Previous studies revealed significant phenotypic plasticity and between-population differentiation in flower morphometric traits of Iris pumila in response to environmental variability between natural shade and exposed habitats. Since these habitats differed in flowering times as well, in this work we investigated at which stages of flower bud development differences between open and shaded habitats start to appear. Our analysis detected several groups of trait development patterns through the I. pumila bud development in two contrasting habitats, with stem length being the most suitable trait for application in further analyses of so-called 'shade avoidance syndrome'.Projekat ministarstva br. 17302

Avramov, Stevan

Barišić Klisarić, Nataša

Miljković, Danijela

Tarasjev, Aleksej

Živković, U.

English

 Digital Repository of Archived Publications - Institute for Biological Research Sinisa Stankovic (RADaR)

Arch. Biol. Sci., Belgrade, 64 (1), 77-83, 2012 DOI:10.2298/ABS1201077L
77
STAGES OF FLOWER BUD DEVELOPMENT IN IRIS PUMILA AND  
BETWEEN-HABITAT MORPHOLOGICAL DIFFERENCES
NATAŠA BARIŠIĆ KLISARIĆ, DANIJELA MILJKOVIĆ, S. AVRAMOV,  
U. ŽIVKOVIĆ and A. TARASJEV
Institute for Biological Research, “Siniša Stanković”, University of Belgrade, 11000 Belgrade, Serbia 
Abstract - Previous studies revealed significant phenotypic plasticity and between-population differentiation in flower 
morphometric traits of Iris pumila in response to environmental variability between natural shade and exposed habitats. 
Since these habitats differed in flowering times as well, in this work we investigated at which stages of flower bud develop-
ment differences between open and shaded habitats start to appear. Our analysis detected several groups of trait develop-
ment patterns through the I. pumila bud development in two contrasting habitats, with stem length being the most suitable 
trait for application in further analyses of so-called “shade avoidance syndrome”. 
Key words: Phenotypic plasticity, between-population differentiation, “shade avoidance syndrome”, flower morphometric 
traits, stage allometry, Iris pumila
INTRODUCTION
The evolution of adaptation depends not only on 
the genetic variation that leads to between-popu-
lation differentiation for the traits in question, but 
also on their phenotypic plasticity (Schlichting and 
Pigliucci, 1998; Pigliucci, 2001; de Jong, 2005). Pre-
vious studies of Iris pumila flower morphometric 
traits in natural habitats detected significant envi-
ronmental heteroscedascity (Tucić et al., 1990) and 
revealed both significant plastic responses as well as 
between-population differentiation between shad-
ed and open habitats (Tarasjev et al., 2009). Plastic-
ity and differentiation were mostly consistent with 
responses expected in the case of so called “shade 
avoidance syndrome” (Schmitt and Wulff, 1993; Von 
Wettberg, et al., 2008; McGoey and Stinchcombe, 
2009; Tarasjev et al., 2009). On the other hand, the 
study of I. pumila phenology revealed similar sig-
nificant phenotypic plasticity as well as population 
differentiation in the flowering time of populations 
occupying the same contrasting habitats (Tarasjev, 
1997). The possible influence of flowering time on 
detected differences in flower morphometric traits 
in previous studies must also be examined. To ap-
proach this relationship, the ontogenic aspect must 
be covered since plasticity in plants can be the re-
sult of the direct response of a trait to environmen-
tal cues during development, or a passive result of 
changes in the plant growth rate due to various fac-
tors (Schlichting and Pigliucci, 1998; Wright and 
McConnaughay, 2002; Varin et al., 2009), with the 
phenological response influencing other traits being 
a significant one (Diggle, 1999; Forrest and Miller-
Rushing, 2010). Previous studies of the flower buds 
in populations from open and shaded habitats, but 
performed at different time points (Barišić Klisarić 
et al., 2011), revealed that stem length was the only 
trait that showed higher values in shaded habitats 
for the very beginning. Most of the other traits had 
higher values in the bud stage in habitats that will 
flower earlier (open habitats) compared to habitats 
78 NATAŠA BARIŠIĆ KLISARIĆ   ET AL.
that have higher values for open flowers (shaded). 
To supplement the analysis of the flower’s morpho-
metric traits at the same time periods, we studied 
the morphometric traits in the same stages of flower 
bud development.
The goals of this study were to analyze the mor-
phometric flower traits at different stages of flower 
ontogeny (stage allometry) on naturally growing 
clones of I. pumila occupying contrasting light hab-
itats (open vs. shaded) in order to: analyze traits at 
different stages during the flower bud ontogenetic 
development and detect the stages at which the 
growth of certain flower parts occurs; analyze traits 
at different stages during the flower bud ontoge-
netic development and to detect the stages at which 
differences between habitats in the size of certain 
flower parts occur, and to compare the detected 
differences in the flower bud stages between clones 
from the two habitats with differences described in 
previous studies in which plants in the flowering 
stage were used.
MATERIALS AND METHODS
The dwarf bearded iris Iris pumila L. is a small mono-
cot that occurs in the lowlands of Central and East-
ern Europe (Mathew, 1981). I. pumila is perennial 
clonal plant, which spreads vegetatively through un-
derground rhizomes. Therefore, the same genotypes 
can be studied over different years and sufficient 
numbers of replicas of the same genotype established 
in the natural population can be obtained for experi-
mentation. 
This study was conducted in the Deliblato 
Sands, a protected sandy area 40 km NE from Bel-
grade, Serbia (44°48’N, 200 58’ E), on 64 naturally 
growing clones in two I. pumila habitats (47 from 
open habitats and 18 from shaded ones). In an 
open habitat the plants are exposed to direct sun-
light compared to plants in shaded habitats (e.g. 
planted pine forest) created under anthropogenic 
influence. The contrasting habitats are located in 
the northwestern part of the Deliblato Sands and 
they differ in many ecological indices (Kojić et al., 
1994), foremost in the quality and intensity of light. 
In the sand-steppe habitat, the radiation intensity 
is 666.1 µmol m-2s-1 and red/far red ratio (R/FR) is 
1.16 ((E), estimated with a LiCor 1000 (D) datalog-
ger and Li190SA and Skye SKR-110 sensors). In the 
forest site, clones of I. pumila grow in the vegeta-
tion shade (radiation intensity of 170.9 µmolm-2s-1 
and R/FR ratio of 0.96).
Detailed descriptions of the parts of the I. pumila 
flower, as well as their graphical representation, are 
given in Tarasjev et al. (2009). During the beginning 
of the flowering season, buds were collected from 
selected clones in four stages of flower bud develop-
ment (two buds per stage/clone combination). Up 
to two opened flowers per clone were also collected. 
The analyzed stages are presented in Fig. 1, and these 
are: 1. Hidden bud stage (H) with no visible flower 
parts, but the bud is palpable between the leaves; 2. 
Spathe stage (S) where two spathes are visible out-
side the leaf “pocket”, but no other parts of the flower 
bud are visible yet; 3. Colored bud stage (C) where 
the colored parts of perianth are visible outside the 
leaf “pocket” between the spathes; 4. Final bud stage 
(B) – the flower has not yet opened, but most of the 
perianth parts are already outside the spathes, and 5. 
Open flower stage (F).
A total of 65 clones (47 from open and 18 from 
shaded habitats) were analyzed with up to two sam-
ples per each stage on each clone resulting in a to-
tal of 423 analyzed buds and flowers. All measure-
ments of the vegetative and floral parts were made 
to the nearest millimeter using fresh material. The 
following traits were utilized in this analysis: length 
of the first spathe (LIS), length of the second spathe 
(LIIS), stem length (LS), ovary length (OL), ovary 
width (OW), tube length (TL), tube radius (TR), fall 
length (FL), fall width (FW), standard length (SL), 
standard width (SW), stigma length (SML), crest 
length (CL), crest width (CW), stamen length (STL) 
and anther length (AL). Morphometric data were 
transformed to natural logarithms as the appropri-
ate transformation determined by the method given 
by Box et al. (1978) and the SAS program given by 
Fernandez (1992). Statistical analyses were carried 
STAGES OF FLOWER BUD DEVELOPMENT IN IRIS PUMILA 79
out on clone means by ANOVA with Habitat and 
Stage as the main factors. For pairwise comparisons 
between stages, the Scheffe test was used. All statis-
tical testing was done by SAS program (SAS 1989) 
PROC MEANS and PROC GLM procedures. 
RESULTS AND DISCUSSION
Several studies of the morphology of the flowering 
dwarf bearded iris Iris pumila L. have previously 
been conducted on plants and populations of this 
perennial monocot from open and shaded localities 
in Natural Protected Reserve of Deliblato Sands, and 
the results of comparison of plants occupying open 
and shaded habitats were mostly consistent with the 
responses expected in the case of so called “shade 
avoidance syndrome” (Tarasjev, 2009). 
The results of this study are presented in Fig. 2 
for the outer parts of the I. pumila blossom and in 
Fig. 3 for the perianth parts. Analysis indicated that 
most of the development occurred in the earlier stag-
es of bud development compared to the later ones. 
No differences between B and F stages were detect-
ed in this study for all traits and both habitats, and 
significant differences between C and B stages were 
detected only for stem length and tube length, both 
in the open habitat. However, a large number of sig-
nificant differences between the C and F stages (Fig 
2 and 3) indicate that a certain amount of growth 
occurs in the later stages, too. However, growth was 
more profound in the earlier stages of flower bud de-
velopment, with all but one analyzed trait showing 
significant differences between the H and S stages 
in both habitats, while 11 out of 16 analyzed traits 
showed significant differences between the S and C 
stages. The lengths of the first and second spathes, as 
well as anther length, finished their development in 
S stage.
Regarding the differences between habitats, they 
were significant for only two traits (stem and ovary 
lengths) in the earliest stage of flower bud develop-
ment. The number of traits with significant between-
habitat differences gradually increased in the later 
stages: five traits showed significant between-habitat 
Fig. 1. Analyzed bud stages in development of I.pumila flower: Hidden bud stage (H), Spathes stage (S), Colored bud stage (C), Final bud 
stage (B) and Open flower stage (F). For description of each stage see Matherial and methods section
80 NATAŠA BARIŠIĆ KLISARIĆ   ET AL.
Fig. 2. Morhometric change through developmental stages of outer parts of I.pumila blossom in two habitats (open and shaded one). 
Developmental stages within each habitat that share the same letter (capital letters for shaded habitat, small letters for open habitat) do 
not differ significantly on the basis of Scheffe test.  Siginificant differences between habitats for particular stage is indicated by asterisks. 
For character abbreviations see Matherial and methods section.
STAGES OF FLOWER BUD DEVELOPMENT IN IRIS PUMILA 81
differences in the S stage, eight traits in the C and B 
stages, and twelve traits in the open flower stage (F). 
No differences between habitats were detected for FL 
CW, SML and STL.
On the basis of these findings, the analyzed 
traits can be divided into several groups and one 
representative trait for each group can be utilized in 
further studies. The first group consists of traits that 
showed no differences between habitats and ended 
their development in the C stage (FL, CW, SML and 
STL). This group could be excluded from further 
analyses of shade avoidance syndrome of Iris pumi-
la. Ovary width (OW) showed a continuous pattern 
of growth but with differences that were detectable 
only in the open flower stage with no differences 
Fig. 3. Morhometrical change through developmental stages of perianth parts of I.pumila blossom in two habitats (open and shaded 
one). Developmental stages within each habitat that share the same letter (capital letters for shaded habitat, small letters for open habitat) 
do not differ significantly on the basis of Scheffe test. Siginificant differences between habitats for particular stage is indicated by aster-
isks For character abbreviations see Matherial and methods section.
82 NATAŠA BARIŠIĆ KLISARIĆ   ET AL.
in the bud stages, which was similar to the previ-
ous group. The other group consists of traits that 
start to appear between-habitat differences from the 
C stage (TL, SL, CL FW and SW) but continue to 
grow through the last two stages. The third group 
begins to diverge in the S stage (LIS, LIIS, TR, AL) 
with only the tube radius (TR) continuing to grow 
further through the later stages.
A special case of between-habitat difference is the 
length of the stem (LS). In previous analysis of flower 
bud morphometric traits in different time periods 
(Barišić Klisarić, 2011), the LS was the only trait that 
showed divergence between habitats in all time peri-
ods, and the direction of this difference was the same 
as in analyses of I. pumila flowers (Tarasjev, 2009). 
In this study, it was one of only two traits (together 
with ovary length) that showed between-habitat dif-
ferences from the earliest bud stage. However, while 
the difference for OL was small and even undetect-
able in the following S stage, the LS was two times 
larger in the shaded habitat compared to the open 
one in the “Hidden bud” (H) stage. This size differ-
ence increases threefold in later stages. This makes 
stem length the most usable trait in further studies 
of flower morphological differences between open 
and shaded habitats that might by accompanied by 
selected representative traits from two other groups. 
The reduction of the number of traits that is suggest-
ed by the results of this study can therefore enable 
larger and more extensive experiments compared 
to those already performed on the I. pumila flower 
morphometric traits (Tucić et al., 1990; Tarasjev, 
1994; Tarasjev, 1995; Tarasjev et al., 2006; Tarasjev 
et al., 2009).
Acknowledgements - This work was supported by the Ministry 
of Science and Education of the Republic of Serbia Research 
Grant no. 173025
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Stages of flower bud development in Iris pumila and between-habitat morphological differences

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Stages of flower bud development in Iris pumila and between-habitat morphological differences

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