PHYTOLOGIA BALCANICA 19 (2): 201 – 208, Sofia, 2013
Breeding systems of Haberlea rhodopensis (Gesneriaceae), a
Tertiary relict endemic to the Balkan Peninsula
Katerina Bogacheva-Milkoteva1, Ekaterina Kozuharova1, Regine Claßen-Bockhoff 2
1 Department of Pharmacognosy, Faculty of Pharmacy, Medical University of Sofia,
2 Dunav Str. 1000 Sofia, Bulgaria, e-mail: email@example.com, e-mail: ina_
firstname.lastname@example.org (corresponding author)
2 Johannes Gutenberg-University, Institut für Spezielle Botanik, 55099 Mainz, Germany,
Received: May 26, 2013 ▷ Accepted: July 28, 2013
Abstract. This study presents the preliminary results on sexual reproduction of Haberlea rhodopensis, Gesneriaceae – a
Tertiary relict and endemic species to the Balkan Peninsula. Our experiments have shown that H. rhodopensis
is self-compatible, but not autogamous. Phenology of the populations, herkogamy and weak protrandry are
mechanisms which favour the outcrossing in natural populations. Pollination success is characterized by
high seed production. Seeds germinate readily, but grow extremely slowly and few of them survive to adult
plants in culture and in natural populations.
Key words: breeding systems, floral mechanism, Haberlea rhodopensis, phenology, seed set
Haberlea rhodopensis Friv. (Gesneriaceae) is a Ter-
tiary relict and Balkan endemic. In Europe, there
are only three genera in the family: Haberlea (in
Bulgaria and Greece), Ramonda (Balkans and Ibe-
rian Peninsula) and the monotypical Jankaea (in
Greece) (Ganchev 1950).
Haberlea rhodopensis is a medicinal plant, list-
ed in the Bulgarian Medicinal Plants Act (2000).
It is also an amazing resurrection plant; therefore,
recently most investigations have focussed on its
desiccation tolerance mechanisms and potential
for multipurpose usage. Efforts at conservation of
Haberlea rhodopensis are based mainly on in vitro
cultures, ex situ collections and habitat exploring
(Djiljanov & al. 2009). The plant propagates vege-
tatively and it is possible to cultivate it from seeds
(Ganchev 1950; Djiljanov & al. 2005), but there are
no studies on its breeding system.
The aim of our study was to throw light on the
breeding systems of Haberlea rhodopensis: 1) to test
outcrossing of the species and its mechanism; 2) to
test seed germination and early stages of ontogen-
esis; 3) to compare native populations and plants
grown ex situ.
Material and methods
Material and methods
We have used plant material collected from five
natural localities in Central and Eastern Rhodopi
Mts, Bulgaria and an ex situ collection from the Bo-
tanical Garden at Johannes-Gutenberg University
of Mainz, Germany. Observations were conducted
in 2008–2010 and 2012, during the flowering pe-
202 Bogacheva-Milkoteva, K. & al. • Breeding systems of Haberlea rhodopensis
riod from April to July. For laboratory works, we
used the Leitz Diaplan light microscope with flu-
orescence equipment, Binokular Leica MZ 16A,
Fuchs-Rosenthal counting chamber, Philips XL 30
scanning electron microscope (SEM), and Leica
Application Suite software and Image Tool MT 3.00
for photographs, pollen and ovule counting. The
authors have followed the manufacturers’ instruc-
tions and standard protocols.
Phenology. In the flowering period (April–July) of
2008–2010 and 2012, in situ (natural populations)
and ex situ observations have been carried out at
different altitudes and slope exposures. Life du-
ration of free and bagged-flower plants was com-
pared. For the purpose, two sites were chosen with
different sun exposure, so as to trace out any dif-
ferences in phenology, according to the autecolog-
Breeding systems. In order to test autogamy, the
protocols of Dafni (1992) were considered and con-
sequently were bagged: a) a total of 123 flowers of
29 plants for spontaneous self pollination, b) 38
hand-selfed flowers from six plants, c) four hand-
crossed flowers from four plants, and d) 10 untreat-
ed control flowers from three plants. Plant rosettes
were transferred to the experimental plot of the
Faculty of Pharmacy at different time of the year. To
test ability of propagation by leaves, 10 leaves were
put into a light soil mix. Ovaries and anthers were
sampled from three different sampling sites: Beden
village in 2008 (Central Rhodopi), river Borovitsa
in 2010 (Eastern Rhodopi), Ustovo district in 2010
(Central Rhodopi), and the Botanical Garden of
Mainz University, Germany in 2012. Pollen grains
and ovules were counted and calculations were per-
formed according to the standard methodology
(Cruden 1977; Daphni 1992).
Floral mechanism. To investigate dichogamy
and/or herkogamy, we have dissected and photo-
graphed flowers in different stages.
Stigma receptivity. To evaluate changes on the stig-
ma surface indicating receptivity, we have collected
stigmas from flowers in different phases of develop-
ment, and photographed them with SEM.
Pollen tube growth. Six flowers in bud stage were
bagged and hand-selfed. We sampled the stigmas 3,
4 and 20 hours after pollination and have observed
the pollen tube growth under a fluorescence micro-
Pollination success. Seed set per fruit of a random
sample in plants from different sampling sites was
Seed germination and mycorrhiza. Fifty fresh
seeds from the Ustovo district and the Botanical
Garden of Mainz and 50 two-year old seeds from
Yagodina and Mugla villages were placed in Petri
dishes on wet filter paper, or directly in soil, with-
out additional treatment. Three weeks later the
two-year old seeds were scarificated. Squash micro-
scopic preparations of roots were made following
the standard method, so as to test the plant for my-
Results and discussion
Results and discussion
Phenology. Anthesis is most prolonged in the natu-
ral populations (22 days) and the shortest in the shady
population from the Botanical Garden (12 days). The
exposed to sunshine ex situ population had an 18-days
anthesis. In spite of the same altitude, the anthesis in
the shady population of the Botanical Garden began
a week later, but ended at the same time. Compared
with the natural habitat at 800 m, anthesis in the ex si-
tu population had begun a month earlier at about 100
m. A comparison of the results in the natural habitat
for the years 2010 and 2012 has shown that in 2010
anthesis began on 15th May, and in 2012, after a very
long and cold winter, it began on 24th May (Fig. 1).
Life duration of a flower was prolonged between 7–10
days and no difference was traced out between free
and bagged flowers. Observation also showed that the
flowers in inflorescence of one individual were in dif-
ferent phases of flowering, as well as the individuals in
On 24th August 2008, secondary flowering was ob-
served in the area of Chudnite Mostove (Central Rho-
dopi, at 1500 m). The flowering buds were laid in au-
tumn and were preserved in winter time.
203Phytol. Balcan. 19(2) • Sofia • 2013
Fig. 1. Comparative phenology of three sites: a) population exposed to sunshine from the Botanical Garden of Mainz University, 100 m a.s.l.;
b) population in shadow from the Botanical Garden of Mainz University; c) natural population in Ustovo district, Central Rhodopi , 800 m a.s.l.
youn g flowers
204 Bogacheva-Milkoteva, K. & al. • Breeding systems of Haberlea rhodopensis
Breeding system. Haberlea rhodopensis is a long-
living perennial (Plate 1, Fig. 1). Plants of the white
variety ('Virginalis') are grown in the UK since
1938, and still grow strongly in the course of 75
years. This corresponds to the reports concerning
the age of plants from the sister species Ramonda
myconi. Data on the accumulated individual growth
in size (number of leaves and rosette diameter) sug-
gest that under the current environmental condi-
tions the time from germination to attainment of
the minimum size for reproduction is ca. 70 years.
Similarly, the estimated age of a plant with 11 leaves
and rosette diameter of 12 cm (the median size of
plants in all the regions) is 200–250 years (Dubreuil
& al. 2008).
Haberlea rhodopensis has good ability for vege-
tative propagation (Plate 1, Fig. 1, Plate 2, Fig. 1).
In situ the big plants shoot thin pale-yellow hori-
zontal rhizomes, running under the moss around
the plants, and bear small rosettes. Such rhizomes
have never been seen in cultivated plants, however,
it was certainly easy to divide the large plants and
grow up many of the divisions ex situ. Even some ex
situ propagation by leaf was possible: three of the
leaves produced daughter rosettes, although they
remained extremely small and did not develop in-
to big plants. Plants transferred in spring and early
summer, especially if taken with the moss cushion,
were doing very well ex situ (Plate 1, Fig. 4, On the
other hand, if the plants were transferred in the au-
tumn they had zero ability to survive.
The flowers bear features of bee pollination syn-
drome (Plate 1, Fig. 2, Plate 2, Fig. 2,). The tested
flowers did not self-pollinate spontaneously. None
of the 123 bagged flowers tested for spontaneous
self-pollination set seeds. There was no self incom-
patibility, as 16 flowers (50 %) of the hand-selfed
ones produced seeds. The control hand-crossed
four flowers (100 %) produced seeds, which have
proved that bagging does not effect the process of
pollination and fertilization. Eight of the ten un-
treated flowers set seeds (80 %).
An alternative method for testing the breed-
ing system by calculation of P/O ratio correspond-
ed partially to the field tests. Interestingly, differ-
ent values of pollen-to-ovule ratio were recorded
for the different sampling sites: Beden village 308:1,
river Borovitsa 94:1, Ustovo district 141:1, and
Mainz Botanical Garden 141:1. Following Cruden`s
classification of the breeding system P/O ratio, var-
ying in the populations between 94:1 and 308:1, in-
dicates an obligate to facultative autogamy (Cruden
1977). If taken into account that pollination success
is not dependent solely on pollen limitation, but is
due to sex allocation, it could be maintained that
the increased number of ovules is not just an auto-
gamy indicator, but an attempt at ensuring a maxi-
mum seed set (Etcheverry & al. 2012).
Pollen tube growth. Our results show that self pol-
len germinates on the stigma of the same individu-
al. Three hours after pollination, pollen germina-
tion is at its very beginning. After four hours the
pollen tubes can be clearly distinguished and twen-
ty hours after pollination the pollen tubes reach
about 2–3 mm in the pistil (Plate 1, Fig. 6).
Floral mechanism. In our opinion, outcrossing was
favoured under natural conditions: weak protandry
was observed and strong herkogamy, as mecha-
nisms which support outcrossing. In the bud stage,
the anther length exceeded the length of the pistil
and they were situated above the stigma (Plate 1,
Figs 3, 5). In many cases, the extrorse pollen-shed-
ding anthers opened in the bud stage, thus self pol-
len deposition on the stigma was possible, but at
that time the stigma was not receptive.
Stigma receptivity. Changes in the stigma area in-
dicating receptivity are visible on the microscopic
pictures 2–3 days after the flower opening (Plate 2,
Figs 5, 6). During bud development the pistil grows
and, when the flower opens, the pistil length ex-
ceeds the length of anthers. Anthers remain fas-
tened in pairs about 3 mm under the stigma level
on the pistil.
Pollination success. Eighty percent of the random-
ly collected fruits for estimation of the free pollina-
tion success had seeds. Sixteen percent of the flow-
ers had not been pollinated and four percent were
predated. Seed set in a fruit of a random sample
showed that on the average 70 % of the ovules per
fruit have matured to seeds (Fig. 2).
Obviously, when self pollen reaches the receptive
stigma, pollination starts and seeds are formed, but
a pollen-transporting agent is needed. The relative-
ly high fruiting success of the control flowers shows
205Phytol. Balcan. 19(2) • Sofia • 2013
Seed set per fruit
428,2 632,8 507,5
ovules damaged seeds
Fig. 2. Seed set per capsule of random
samples from four di erent natural popu-
lations in the Central Rhodopi Mountains.
that pollination is efficient, but bagging experiments
reveal that Haberlea is self-compatible, though not
generally autogamous. Our results correspond to the
data obtained for the related species Jankaea heldre-
ichii (Vokou & al. 1990) in Greece, which also pro-
duces a high number of seeds per capsule and is not
autogamous, as well as for Ramonda myconi on the
Iberian Peninsula (Pico & Riba 2002).
Seed germination and mycorrhiza. The seeds
of H. rhodopensis readily germinate after 7–12
days. Seedlings grow extremely slowly, reaching a
length of less than 4 mm in five months as shown
in Plate 2 Fig. 6. After five months, less than 1 %
of the seeds survived. Two-year old seeds did not
germinate, even after being scarificated for a week.
The root microscope preparations clearly show the
structures, which indicate the mycorrhiza (Plate 2,
Fig. 4). Mycorrhizas have been observed also in the
related species R. serbica (Rakic & al. 2009).
Observations of the natural populations have
shown that they are dominated by adult plants.
Young plants were very few and no seedlings could
be found. Demographic studies of the sister species
Ramonda myconi are in concordance with the pre-
sent study: populations of this species exist due to
the adult, well-adapted plants (Pico & Riba 2002).
Young plants of H. rhodopensis seldom survive, so
vegetative reproduction and preservation of the ex-
isting plants is of extreme importance for conserva-
tion of the species. In spite of the desiccation toler-
ance of adult plants, it is not certain that seedlings
are desiccation tolerant. Thus drought may be a
limiting factor in nature. Another possible explana-
tion is the lack of suitable surfaces, where the seed-
lings can get established, supposedly as in the case
of R. myconi (Pico & Riba 2002) – the steep, rocky
slopes are often flooded. Taking into account that
the species is self compatible, other possible expla-
nation of its low survival rate can be potential in-
breeding depression. This could be regarded as a di-
rection for future studies. The low survival rate of
the seedlings in culture could be explained by the
lack of symbiotic fungi.
Although the populations are not endangered pres-
ently and the species is categorized as Least Concern
(Petrova & Vladimirov 2009), picking of adult plants
is the main hazard factor for the balance in the popu-
lations. Ex situ collections and in vitro cultures are ex-
tremely important for investigation, but future stud-
ies should also include aut- and sinecological factors,
such as pollinating agents, symbiotic or antagonistic
relationships, demographic structure of the popula-
tions, possible genetic sequences of self-compatibility,
and effectiveness of the mechanisms preventing auto-
gamy. The protection status of Haberlea rhodopensis
should also correspond to the tendencies in the devel-
opment of natural populations.
206 Bogacheva-Milkoteva, K. & al. • Breeding systems of Haberlea rhodopensis
Plate I. Fig. 1. Typical microhabitat of Haberlea rhodopensis; Fig. 2. e owers bear features of bee pollination syndrome; Figs 3 and
5. Floral mechanism – weak protandry and strong herkogamy; Fig. 4. Plants transferred in spring and early summer, especially if taken
with the moss cushion, were doing very well ex situ; Fig. 6. Pollen tube growth.
207Phytol. Balcan. 19(2) • Sofia • 2013
Plate II. Fig. 1. Haberlea rhodopensis has good ability for vegetative propagation; Fig. 2. e owers bear features of bee pollination
syndrome; Fig. 3. Seedling of age about four ve months; Fig. 4. Root microscope preparations clearly show the structures, which indi-
cate the mycorrhiza; Figs 5 and 6. Changes in the stigma area indicating receptivity are visible on the microscopic pictures.
208 Bogacheva-Milkoteva, K. & al. • Breeding systems of Haberlea rhodopensis
Acknowledgements. The study was conducted with the fi-
nancial support of the Deutsche Bundesstiftung Umwelt project,
Medical University of Sofia and Johannes Guthenberg University
of Mainz. The authors extend special thanks to Prof. A.J. Richards
for the interest in their work and his valuable comments.
Bulgarian Medicinal Plants Act 2000. Promulgated, SG
No. 29/7.04.2000, Amended, SG No. 23/1.03.2002, 91/25.09.2002
Cruden, W. 1977. Pollen-ovule ratios: a conservative indicator of
breeding systems in flowering plants. – Evolution, 31(1): 32-46.
Dafni, A. 1992. Pollination Ecology : A Practical Approach. Oxford
Djiljanov, D., Genova, G., Parvanova, D., Zapryanova, N.,
Konstan tinova, T. & Atanassov, A. 2005. In vitro culture of the
resurrection plant Haberlea rhodopensis. – Pl. Cell Tissue Organ
Cult., 80(1): 115-118.
Djiljanov, D., Ivanov, S., Georgieva, T., Monyakova, D., Berkov, S.,
Petrova, G., Mladenov, P., Christov, N., Hristozova, N., Peshev,
D., Tchorbadjieva, M., Alexieva, V., Tosheva, A., Nikolova,
M., Ionkova, I. & van den Ende, W. 2009. A holistic approach
to resurrection plants. Haberlea rhodopensis – a case study. –
Biotechnology & Biotechnological Equipment, 23(4):1414-1416.
Dubreuil, M., Riba, M. & Mayol, M. 2008. Genetic structure and
diversity in Ramonda myconi (Gesneriaceae): effects of historical
climate change on a pre-Glacial relict species. – Amer. J. Bot.,
Etcheverry, A., Aleman, M., Figueroa-Fleming, T., Lopez-Spahr,
D., Gomez, C., Yanez, C., Figueroa-Castro, D. & Ortega-Baes, P.
2012. Pollen:ovule ratio and its relationship with other floral traits
in Papilionoideae (Leguminosae): an evaluation with Argentine
species. – Pl. Biol, 14(1): 171-178.
Ganchev, I. 1950. Anabiotic desiccation resistance and other
biological traits of Haberlea rhodopensis Friv. – Izv. Bot. Inst.
(Sofia), 1(1): 191-214.
Petrova, A.& Vladimirov, V. (eds) 2009. Red list of Buglarian
vascular plants. – Phytol. Balcan., 15(1): 63-94.
Pico, F. & Riba, M. 2002. Regional-scale demography of Ramonda
myconi: Remnant population dynamics in a pre-Glacial relict
species. – Pl. Ecol., 161(1): 1-13.
Rakic, T., Quartacci, M F., Cardelli, P., Navari-Izzo, F. & Ste-
fa no vic, B. 2009. Soil properties and their effect on water and
mineral status of resurrection plant Ramonda serbica. – Pl. Ecol.,
Vokou, D., Petanidou, Th. & Bellos, D. 1990. Pollination ecology
and reproductive potential of Jankaea heldreichii (Gesneriaceae):
a tertiary relict on Mt Olympus, Greece. – Biol. Conservation,