American Journal of Plant Sciences, 2012, 3, 1588-1593
http://dx.doi.org/10.4236/ajps.2012.311192 Published Online November 2012 (http://www.SciRP.org/journal/ajps)
Comparison of Photosynthetic Pigment Contents of the
Resurrection Plants Ramonda serbica and Ramonda
nathaliae of Some Different Populations from Kosovo,
Albania and Macedonia
Bekim Gashi1,2, Kasamedin Abdullai1, Efigjeni Kongji k a3
1Department of Biology, Faculty of Mathematics and Natural Sciences, University of Pristina, Pristina, Republic of Kosovo;
2Department of Biotechnology, Faculty of Natural Sciences, University of Tirana, Tirana, Albania; 3Section of Natural and Technical
Sciences, Academy of Sciences of Albania, Tirana, Albania
Received August 17th, 2012; revised September 19th, 2012; accepted October 9th, 2012
ABSTRACT
The resurrection plants Ramonda serbica and Ramonda nathaliae are the physiological model plant and endemic spe-
cies from Balkan Peninsula. Study was carried out to assess the impact of different populations on photosynthetic pig-
ment contents and their effect on physiological activity of these species. The experiment was conducted with leaves of
R. serbica collected from nine populations in Kosovo, three in Albania and two in Macedonia, while for R. nathaliae
four populations in Macedonia. For this purpose plants after collection from their natural populations were kept for 24
hours at constant temperature and humidity and in natural photoperiod. According to our data the pigment contents
(Total chl + Carot) of R. serbica, measured as mg per gram dry weight (DW), were higher (7.06 mg·g−1 DW) in Radaci
populations (Kosovo) and lower (4.63 mg·g−1 DW) in Jukniu Mountain-Kruja (Albania). On the other hand, the higher
(5.28 mg·g−1 DW) of pigment contents (Total chl + Carot) of R. nathaliae were observed in Vorca populations and the
lower (4.24 mg·g−1 DW) in Kaparllëk populations. Ratio chlorophyll a/b of R. serbica in Kruja Castle populations from
Albania was the higher (3.68) comparing with Zhlebi populations from Kosovo (1.68). Similarly in case of R. nathaliae
the higher (3.36) value was in Kaparllëk populations and the lower (3.12) in Matka populations. The data obtained were
further analyzed using one-way ANOVA and a significant change was recorded in the different populations. These
studies clearly indicate that the Ramonda plants from different ecological habitats there have been changes of photo-
synthetic pigment contents.
Keywords: Resurrection Plants; Physiological Model; Populations; Photosynthetic Pigments
1. Introduction
A small number of higher plant species, not closely re-
lated, have adapted to environments with rapidly devel-
oping and often extended periods of extreme dryness,
followed by sudden water availability. These are called
desiccation-tolerant or resurrection plants. Examples are
Ramonda serbica and Ramonda nathaliae, a rare resur-
rection plant of the Balkan Peninsula, an endemic relicts
of the tertiary period.
The species of family Gesneriaceae are good exam-
ples of ecophysiological divergence regarding the plant
water relations and the adaptations to the conditions of
water regime in the habitat. Under the conditions of wa-
ter deficit in the habitat, these plants gradually wilt and
pass to anabiosis. Resurrection plants are usually subdi-
vided into homoiochlorophyllous plants retaining their
chlorophyll (Chl) during desiccation and poikilochloro-
phyllous plants where desiccation results in the loss of
Chl which must be re-synthesized following rehydration
[1]. However, pigment loss and destruction of the other
thylakoid pigments are highly organised responses to
desiccation, realised via a well-defined metabolic path-
way [1]. From an ecological point of view, R. serbica is a
perennial, herbaceous, shade-adapted species belonging
to the group of homoiochlorophyllous poikilohydric
plants which preserve more than 80% of the chlorophyll
content during dehydration [2,3].
Its ability to survive in harsh environmental conditions
has so far been studied in terms of morphologic, physic-
ologic, and biochemical as well as the propagation
through in vitro cultivation. This research has brought the

Comparison of Photosynthetic Pigment Contents of the Resurrection Plants Ramonda serbica and
Ramonda nathaliae of Some Different Populations from Kosovo, Albania and Macedonia
1589
evidence of adaptive features necessary to preserve cell
membrane integrity [4], antioxidative capacity [5,6],
photosynthetic activity [1], CO2 fixation and chlorophyll
a fluorescence [7], genome size variation and polyploidy
[8], osmotic adjustment [9], seed germination [10] and in
vitro cultivation from seeds of Ramonda plants [11-13].
Nevertheless, up to now there is only a few data from
other authors for photosynthetic pigments content of
Ramonda serbica or similar species from Geseneriaceae
in different populations. Right now still are missing the
study belonging to Ramonda species on concentration of
photosynthetic pigments.
We are incited to find the concentration of chlorophyll
a, b, total chlorophyll, carotenoids and their ratio from
different ecological habitats conditions and their impact
on ecophysiological activity for these plants.
2. Materials and Methods
The resurrection plants Ramonda serbica and Ramonda
nathaliae were collected from their natural habitat. R.
serbica in nine populations in Kosovo, three in Albania
and two in Macedonia, while for R. nathaliae four popu-
lations in Macedonia (Table 1). Plants of the same age
were harvested together with the layer of soil on which
they grew.
For the photosynthetic pigments content study, three
leaves of similar ages, comparable in size, were collected
from five plants from the middle of the rosette.
2.1. Chlorophyll (a, b and Total) and
Carotenoids Analysis
Pigments were extracted by grinding 60 - 80 mg freshly
sampled leaves in 80% (v/v) acetone/water containing
MgCO3 (0.5% w/v) at room temperature for 24 h in the
dark. Photosynthetic pigments of all the samples were
extracted in triplicate to minimize experimental errors.
Concentration of chlorophyll and carotenoid contents
were measured by using absorbance recorded at 663 nm,
644 nm and 452.5 nm for maximum absorption of chlo-
rophyll “a” (Chl a), chlorophyll “b” (Chl b) and Carote-
noids, respectively. The extinction coefficients were de-
termined by a UV-Vis spectrophotometer. Pigment con-
tents were calculated in mg·g−1 dry leaf weight (DW) by
applying the absorption coefficient equations described
Table 1. Geographic origin and descr iption of Ramonda plants.
Code of populations Species Locality of populations Directions Altitude (m) Country
1 Ramonda nathaliae Gorge of river Matka N 348 Macedonia
2 Ramonda nathaliae Kaparllëk N and NE 448 Macedonia
3 Ramonda nathaliae Cerovë N and NE 591 Macedonia
4 Ramonda nathaliae Vorcë N and NE 407 Macedonia
5 Ramonda serbica Gorge of River Radika N 920 Macedonia
6 Ramonda serbica Llukovo N 630 Macedonia
7 Ramonda serbica Gorge of Zhlebi N and NE 1250 Kosovo
8 Ramonda serbica Radac N 545 Kosovo
9 Ramonda serbica Gorge river of Sushica N 676 Kosovo
10 Ramonda serbica Canyon of Rugova N 800 Kosovo
11 Ramonda serbica Gorge of Koprivnik N 750 Kosovo
12 Ramonda serbica Gorge of River Prizren N 530 Kosovo
13 Ramonda serbica Gorge of Rusenica N 1340 Kosovo
14 Ramonda serbica Gorge of Matosi N 910 Kosovo
15 Ramonda serbica Shkëmbi i përgjakur N 1170 Kosovo
16 Ramonda serbica Kruja Castle N and NE 565 Albania
17 Ramonda serbica Jukniu Mountain N and NE 827 Albania
18 Ramonda serbica Petrela Castle N 340 Albania

Comparison of Photosynthetic Pigment Contents of the Resurrection Plants Ramonda serbica and
Ramonda nathaliae of Some Different Populations from Kosovo, Albania and Macedonia
1590
by Lichtenthaler (1986) [14]:

 
1
Chl mggDW
10.3OD6630.918OD644V100FWDW.
a 
  




1
Chl mggDW
19.7OD6443.87OD663V100 FWDW.
b 







1
Carotenoids mg gDW
4.75 OD452.50.226 ChlChl
V100FW DW.
ab


 

 


where
DW = Dry leaf weight.
FW = Fresh leaf weight.
OD = Optical density.
V = Volume of sample.
2.2. Data Analysis
The experiment was performed in a randomized design
with five replicates. Differences among parameters and
between the populations were tested using SPSS 17 sta-
tistical program. Statistical variance analysis of the all
data was performed using one-way ANOVA and com-
pared with least significant difference (LSD) at the 5%
and 1% level. Mean comparison was performed with
Duncan’s test at the 5% level of significance.
3. Results and Discussions
Based on the obtained results and presented on the Ta-
bles 2 and 3, high significant differences at P < 0.01 have
been ascertained between populations, for all researched
parameters of Ramondas plants on different populations.
The effect of different ecogeographic factors combine on
highest differences on photosynthetic pigment concentra-
tion and their ratio between different populations.
At R. nathaliae, the highest content of Chl a, Carete-
noids and Total Chlorophyll + Carotenoids has been as-
certained at Vorca population, whereas the content of Chl
b and Total Chl was higher at Gorge of river Matka
population. The lowest content of all photosynthetic
pigments was obtained at Kaparllëk population (Table 2).
At Gorge of river Matka population, the Ratio Chl a/b
(Figure 1) was the lowest (3.12), whereas the Ratio Total
Chl/Carot was the highest (4.53) compared to other
populations. Based on the results collected in the field,
the Gorge of River Matka population had less sunlight
due to the hindrance by the forest trees. For this reason
the Chl a/b ratio has been frequently used as an indicator
of plant response to shading [15].
Based on the gained and analysed results with One-
way ANOVA and compared with Duncan test, at R. ser-
bica leaves, the highest photosynthetic pigments content
(Table 3) was ascertained at Radac and Llukovo popula-
tions. The similar results were gained for the Gorge of
Matosi population, too. Out of all investigated popula-
tions, it is worth mentioning that these three populations
have been with less sunlight compared with other popu-
lations. This increase of the photosynthetic pigments
content is justified by the fact which for many plant spe-
cies that grow under environmental shade conditions will
result in growth of pigment content. In addition, Gon-
çalves et al. (2001) [16] at Mahogany and Tonka bean,
leaf chlorophyll concentrations (Chl a, Chl b, Chl tot) on
a fresh mass basis were higher in shade leaves than in
sun leaves. According to Tan et al. (2000) [17] there is
widespread interest in chlorophylls and their degradation
reactions. Chlorophyll is synthesized and degraded
(photo oxidation) under irradiation. At high irradiance,
however, the degradation rate overtakes the rate of syn-
thesis, therefore, a lower chlorophyll concentration is
observed. Due to this fact, shade leaves in comparison
with sun leaves tend to show higher chlorophyll concen-
trations per unit leaf weight [18].
Lower photosynthetic pigments content was observed
at R. serbica population from Albania, especially in
Jukniu Mountain and Kruja Castle populations. These
populations are with more intensity of the sunlight com-
pared with other populations from Albania, Kosovo and
Macedonia (Table 3). Based on statistical analysis re-
sults with Duncan test, the populations’ alignment with
approximate content of photosynthetic pigments clearly
indicates the similar ecological conditions of these popu-
lations.
The highest content of Chl a, Total Chl and Total Chl/
Carot (Table 3) for all the investigated population of R.
serbica was ascertained at Radac population (4.47, 5.90,
and 7.06 mg·g−1 DW, respectively), whereas the lowest
at Jukniu Mountain (2.86, 3.70, and 4.63 mg·g−1 DW,
respectively). For the Chl b the highest content was at
Gorge of Zhlebi population (1.76 mg·g−1 DW), whereas
the lowest at Shkembi i pergjakur population (1.42
mg· g −1 DW). Our results of higher Chl b in the shade
environment are in accordance with other authors results.
Gonçalves et al. (2001) [16] showed that Chl b concen-
tration on a mass basis of tonka bean and mahogany was
higher in the shade environment.
The highest Carotenoids concentrations was at Gorge
of Matosi population (1.26 mg·g−1 DW), whereas the
lowest at Gorge of Zhlebi population (0.81 mg·g−1 DW).
Another very important indicator for the intensity of
sunlight is also Ratio Chl a/b, where the highest values
for this parameters (Figure 1) were shown at Kruja Cas-
tle and Jukniu Mountain populations (3.68 and 3.42, re-

Comparison of Photosynthetic Pigment Contents of the Resurrection Plants Ramonda serbica and
Ramonda nathaliae of Some Different Populations from Kosovo, Albania and Macedonia
1591
Table 2. The effects of different populations of R. nathaliae from Macedonia on photosynthetic pigment content in mg per
gram dry weight (DW).
Chl a Chl b Total Chl Carotenoids Total Chl + Carot
Code of populations
Mean ± SX Mean ± SX Mean ± SX Mean ± SX Mean ± SX
1 3.27 ± 0.16 a 1.06 ± 0.06 a 4.33 ± 0.22 a 0.95 ± 0.04 b 5.28 ± 0.25 a
2 2.58 ± 0.10 b 0.80 ± 0.05 bc 3.38 ± 0.14 b 0.86 ± 0.03 b 4.24 ± 0.17 b
3 2.82 ± 0.15 b 0.90 ± 0.06 ab 3.72 ± 0.20 b 0.94 ± 0.04 b 4.66 ± 0.24 b
4 3.32 ± 0.08 a 1.01 ± 0.03 b 4.33 ± 0.11 a 1.06 ± 0.02 a 5.39 ± 0.13 a
P ≤ 0.05 0.388 0.135 0.478 0.091 0.566
LSD
P ≤ 0.01 0.588** 0.204** 0.727** 0.138** 0.858**
Chl—Chlorophyll; Carot—Carotenoides; *Significant at 0.05 probability level; **Significant at 0.01 probability level. Columns with different letters differ sig-
nificantly at P < 0.05 by one-way ANOVA with Duncan’s multiple range test.
Table 3. The effects of different populations of R. serbica from Kosovo, Albania and Macedonia on photosynthetic pigment
content in mg per gram dry weight (DW).
Chl a Chl b Total Chl Carotenoids Total Chl + Carot
Code of populations
Mean ± SX Mean ± SX Mean ± SX Mean ± SX Mean ± SX
5 3.43 ± 0.09 de 1.14 ± 0.04 d 4.57 ± 0.13 c 1.11 ± 0.03 bcd 5.68 ± 0.15 cd
6 4.38 ± 0.08 a 1.43 ± 0.05 c 5.81 ± 0.12 a 1.19 ± 0.05 ab 7.00 ± 0.15 a
7 2.86 ± 0.08 g 1.76 ± 0.06 a 4.61 ± 0.13 c 0.81 ± 0.02 f 5.43 ± 0.15 cd
8 4.47 ± 0.13 a 1.43 ± 0.04 c 5.90 ± 0.16 a 1.16 ± 0.05 bc 7.06 ± 0.20 a
9 3.70 ± 0.09 c 1.34 ± 0.03 c 5.04 ± 0.12 b 1.07 ± 0.02 cd 6.12 ± 0.14 b
10 3.49 ± 0.07 cde 1.10 ± 0.03 d 4.58 ± 0.08 c 1.08 ± 0.02 cd 5.66 ± 0.10 cd
11 3.42 ± 0.11 de 1.19 ± 0.04 d 4.62 ± 0.10 c 1.04 ± 0.05 d 5.66 ± 0.11 cd
12 3.37 ± 0.07 e 1.10 ± 0.02 d 4.46 ± 0.09 c 1.08 ± 0.02 cd 5.55 ± 0.11 cd
13 3.13 ± 0.07 f 1.20 ± 0.02 d 4.33 ± 0.09 c 1.05 ± 0.02 d 5.37 ± 0.12 d
14 4.02 ± 0.08 b 1.58 ± 0.06 b 5.61 ± 0.13 a 1.26 ± 0.03 a 6.86 ± 0.14 a
15 3.67 ± 0.08 cd 1.42 ± 0.03 c 5.09 ± 0.11 b 1.11 ± 0.03 bcd 6.20 ± 0.13 b
16 2.96 ± 0.06 fg 0.81 ± 0.02 e 3.76 ± 0.07 d 0.93 ± 0.02 e 4.69 ± 0.09 e
17 2.86 ± 0.06 g 0.84 ± 0.03 e 3.70 ± 0.09 d 0.93 ± 0.02 e 4.63 ± 0.11 e
18 3.59 ± 0.09 cde 1.10 ± 0.03 d 4.68 ± 0.12 c 1.13 ± 0.03 bcd 5.81 ± 0.15 bc
P ≤ 0.05 0.339 0.149 0.431 0.098 0.492
LSD
P ≤ 0.01 0.458** 0.201** 0.582** 0.132** 0.470**
Chl—Chlorophyll; Carot—Carotenoides; *Significant at 0.05 probability level; **Significant at 0.01 probability level. Columns with different letters differ sig-
nificantly at P < 0.05 by one-way ANOVA with Duncan’s multiple range test.
spectively).
At the Gorge of Zhlebi population was observed the
lowest value of Ratio Chl a/b and the highest the Ratio of
Total Chl/Carot (5.67). Gonçalves et al. (2001) [16] con-
firm a slight differences in Chl a/b ratio and chlorophyll/
carotenoid ratio between species and environments. In
the open site, the Chl a/b ratio was higher and chloro-
phyll/carotenoid ratio was lower in tonka bean and ma-

Comparison of Photosynthetic Pigment Contents of the Resurrection Plants Ramonda serbica and
Ramonda nathaliae of Some Different Populations from Kosovo, Albania and Macedonia
1592
Figure 1. Ratio chlorophyll a/b and Total chlorophyll/Carotenoids of Ramonda plants from different populations.
hogany.
4. Conclusions
The populations with north-east direction and the highest
exposition to the sunlight have lowest photosynthetic
pigment content compared to populations with less sun-
light. The lowest Chl a/b ratio is at populations with less
sunlight whereas the highest Chl/Carot ratio.
According to statistical analysis results with Duncan
test, the populations’ alignment with approximate content
of photosynthetic pigments clearly indicates the similar
ecological conditions of these populations.
There are no significant differences of photosynthetic
pigments content between populations and their eleva-
tion.
5. Acknowledgements
We would like to thank Prof. Fadil Millaku for literature
and professional collaboration.
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Ramonda nathaliae of Some Different Populations from Kosovo, Albania and Macedonia
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