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Journal of Arid Environments 119 (2015) 27e30
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Journal of Arid Environments
journal homepage: www.elsevier.com/locate/jaridenv
Short communication
In situ study of deep roots of Capparis spinosa L. during the dry season:
Evidence from a natural “rhizotron” in the ancient catacombs of Milos
Island (Greece)
Sophia Rhizopoulou*, Georgios Kapolas
Department of Botany, Faculty of Biology National & Kapodistrian University of Athens Panepistimiopolis, Athens 15784, Greece
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 5 June 2014
Received in revised form
22 January 2015
Accepted 18 March 2015
Available online
The growing period of the deep-rooted, winter-deciduous Capparis spinosa (caper) coincides with the dry
season, in the Mediterranean Basin. Roots of wild shrubs of C. spinosa penetrated cracks of a pumice
substrate and lengthened in the subterranean environment of the ancient catacombs of Milos Island
(Greece); therein, they reached depths of up to 20 m and exploited water present in deep soil layers.
Elevated water potential (J) was measured in root segments (Jr ¼ 0.35 ± 0.06 MPa), a few centimetres
from the root apices of C. spinosa found in the catacombs during the dry season. Also, estimates of foliar
predawn water potential and gas exchange components, as well as of nocturnal floral water potential of
C. spinosa grown on slopes above the subterranean monument were high and reflected the efficiency of
roots in retaining acquisition of water from deep soil layers, during the dry season.
© 2015 Elsevier Ltd. All rights reserved.
Keywords:
Caper
Catacombs
Deep roots
Dry season
Natural rhizotron
Water uptake
1. Introduction
The catacombs of Milos Island (24 250 3300 E, 36 440 2100 N;
altitude 70.5 m) in Greece (https://www.google.com/maps/place/
CatacombsþofþMilos) were excavated in loose, volcanic rock of
pumice tuff and consist of corridors (length: 184 m) and chambers
(width: 1.2e5.5 m and height: 1.6e2.5 m). This was a place of
worship, a burial site and a refuge in times of persecution of early
Christians (1ste6th century). The catacombs of Milos (www.
catacombs.gr/map), officially revealed in the 19th century
(Sotiriou, 1928), are among the most important discovered worldwide along with the catacombs of Rome and the Holy Land. Also, it
may be worth noting that near the catacombs and inside a buried
niche, the Venus of Milos eone of the most famous works of art of
all timee was found in the 19th century (Curtis, 2004).
The natural vegetation on slopes and abandoned terraces at
approximately 10e25 m above the catacombs (Fig. 1) consists of
Artemisia absinthium L. (Asteraceae), Capparis spinosa L. (Capparaceae), Ficus carica L. (Moraceae), Phlomis fruticosa L. (Lamiaceae),
Pistacia lentiscus L. (Anacardiaceae) and Salsola L. spp.
* Corresponding author.
E-mail address: [email protected] (S. Rhizopoulou).
http://dx.doi.org/10.1016/j.jaridenv.2015.03.010
0140-1963/© 2015 Elsevier Ltd. All rights reserved.
(Chenopodiaceae). We observed lateral spread of roots at shallow
depths in the pumice horizons, roots grown in fissures and soil-rock
interfaces and roots expanded under stones. Also, we observed
roots entering the catacombs and elongating for many meters
laterally attached to the ceiling of chambers and corridors, in
cohesiveness with the pumice medium. Roots were growing along
paths, picking their way through stony particles, decaying fibre and
the texture of the volcanic substrate (Andronopoulos and Tzitziras,
1988). It is well known that the physical characteristics of the
substrate affect the development of roots; it seems likely that root
penetration of the roof of the Milos catacombs was greatly influenced by the compaction of the pumice tuff. Roots growing over
large distances horizontally and vertically, within chambers and
corridors, most probably responding to moisture gradient and
gravity vector, eventually reached the floor of the catacombs.
2. Materials and methods
The expansion rate of ten tips of roots grown in the catacombs
was measured using a ruler and marks made with small paint
brushes.
The water potential (J) was measured in ten root segments,
collected a few centimetres above the same root apices used for
28
S. Rhizopoulou, G. Kapolas / Journal of Arid Environments 119 (2015) 27e30
Fig. 1. Schematic section of the catacombs of Milos Island (Greece), illustrating plant cover on slopes and terraces (altitude: 79e97 m) above chambers and a corridor of the
catacombs (altitude: 70.5 m), and indicating rooting profiles; the numbers represent altitude (m) above the sea level.
elongation measurements, using C-52 sample chambers (Wescor,
Inc. Logan, Utah, USA) attached to a Dew point psychrometer
microvoltmeter (Wescor, HR-33T, Inc. Logan, Utah, USA). Also,
predawn J was measured periodically on discs (6 mm in diameter)
of fully expanded leaves (three leaves per shrub at the 4th to 5th
position from shoot apex), as well as nocturnal J of expanded
flower petals (Rhizopoulou et al., 2006) collected from five shrubs
of C. spinosa L. (Capparaceae), grown on slopes approximately
18e20 m above the catacombs of Milos (Fig. 1), during the summer
flowering period of C. spinosa.
Root samples were cut from ten roots found elongating in
different sites in the catacombs of Milos, and used for elongation
and water potential measurements; the samples were carefully
transferred to the laboratory, where they were cut in pieces and
fixed in 3% glutaraldehyde in Na-phosphate buffer at pH 7, at room
temperature, for 2 h. The plant material was washed three times by
immersion in buffer for 30 min each time; then, it was post fixed in
1% OsO4 in the same buffer at 4 C for 5 h and dehydrated in acetone
solutions. Dehydrated tissues were embedded in SPURR (Serva)
resin. Semi-thin sections of embedded tissues (LKB Ultratome III
microtome) were stained in Toluidine Blue ‘0’, in 1% borax solution,
photographed and digitally recorded using a Zeiss Axioplan light
microscope (Carl Zeiss Inc., Thornwood, N.Y.) equipped with a
digital camera (Zeiss AxioCam MRc5).
Net CO2 assimilation and transpiration were obtained from ten
fully expanded leaves of selected from shrubs of C. spinosa grown
above the catacombs of Milos (Fig. 1) using a portable gas analyzer
(Li-6400, Li-Cor Inc., Lincoln, NE, USA) equipped with a light source
(6200-02B LED, Li-Cor), at midday, throughout July. Conditions in
the leaf cuvette consisted of a photosynthetic photon flux density of
1600 mmol m2 s1, leaf temperature was maintained at 25 C, leaf
to air water vapour pressure deficit was kept <1.1 kPa, and the
ambient CO2 concentration was 340 mmol mol1 air; measurements
were made after steady state conditions were obtained, which
required around 2 min (Meletiou-Christou and Rhizopoulou, 2012).
Soluble sugars were extracted from the dried (48 h at 80 C),
powdered plant material collected from shrubs of C. spinosa grown
on the slopes above the catacombs of Milos, as well as from roots
expanded within the catacombs, and used for physiological an
anatomical estimates, and were quantified in triplicate samples,
according to the colorimetric method of Dubois et al. (1956); Dglucose (Serva) solutions were used for the standard curve.
Values of monthly precipitation and air temperature were obtained from a meteorological enclosure in the research site, over 15
years. Microclimatic variables in the indoor air of the hypogeal
network of the catacombs of Milos were measured using a portable
Li-6400 system (Li-Cor, Lincoln, NE, USA), throughout a year.
3. Results and discussion
In the indoor air of the hypogeal network of the catacombs of
Milos elevated CO2 concentration (0.05e0.07 %), enhanced relative
humidity (60e80 %) and temperatures in the range of 15e25 C
were detected.
S. Rhizopoulou, G. Kapolas / Journal of Arid Environments 119 (2015) 27e30
29
Fig. 2. Representative light micrographs of transverse sections of roots (left) grown in the catacombs of Milos and (right) belonging to wild shrubs of C. spinosa according to Psaras
and Sofroniou (1999).
We identified those roots grown in the dark and humid microenvironment of the catacombs as belonging to C. spinosa L. (Fig. 2),
based on quantitative features of root xylem and vessel distribution
(Psaras and Sofroniou, 1999; Selmeier, 2005; Gan et al., 2013). It is
likely that quantitative traits of C. spinosa such as vessel diameters
are highly diagnostic (Selmeier, 2005). The mean diameter of root
vessels was approximately 90 ± 6 mm; the conducting capacity of a
capillary is proportional to the 4th power of its radius (HagenPoiseuille law) (Zimmermann, 1983). The conducting elements of
the well developed root xylem constitute a pathway for longdistance water transport and may be considered an adaptation of
C. spinosa to the arid environment (Rhizopoulou, 1990; Gan et al.,
2013). The xylem reflects the root segment history, i.e. its development in relation to the environment (Lobet et al., 2014).
C. spinosa (caper) is a winter-deciduous, herbaceous, perennial,
branched bush growing close to the ground and in crevices and
walls, creeping along steep rocky cliffs and stony slopes, in dry and
arid environments (Sozzi, 2001), and widely distributed in arid and
semi-arid landscapes in the Mediterranean region and adjacent
areas. The pickled flowers buds of this species are well known as
caper appetizers and the commercial spice caper. Although water
scarcity limits plant productivity in the Mediterranean region
during summer, the growth of the above ground tissues and the
completion of the life-cycle of C. spinosa (from May to September)
coincide with the dry season (Rhizopoulou and Psaras, 2003; Gan
et al., 2013), which is considered the most stressful for plant life
in this region. In particular, during the study period the average
daily mean air temperature and average monthly rainfall was
24 ± 2 C and 2 ± 0 mm in June, 25 ± 5 C and 0 ± 0 mm in July, and
27 ± 4 C and 1 ± 0 mm in August, respectively. The vegetative
activity of C. spinosa starts by forming new expanding stems and
leaves in the beginning of the dry period (Rhizopoulou et al., 1997),
i.e. in May, when average daily mean air temperature and monthly
rainfall are 22 ± 8 C and 13 ± 3 mm, respectively. The aboveground parts of C. spinosa wilt in the beginning of the wet period
in October, when average daily mean air temperature and monthly
rainfall are 18 ± 2 C and 43 ± 4 mm, respectively. Another striking
feature of C. spinosa is the nocturnal, large, short-lived flowers that
expand during the summer drought (Rhizopoulou et al., 2006;
Chimona et al., 2012).
Elevated values of water potential (J) were obtained from root
segments (Jr ¼ 0.35 ± 0.06 MPa) located a few centimetres from
the root apices of C. spinosa found in the catacombs, during the dry
season (Table 1). Also, although roots expanding in the catacombs
cannot be directly linked with aboveground plant tissues, predawn
J of fully expanded leaves (Jl ¼ 1.18 ± 0.05 MPa) and nocturnal J
of expanded flower petals (Jf ¼ 0.82 ± 0.03 MPa) from shrubs of
C. spinosa (Table 1) growing on slopes approximately 18e20 m
above the catacombs of Milos (Fig. 1) indicated access to water
sources.
The expansion rate of root tips of C. spinosa growing in the
catacombs was found to be approximately 2.5 mm h1, during the
drought summer season (Table 1). Also, enhanced soluble sugar
content was estimated in roots (18 mg g1 d.w.) collected from the
catacombs, as well as in leaves (53 mg g1 d.w.) and shoots
(30 mg g1 d.w.) of shrubs of C. spinosa growing above the catacombs (Table 1). The expansion of plant tissues depends on carbohydrate supply and sugar translocation from source (i.e. distant
photosynthetic tissues) to sink indicates transport activity (Yili
et al., 2006); increased allocation to roots will increase the probability of reaching deeper water stores necessary for survival
through a dry period.
Transpiration and net CO2 assimilation rates (Table 1) of the
adaxial (8.2 mmol m2 s1 and 17.5 mmol m2 s1, respectively) and
the abaxial (5.6 mmol m2 s1 and 11.8 mmol m2 s1, respectively)
leaf surfaces of C. spinosa grown above the catacombs are consistent
with earlier results (Rhizopoulou et al., 1997; Levizou et al., 2004;
Gan et al., 2013).
Plant rooting depth and functional root morphology are keyelements of plant response to water deficit conditions (White
et al., 2013). It is likely that long roots of C. spinosa, extending
deep into the catacombs of Milos (total length > 50 m) and reaching
depths of approximately 20 m below the above-ground plant parts
(Fig. 1), tap water from very deep soil layers, enabling the plant to
Table 1
Water potential, root elongation rate, soluble sugar content and gas exchange traits in tissues of shrubs of Capparis spinosa grown in the catacombs of Milos Island.
Parameters
Water potential (MPa)
Elongation rate (mm h1)
Soluble sugars (mg g1 d.w.)
Roots
0.35 ± 0.06
2.50 ± 0.20
18.00 ± 0.60
Shoots
30.00 ± 0.40
Leaves
Petals
1.18 ± 0.05
0.82 ± 0.03
53.00 ± 0.80
Leaf surface
Net CO2 assimilation (mmol m2 s1)
Transpiration (mmol m2 s1)
Adaxial
17.5 ± 0.40
8.2 ± 0.80
Abaxial
11.8 ± 0.60
5.6 ± 0.30
30
S. Rhizopoulou, G. Kapolas / Journal of Arid Environments 119 (2015) 27e30
sustain leaf and floral water demand during the prolonged dry
period. C. spinosa has evolved adaptations to water stress during
the progressive drought, as the soil water reserve is not refilled
during its growing season, and is gradually restricted to deeper soil
layers. Roots of wild shrubs of C. spinosa, initially tracing their way
through upper soil horizons, penetrated cracks in pumice substrate
and exhibited horizontal and vertical spread in the catacombs of
Milos, allowing plants to effectively exploit larger volumes of the
pumice substrate and facilitating acquisition of water from deep
soil layers.
Despite recent technological advances and innovations, the
study of deep roots in natural ecosystems is still fragmentary and
remains time-consuming, technically demanding and costly
(Maeght et al., 2013). Excavation and coring techniques used to
obtain data concerning deep root distribution are particularly labour intensive. The cost of establishing deep trenches lead researchers to use available soil profiles created by road cuts, exposed
stream cut-banks or landslides, in order to determine vertical
rooting pattern. Also, roots of plants are regularly found in caves
and mine shafts; however, such observations have merely been
mentioned in the literature as curiosities. Rhizotrons, promising
tools for laboratory studies and relatively small soil volumes,
remain of limited use for deep root observation; while, sampling
depths are often decided arbitrarily and set to values that are too
shallow to allow reliable estimates of rooting depth, under ambient
conditions. In other words, research on roots is mostly confined to
the uppermost soil horizons, while research on deep roots remains
laborious.
The particularly deep roots of C. spinosa found in the catacombs
of Milos draw water from soil layers, where direct sampling of root
specimens and measurements of root water status are reported for
the first time. In this respect, the study of roots of C. spinosa has
been facilitated by in situ monitoring of root development in the
ancient monument.
Acknowledgements
The research in the Catacombs of Milos was financially supported
by the British Council (ELKE 70/3/4326) and the Greek Ministry of
Civilization. We thank Assoc. Prof. N. Christodoulakis, Dr. A. Argiropoulos, C. Chimona and J. Pouris for technical assistance, Prof. W. J.
Davies for advice, and D. Koukos for editing the text.
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