Aggregation protects Xexible-shelled reptile eggs from severe
hydric stress
Adolfo Marco · Carmen Díaz-Paniagua
Abstract Many reptiles lay eggs with Xexible shells that
can progressively lose water until lethal dehydration under
dry soil conditions. The number of eggs that develop
together may inXuence the water exchange in the nest. We
hypothesise that egg aggregation could reduce water lost
under dry conditions. We exposed aggregated and isolated
eggs to severe hydric stress followed by a period of rehydration. Hydric stress caused a general loss of water in
common chameleon eggs. Initial egg mass did not aVect
survival but eggs that had lost more water had higher mortality and produced smaller hatchlings. Mass loss was
higher and even lethal for isolated Chamaeleo chameleon
eggs. However, aggregated eggs lost less water and most
survived this period. After hydric stress, all surviving eggs
gained mass via water absorption, and aggregation negatively aVected water uptake. Isolated eggs hatched at
smaller sizes than aggregated eggs. Aggregation also
favoured hatching synchrony. Large clutches may favour
hatching success of terrestrial Xexible-shelled eggs incubated under severe drought conditions.
Keywords Chameleons · Eggs · Incubation ·
Water potential · Hatching synchrony
Communicated by H.V. Carey.
A. Marco (&) · C. Díaz-Paniagua
Doñana Biological Station,
Spanish Council for ScientiWc Research,
CSIC, Apartado 1056, 41013 Seville, Spain
e-mail: amarco@ebd.csic.es
Introduction
Many reptiles, some amphibians, and many invertebrates
oviposit terrestrial eggs that are permeable to water and
thus sensitive to the water potential of the nest environment
(review in Belinsky et al. 2004). Water exchange of Xexible-shelled eggs has been extensively studied in reptiles
(Thompson 1987; Packard and Packard 1988). Flexibleshell eggs of squamate reptiles typically absorb water from
the soil during incubation, leading to an increase of egg
mass of up to three or four times (Packard 1991; Overall
1994).
For many squamates, absorption of water is necessary
for successful completion of embryonic development
(Packard 1991). Females of species that inhabit arid or
semi-arid areas must Wnd nesting sites where moisture
remain suitable during the entire incubation period, since
under prolonged exposure to dry conditions progressive
loss of water can lead to embryo death (Muth 1980; Packard 1991). Rate of water loss is positively related to the egg
surface size in contact with dry soil (Tracy and Snell 1985;
Packard 1999). Thus, the number of eggs that develop
together may inXuence water exchange (Marco et al. 2004).
Egg aggregation can reduce the rate of water absorption in
wet soils, as shell contact with other eggs reduces the area
in contact with the substrate. In substrates at ¡650 kPa, lizard eggs aggregated in small clutches absorbed less water
and produced smaller hatchlings than did isolated eggs
(Marco et al. 2004). However, we are not aware of evidence of the eVect of egg aggregation in very dry substrate
conditions.
The aim of this study is to test the hypothesis that aggregation protects Xexible-shelled eggs from severe hydric
stress. We incubated Xexible-shelled eggs of common chameleons, Chamaeleo chamaeleon (Linnaeus, 1758) in two
123
diVerent levels of aggregation and exposed them to a period
of severe hydric stress followed by incubation under two
diVerent and typical soil water potentials. Aestival periods
of hydric stress and warm temperatures, very common in
semiarid areas, can cause severe egg dehydration in natural
nests (personal observations). In this study, however, we
analyse the eVect of severe drought periods during the cold
season (autumn and winter) on chameleon egg incubation.
Over the last decades, autumnal droughts have not been
infrequent and have had detrimental eVects on chameleon
reproduction, through an increase in mortality of reproducing females or a decrease in their fecundity (Blázquez et al.
2000; Díaz-Paniagua et al. 2002).
The common chameleon in Spain would appear to be
particularly challenged by dry soil conditions during incubation. In south-western Spain, females excavate nests during September and October in sandy substrates that can
undergo extreme variations of water potential. Clutch size
ranges from 4 to 40 Xexible-shelled eggs (Díaz-Paniagua
et al. 2002). Incubation takes nearly 10 months and hatchlings emerge during the summer of the year following oviposition (Bons and Bons 1960; Blasco et al. 1985; DíazPaniagua et al. 2002). Embryos experience a physiological
diapause during the Wrst 3 months of incubation and a
period of cold torpor of approximately two more months
(Díaz-Paniagua 2007; Andrews et al. 2008). During this
period of 5 months, all embryos remain at the gastrula
stage. During incubation, eggs may double or triple their
initial mass by absorption of water from nest surroundings
(Díaz-Paniagua and Cuadrado 2003).
Materials and methods
Egg collection
We collected 17 gravid chameleon females (C. chamaeleon) from several sites in Cadiz province (southwest
Spain) between 2 and 17 October 2000. They were kept
outside on native vegetation at the Weld sites where captured, in ten acetate paper enclosures 2 m in diameter and
40 cm high, surrounding shrubs and preventing their
escape. Natural food was supplemented with live crickets.
Egg laying took place after a maximum of 15 days of captivity (3–31 October 2000). After egg laying, females were
released where collected. Females laid an average of 13.3
eggs per clutch. Immediately after being laid, eggs were
extracted from the soil, cleaned with a soft brush, weighed
(initial egg mass) on a digital scale (§0.01 g) and measured
with a digital caliper (§0.1 mm). In order to provide an initial pre-experimental period (period 0) of water absorption,
eggs were then kept buried in wet sand within closed plastic
containers until the beginning of the experiment. Eggs were
123
aggregated by clutch during this period. This pre-experimental period lasted an average of 36 days varying from 23
to 51 (SD = 9.57) depending on the laying date.
Experimental procedures
At the beginning of the experiment, 120 eggs from 17
females were assigned to two hydric treatments (dry and
wet) and two levels of aggregation (aggregated and isolated). From eight females with large clutches, we selected
12 eggs, and assigned one egg for each one of the three replicates of each aggregation and moisture treatments. From
six females, we obtained eight eggs assigned in all treatments but only in two replicates, and from eight females we
obtained only four eggs, one for each diVerent treatment.
Eggs were completely buried in six plastic containers
(40 £ 40 £ 10 cm) half Wlled with sand. To evaluate the
inXuence of aggregation on embryonic development, we
placed 20 eggs in each container, 10 aggregated compactly
and 10 isolated. Both groups of eggs were placed 10 cm
apart to avoid interaction. We placed aggregated and isolated eggs in the same containers to reduce the eVect of the
container on their embryonic development. Aggregated egg
contact was maximized by random placement in two layers
of Wve eggs, minimizing their separation. Isolated eggs
were placed 1 cm apart and at depths similar to aggregated
egg depths. All eggs were placed at least 2 cm from container walls. Three containers were assigned to the dry
treatment and the other three to the wet treatment.
The study involved one pre-experimental period 0
(explained in the egg collection section) and three experimental periods (Table 1): period 1 was of 110 days of
hydric stress during which all eggs were exposed to a water
potential of approximately ¡1,650 kPa in the wet treatment
and ¡1,950 in the dry treatment. Containers were buried at
40 cm depth in a very sunny place close to the sites where
females oviposited. Period 2 was of roughly 100 days of
rehydration (days 110–210), in which water potentials were
¡150 kPa for the wet treatment, and ¡650 kPa for dry
treatment. Containers were buried at the same Weld location
and depth as in period 1. Water potentials in period 3 were
the same as in period 2, during approximately 2 months,
until hatching (days 210 to 270). In order to monitor hatching, containers were returned to our laboratory, with an
ambient temperature of 23.5–31°C, shaded until hatching,
with moisture similar to that of period 2. During this Wnal
phase, we monitored containers daily in order to detect
hatchling emergence.
Hydric experimental conditions imitated the sequence of
rainfall in early autumn (that triggers chameleon egg-laying), followed by a prolonged dry autumn and winter,
Wnishing with rainfall again in late winter or spring. During
period 0 and the Wrst third of period 1, embryos were in
Table 1 Description of details
of the experimental conditions
Period
Description
0
Pre-experimental
Treatment
Water potential
Location
Time (days)
Wet sand
Laboratory
23–51 days
water absorption
1
Hydric stress
2
Rehydration
3
Rehydration
and pre-hatching
diapause (Díaz-Paniagua 2007; Andrews et al. 2008). To
obtain the selected soil water potentials, clean sand was initially dried at 80°C until there was no further weight loss.
Then, dechlorinated water was added to sand until the
selected water potential was obtained. The quantity of
water required to obtain the three selected soil water potentials was determined by vapor psychrometry using a Dew
Point Microvoltimeter (Wescor, HR 33T, Wescor Inc.,
Utah). Containers were then sealed to reduce water evaporation. Sand substrate was replaced at day 110, 150 and
210. At laying, at the beginning of hydric stress, and at substrate replacement, we recorded egg survival, measured
maximum egg length and width and weighed eggs in a portable digital balance to the nearest 0.01 g. Immediately after
hatching, we measured hatchling mass to the nearest 0.01 g
and hatchling snout-vent length (SVL) to the nearest 1 mm
using a digital calliper with a precision of 0.1 mm. In order
to compare hatching synchrony among treatments, we calculated as indicative of hatching synchrony for each egg
group (isolated and aggregated eggs) in each container:
both number of days elapsed between hatching of the Wrst
and last egg (hatching duration), and mean hatching-day
diVerence from the date of the Wrst hatching. We considered
incubation duration as the total number of days elapsed
from the start of the experiment to hatching of eggs.
Hatchlings were housed in 100 £ 40 £ 40 cm glass laboratory terraria at approximately 26°C under a natural light
cycle, fed crickets ad libitum, and were released within
2 weeks of hatching in the area where females had been
collected, in an attempt to minimize the impact on individuals and population of origin.
Analysis of data
Analyses of treatment eVects were based upon mean values
for each replicate of ten aggregated and isolated eggs. To
evaluate container eVect on incubation we used nested
ANOVAs for all dependent variables, with substrate water
potential and the container (nested in the former) as categorical factors. To determine the eVect of substrate water
potential or the level of egg aggregation on egg mass varia-
Wet
¡1,650 kPa
Buried in Weld
110
Dry
¡1,950 kPa
Buried in Weld
110
Wet
¡150 kPa
Buried in Weld
100
Dry
¡650 kPa
Buried in Weld
100
Wet
¡150 kPa
Laboratory
Until hatching
Dry
¡650 kPa
Laboratory
Until hatching
tion during incubation we used repeated measures two-way
ANOVAs, considering egg mass at days 90, 180 and 270 as
dependent variables. To determine the overall eVect of substrate water potential or egg aggregation on incubation, we
used multivariate two-way analysis of variance (MANOVA),
considering hatching success, and hatchling mass and SVL
as dependent variables. Independent factors were water
potential (wet or dry) and level of aggregation (isolated or
aggregated). We used two-way ANOVAs to determine the
eVect of each selected factor and the interaction between
them on each dependent variable. We also used Tukey honestly signiWcant diVerence for post-hoc pairwise comparisons. All analyses were performed using STATISTICA 6.0.
Results
For all dependent variables used in the study, nested ANOVAs indicated that the container had no eVect on results
(for example: mortality: F5,6 = 0.575, P = 0.692; egg mass
at hatching: F5,6 = 0.760, P = 0.587). For that reason, we
considered the two groups of eggs in each container as
independent.
Variation in egg mass
Mean egg mass at hatching was 1.21 g (SD = 0.16) and all
eggs absorbed water during the pre-experimental period 0
of hydration (Fig. 1). Duration of this period was similar
for all treatments (ANOVA: F3,116 < 0.001, P > 0.99). At
the beginning of the period of hydric stress (period 1),
mean egg mass was 1.50 g and there were no diVerences
among eggs of the two hydric treatments (Table 2).
Repeated measures ANOVA indicated an overall eVect of
soil water potential and egg aggregation on egg water
exchange (Table 2). During the period of hydric stress, all
eggs lost mass and there was no relationship between mass
lost and initial egg mass (product moment correlation:
r = 0.120, F1,10 = 0.146, P = 0.710). At the end of period 1,
mean mass of isolated eggs was lower than the mean egg
mass immediately after laying at both water potential
123
Fig. 1 Variation in mean mass
of chameleon eggs (§SE) during
incubation under a 110-day peri-
od of hydric stress. Eggs were
exposed to two levels of soil
water potential and two levels of
egg aggregation. Egg measurements of the two aggregation
treatments were recorded on the
same days. Open triangles and
continuous line correspond to
groups of ten eggs; full squares
and dashed line correspond to
isolated eggs
Table 2 Results of repeated measures two-way ANOVAs that ana-
two-way ANOVAs (F values with P values) that analyse the inXuence
lyse the inXuence of the substrate water potential and the level of egg
aggregation on egg mass variation, and multivariate and univariate
of the substrate water potential and the level of egg aggregation on
embryonic development and hatchling characteristics of chameleons
Water potential
Wilks
Egg mass at day 0
F
0.17
Repeated measures ANOVA
Overall eVect
0.081
Aggregation
P
Wilks
0.693
F
P
0.60
0.461
6.701
0.024
Wilks
P
1.29
0.288
22.75
0.0011
2.805
0.131
4.10
0.077
26.40
0.0009
1.510
0.254
Egg mass at day 150
23.34
0.0013
0.09
0.767
0.29
0.607
Egg mass at day 210
MANOVA
45.66
0.0001
6.70
0.032
1.49
0.256
0.105
0.229
0.416
F
Egg mass at day 110
Overall eVect
0.230
Water potential £ aggregation
14.19
0.007
5.62
0.047
1.10
0.431
Final egg mortality
19.97
0.0033
10.149
0.015
0.419
0.538
Hatchling mass
55.88
0.0001
11.62
0.011
1.75
0.228
Hatchling SVL
38.63
0.0004
20.86
0.0026
4.56
0.070
levels. However, aggregated eggs lost less water (Fig. 1)
and mean values were not lower than the mean egg mass
immediately after laying (Fig. 2).
Aggregation aVected water exchange of eggs (Table 2,
Fig. 1). Aggregated eggs lost an average of 16.5% of their
mass while isolated eggs lost an average of 31.3% during
the period of hydric stress (Fig. 2). During periods 2 and 3,
all eggs gained mass. There were, however, signiWcant
diVerences in mass gain among water potential and aggregation treatments (Fig. 1). Isolated eggs gained more mass
than aggregated eggs and eggs in the wet treatment gained
123
0.603
more water than eggs in the dry treatment (Fig. 1). By the
end of period 2, the earlier diVerences between aggregated
and isolated egg mass had been eliminated (Table 2). During period 3, isolated eggs gained more water than aggregated ones (Table 2).
Incubation duration and hatching synchrony
Aggregated eggs hatched slightly earlier than isolated ones
(two-way ANOVA: F1,7 = 6.892, P = 0.034). However,
neither the level of substrate moisture (two-way ANOVA:
hatched on the same day. Aggregated-dry incubation eggs
hatched within a period of 3 days (in one box all eggs
hatched on the same day). Isolated eggs showed a wider
range of days between Wrst and last hatching, with a delay
of up to 9 days after the Wrst hatching.
Mortality and hatchling phenotype
Fig. 2 InXuence of aggregation of chameleon eggs on water exchange
during a period of severe hydric stress. Unbroken line is an isocline that
corresponds to the absence of mass variation during hydric stress.
Open triangles and dotted line correspond to live aggregated eggs; solid triangles dead eggs; open squares and dashed line correspond to live
isolated eggs; solid squares dead isolated eggs
F1,7 = 0.092, P = 0.771), nor the interaction with aggregation had any signiWcant eVect on incubation duration (twoway ANOVA: F1,7 = 1.117, P = 0.326). All eggs hatched
within an interval of 8 days (Fig. 3). Water potential had no
eVect on the level of synchronicity at hatching (2-sided
Mann–Whitney U test: hatching duration: Z = 0.091,
P = 0.931; hatching synchrony: Z = 0.280, P = 0.792).
However, a signiWcant eVect of aggregation was observed
on the hatching synchrony within each egg group (2-sided
Mann–Whitney U test, hatching duration: Z = 2.435,
P = 0.017; hatching synchrony: Z = 2.616, P = 0.009). In
aggregated wet incubation, all eggs from the same box
Hatching success varied among treatments (Table 2), and
63.3% of the eggs hatched successfully. Both aggregation
and water potential aVected survival to hatching. Immediately after hydric stress, the percentage of mortality of isolated eggs was 15% while mortality of aggregated eggs was
1.7% (two-tailed Fisher exact test: P = 0.017). At hatching,
mortality was especially high for isolated-dry treatment
eggs (Fig. 4). Under wet conditions, all aggregated eggs
survived and only six isolated eggs did not (Fig. 4).
Mortality was not higher in smaller eggs. Mean initial
egg mass of survivors was 1.50 g, while eggs that died during the experiment had a mean initial egg mass of 1.57 g
(Student’s t test: t = 0.704, P = 0.483). An ANCOVA
where egg mass before hydric stress was considered as a
covariate indicated that mass lost was related to egg survival (ANCOVA: F = 70.59, P < 0.001). Eggs that died
after hydric stress had lost an average of 46.4%
(SD = 13.42) of mass while eggs that survived had lost an
average of 21.8% (SD = 7.87) of their initial mass (Fig. 2).
Both hydric and aggregation treatments aVected hatchling length and mass. Wet treatment eggs produced larger
hatchlings than eggs incubated in the dry treatment (Fig. 5,
Table 2). Similarly, aggregated eggs produced larger,
heavier hatchlings than isolated eggs. A partial correlation
analysis where the initial egg mass was included in the
Fig. 3 Hatching duration and synchrony of egg clusters incubated at
diVerent levels of substrate water potential and egg aggregation. Horizontal bars indicate the hatching period of each egg mass. Horizontal
bars correspond to the hatching period of each group of aggregated (in
black) and isolated (in gray) eggs
Fig. 4 Mean values of the proportion of Wnal mortality (§SD) of chameleon eggs incubated under diVerent levels of egg aggregation and
soil water potential. Open circles correspond to aggregated eggs; solid
squares isolated eggs
123
Fig. 5 Mean values of hatchling mass and SVL (§SD) of chameleons
incubated under diVerent levels of egg aggregation and soil water potential. Open circles correspond to aggregated eggs; solid squares iso-
lated eggs
model showed that hatchling SVL was correlated with the
rate of water exchange during hydric stress (beta = 0.654,
F = 2.767, P = 0.024). Eggs that lost more water during
hydric stress produced smaller hatchlings.
Discussion
During the long incubation period of chameleons, nests
may experience drastic changes in substrate hydric conditions that can deeply aVect egg water exchange. When
exposed to a wet environment immediately after oviposition, eggs gained in mass; when exposed to drought, they
lost mass. During the period of hydric stress, eggs lost up to
0.32% of their mass daily. This is a common characteristic
in Xexible-shelled reptile eggs that may undergo conditions
ranging from extreme dehydration to hyperhydration
(Tracy and Snell 1985; Packard and Packard 1988; Packard
1991; Vleck 1991).
Eggs can easily lose water under dry substrate conditions
and such dehydration can aVect embryonic survival (Muth
1980; Packard 1991). In turtles, the amount of water
absorbed may inXuence hatching success or hatchling survival and Wtness (Gutzke et al. 1987; Janzen et al. 1995;
Finkler 1999). The inXuence of the hydric environment on
hatching success has also been demonstrated in snakes
(Black et al. 1984; Gutzke and Packard 1987; Ji and Du
2001). The risk of dehydration is usually associated with
high temperatures. However, in this study we found severe
123
chameleon egg dehydration during the cold season. In fact,
water loss of the eggs during vernal hydric stress reached
lethal levels in many eggs. Survivors were able to absorb
water during a later exposure to wetter conditions. Absorption of water replacing that lost during preceding periods
has been previously reported in Xexible-shelled eggs of turtles (Gutzke and Packard 1987; Packard and Packard 1988).
This compensatory water exchange was explained as a
response of eggs to the availability of water provided by
occasional rainfall recharges of the substrate (Packard and
Packard 1988). In chameleon eggs, we found that mortality
was associated with higher proportions of water loss, especially for eggs that reached values lower than the initial
mass at oviposition. Eggs that lost less water could recover
after posterior rehydration and survived to hatching.
Severe hydric stress after previous hydration caused only
occasional mortality in aggregated eggs but most of the isolated eggs exposed to the driest water potential, died.
Aggregation clearly protected eggs from dehydration. Factors such as egg surface area, characteristics of the eggshell,
soil water potential or temperature aVect the exchange of
water between a reptilian egg and its environment (Packard
and Packard 1988; Ackerman 1991; Díaz-Paniagua and
Cuadrado 2003). The proportion of the eggshell in contact
with the substrate can also inXuence water exchange. Eggs
having a large proportion of their eggshell surface in contact with the substrate can absorb more water than eggs
with smaller fractions of their shells in contact with the soil
(Tracy and Snell 1985; Packard 1991). In aggregated eggs,
physical contact with each other reduces contact with the
substrate, because only a very small proportion of shell of
those eggs in the middle of the cluster can be in contact
with the substrate.
In a previous study, Marco et al. (2004) demonstrated
that under wet conditions, aggregated lizard eggs could
compete for available water within the nest, so that aggregation can have a negative eVect on the rate of water
absorption. During incubation, large clusters of eggs would
absorb more water than smaller ones or isolated eggs and
could deplete water around the nest. Similarly, in this
study, during the rehydration phase, isolated chameleon
eggs recovered initial mass more quickly, even though their
mass had been smaller than that of aggregated eggs after
hydric stress. During the rehydrating phase, isolated eggs
compensated for these diVerences and became even
heavier. However, under very dry soil conditions, water
potential inside eggs can be higher than in the substrate.
DiVerences in the rate of egg rehydration between aggregated and isolated eggs could be due to the level of aggregation or also due to the level of hydration after the period
of hydric stress that diVered among aggregated and isolated
eggs. The results of this study suggest that with little water
available, aggregated eggs would lose less water than
isolated eggs. In extremely dry conditions, large clutch size
would increase the probability of survival of at least some
eggs of the total clutch, as the layer of eggs in contact with
nest substrate would protect inner eggs against desiccation.
Clutch size is the main determinant of the number of
eggs in physical contact. In reptiles with Xexible-shelled
eggs, species with small clutch sizes would be more vulnerable to dehydration than species with large clutch sizes,
although in our study we tested only large-size aggregations versus isolated eggs. In C. chamaeleon, we did not
detect an eVect of egg size on embryo survival, suggesting
that in this species egg survival is favoured by laying more
eggs, rather than larger eggs, while even small eggs would
have water enough to resist moderate hydric stress. C. chamaeleon has a relatively large sized egg in comparison with
other chameleons, as for example, Chamaeleo calyptratus,
a much larger species. Both species lay eggs of roughly the
same size (1–1.4 g) (Andrews and Donoghue 2004).
Egg aggregation also caused a signiWcant hatching synchrony that reached a maximum in aggregated eggs incubated in wet conditions (1 day). Under dry conditions, all
aggregated eggs of every clutch hatched within 1–2 days.
The lower synchronicity of isolated eggs (up to 8 days)
may hardly be explained by slight diVerences in incubation
conditions within containers. Aggregated and isolated eggs
were incubated within the same containers and at the same
temperature and water potential. Mobility of Wrst hatchlings
could have triggered hatching of eggs in contact. There is
not much information about temporal plasticity or synchrony of hatching in reptiles. In the turtle, Emydura macquarii, synchronous hatching is considered to facilitate a
quicker emergence from the nest (Spencer et al. 2001). In
nests dug deep in the ground, hatchlings may require a concerted digging eVort to reach the surface. This behaviour
has been reported for eggs of Mona Iguanas (Cyclura cornuta) that hatch in synchrony (Wiewandt 1982). Chameleon
eggs are located deep in the substrate (Díaz-Paniagua et al.
2002), and hatchlings emerge in summer, when the substrate is very dry and does not facilitate digging to the surface. Hatching synchrony could increase the success of
hatchlings in reaching the surface. If this were true, egg
aggregation would facilitate hatching synchrony and hatchling emergence.
In summary, this study indicates that chameleon eggs are
vulnerable to dry substrate incubation conditions, but their
mortality rates may be reduced by grouping eggs in a compact cluster, due to reduced water loss during periods of
hydric stress. Adaptations that may reduce the impact of
arid environments on egg survival are, for example,
reduced eggshell permeability, larger egg size or albumen
content making eggs less susceptible to desiccation, maternal selection of deep and wet nest sites, or by delaying nesting until wet periods enhance incubation conditions (Tracy
and Snell 1985; Vleck 1991). Egg aggregation may also be
considered to favour resistance to drought periods.
Acknowledgments Thanks to Ana Andreu, Juan José Gómez, Antonio Conejo and Wouter de Vries for their help and to Robin Andrews
for editorial assistance. The Centro de Recuperación de Especies Amenazadas de El Puerto de Santa María allowed us to use their facilities
to incubate chameleon eggs under natural conditions. The Spanish
Ministry of Science and Technology funded this study (CICYT PB971162). The study was conducted with the permission of the Consejería
de Medio Ambiente, Junta de Andalucía, Spain and complies with cur-
rent Spanish legislation.
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