Abstract. The chromosome number 2n = 30, and nuclear DNA amount 2C = 0.79 pg, are determined for the first time for Micromeria acropolitana, a rare and endangered species from the Acropolis in Athens, Greece. The plant was considered extinct but rediscovered in 2006, a hundred years later. Its current status in the original habitat is assessed, and proposals for ensuring its survival presented

Gregory Tsounis

Kit Tan

Lambros Tsounis

Sonja Siljak-Yakovlev

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201PHYTOLOGIA BALCANICA 17 (2): 201 – 204, Sofia, 2011
Genome size and chromosome number of Micromeria 
acropolitana (Lamiaceae), a steno-endemic from Greece 
Sonja Siljak-Yakovlev1, Kit Tan2, Gregory Tsounis3 & Lambros Tsounis3
1 Univ Paris-Sud, AgroParisTech, UMR 8079, Ecologie, Systématique, Evolution, 
Université Paris-Sud, Bâtiment 360, 91405 Orsay Cedex, France, 
e-mail : sonia.yakovlev@u-psud.fr (corresponding author)
2 Institute of Biology, University of Copenhagen, Øster Farimagsgade 2D, DK-1353 
Copenhagen K, Denmark, e-mail: kitt@bio.ku.dk 
3 Makriyanni Str. 7, Acropolis, Athens 11742, Greece, e-mail: greenapple@greenapple.gr
 Received: May 28, 2011 ▷ Accepted: July 04, 2011
Abstract. The chromosome number 2n = 30, and nuclear DNA amount 2C = 0.79 pg, are determined for the first 
time for Micromeria acropolitana, a rare and endangered species from the Acropolis in Athens, Greece. The 
plant was considered extinct but rediscovered in 2006, a hundred years later. Its current status in the original 
habitat is assessed, and proposals for ensuring its survival presented.
Key words: Acropolis, chromosome number, conservation, Micromeria, nuclear DNA content
Introduction
Micromeria acropolitana Halácsy belongs to the fam-
ily Lamiaceae, subfam. Nepetoideae, tribe Mentheae 
and subtribe Menthinae (Bräuchler & al. 2008). Its lo-
cus classicus is the famed archaeological site of Greece, 
the Acropolis of Athens where it inhabits both the nat-
ural limestone rock of the hill and the quarried marble 
used for the buildings. It was considered extinct when 
it had not been sighted for a hundred years. It was re-
discovered in 2006, a hundred years later (Tan, Kit & 
al. 2010). The species is endangered as the population 
size is small and the habitat in a densely populated ar-
ea of urban activity. Thousands of visitors invade the 
site at the height of the tourist season including 11,000 
people daily during July 2010 when the new Acropolis 
Museum was opened. The Acropolis is the great his-
toric monument to Greek civilization and regarded as 
part of the Greek heritage. In order to protect both the 
monument and Micromeria acropolitana, the one and 
only endangered endemic plant of the Acropolis, we 
realised the biology of species should be better known. 
Various studies were carried out and the chromosome 
number and genome size determined for the first time 
in this paper.
Material and methods
Chromosome preparation
Seeds were collected from a natural population on the 
Acropolis, Athens. They were laid on moist filter paper 
in Petri dishes and kept at 28 °C in a thermostat germi-
nator. The root tip meristems were removed from ger-
minated seedlings and treated with 8-hydroxy-quin-
oline solution for 3 h at 16 °C, and subsequently fixed 
in fresh ethanol:acetic acid (3:1) before storing at 4 °C 
for 24–48 h. The meristems were stained in Schiff ’s re-
agent after hydrolysis in 1 N HCl for 12 min at 60 °C, 
and squashed in a drop of acetic carmine following 
Östergren & Heneen’s technique (1962). Chromosome 
counts were made on well-spread meta phase plates 
from a minimum of 10 seedlings and approximately 10 
202 Siljak-Yakovlev, S. & al. • Genome size and chromosome number of Micromeria acropolitana
cells per slide. The slides were analyzed using a Zeiss 
Axiophot microscope coupled with a highly sensitive 
CCD camera (RETIGA 2000R; Princeton Instruments, 
Every, France) and an image analyser (Metavue, Eve-
ry, France).
Flow cytometry for 2C DNA value
The DNA amount was determined by flow cytometry 
following the technique of Marie & Brown (1993). The 
leaves of the Micromeria and an internal standard (Pe-
tunia hybrida (Hook) Vilm. cv. 'PxPC6'; 2C =  2.85 pg) 
were chopped together using a razor blade in Gal-
braith buffer (Galbraith & al. 1983) containing 0.1 % 
(w/v) Triton X-100 with addition of fresh 10 mM sodi-
um metabisulphite and 1 % polyvinylpyrrolidone. Nu-
clei were stained with 30 μg/ml propidium iodide (Sig-
ma Chemical Co. St. Louis, USA), a DNA intercalating 
dye used after RNAse treatment (2.5 units/ml; Roche). 
At least 5 individuals per population and 5000 to 10,000 
nuclei per individual were analysed on a flow cytome-
ter (CyFlow SL3, Partec, Munster, Germany) to obtain 
the mean DNA content. The 2C DNA value was calcu-
lated using the linear relationship between the fluores-
cent signals from stained nuclei of Micromeria leaves 
and the internal standard.
Results and discussion
Due to an excellent 100 % germination success the chro-
mosome number 2n =  30 was determined in ten seed-
lings. This number was stable in each meristem. The 
chromosomes are small in size (1–2 μm). One pair with 
secondary constrictions bearing satellites was observed 
(Fig. 1A & B, arrows). The observed chromosome 
number (2n =  30) is the more frequently encountered 
one in the genus Micromeria. The number 2n =  20 has 
been reported for M. thymifolia (Scop.) Fritsch, and M. 
dalmatica Benth. (Papes & Silic 1981) and 2n =  60 for 
M. graeca (L.) Benth. (Luque & Lifante 1991) and M. 
fontanesii Pomel (Vogt & Oberprieler 1994).
The DNA amount of M. acropolitana was 2C =  0.79 
(± 0.02) pg (Fig. 1C) which represents the very small 
genome size (1C ≤ 1.40 pg) according to the classifica-
tion of Leitch & al. (1998). 
Despite the size and great economic importance of 
the Lamiaceae (225 genera and 6170 species accord-
ing to Wielgorskaya 1995) there is not much data con-
cerning chromosome number or genome size of the 
member species. In the Plant DNA C-values Database 
of Bennett & Leitch (http://data.kew.org/cvalues/, lat-
est release 5.0, December 2010) the DNA content for 
the Lamiaceae was only presented for 46 species be-
longing to 19 genera. The genome size (2C DNA) rang-
es from 0.55 pg for Nepeta teydea (Suda & al. 2003) 
to 12.47 pg for Stachys grandiflora (Barow & Meister 
2003). Only five out of 221 known species of the genus 
Micromeria have been measured up to now, and their 
genome size ranges from 0.72 pg for Micromeria hys-
sopifolia Webb & Berthel. to 0.88 pg for M. glomera-
ta P. Pérez (Suda & al. 2003). The value of M. acropo-
litana (2C =  0.79) is intermediate. Two other species 
with 2n =  30 (M. lachnophylla Webb & Berthel. and 
M. varia Benth.) presented 2C DNA values of 0.74 and 
Fig.1. A & B, Micromeria acropolitana: Two mitotic metaphases showing 2n =  30 with one pair of satellite chromosomes (arrowed); 
C, Histogram of nuclear DNA content of M. acropolitana and internal standard Petunia hybrida. Th eir nuclei form two distinct peaks 
whose positions indicate relative nuclear DNA content. 
A B
10 μm
100
80
60
40
20
0
C
N
uc
le
i n
um
be
r
Propidium Iodide intensity
Micromeria
Gate: R1
FL2 IP
Petunia
0                             100                           200                           300                          400                          500
A B
C D
203Phytol. Balcan. 17(2) • Sofia • 2011 
Fig. 2. A, Micromeria acro-
politana growing in natural 
rock of the Acropolis; B, Plant 
in crevice of large limestone 
block (porolithos, not natural 
rock); C, Plant in crack formed 
by porolithos and Pendelis 
marble; D, ripe seeds.
but also in wall crevices and marble blocks of the 
temples and other monuments. Here, we must make 
mention of the habitat and substrate. The sacred 
hill of the Acropolis was never quarried, the lime-
stone for the buildings and walls was cut from sever-
al of the adjacent hills or from Piraeus, Mt Hymettos 
and the island of Aegina. The famous white marble 
of the buildings came from Mt Pendeli to the north-
east. The large limestone blocks forming the walls of 
the monuments are not natural rock but porolithos. 
In Fig. 2C, a plant is 
shown growing be-
tween a crack formed 
by porolithos and mar-
ble. Plants thrive in 
such habitats, in wall 
crevices and broken 
marble with only a lit-
tle soil. Fig. 2A shows 
a plant growing on the 
natural rock of the hill 
of Acropolis which is 
Late Cretaceous lime-
stone overlying marl 
and sandstone, and 
Fig. 2B, a plant on po-
rolithos.
0.75 pg respectively (Suda & al. 2003). Unfortunately, 
no data for the chromosome number and genome size 
of four closely related species (M. microphylla Benth., 
M. sphaciotica Boiss. & Heldr., M. carpatha Rech., and 
M. hispida Boiss. & Heldr.) were available for compar-
ison which would be very useful.
Additional plant sites on the Acropolis
Since the publication of the rediscovery several new 
sites have been located, not only on the natural rock 
6 • Phytol. Balcan. 17(2) • 2011 
204 Siljak-Yakovlev, S. & al. • Genome size and chromosome number of Micromeria acropolitana
An increase in size of the populations
In June 2009 we noted c. 150 plants and in July 2010, 
after plentiful and prolonged winter rains we esti-
mated that the total number of plants on the Acrop-
olis must be at least 400. At the time of sending this 
paper to press (May 2011) we counted c. 450 plants. 
The populations have not only remained steady but 
have shown a slight increase in numbers of individ-
uals. The plant was first discovered in flower on 30 
August 1906 by Maire and Petitmengin, two French 
botanists and explorers. 2011 was an unusual year as 
the plants flowered in February instead of May and 
June. From floristic notes kept by resident amateur 
botanists there is a tendency for species to come in-
to flower earlier and earlier over a period of years, 
e.g., Crocus beginning to flower in late December 
instead of the usual February or March. Seed set of 
M. acropolitana was good in 2011 despite unsettled 
weather in the late spring with almost daily “tropi-
cal” rain.
Conservation measures taken 
Seed from various populations on the natural rock 
and on the quarried limestone and marble blocks 
(Fig. 2D) has been collected and stored at the Insti-
tute of Biology, University of Copenhagen at a room 
temperature of 20 °C.
Conservation measures proposed
Clearly, the greatest threat within the archaeological 
site is human disturbance.
Thus the populations should be periodically 
moni tored.
The areas where the plants are most at risk of be-
ing trodden by visitors should be fenced off or sur-
rounded. Bilingual signs in Greek and English should 
be displayed to prohibit the collection of plants and 
animals, e.g., butterflies.
Certain areas of the stonework not in the direct eye 
of the public should undergo less thorough spring-
cleaning so as not to eradicate established plants. The 
presence of plants, soil and loose material help retard 
water run-off and should not be removed.
A small rock garden with established Micromer-
ia populations can be created near the entrance of 
one of the theatres. In this way all visitors can see the 
rare endemic plant of the Acropolis without disturb-
ing the wild populations.
Acknowledgements. The authors gratefully acknowledge M. 
Bourge and S. Brown for their assistance on the cytometry Imag-
if Platform of the IFR87 La plante et son environnement, Gif-sur-
Yvette, France.
References
Barow, M. & Meister, A. 2003. Endopolyploidy in seed plants 
is differently correlated to systematics, organ, life strategy and 
genome size. – Pl. Cell Environm., 26: 571-584.
Bräuchler, C., Ryding, O. & Heubl G. 2008. The genus Micromeria 
(Lamiaceae), a synoptical update. – Willdenowia, 38: 363-410.
Galbraith, D., Harkins, K., Maddox, J., Ayres, N., Sharma, D. & 
Firoozabady, E. 1983. Rapid flow cytometric analysis of the cell 
cycle in intact plant tissues. – Science, 220: 1049-1051.
Leitch, I.J., Chase, M.W. & Bennett, M.D. 1998. Phylogenetic 
analysis of DNA C-values provides evidence for a small ances-
tral genome size in flowering plants. – Ann. Bot., 82 (Suppl. A): 
85-94.
Luque, T. & Lifante, Z.D. 1991. Chromosome numbers of plants 
collected during Iter Mediterraneum I in the SE of Spain. – 
Bocconea, 1: 303-364. 
Marie, D. & Brown, S.C. 1993. A cytometric exercise in plant 
DNA histograms with 2C values for 70 species. – Biol. Cell, 78: 
41-51.
Östergren, G. & Heneen, K.W. 1962. A squash technique for chro-
mosome morphological studies. – Hereditas, 48: 332-341.
Papes, D. & Silic, C. 1981. In: Chromosome number reports 
LXX. – Taxon, 30: 70. 
Suda, J., Kyncl, T. & Freiova, R. 2003. Nuclear DNA amounts in 
Macaronesian angiosperms. – Ann. Bot., 92: 153-164.
Tan, Kit, Tsounis, G. & Tsounis, L. 2010. Micromeria acropolitana 
(Lamiaceae) rediscovered in Athens (Greece). – Phytol. Balcan., 
16: 237-242. 
Vogt, R. & Oberprieler, C. 1994. Chromosome numbers of North 
African phanerogams. IV. – Candollea, 49: 549-570. 
Wielgorskaya, T. 1995. Dictionary of generic names of seed 
plants. Consulting Editor Takhtajan A. Columbia Univ. Press, 
New York.


Genome size and chromosome number of Micromeria acropolitana (Lamiaceae), a steno-endemic from Greece

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Genome size and chromosome number of Micromeria acropolitana (Lamiaceae), a steno-endemic from Greece

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