that the overall higher PPFD in the terrestrial
environment (Table 1 ; Table 2, Robe & Griffiths,
1990) might have contributed to higher rates of
photosynthesis, although CO uptake by aquatic L.
#
uniflora responds very little to increased light
intensity (Robe & Griffiths, 1990). However, in July,
terrestrial leaves showed an eightfold higher activity
of Ribulose bisphosphate carboxylase-oxygenase
(Rubisco) than aquatic leaves sampled in June, and
no CAM (W. E. Robe & H. Griffiths, unpublished
results). Greater Rubisco activity in emergent than
in submersed leaves has also been found in other
amphibious species (Maberly & Spence, 1989). Thus
it seems very likely that changes in photosynthetic
biochemistry, triggered by emersion and resulting in
higher rates of carbon fixation, were a major factor in
the rapid new leaf growth which preceded flowering.
The abrupt decline in the growth of emersed L.
uniflora between July and August might have been
partly a seasonal phenomenon, related to day length,
since growth of permanently submersed plants also
showed a July maximum (Fig. 5 b). However, water
stress can cause a marked reduction in the growth of
true terrestrial species (Smith & Griffiths, 1993), and
although the sediments dried out gradually at
Thirlmere the water potential which develops is
dependent on sediment type (see Meidner & Sherrif,
1976) and maintenance of leaf turgor will depend on
the capacity for osmotic adjustment.
Reproduction : costs and benefits, and induction of
flowering
At Esthwaite and Red Tarn, submersed plants
produced 1n8 and 1n6 ramets respectively between
May and September, a number similar to that found
in other habitats (Boston & Adams, 1987). This level
of vegetative reproduction in the aquatic environment seems to be considerably more expensive in
terms of C and N investment by parent plants than
is terrestrial flowering and seed set. To be more
precise about the relative costs, more information is
needed on the extent of physiological integration
between parent and connected ramets, stolon length
and branching pattern, and the stage at which ramets
become independent under water. However, although possibly costly, vegetative reproduction has
21
many potential advantages for a species in a heterogeneous, disturbed environment such as that
inhabited by L. uniflora. These include rapid local
colonization and invasion, ease of space and resource
capture, active habitat selection, exclusion of competitors and persistence during adversity (Pitelka &
Ashmun, 1985 ; Silander, 1985). That ramet production is very cost-effective for L. uniflora is
suggested by the extensive, dense mats of plants
often seen at lake margins (Farmer & Spence, 1986).
Information on the size of clones and their life span
would enable greater understanding of the evolutionary implications.
At Thirlmere, emersion resulted in the rapid
induction of flowering. Many factors, both environmental and internal, can interact to affect flower
initiation in plants. Although day length is the most
common controlling factor, sensitivity to day length
can be altered by light intensity, temperature, root
removal, presence of inhibitors, and application of
growth substances (Bernier, 1988 ; Vince-Prue,
1994). It seems possible that in L. uniflora a
requirement for emersion has been superimposed on
a requirement for long days (associated with summer
flowering) in the terrestrial ancestor, and that the
same stimulus which triggers the development of
terrestrial leaves (with their higher Rubisco activities) also initiates flowering.
Flowering L. uniflora produced on average 8n5
relatively large seeds per plant, a very low level of
seed production ; some aquatic macrophytes produce
thousands per plant (Sculthorpe, 1967). Furthermore, in L. uniflora, seed production is very
unpredictable. Although at Thirlmere most plants
flower almost every year, in many habitats flowering
is much less frequent and confined to a small
percentage of plants during a short period of
emersion. However, it seems that even these low
levels of sexual reproduction and recombination can
be sufficient to maintain genetic variation and
evolutionary potential (Silander, 1985). That this is
the case for L. uniflora is suggested by the wide range
of habitats occupied (see Pearsall, 1920 ; Spence,
1964 ; Robe, 1989).
Comparison with L. uniflora at Lake Hampen,
Denmark
In contrast with our findings, Nielsen & SandJensen (1997) found little difference in the appearance or growth rate of L. uniflora in 30 cm of
clear water and 40 cm above the water level at Lake
Hampen, Denmark. They suggested that, in comparison with other amphibious species, L. uniflora
shows only slight heterophylly and no growth
stimulation on emersion because the CO environ#
ment of the plants is unchanged (owing to continued
uptake from the sediments) and there are only
small differences in light intensity and temperature
Printed from the C JO service for personal use only by...
22
W. E. Robe and H. Griffiths
between shallow water and air. However, our study
clearly confirms (Figs 1, 3 ; Table 2) observations in
the earlier literature (Gluck, 1911 ; Arber, 1920) that,
in response to emersion, L. uniflora growing undisturbed in situ can be distinctly heterophyllous,
and establishes that changes in leaf form and growth
rate occur rapidly and precede flowering. Furthermore, these changes occur despite the likely continued use of sediment CO and small differences in
#
light intensity, and temperature (Table 1). At Lake
Hampen, leaves of submersed and emersed plants
were very small, but in other respects, including N
content, seemed to be intermediate between the
aquatic and terrestrial types found at Thirlmere.
However, the plants did not appear to flower. It
seems possible that at Thirlmere, where the spring\
early summer fall in water level was large and very
rapid, L. uniflora displayed maximum phenotypic
potential for acclimation from deep-water aquatic
conditions to dry-land, terrestrial conditions, whereas at Lake Hampen, where the plants remained close
to the water’s edge on gravel sediments, correspondingly small phenotypic changes took place. At
Lake Hampen, the stimulus or stimuli necessary to
trigger the co-ordinated changes in morphology,
growth, photosynthetic pathway and reproduction
seen at Thirlmere were perhaps absent or not
effective, possibly because of the stressed condition
of the plants. Alternatively, populations from different sites might differ in their capacity for
plasticity. Clearly, there is a need to identify the
external or internal factors which regulate phenotypic plasticity and flowering in L. uniflora.
Summary and conclusion
In conclusion, the success of this amphibious species
can be accounted for by a combination of continuous
vegetative reproduction when submersed, and a high
degree of phenotypic plasticity in response to
emersion, which allows some flower and seed
production to occur during often brief periods in the
terrestrial environment. The physiological and biochemical process underlying the changes in growth
form, growth rate, and C and N investment which
followed emersion are currently under investigation.
              
We are grateful to the Natural Environment Research
Council for Research Grant GR3\8930, and to North
West Water who kindly gave permission for us to work at
Thirlmere Reservoir. We would also like to thank Gordon
Beakes of the Department of Biological and Nutritional
Sciences, University of Newcastle upon Tyne, for allowing
us to use his microscope and photographic facilities, and
Chris Quarmby and Darren Sleep of the Institute of
Terrestrial Ecology, Merlewood Research Station,
Cumbria, for carrying out the total C and N analysis.

Anderson LWJ. 1978. Abscisic acid induces formation of floating
leaves in the heterophyllous aquatic angiosperm Potamogeon
nodosus. Science 201 : 1135–1138.
Arber A. 1920. Water plants. Cambridge, UK : Cambridge
University Press.
Aulio K. 1986. CAM-like photosynthesis in Littorella uniflora
(L.) Aschers. : the role of humidity. Annals of Botany 58 :
273–275.
Beer S, Sand-Jensen K, Madsen TV, Nielsen SL. 1991. The
carboxylase activity of Rubisco and the photosynthetic performance in aquatic plants. Oecologia 87 : 429–434.
Bernier G. 1988. The control of floral evocation and morphogenesis. Annual Review of Plant Physiology and Plant
Molecular Biology 39 : 175–219.
Bodkin PC, Spence DHN, Weeks DC. 1980. Photoreversible
control of hetetophylly in Hippuris vulgaris L. New Phytologist
84 : 533–542.
Boston HL, Adams MS. 1987. Productivity, growth and
photosynthesis of two small ‘ isoetid ’ plants, Littorella uniflora
and Isoetes macrospora. Journal of Ecology 75 : 333–350.
Boston HL, Adams MS, Pienkowski TP. 1987. Utilization of
sediment CO by selected North American isoetids. Annals of
#
Botany 60 : 485–494.
Bowes G, Salvucci ME. 1989. Plasticity in the photosynthetic
carbon metabolism of submersed aquatic macrophytes. Aquatic
Botany 34 : 233–266.
Bradshaw AD. 1965. Evolutionary significance of phenotypic
plasticity in plants. Advances in Genetics 13 : 115–155.
Bristow JM. 1969. The effects of carbon dioxide on the growth
and development of amphibious plants. Canadian Journal of
Botany 47 : 1803–1807.
Christensen PB, Revsbech NP, Sand-Jensen K. 1994. Microsensor analysis of oxygen in the rhizosphere of the aquatic
macrophyte Littorella uniflora (L.) Ascherson. Plant Physiology
105 : 847–852.
Clapham AR, Tutin TG, Warburg GF. 1981. Excursion Flora of
the British Isles. Cambridge, UK : Cambridge University Press.
Cook CDK. 1969. On the determination of leaf form in Ranunculus
aquatilis. New Phytologist 68 : 469–480.
Deschamp PA, Cooke TJ. 1983. Leaf dimorphism in aquatic
angiosperms : significance of turgor pressure and cell expansion.
Science 219 : 505–507.
Farmer AM, Maberly SC, Bowes G. 1986. Activities of
carboxylation enzymes in freshwater macrophytes. Journal of
Experimental Botany 37 : 1568–1573.
Farmer AM, Spence DHN. 1986. The growth strategies and
distribution of isoetids in Scottish freshwater lochs. Aquatic
Botany 26 : 247–258.
Gluck H. 1911. Biologische und morphologische Untersuchungen u$ ber Wasser- und Sumpfgewachse. III. Die Uferflora. Jena.
Goliber TE, Feldman LJ. 1989. Osmotic stress, endogenous
abscisic acid and the control of leaf morphology in Hippuris
vulgaris L. Plant, Cell and Environment 12 : 163–171.
Groenhof AC, Smirnoff N, Bryant JA. 1988. Enzymic activities
associated with the ability of aerial and submerged forms of
Littorella uniflora (L.) Aschers. to perform CAM. Journal of
Experimental Botany 29 : 353–361.
He C-J, Morgan W, Drew MC. 1996. Tranasduction of an
ethylene signal is required for cell death and lysis in the root
cortex of maize during aerenchyma formation induced by
hypoxia. Plant Physiology 112 : 463–472.
Hellwege EM, Dietz K-J, Hartung W. 1996. Abscisic acid
causes changes in gene expression involved in the induction of
and land form of the liverwort Riccia fluitans L. Planta 198 :
423–432.
Hostrup O, Wiegleb G. 1991. Anatomy of leaves of submerged
and emergent forms of Littorella uniflora (L.) Oecologia 68 :
615–622.
Keeley JE. 1996. Aquatic plant photosynthesis. In : Winter K,
Smith JAC, eds. Crassulacean Acid Metabolism Berlin, Germany : Springer-Verlag.
Larcher W. 1983. Physiological plant ecology. London, UK :
Longman.
Maberly SC, Spence DHN. 1989. Photosynthesis and photorespiration in freshwater organisms : amphibious plants.
Aquatic Botany 34 : 267–286.
Printed from the C JO service for personal use only by...
Littorella uniflora : adjustment to emersion
Madsen TV. 1987 a. Interaction between internal and external
CO pools in the photosynthesis of the aquatic CAM plants
#
Littorella uniflora (L.) Aschers and Isoetes lacustris L. New
Phytologist 106 : 35–50.
Madsen TV. 1987 b. Sources of inorganic carbon acquired
through CAM in Littorella uniflora (L.) Aschers. Journal of
Experimental Botany 38 : 367–377.
Madsen TV, Breinholt M. 1995. Effects of air contact on
growth, inorganic carbon sources and nitrogen uptake by an
amphibious freshwater macrophyte. Plant Physiology 107 :
149–154.
Madsen TV, Sand-Jensen K. 1991. Photosynthetic carbon
assimilation in aquatic macrophytes. Aquatic Botany 41 : 5–40.
Meidner H, Sheriff DW. 1976. Water and plants. London, UK :
Blackie.
Milburn JA. 1979. Water flow in plants. London, UK : Longman.
Misra RD. 1938. Edaphic factors in the distribution of aquatic
plants in the English Lakes. Journal of Ecology 26 : 41–51.
Nielsen SL. 1993. A comparison of aerial and submerged
photosynthesis in some Danish amphibious plants. Aquatic
Botany 45 : 27–40.
Nielsen SL, Gacia E, Sand-Jensen K. 1991. Land plants of
amphibious Littorella uniflora (L.) Aschers. maintain utilization
of CO from the sediment. Oecologia 88 : 258–262.
#
Nielsen SL, Sand-Jensen K. 1997. Growth rates and morphological adaptations of aquatic and terrestrial forms of
amphibious Littorella uniflora (L.) Aschers. Plant Ecology 129 :
135–140.
Parker RE. 1979. Introductory statistics for biology. London, UK :
Edward Arnold.
Pearsall WH. 1920. The aquatic vegetation of the English Lakes.
Journal of Ecology 8 : 163–199.
Pedersen O, Sand-Jensen K. 1992. Adaptations of submerged
Lobelia dortmanna to aerial life form : morphology, carbon
sources and oxygen dynamics. Oikos 65 : 89–96.
Pedersen O, Sand-Jensen K. 1997. Transpiration does not
control growth and nutrient supply in the amphibious Mentha
aquatica. Plant, Cell and Environment 20 : 117–123.
Pitelka LF, Ashmun JW. 1985. Physiology and integration of
ramets in clonal plants. In : Jackson JBC, Buss LW, Cook RE,
eds. Population Biology and Evolution of Clonal Organisms. New
Haven, CT, USA & London, UK : Yale University Press.
Raven JA. 1984. Energetics and transport in aquatic plants. MBL
Lectures in Biology, Vol. 4. New York, USA : Alan R. Liss.
Raven JA, Handley LL, MacFarlane JJ, McInroy J, McKenzie L, Richards JHH, Samuelsson G. 1988. The role of
CO uptake by roots and CAM in acquisition of inorganic C by
#
plants of the isoetid life form : a review, with new data on
Eriocaulon decangulare L. New Phytologist 108 : 125–148.
Raven JA, Osborne BA, Johnston AM. 1985. Uptake of CO by
#
aquatic vegetation. Plant, Cell and Environment 8 : 417–425.
Richardson K, Griffiths H, Reed ML, Raven JA, Griffiths
NM. 1984. Inorganic carbon assimilation in the isoetids, Isoetes
lacustris L. and Lobelia dortmanna L. Oecologia 61, 115–121.
Robe WE. 1989. Ecophysiology of Littorella uniflora : interactions
between inorganic carbon supply and C and CAM photosynthetic
$
characteristics. Ph.D thesis, University of Newcastle upon
Tyne, UK.
Robe WE, Griffiths H. 1988. C and CAM photosynthetic
$
characteristics of the submerged aquatic macrophyte Littorella
uniflora : regulation of leaf internal CO supply in response to
#
variation in rooting substrate inorganic carbon concentration.
Journal of Experimental Botany 39 : 1897–1410.
23
Robe WE, Griffiths H. 1990. Photosynthesis of Littorella uniflora
grown under two PAR regimes : C and CAM gas exchange and
$
the regulation of internal CO and O concentrations. Oecologia
#
#
85 : 128–136.
Robe WE, Griffiths H. 1992. Seasonal variation in the ecophysiology of Littorella uniflora (L.) Ascherson in acidic and
eutrophic habitats. New Phytologist 120 : 289–304.
Robe WE, Griffiths H. 1994. The impact of NO − loading on the
$
freshwater macrophyte Littorella uniflora : N utilization strategy
in a slow-growing species from oligotrophic habitats. Oecologia
100 : 368–378.
Roelofs JGM. 1983. Impact of acidification and eutrophication
on macrophyte communities in soft waters in The Netherlands.
1. Field Observations. Aquatic Botany 17 : 139–155.
Rowell DL. 1994. Soil science : methods and applications. London,
UK : Longman.
Russell EW. 1973. Soil conditions and plant growth. London, UK :
Longman.
Sand-Jensen K, Prahl C, Stokholm H. 1982. Oxygen release
from roots of submerged aquatic macrophytes. Oikos 38 :
349–354.
Sand-Jensen K, Sondergaard M. 1978. Growth and production
of isoetids in oligotrophic Lake Kalgaard, Denmark. Verhandlungen Internationale Vereinigung fur Theoretische und Angewandte Limnologie 20 : 659–666.
Schuette JL, Klug MJ, Klomparens KL. 1994. Influence of
stem lacunar structure on gas transport : relation to the oxygen
transport potential of submersed vascular plants. Plant, Cell
and Environment 17 : 355–365.
Schuurkes JAAR, Kok CJ, Den Hartog C. 1986. Ammonium
and nitrate uptake by aquatic plants from poorly buffered and
acidified waters. Aquatic Botany 24 : 131–146.
Sculthorpe CD. 1967. The biology of aquatic vascular plants.
London, UK : Edward Arnold.
Silander JJ. 1985. Microevolution in clonal plants. In Jackson
JBC, Buss LW, Cook RE, eds. Population Biology and Evolution
of Clonal Organisms. New Haven, CT, USA & London, UK :
Yale University Press.
Smith H. 1994. Sensing the light environment. In : Kendrick RE,
Kronenberg GHM, eds. Photomorphogenesis in Plants. Dordrecht, The Netherlands, Boston, MA, USA, London, UK :
Kluwer Academic Publishers.
Smith JAC, Griffiths H. 1993. Water deficits. Oxford, UK : Bios
Scientific Publishers.
Spence DHN. 1964. The macrophyte vegetation of freshwater
lochs, swamps and associated fens. In : Burnett JH, ed. The
Vegetation of Scotland. London, UK : Oliver & Boyd.
Spence DHN. 1981. The zonation of freshwater plants. Advances
in Ecological Research 12 : 37–125.
Spence DHN, Bartley MR, Child R. 1987. Photomorphogenic
processes in freshwater angiosperms. In : Crawford RMM, ed.
Plant Life in Aquatic and Amphibious Habitats. London, UK :
Blackwell Scientific Publications.
Steer MW. 1981. Understanding cell structure, Cambridge, UK :
Cambridge University Press.
Trewavas AJ, Mulho! R. 1997. Signal perception and transduction : the origin of the phenotype. The Plant Cell 9 :
1181–1195.
Vince-Prue D. 1994. The duration of light and photoperiodic
responses. In : Kendrick RE, Kronenberg GHM, eds. Photomorphogenesis in Plants. Dordrecht, The Netherlands, Boston,
MA, USA, London, UK : Kluwer Academic Publishers.
Willmer CM. 1983. Stomata. London, UK : Longman.
Printed from the C JO service for personal use only by...