Austral Ecology (2009) 34, 780–792

Morphological variation and floral abnormalities in a

trigger plant across a narrow altitudinal gradient aec_1984 780..792

ARY A. HOFFMANN,* PHILIPPA C. GRIFFIN AND ROGER D. MACRAILD

Centre for Environmental Stress and Adaptation Research (CESAR), Bio21 Molecular Science
Institute, Department of Genetics, The University of Melbourne, Parkville,Victoria 3010, Australia
(Email: ary@unimelb.edu.au)

Abstract Local adaptation in alpine plants has been demonstrated across wide altitudinal gradients, but has
rarely been examined across the alpine-to-montane transition that often encompasses only a few hundred metres.
Here we characterize morphological variation in leaf and floral characteristics of the trigger plant Stylidium armeria
along a narrow altitudinal gradient in the Bogong High Plains in Victoria. Across this gradient, which encompasses
the high-elevation limit of this species, linear changes were found for floral scape height, leaf length and flower
number. All these traits decreased with increasing altitude, whereas the frequency of abnormal flowers increased.
When plants were grown in a common garden environment, an altitudinal pattern for flower abnormalities was no
longer detected. However, altitudinal patterns for leaf length and scape height were maintained, albeit weaker than
in the field. This indicates heritable variation for these morphological traits; the altitudinal patterns are likely to
reflect the effects of selection by environmental factors that vary with altitude. Selection pressures remain to be
identified but have generated both cogradient and countergradient patterns of variation.

Key words: adaptation, altitude, cline, floral abnormality, Stylidium.

INTRODUCTION Australia’s alpine and subalpine regions cover only

Morphological, physiological and genetic variation has
been described in many plant species across altitudinal
gradients encompassing tens to hundreds of kilome-
tres, beginning with the classic studies of Clausen,
Turesson and others (e.g. Turesson 1922; Clausen
et al. 1941). Sometimes this variation is mostly geneti-
cally based, but in other cases environment plays
the main role (Cordell et al. 1998; Hassel et al. 2005).
There is much less information about adaptation
across narrow altitudinal gradients of just a few kilo-
metres in length even though many alpine plants are
only found in a narrow range of altitudes (Byars et al.
2007; Gimenez-Benavides et al. 2007). In alpine envi-
ronments, small changes in altitude can correspond to
large shifts in temperature, humidity, exposure and
other abiotic variables (Korner 1999; Hovenden &
Vander Schoor 2004). This produces sharp changes
in selection pressures across short distances, and the
potential for local adaptation to altitude over small
scales. Understanding this level of adaptation is impor-
tant particularly for species that occupy a narrow alti-
tudinal range. Such species are likely to be especially
prone to extinction if they are unable to adapt to rapid
environmental changes.
*Corresponding author.
Accepted for publication October 2008.
© 2009 The Authors
Journal compilation © 2009 Ecological Society of Australia
0.15% of the country, and true alpine ecosystems
are only restricted to a maximum altitudinal range
of around 300 m (Costin et al. 2000). The regions
support a high level of biodiversity (McDougall 2003),
which is threatened by warming and precipitation
changes resulting from global climate warming
(Williams et al. 2006). Climate changes will theoreti-
cally allow invasion of plants and animals from lower
elevations (Jump & Penuelas 2005). Subalpine wood-
land species have already been documented encroach-
ing on alpine herbfields in Australia and elsewhere
(Grabherr et al. 1994; Lloyd & Fastie 2003). The
persistence of many alpine species may depend on
whether they possess genetic variation for adaptive
traits, indicated by locally adapted ecotypes, unless
they possess a high level of plasticity that allows them
to track environmental changes.
Adaptive differentiation in plants can be evaluated
through common garden experiments or transplants.
Classic experiments with these techniques on grasses
and other plants growing on mine tailings were among
the first to demonstrate local adaptation to environ-
ments across only a few hundred metres (Macnair
1987). These types of studies suggest that in some
cases strong selection can maintain genetic differences
among nearby populations despite gene flow (Linhart
& Grant 1996). In other cases, however, gene flow
between adjacent populations appears strong enough
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 M O R P H O L O G I C A L VA R I AT I O N A N D F L O R A L A B N O R M A L I T I E S
to prevent local adaptation unless sites are separated
by several kilometres (Dawson et al. 2007). In the
Bogong High Plains of Victoria, both transplants and
common garden designs have been used to demon-
strate local genetic differences in the alpine grass Poa
hiemata (Byars et al. 2007). This species showed a
pattern typically seen in alpine plants (Clausen et al.
1941; Conover & Schultz 1995; Angert & Schemske
2005) – a decrease in plant width but an increase in
leaf length at higher elevations – over a gradient span-
ning only 1 km in length and 200 m in elevation.These
differences in P. hiemata had a large heritable compo-
nent and contributed to survival differences between
plants from various locations along the gradient.
In this study, we describe altitudinal variation in
growth form of the trigger plant, Stylidium armeria
(Labill.) Labill. Trigger plants are among the most
striking flowering plants of alpine meadows because
they develop long flowering scapes. Stylidium armeria
on the Bogong High Plains, Victoria, occurs up to
about 1800 m above sea level. At this elevation, plants
grow in alpine meadows, while at lower elevations
plants are found among alpine shrubs and among
snowgums (Eucalyptus pauciflora subsp. niphophila)
below the treeline. The inclusion of upper-limit S.
armeria populations in this study was of particular
interest because marginal plant populations often have
higher levels of floral variability than do mid-range
populations (Siikamaki & Lammi 1998).
We address several questions. What morphological
patterns occur in S. armeria across the alpine and
subalpine zone? Is there any evidence for an increase
in morphological variability or abnormalities in high-
altitude populations? Do any of these patterns have
a genetic basis? We use common garden experiments
to test whether patterns in morphological traits
have a genetic basis or whether they are environmen-
tally determined. The findings are compared with
those for other alpine species and interpreted in
the context of conserving genetic resources more
generally.
METHODS
Taxonomy and sites
Raulings and Ladiges (2001) identified two species of
Stylidium in Australian alpine areas, distinguished on
the basis of scape length, flower number and other
characteristics. Stylidium armeria (as described by
Raulings & Ladiges 2001) is widespread throughout
south-eastern Australia and morphologically variable,
and distinguishable from Stylidium montanum that is
restricted to peaty bogs. However, Jackson and Wilt-
shire (2001), focusing on Tasmanian material, applied
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Journal compilation © 2009 Ecological Society of Australia
781
the name S. armeria to a tetraploid species (2n = 60)
with wide spathulate leaves and a strictly littoral
habitat along the rough water coasts of Tasmania.
Because S. armeria from alpine Victoria is diploid
(2n = 30) (Griffin and Holmes, unpub. data 2008), we
have followed the description and criteria in Raulings
and Ladiges (2001). In S. armeria and other members
of the genus Stylidium, the number of petals per flower
is canalized at 5. This comprises four large petals
arranged in pairs and a fifth petal modified to a very
small labellum with a specialized function (Laurent
et al. 1998). Deviations from four large petals are occa-
sionally found in abnormal Stylidium flowers and are
canalized in related species; for instance Oreostylidium
subulatum, a New Zealand species closely related to
Stylidium, has five full-sized petals (Raulings 2000).
Plants were sampled from two south-easterly slopes
on the Bogong High Plains, Alpine National Park,
Victoria, Australia. The Bogong High Plains cover
a high-elevation area of approximately 120 km2
(McDougall 1982) and are part of the Eastern High-
lands (or Great Dividing Range) of Australia. The
alpine and subalpine areas of the Bogong High
Plains are home to approximately 200 plant species
(McDougall 1982). Grasses (Poaceae), sedges (Cyper-
aceae) and daisies (Asteraceae) make up about 40% of
this flora. Most of this region’s geological units are of
Palaeozoic age (Rosengren 2004), typically based on
the Omeo metamorphic complex comprising schist
and gneiss, slate and quartzite (McDougall 1982;
Rosengren 2004). As a result of their age and extensive
weathering over geologically stable periods, Australian
mountains tend to be flat-topped, gently sloped and of
relatively low elevation (Costin et al. 2000).
Mount Nelse has four peaks; in this study, the
southern peak (‘Mount Nelse’) and the northern
peak (‘New Country Spur’) were chosen for altitudi-
nal transects (Fig. 1). The transect on Mount Nelse
ran from 36°50′51″S, 147°20′31″E to 36°51′03″S,
147°21′29″E. The transect on New Country Spur
ran from 36°49′30″S, 147°20′39″E to 36°49′50″S,
147°21′22″E. Transects ranged from the S. armeria
upper distribution limit down to a natural barrier.
The barrier on Nelse was a watercourse, and there
were 20 sites in total. On New Country Spur the
barrier was a cliff and there were 18 sites. The lowest
sites of both transects occurred at the border of the
subalpine and montane ecosystems. In the growing
season (October–March), mean temperatures are
consistently at least 1°C higher at the lowest sites
than at high-altitude sites (Byars et al. 2007). In this
period, the mean daily min/max temperatures (per
month) range from 1.3/8.8°C (October) to 8.4/
17.4°C (January), with the absolute min/max tem-
peratures recorded measuring -7/27.7°C (Bureau of
Meteorology, http://www.bom.gov.au data from Falls
Creek Weather Station, 1990–2008).
doi:10.1111/j.1442-9993.2009.01984.x
782 A . A . H O F F M A N N ET AL.
Fig. 1. Location of transects (dotted lines) used in this study in the Bogong High Plains in Victoria, Australia.
Field measurements
Measurements were initially undertaken in mid-
February 2004, during the peak flowering season of S.
armeria. Sites were spaced along each transect every
25 m for the top 100 m, every 50 m for the next
100 m, then every 100 m to the bottom of the transect.
The close spacing at the top of both transects was
included to increase the probability of detecting mor-
phological changes that might be associated with mar-
ginality of the populations. At each site, 10 S. armeria
clumps were measured. As S. armeria is rhizomatous,
where there was no ground visible between leaf
rosettes, joined rosettes were considered to be one
clump. Points for sampling were randomly selected
within a 10 m ¥ 10 m area. Where less than 10 plants
were present, all plants were measured. Plant measure-
ments recorded for each of the clumps were: the width
of the clump at its widest point from leaf tip to leaf tip;
the length and width of the largest leaf; the number of
scapes per clump; the height, width and number of
flowers of the tallest scape; and flower abnormalities
(deviations from four large petals).The largest leaf was
determined by eye by comparing the leaves extending
side by side with other leaves coming from the same
rosette. Scape length measurements were made with a
doi:10.1111/j.1442-9993.2009.01984.x
Journal compilation © 2009 Ecological Society of Australia
tape (to the nearest mm), leaf length was measured
with a ruler, while scape width and leaf width were
measured with Vernier digital calipers (to the nearest
0.1 mm).
Field sampling was repeated in 2008 in mid-March
when flowering was coming to an end. In this set of
measurements, both transects were revisited and only
the number of abnormal flowers was scored (as a per-
centage of the total number of flowers visible in an
inflorescence). For the Mount Nelse transect, plant
clumps (5–10 per site) were scored every 5–10 m of
elevation randomly within 5 m ¥ 5 m areas. For the
New Country Spur transect, flowers from plant
clumps were only scored every 20–30 m of elevation
within 10 m ¥ 10 m areas because of limited time.
Glasshouse culture
Seed was collected from more than five plants in
March 2005 from each elevation sampled in 2004 and
planted in seed trays with a mix of five parts medium-
grade pinebark, five parts fine pinebark, one part
coarse sand and one part sieved peat. Additives
(per m3) were 1500 g Saturaid (Debco, Tyabb, VIC,
Australia) and 750 g dolomite. Seed trays were
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 M O R P H O L O G I C A L VA R I AT I O N A N D F L O R A L A B N O R M A L I T I E S
covered with vermiculite and placed in a glasshouse at
the University of Melbourne. Watering in the glass-
house was automatic and was adjusted to water to
container capacity once a day or less often, depending
on climatic conditions. After germination in May
2005, plants were transferred individually to 8-cell
seed raising trays, with one plant grown in each cell.
Once seedlings had reached 2 cm in height, they were
transplanted to 80-mm pots with a soil mix involving a
ratio of four parts ground medium-grade pinebark to
one part coarse mined sand, with several additives:
4000 g Debco Green Jacket No.2 (N : P : K of
16.5:14.1:9.6), 1500 g Saturaid and 1000 g dolomite
(all per m3). The air-filled porosity of this mix was
24%, similar to alpine soils. Seedlings and mature
plants were always arranged randomly in the glass-
house and each plant was therefore treated as a sepa-
rate data point for assessing site and transect effects.
Plants were grown under 70% shadecloth outdoors
until they reached maturity. For scoring morphology
and flowering, plants were labelled with a randomly
assigned number, which enabled blind scoring of flow-
ering and morphology.
Flowering was observed at least once every 2 weeks
between September 13 and December 20, 2007,
which encompassed the entire first flowering season.
Temperatures in this period ranged from 5.7°C to
36.9°C, with the average daily min/max temperatures
13.0/22.9°C (Bureau of Meteorology, http://www.
bom.gov.au data from Melbourne Weather Station,
2007). The date of each observation was recorded and
converted to an integer representing the day after Sep-
tember 13. At each observation, the following flower
types were counted on each scape of each plant: open
flowers (with petals expanded enough to count them),
dead flowers (petals withered), new (previously unob-
served) flowers and ‘abnormal’ flowers (with large
petal number other than 4). For new abnormal
flowers, the petal number and position on the stem
(counted up from the lowest flower, which was usually
the first to open and numbered ‘one’) were also noted.
At the end of the flowering season, the length and
diameter of each fully extended scape, and length and
width of the longest leaf (as selected by eye) on each
plant were measured as described above. Measure-
ment repeatability (R2 from correlation analysis) based
on 30 individuals was high (scape height, 0.999; leaf
length, 0.994; leaf width, 0.972; scape width, 0.95).
Analysis
The incidence of floral abnormalities was expressed as
a percentage of the total number of flowers in an
inflorescence to correct for any changes in the total
number of flowers with altitude. For the 2004 (Nelse
and New Country Spur) and 2008 (Nelse) field data,
© 2009 The Authors
Journal compilation © 2009 Ecological Society of Australia
783
the mean incidence of abnormalities at a site was
tested against altitude using non-parametric correla-
tions because the data were not normally distributed.
We combined all abnormalities in this analysis,
although the 5-petal abnormality predominated. For
the 2008 data from New Country Spur and the glass-
house data, parametric correlations were computed to
investigate the association between altitude and abnor-
mality incidence because these data were normally
distributed. We also undertook logistic regressions on
these datasets where the number of abnormal flowers
was weighted by the total number of flowers scored for
plants derived from that site. We tested the effects of
altitude on the incidence of abnormal flowers at a site
with a logit model, where number of abnormal flowers
as a proportion of the total number of flowers scored
was the summed binary response variable and eleva-
tion was the explanatory variable.
Almost all flowers on a plant were scored for abnor-
malities in the glasshouse experiment, so there was an
opportunity to link the position of flowers on an inflo-
rescence with the presence of abnormalities. We first
scored whether abnormalities occurred randomly
along the length of an inflorescence by dividing the
inflorescence in quarters from bottom to top and car-
rying out a chi-squared test with the expectation of an
equal number of abnormal flowers in each quarter. We
then computed whether the two most frequent abnor-
malities (three and five petals) occurred in different
positions along the inflorescence, using plants that
produced only one type of abnormality. Mean position
along a scape was treated as the dependent variable in
an anova, and type of abnormality as a factor. In the
few (11) plants that produced both types of abnor-
malities, the position of the 3- and 5-petal flowers
along a scape was treated as the dependent variable
and compared between the abnormality types using
paired t tests.
To assess the effects of transect and altitude of origin
on morphological traits in the field, the morphological
traits were treated as dependent variables, transect was
treated as a fixed factor and altitude as a covariate
in analyses of covariance (ancovas). The interaction
between transect and altitude indicated whether
altitudinal patterns were the same across the two
transects. These analyses were run on average values
for each site (rather than individual plants) because
plants at each site shared the same environment. We
also undertook linear regressions to determine predic-
tive equations for changes in plant morphological
traits with altitude in each transect. Polynomial terms
for altitude were initially included in these regression
equations, but polynomial terms were never significant
and therefore excluded.
In the common garden experiments, ancovas were
also carried out on the morphological traits to test
whether there were significant effects of altitude and
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784 A . A . H O F F M A N N ET AL.

a b

c d

Fig. 2. Floral morphology in alpine Stylidium. (a) Normal flowers on scape. Each flower carries four large petals. (b) Abnormal
flower with five petals, the most commonly observed abnormality. (c) Abnormal flower with six petals. (d) Rare inflorescence with
white flowers.

transect from which the seeds originated. In these
analyses, individual plants (rather than sites) were
treated as data points because plants representing seed
collected from sites had been reared individually in a
randomized design, as described above. Linear regres-
sions were used to calculate equations predicting
changes in morphological traits with altitude; these
analyses were undertaken separately for each transect.
To directly compare genetic (G) and environmental
(E) contributions to mean differences among plant
populations at the ends of the gradients (at 1530 m
and 1800 m), we computed the phenotypic difference
from the estimated regression line obtained for the
field data. The field phenotypic difference includes
genetic and environmental contributions. We com-
pared this with the estimated difference between plant
material derived from 1530 m and 1800 m observed
when plants were grown from seed in the glasshouse,
again obtained from regression lines. The glasshouse
difference reflects genetic influences (G). For those
traits that showed similar patterns in the field and
laboratory, we could then compute the contribution of
G and E to the difference between plants from 1530 m
and 1800 m.These values were expressed as a percent-
age of combined difference (G + E) for each site. Note
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Journal compilation © 2009 Ecological Society of Australia
that we are comparing genetic and environmental con-
tributions with mean differences in this analysis, and
not their contributions to trait variation within
populations.
RESULTS
Floral abnormalities
In 2004, a total of 872 plants were assessed at 34 of the
38 sites, 404 plants at Mount Nelse and 468 at New
Country Spur. The number of large petals on each
flower ranged from 3 to 9, with four large petals being
the canalized phenotype. Some examples of floral
abnormalities are shown in Figure 2. For both
transects, altitude was positively correlated with the
occurrence of abnormalities, which averaged 3% at
Nelse and 1% at New Country Spur (Table 1). The
effect of altitude was also significant in logistic regres-
sions where the number of abnormal flowers
(weighted by the total number of flowers) was exam-
ined across the different altitudes at both Nelse
© 2009 The Authors
 M O R P H O L O G I C A L VA R I AT I O N A N D F L O R A L A B N O R M A L I T I E S
Table 1. Correlations between incidence of flower abnormalities and altitude in the field (2004 and 2008) and in the
glasshouse
Collection Transect
Field, 2004 Nelse
New Country Spur
Field, 2008 Nelse
New Country Spur
Glasshouse Nelse
New Country Spur
Non-parametric correlations (first three entries) or parametric correlations (last three entries) were used depending on
normality of the data.
(Wald = 89.56, d.f. = 1, P < 0.001) and at New
Country Spur (Wald = 30.64, d.f. = 1, P < 0.001).
In 2008, 317 plants were assessed at 39 sites on
Mount Nelse, and 250 plants were assessed at 14 sites
on New Country Spur. Abnormality–altitude cor-
relations were significant at both sites (Table 1) and
patterns were consistent with those found in 2004
(Fig. 3), although the incidence of abnormalities was
higher at both sites in 2008 than in 2004, particularly
at New Country Spur where the average was 14% (at
Mount Nelse it was 4%). Plants with five petals re-
presented 62% of abnormalities, and plants with three
petals, 31%. As in 2004, altitude had a significant
effect on the incidence of abnormal flowers in logistic
regressions carried out on data from both Nelse
(Wald = 13.75, d.f. = 1, P < 0.001) and New Country
Spur (Wald = 28.77, d.f. = 1, P < 0.001).
In the glasshouse trials, petal numbers varied from 2
to 8. The percentage of abnormal flowers was compa-
rable with the incidence of abnormalities found in the
field at sites from low altitudes (Fig. 3). Flowers with
five petals represented 81% of the glasshouse abnor-
malities, and 3-petal flowers 14%. There was no cor-
relation between the altitude of the seed source and
the incidence of abnormalities (Table 1). A logistic
regression analysis on the number of abnormal flowers
weighted by the total number of flowers showed no
evidence for an effect of altitude at either Nelse
(Wald = 0.329, d.f. = 1, P = 0.566) or New Country
Spur (Wald = 0.333, d.f. = 1, P = 0.564).
The higher proportion of 5-petal flowers in the
glasshouse experiment than in the field may derive
from the fact that flowers with particular abnormalities
appeared at different times during the development of
the inflorescence, whereas flowers in the field were
only scored on one occasion. Both types of abnormali-
ties were non-randomly distributed along the inflores-
cence, as indicated by chi-squared tests on position
data when the inflorescence was divided into quarters
(3-petal flowers, c2 = 10.44, d.f. = 3, P = 0.01; 5-petal
flowers, c2 = 135.18, d.f. = 3, P < 0.001). The 3-petal
flowers occurred most frequently in the top quarter of
© 2009 The Authors
Journal compilation © 2009 Ecological Society of Australia
785
Number of
sites compared r P
18 0.589 0.010
15 0.802 <0.001
39 0.593 <0.001
14 0.537 0.045
10 0.020 0.957
11 -0.214 0.526
flowers produced (46%), while the 5-petal flowers
were most common in the bottom quarter (73%). To
directly compare the position of 3-petal and 5-petal
flowers, we examined the position of abnormalities in
the inflorescence relative to the total number of flowers
on that scape. For flowers that only produced a single
type of abnormality, the mean position for the 3-petal
flowers was 72.4% along the inflorescence, compared
with 25.4% for the 5-petal abnormality. This differ-
ence was significant by an anova (F(1,170) = 51.56,
P < 0.001). For the 11 plants that had both types of
abnormalities, the difference in position between the
3- and 5-petal abnormalities was also significant by a
paired t test (t = 4.10, d.f. = 10, P = 0.002).Thus while
both types of abnormalities were found scattered along
the scape, plants tended to produce 5-petal flowers
early in flowering, and 3-petal flowers later.
Flower emergence might confound the altitudinal
patterns because inflorescences in one part of the
transect might be more fully developed (and therefore
have relatively fewer early-appearing 5-petal abnor-
malities) than those in another part. Because plants
sampled in 2004 and 2008 were past the initial stage
of flowering, we suspected that this problem was
minimized. To test it directly, we calculated correla-
tions between the percentage of inflorescences with
3-petal and 5-petal abnormalities at the different alti-
tudes for the 2008 data. If inflorescence development
was important, we might expect that a negative rela-
tionship should exist between the incidence of the two
types of abnormalities. However, this relationship was
positive for both transects (Spearman rank correlation
coefficients, r = 0.32, n = 39, P = 0.04 for Nelse, r =
0.34, n = 14, P = 0.24 for New Country Spur) and
both the 3-petal and 5-petal abnormalities showed an
increase with altitude.
Plant measurements
Several of the morphological characters varied with
altitude.These were scape length, flower number on an
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786 A . A . H O F F M A N N ET AL.

2004 data
0.4

0.35

0.3

0.25

0.2

0.15

0.1

0.05

0
1400 1500 1600 1700 1800 1900

2008 data
Nelse
proportion flowers abnormal

0.4
New Country Spur
0.35

0.3

0.25

0.2

0.15

0.1

0.05

0
1400 1500 1600 1700 1800 1900
altitude (m)

glasshouse
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
1400 1500 1600 1700 1800 1900

Fig. 3. Altitudinal patterns for incidence of floral abnormalities at Mount Nelse, New Country Spur and in the glasshouse
trials.

inflorescence and leaf length (Table 2). For these
traits, the altitude effect was significant in the ancova
but no significant interactions were detected between
altitude and transect, meaning trends were consistent
across the two transects. Trait values decreased with
increasing altitude (Fig. 4). In all cases, a linear rela-
tionship between altitude and the trait produced a
higher R2 than did a quadratic relationship. Two data
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Journal compilation © 2009 Ecological Society of Australia
points in the Mount Nelse transect at around 1600 m
were outliers for all three traits (Fig. 4).
In contrast, for the number of scapes per plant, there
was a significant altitude–transect interaction, because
the altitudinal pattern evident for Nelse was not
present at New Country Spur (Fig. 4). Scape numbers
were also lower at New Country Spur than at Nelse
overall.
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 M O R P H O L O G I C A L VA R I AT I O N A N D F L O R A L A B N O R M A L I T I E S
Table 2. ancova on mean trait measurements for field plants collected at different altitudes in 2004
Transect
Trait (d.f. = 1)
Scapes per plant 13.59**
Scape length (mm) 0.44
Scape width (mm) 0.64
Number of flowers 18.52
Leaf length (mm) 28.78
Leaf width (mm) 4.78
**P < 0.01, ***P < 0.001. †Mean Squares divided by 1000 for width of clump, scape length, divided by 10 for flower number,
leaf length, multiplied by 10 for scape width.
Two of the three traits that exhibited altitudinal
associations in the field showed a consistent pattern in
the glasshouse. Both leaf length and scape length were
shorter in plants grown from high-altitude seed in the
common glasshouse environment (Fig. 5) and this
effect was significant in an ancova (Table 3), though
weaker than that seen in the field. For the field data on
scape length, the regression coefficients for Nelse and
New Country Spur were -1.01 and -0.94 respectively,
compared with coefficients for the glasshouse of -0.28
and -0.29. For leaf length, regression coefficients were
-0.118 and -0.186 in the field for Nelse and New
Country Spur respectively, compared with equivalent
glasshouse values of -0.06 and -0.04. Similar signifi-
cant patterns were found if site means (rather than
individual plants) were used in analyses of the glass-
house data.
We estimated the genetic and environmental contri-
butions to the mean difference between the 1530-m
and 1800-m ends of the transect, for the traits that
showed altitudinal variation in the same direction in
the glasshouse as in the field. These analyses (Table 4)
suggest that the environmental contribution to the alti-
tudinal gradient tended to be more than twice as large
as the genetic contribution, with the exception of leaf
length at Nelse, where the genetic and environmental
contributions were approximately equal.
Finally, some altitudinal trends evident in the glass-
house data were not apparent in the field comparisons.
Scape width increased significantly with altitude
(Fig. 5), whereas no such pattern was evident in the
field. Leaf width showed no pattern in the field but a
pattern in the glasshouse, increasing with source alti-
tude in the Nelse plants but decreasing for the New
Country Spur plants (Fig. 5). This led to a significant
interaction between altitude and transect (Table 3).
Flower number also showed a significant interaction
in the ancova, and increased with altitude at New
Country Spur, in contrast to the pattern at Mount
Nelse and in the field (cf. Fig. 3).
© 2009 The Authors
Journal compilation © 2009 Ecological Society of Australia
787
Mean squares†
Altitude Interaction Error
(d.f. = 1) (d.f. = 1) (d.f. = 31)
26.18*** 22.49** 2.92
268.81*** 0.20 6.54
1.21 0.31 0.81
293.03*** 18.44 9.73
662.58** 32.90 62.18
0.22 0.07 0.58
DISCUSSION
The field data from two collections in different years
suggest that flower abnormalities increase in frequency
at higher elevations, but this pattern appears to be
completely environmentally determined because it was
not evident in the glasshouse-raised trigger plants.
Abnormal flowers were particularly common on some
plants. In the field, we observed one plant with about
70% of its flowers abnormal. Even in the glasshouse,
one individual had 41% of its flowers abnormal. Petal
numbers were also variable and ranged from 2 to 8
even though the vast majority of abnormal flowers had
five or three petals. The absence of an altitudinal
pattern suggests that abnormalities reflect the effects
of environmental conditions at high elevations. These
might include temperature effects, ultraviolet irradia-
tion and/or other factors that vary with altitude. The
lack of an abnormality pattern in the glasshouse plants
suggests that there is no heritable component to this
trait, although we did not directly rear seed from plants
with a high number of abnormal flowers. Data span-
ning the entire field flowering season of S. armeria are
required to directly assess any potential confounding
effect of elevation on flowering time.
Because floral abnormalities appear to represent an
environmental response, the incidence of abnormali-
ties might reflect levels of stress experienced by plants
in a population. Huether (1969) found that stressful
conditions also triggered floral abnormalities in closely
related species of Linanthus; a survey of corolla lobe
number indicated that populations contained 1–4% of
flowers deviating from the normal lobe number, but
this increased substantially under herbivory and late
flowering.The 1–4% abnormality occurrence is similar
to that seen in S. armeria in the present study. There
has been much discussion in the literature about using
the loss of developmental stability as a measure of
stressful conditions, focussed mostly around scoring
deviations from bilateral symmetry or developmental
doi:10.1111/j.1442-9993.2009.01984.x
788 A . A . H O F F M A N N ET AL.

plant width (in mm) scapes per plant

450 14
400
12
350
10
300
250 8
200 6
150
4
100
50 2

0 0
1400 1500 1600 1700 1800 1900 1400 1500 1600 1700 1800 1900

scape length (in mm) scape width (in mm)

1000 4
900
800 3.5

700
3
600
500 2.5
400
2
300
200 1.5
100
0 1
1400 1500 1600 1700 1800 1900 1400 1500 1600 1700 1800 1900

flower number leaf length (in mm)

70 250

60
200
50
150
40

30 100

20
50
10

0 0
1400 1500 1600 1700 1800 1900 1400 1500 1600 1700 1800 1900

leaf width (in mm)

10

8
Nelse

7
New Country
Spur
6

4
1400 1500 1600 1700 1800 1900

Altitude (m)
Fig. 4. Change in morphological traits with altitude in field samples taken from Mount Nelse and New Country Spur. Each
data point represents the average measurements of 8–10 plant clumps. Linear regression lines are included where these are
significant.
doi:10.1111/j.1442-9993.2009.01984.x © 2009 The Authors
Journal compilation © 2009 Ecological Society of Australia
 M O R P H O L O G I C A L VA R I AT I O N A N D F L O R A L A B N O R M A L I T I E S 789

scape width (in mm) - Nelse scape width (in mm) - NCS

5 5

4 4

3 3

2 2

1 1

0 0
1400 1500 1600 1700 1800 1900 1400 1500 1600 1700 1800 1900

scape length (in mm) - Nelse scape length (in mm) - NCS
1000 1000
900 900
800 800
700 700
600 600
500 500
400 400
300 300
200 200
1400 1500 1600 1700 1800 1900 1400 1500 1600 1700 1800 1900

flowers per scape - Nelse flowers per scape - NCS

120 120
100 100
80 80
60 60
40 40
20 20
0 0
1400 1500 1600 1700 1800 1900 1400 1500 1600 1700 1800 1900

leaf length (in mm) - Nelse leaf length (in mm) - NCS
300
300
250
250
200
200
150
150
100
100
50
50
1400 1500 1600 1700 1800 1900
1400 1500 1600 1700 1800 1900

leaf width (in mm) - Nelse leaf width (in mm) - NCS

12
12
10 10

8 8

6 6

4 4

2 2
0 0
1400 1500 1600 1700 1800 1900 1400 1500 1600 1700 1800 1900

Fig. 5. Morphological traits measured on plants grown in the common garden glasshouse. Each point represents a plant from
the altitudinal origin and transect indicated. Linear regression lines are included where these are significant.

© 2009 The Authors doi:10.1111/j.1442-9993.2009.01984.x

Journal compilation © 2009 Ecological Society of Australia
790 A . A . H O F F M A N N ET AL.

Table 3. ancova on trait measurements for individual glasshouse plants grown from seed collected from different altitudes and
transects

Mean squares†

Transect Altitude Interaction Error

Trait (d.f. = 1) (d.f. = 1) (d.f. = 1) (d.f. = 414–425)

Scape length (mm) 0.33 246.29*** 0.06 16.50

Scape width (mm) 2.88 29.95*** 3.10 2.64
Number of flowers 592.37 114.83 576.37*** 38.52
Leaf length (mm) 9.67 801.61** 13.39 89.46
Leaf width (mm) 28.57*** 0.04 26.15*** 1.78

**P < 0.01, ***P < 0.001. †Mean Squares divided by 1000 for width of clump, scape length, divided by 10 for flower number,
leaf length, multiplied by 10 for scape width.

Table 4. Relative contribution of genetic (G) and environ-
mental (E) components (as a percentage) for traits where
field and glasshouse patterns with altitude were in the same
direction
Transect Trait G E
Mount Nelse Scape length 27.59 72.41
Number of flowers 31.31 68.69
Leaf length 48.43 51.57
New Country Spur Scape length 30.67 69.33
Leaf length 23.74 76.26
complexity rather than floral abnormalities (Moller &
Shykoff 1999). In some studies, asymmetry in floral
characters has been linked to marginal environmental
conditions. In particular, Siikamaki and Lammi
(1998) studied the plant Lychnis viscaria and found
that differences in fluctuating flower asymmetry
between central and marginal populations disappeared
when these were raised in a common garden. Fluctu-
ating asymmetry may serve as an indicator of some
environmental stresses in plants (Moller & Shykoff
1999; Valkama & Kozlov 2001; Llorens et al. 2002)
although this varies across species and the nature of
the stresses being considered. The present results
suggest that floral abnormalities might represent a
simple way of assessing stressful conditions, although
this needs to be explored further in controlled experi-
ments where plants are exposed to different stresses.
We also directly assessed leaf asymmetry in prelimi-
nary work in 2004 but this measure did not vary with
altitude (data not presented).
Floral morphology is thought to be under strong
stabilizing selection mainly because of pressures
imposed by pollinators (Cresswell 1998). The high
incidence of floral abnormalities in Stylidium under
some conditions seems striking, given that petal
number in trigger plants is a canalized trait. It is
unclear whether abnormal flowers set fewer seed, as
might be expected given the tight association between
doi:10.1111/j.1442-9993.2009.01984.x
Journal compilation © 2009 Ecological Society of Australia
Stylidium flower morphology and pollinators that is
likely to drive floral divergence in this group (Cresswell
1998). If this is the case, producing abnormal flowers
at high altitudes is an example of a non-adaptive
plastic response to stress (Ghalambor et al. 2007).This
type of plasticity can facilitate adaptation to new envi-
ronments by exposing otherwise-cryptic genetic varia-
tion to new selection pressures (examples reviewed in
Ghalambor et al. 2007).
Unlike floral abnormalities, two morphological traits
(leaf length, scape length) exhibited an altitudinal
pattern that was partly heritable and repeatable when
plants were grown under similar conditions. In both
cases, the slope of the regression line linking altitude to
mean scape or leaf length was weaker in the glasshouse
than in the field. The genetic components tend to be
smaller than the environmental components, suggest-
ing that environmental effects have a larger impact in
generating the pattern observed in the field.
Patterns of morphological variation where genetic
and environmental components act in the same direc-
tion (‘cogradient’ patterns) were previously described
in P. hiemata collected across similar, though shorter,
transects (Byars et al. 2007). In the case of P. hiemata,
one trait that exhibited cogradient selection (leaf
length) decreased with altitude, as in the case of
S. armeria. The other trait (plant circumference)
increased with altitude. Both these patterns were
linked to survival; transplant experiments indicated
that grasses at high altitudes with relatively shorter
leaves and wider circumferences had a higher survival
(Byars et al. 2007). It remains to be seen whether any
of the Stylidium traits can be linked to survival or
reproductive output.
In contrast to these cogradient patterns, there was
evidence for opposing patterns of genetic and environ-
mental variation in scape width. Countergradient
patterns can arise if selection across a gradient favours
a particular phenotype, but environmental effects
along a gradient cause deviations from this phenotype
(Conover & Schultz 1995). In this case, genetic effects
© 2009 The Authors
 M O R P H O L O G I C A L VA R I AT I O N A N D F L O R A L A B N O R M A L I T I E S
can act counter to the environmental effects – for
instance, the increase in scape width seen in the glass-
house with altitude is likely to counter environmental
effects that decrease scape width at higher altitudes.
The net result is that the same average scape width is
maintained across the entire gradient.
In conclusion, there is ample altitudinal variation in
morphological traits in S. armeria. This variation is
influenced by environmental and genetic factors and
may reflect adaptive patterns of differentiation. The
Stylidium data highlight the fact that alpine species are
likely to be genetically differentiated even across a
relatively narrow range of altitudes, confirming pat-
terns previously described in P. hiemata. In aiming to
conserve a genetically ‘healthy’ species that retains
adaptive potential, populations at high- and low-
altitude sites should be maintained. This ensures that
conserved S. armeria (and other species with similar
distributions) include genotypes adapted to local con-
ditions and capable of responding to different selection
pressures. Further experiments, including reciprocal
transplants, are needed to identify physiological traits
associated with local adaptation and further clarify the
impact of environmental and genetic factors in altitu-
dinal differentiation.
ACKNOWLEDGEMENTS
This research was supported by the Australian
Research Council via their Linkage and Special
Research Centre schemes. We are also grateful to
support from the Department of Sustainability and the
Environment, and to the Victorian National Parks
Association for support and site access.We thank Sean
Byars, Lindy MacRaild, Paul Mitrovski and Gareth
Holmes for assistance with this work and Pauline
Ladiges for discussions.
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