Botanical Journal of the Linnean Society (2001), 136: 231238.

With 8 figures
doi:10.1006/bojl.2000.0430, available online at http://www.idealibrary.com on

Leaf anatomical variation in Alchornea triplinervia

(Spreng) Mull. Arg. (Euphorbiaceae) under distinct
light and soil water regimes
G. ROCAS and F. R. SCARANO FLS
Universidade Federal do Rio de Janeiro, Departamento de Ecologia, CCS, Ilha do Fundao, 21941-970,
Rio de Janeiro, RJ, Brazil

C. F. BARROS
Jardim Botanico do Rio de Janeiro, Laboratorio de Botanica Estrutural, 22460-030, Rio de Janeiro,
RJ, Brazil

Received July 2000; accepted for publication October 2000

Alchornea triplinervia (Spreng.) Mull. Arg. (Euphorbiaceae) is a tree which occurs in a broad range of habitats in
Brazil. In the State of Rio de Janeiro, it occurs from montane forests to swamplands at sea level. A quantitative
approach was used to examine the role of light and soil water regime on the variations found in anatomical traits
of the palisade and spongy parenchyma, outer epidermal cell wall of the abaxial and adaxial surfaces, the percentage
of sclerenchymatous area in relation to the total midrib area and the ratio of palisade to spongy parenchyma for
five distinct ecological populations: M1 late secondary montane forest (shaded, unflooded); M2 early secondary
montane forest (semi-exposed, unflooded); S1 primary swamp forest (semi-exposed, flooded); S2 secondary
swamp forest (exposed, flooded); and D deforestation area (exposed, unflooded). Tukey tests were carried out for
multiple comparisons, while one-way factor variance analysis was used to test for differences among ecological
populations. A principal component analysis was used to order the populations as well as to find the higher variance
component. These populations developed different response levels to the environmental factors studied, namely
light and soil water regime. Light accounted for the variations found in palisade and spongy parenchyma while the
combination of light and soil water determined the variations found regarding the outer epidermal cell wall of the
abaxial surface, the percentage of sclerenchymatous area in relation to the total midrib area and the compaction
of the spongy parenchyma. The separation of S1/M2 and S2/D populations into two groups was due to similar light
regimes, which suggested that light was affecting the leaf anatomical variation of A. triplinervia more than the
interaction of light and soil water regime. 2001 The Linnean Society of London

ADDITIONAL KEY WORDS: Atlantic forest intraspecific variation tropical tree.

INTRODUCTION and anthropogenic impact. These are often highly ad-

aptable plants when faced with environmental vari-
Widespread, common species are not as easily ation, which may be of key relevance in a global change
threatened by extinction as rare and/or endemic spe- scenario (Parsons, 1990).
cies, with the result that conservation initiatives often The Atlantic rainforest complex, one of the worlds
focus on the latter. However, populations of widespread 25 biodiversity hotspots, is notable for its high species
plants can be locally extinguished (McKinney, 1997) diversity and high level of endemisms (Myers et al.,
due to vulnerability to local environmental pressures
2000). However, it also has many interesting cases of
generalist plants which are seldom studied from an
intra-specific variation perspective (Rocas, Barros &
Scarano, 1997). The poor conservation status of this
Corresponding author. E-mail: [email protected] biome and high level of habitat fragmentation (only
231
00244074/01/060231+08 $35.00/0 2001 The Linnean Society of London
232 G. ROCAS ET AL.

7.5% of its original area remains; Myers et al., 2000) In the Poco das Antas Biological Reserve, mu-
demands a deeper scientific knowledge regarding spe- nicipality of Silva Jardim, the plants were collected in
cies acclimation to environmental heterogeneity. a primary swamp forest (S1), a vegetation type which
The euphorb tree Alchornea triplinervia (Spreng.) used to be typical of the Atlantic forest lowlands,
Mull. Arg. is a widespread neotropical species which and is now reduced to a few remnants. The sunlight
occurs in various habitats within the Atlantic rain- penetrates through the canopy semi-exposing the
forest complex. In a previous study we (Rocas et al., understorey. The soil is waterlogged for c. 10 months
1997) compared leaf anatomy of specimens of this of the year (Scarano et al., 1997). Tree mean DBH was
species in relation to light intensity in two remnants c. 15 cm and the mean height c. 15 m.
of secondary forest at distinct successional stages. The Jacarepia State Ecological Reserve, mu-
Subsequently, we found three further populations, two nicipality of Saquarema, is a sandy coastal plain loc-
of them subject to flooding. In this paper, we examine ated between the Atlantic Ocean and the Jacarepia
the role of both periodic flooding and light on leaf lagoon. Specimens were collected in a secondary swamp
anatomical variation for this species, aiming to discern forest (S2). Adult plants had an average height of
the role played by each of these factors on the variations 5 m and DBH was c. 1012 cm. Sunlight penetrated
found. Although acclimation cannot be predicted from through canopy gaps formed by previous anthropogenic
leaf anatomy alone (Strauss-Debenedetti & Berlyn, disturbances. Thus, the understorey was classified as
1994), the variation spectrum provides an indication exposed to sunlight. Here, the soil is waterlogged for
of the species ecological plasticity. 1011 months per year. In a neighbouring impacted
Studies of structural modifications in plants oc- site which suffered deforestation (D), another sample
cupying distinct habitats have often lacked a sound was collected, which was totally exposed to sunlight
quantitative basis (e.g. Zagdanska & Kozdoj, 1994) and growing on unflooded sandy soil. These plants
and more commonly examine the effect of a single were approximately 23 m in height (Sa, 1992).
environmental factor on the character(s) under Thus, A. triplinervia plants in M1 were shaded and
scrutiny (Smirnoff & Crawford, 1987; Sojka, 1992). unflooded, in M2 were semi-exposed and unflooded, in
Currently, a more quantitative approach is being em- D were exposed and unflooded, in S1 were semi-exposed
ployed (Loreti & Oesterheld, 1996; Schreiber & Rie- and flooded and in S2 were exposed and flooded.
derer, 1996; Rozema et al., 1997), although studies
attempting to assess which environmental factor ac-
counts for which variation pattern are still lacking. LEAF ANATOMY
Palisade and spongy parenchyma, and outer epidermal
MATERIAL AND METHODS cell wall of the abaxial and adaxial surfaces were
measured by using sections embedded in methacrylate
STUDY SITES resin (hydroxyethyl methacrylate, Reichert-Jung, his-
Five leaves were taken at the middle-third of the toresin embedding kit), and stained by toluidine blue
canopy of each of 35 mature individuals of Alchornea 0.1% (Feder & OBrien, 1968). These tissues are known
triplinervia (Spreng.) Mull. Arg. in each of five sites to reveal responses related to light and soil water
distributed along three distinct vegetation types in Rio variations (Esau, 1972; Levitt, 1980; Rozema et al.,
de Janeiro State, south-eastern Brazil (Fig. 1). These 1997). Histochemical tests for lignin, tannins, and
sites were chosen because they represent distinct light calcium oxalate crystals were carried out according to
and water regimes within a single vegetational domain, Johansen (1940) and Jensen (1962). The percentage of
the Atlantic rainforest complex. Geographic and cli- sclerenchymatous area in relation to the total midrib
matic features of these sites are depicted in Table 1. area was calculated by using images obtained by the
Leaves were harvested at all sites at the same seasonal camera lucida of a light microscope which were sub-
time and from individuals with similar physiological sequently digitized (Genitizer GT-1212B). The software
age. used was Autocad R13 for Windows. The sample size
Two sites were located at Macae de Cima Ecological established was 25 fields for each measurement (N=
Reserve, municipality of Nova Friburgo: a late-sec- 25), from which were calculated mean and standard
ondary montane forest (M1) and a neighbouring early deviation. Tukey tests were carried out for multiple
secondary montane forest (M2). M1 was characterized comparisons, while one-way analysis of variance was
by a dense canopy cover, a shaded understorey and a used to verify the existence of differences among eco-
humid clay-sandy soil, while M2 had large canopy logical populations. Principal components analysis
gaps, higher light penetration and a dry clay-sandy (PCA) was used to order the populations as well as
soil. The mean trunk diameter at breast height (DBH) to find the higher variance components (Schlichting,
of A. triplinervia was c. 15 cm and the mean height 1986; Zar, 1996). The statistical analyses were done
c. 20 m for both sites (Rocas et al., 1997). with Statistica v. 4.2.
 LEAF ANATOMICAL VARIATION IN ALCHORNEA TRIPLINERVIA 233

Esprito
Santo

410
450
210
210

M1 M2

Minas Gerais

Maca de Cima
Ecological Reserve
Poo das Antas S1
Biological Reserve
Jacarepa State
So Paulo Ecological Reserve
Rio de Janeiro

S2 D
Atlantic Ocean 60 km
State limit

Figure 1. Rio de Janeiro State, south-eastern Brazil, study sites and the leaf size of each sample. Scale bar (inside
the leaves)=3 cm. M1 late secondary montane forest; M2 early secondary montane forest; S1 primary swamp
forest; S2 secondary swamp forest; and D deforestation area.

Table 1. General geographic and climatic information related to the sites where Alchornea triplinervia was sampled
for leaf anatomy analyses in Rio de Janeiro State, Brazil

Sites Macae de Cima Ecological Poco das Antas Biological Jacarepia State Ecological
Reserve Reserve Reserve

Latitude 2230 and 2233S 2230 and 2233S 2247 and 2257S
Longitude 4219 and 4214W 4215 and 4219W 4220 and 4243W
Mean altitude 8801720 m a.s.l. Sea level Sea level
Climate Mesothermic, always Hot and humid Hot and humid
humid
Mean annual rainfall 15002000 mm 2260 mm 1000 mm
Mean minimal temperature 13.8C 18.6C 8C
Mean maximal temperature 21C 30.6C 3032C

RESULTS paracytic stomata and the epidermis was uniseriate.

Stellate multicellular trichomes occurred on both faces
Alchornea triplinervia showed phenotypic variations of all specimens studied.
in regard to leaf morpho-anatomy among the ecological M1, M2 and S1 had a dorsiventral mesophyll. M1
populations studied. M1 (leaves of shaded, unflooded showed a one-layered palisade parenchyma and 35
plants) had mesomorphic characteristics, while the layers of spongy parenchyma. The intercellular spaces
others showed xeromorphic trends. were wide, often occupying all the extension between
M1 leaf length was 2025 cm, while M2 (semi-ex- the palisade and the abaxial epidermis (Fig. 2). M2 and
posed and unflooded) and S1 (semi-exposed and S1 were structurally similar; the palisade parenchyma
flooded) was 1015 cm, and D (exposed and unflooded) contained 12 layers of compactly arranged elongate
and S2 (exposed and flooded), 810 cm (Fig. 1). In all cells and the spongy parenchyma a few small in-
populations the leaves were amphistomatous with tercellular spaces and c. 45 layers. In the latter, which
234 G. ROCAS ET AL.

Figures 27. Transverse leaf section. =palisade parenchyma; _=spongy parenchyma. 2. M1; 3. M2; 4. S1; 5. S2; 6.
d; 7. transverse midrib section of an individual collected in D. gelatinous fibres. Scale bars=50
m.

was closer to the abaxial epidermis, the diameter in- parenchyma layers, in both cases with a compact ar-
creased towards the anticlinal wall, similar to a pal- rangement as compared with the others. They showed a
isade cell (Figs 3, 4). S2 and D frequently showed completely developed second palisade near the abaxial
two palisade parenchyma layers and c. 24 spongy epidermis (Figs 5, 6), characterizing an isobilateral
 LEAF ANATOMICAL VARIATION IN ALCHORNEA TRIPLINERVIA 235

Table 2. Leaf anatomical differences of ecological populations of Alchornea triplinervia. MeanSD and F values
(ANOVA, P<0.05). Means followed by different letters differ at P<0.05 (HSD Tukey test). (+) light exposure or flooded
soil; () shade or unflooded soil; () semi-exposure to light. M1 was used as a control and not included in the
comparative analysis. M1 late secondary montane forest; M2 early secondary montane forest; S1 primary swamp
forest; S2 secondary swamp forest; and D deforestation area

M1 M2 S1 S2 D F
Light + +
Flooding + +

Palisade parenchyma 55.654.96 73.8710.58a 75.115.47a 49.978.54c 55.945.69b 65.02

(PP)
Spongy parenchyma 48.686.72 69.372.54a 68.2311.11a 48.215.46b 51.6846.2b 32.04
(SP)
PP/SP 1.170.21 1.090.18a 1.120.17a 1.050.20a 1.110.24a 0.77
Adaxial epidermis 1.840.49 2.430.46a 2.721.16a 2.700.68a 2.520.57a 0.83
Abaxial epidermis 2.150.4 2.180.4a 1.710.62b 2.210.50a 1.990.42a 5.51
Sclerenchyma 28.913.12 29.85.16b 25.037.79c 34.477.47a 35.443.56a 14.70
percentage

mesophyll. The ratio of palisade to spongy parenchyma significantly from the others. These results indicated
of all populations did not show statistical differences, that the magnitude of the tissue responses was dif-
probably because the ontogeny of these tissues was ferent when they were submitted to distinct en-
not under ecological control (Table 2). Druses of calcium vironmental factors.
oxalate and phenolic substances were found in both The PCA indicated that the anatomical char-
tissues. acteristics varied according to the first two components,
The structural arrangement of the midrib was sim- which together explained 62.96% of the total variance.
ilar in all sites. The uniseriate epidermis is followed PC1, which corresponded to the light factor, was re-
by an angular collenchyma in both faces. The arch- sponsible for 42.46% of the total variance, and was
shaped vascular system showed five bundles disposed strongly influenced by palisade and spongy par-
near the adaxial epidermis (Fig. 7). Different levels of enchyma and sclerenchyma percentage value. PC2,
sclerification and gelatinous fibres were observed. A responsible for 20.46% of the total variance, was mainly
lower proportion of sclerenchyma occurred at S1, fol- influenced by the outer epidermal cell wall of the
lowed by M1-M2 and then S2 and D (Table 2). abaxial surface, and secondarily by the sclerenchyma
The multiple comparison tests and the PCA (Fig. 8A) percentage value (Fig. 8B), and thus corresponded to
showed that M1 formed an isolated and intermediate the interaction of light and water.
group in relation to the other groups (M2/S1 and D/ There was a high positive correlation between pal-
S2). In the field, these individuals were taller (c. 20 m isade and spongy parenchyma, which were negatively
tall), had bigger leaves and a broader canopy, sug- correlated to the sclerenchyma percentage value. The
gesting that these were under less stressful conditions populations were distributed more widely in relation
than the other populations. For this reason, M1 was to PC1 (light) and more narrowly in relation to PC2
used as a natural control and was excluded from the (light/flooding). PC1 was primarily responsible for the
comparative statistical tests. population arrangement and the anatomical character
The phenotypic differences regarding leaf anatomy variation (Fig. 8B). The proximity between the sample-
among the populations of A. triplinervia were tested points M2/S1 and S2/D indicated an intrinsic similarity
by a multiple comparison test (Table 2). M2 and S1 between these groups. Palisade and spongy par-
showed the highest palisade and spongy parenchyma enchyma seemed to play a key role in the ordering of
thickness and S2 the smallest. The outer epidermal PC1, while the outer epidermal cell wall of the abaxial
cell wall of the adaxial surface did not vary significantly surface and the sclerenchyma percentage value best
among the distinct populations. Although M2, S2 and explain the arrangement of PC2. Again, light is the
D differed significantly among them for the outer epi- main factor for the M2/S1 and S2/D grouping.
dermal cell wall of the abaxial surface, they formed
an isolated group in relation to S1. The highest thick- DISCUSSION
ness of the outer epidermal cell wall of the abaxial
surface was found for S2 and M2. D and S2 showed Leaf anatomy of the ecological populations of A. tri-
the highest sclerenchyma percentage values, differing plinervia revealed different magnitudes of response to
236 G. ROCAS ET AL.

6.0
A
5.0

4.0

3.0

2.0
PC2

1.0

0.0

1.0

2.0

3.0
2.5
B

1.5

0.5 2
1
PC2

0.5
3

1.5

2.5
3 2 1 0 1 2 3
PC1

Figure 8. Principal components analysis (PCA): variable ordering (loadings) and population ordering (scores). M1
(); M2 (); S1 (); S2 (+); and D (). (A) M1 included in the analysis; (B) M1 excluded from the analysis.
1=palisade and spongy parenchyma; 2=sclerenchyma percentage; 3=abaxial epidermal cells; 4=adaxial epidermal
cells.

the light and soil water regime. Light accounted for Sprugel & Hinckley, 1996; Rozema et al., 1997; Rocas
variations in the thickness of the outer epidermal et al., 1997), the mesophyll of leaves exposed to high
cell wall (see Schreiber & Riederer, 1996) and in the levels of light was thicker and had a well-developed
thickness and number of cell layers of the palisade palisade layer with columnar-shaped cells, while that
and the spongy parenchyma. Field observations re- of those developed under shade or low irradiance were
vealed that the young leaves remained rolled up to the thin. The columnar shape of palisade cells facilitates
moment of their complete differentiation. During this light penetration to the spongy tissue (Vogelmann &
period, the abaxial epidermis is exposed while the Martin, 1993). The distribution of light through the
adaxial one is protected. Thus, the former is constantly leaf and the acclimation of chloroplasts in habitats
exposed to environmental factors during its de- with high levels of light may maximize the rate of
velopment while the latter is subjected to physical photosynthesis and reduce susceptibility to photo-
factors only when the leaf is already fully grown. The inhibition (De Lucia et al., 1996). As exposure to light
thickness of the outer epidermal cell wall of the former increased, we observed a tendency for the development
varied significantly among the collecting areas, while of another palisade layer near the abaxial epidermis;
that of the latter did not. at its most extreme this resulted in a total development
As in other studies (Chabot & Chabot, 1977; Brooks, of an isobilateral structure at S2 and D. This kind of
 LEAF ANATOMICAL VARIATION IN ALCHORNEA TRIPLINERVIA 237

structure is common in xeromorphic plants (Esau, ACKNOWLEDGEMENTS

1972). In all areas the ratio of palisade to spongy
parenchyma was similar, suggesting that the ontogeny We thank C. G. Costa, D. S. D. Araujo and G. W.
of these tissues under genetic control, while number Fernandes for critical readings of the manuscript; the
of cells, shape, size and compaction of the mesophyll staff at the Plant Anatomy Laboratory of the Rio de
were environmentally controlled. Janeiro Botanical Garden; CNPq (Brazilian Research
According to Esau (1972) and Levitt (1980), light Council) for research grants nos. 520494/93-8 and
enhances the development of palisade parenchyma and 301056/92-7; and the Atlantic Forest Program (Shell,
the compaction of spongy parenchyma, while water Margaret Mee Foundation Amazon Trust, MacArthur
deficit hinders normal cell growth, inducing the ap- Foundation) for funding.
pearance of smaller cells and a higher thickness of
parenchyma due to an accumulation of photosynthetic
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