Plant Syst Evol
DOI 10.1007/s00606-014-1184-8
ORIGINAL ARTICLE
Microscopical investigation of the leaf architecture in greenhouse-
cultivated Ficus (Moraceae)
Yevhen Sosnovsky
Received: 20 April 2014 / Accepted: 24 November 2014
Ó Springer-Verlag Wien 2014
Abstract A high diversity of leaf structural traits in the
genus Ficus is often argued with regard to its taxonomic
implications. Greenhouse-cultivated plants of Ficus are in
special need of identification based on the vegetative
organs since they rarely produce flowers. We studied the
leaf anatomy in 33 Ficus species of six subgenera and 12
sections grown under greenhouse conditions, using light
microscopy and SEM. A great between-species variation of
the leaf traits was revealed as opposed to their stability
within each species. Qualitative leaf traits were found
useful for distinguishing species and some higher taxa of
Ficus. Namely, the features of epidermis, mesophyll, and
vein anatomy were characteristic at the level of sections
and subsections. Quantitative parameters of stomata and
glandular trichomes, as well as the thickness of lamina and
its inner layers, separated well the species and subgenera of
Ficus. The classification of the species based on the
quantitative leaf parameters revealed several groups of
related species, which are placed close in the existing
system of the genus. New data on Ficus leaf architecture
were obtained, including the presence of branched tric-
homes, the features of vein anatomy, and the diagnostic
value of laminar hydathodes. The study shows that many
characters of Ficus leaf architecture are of taxonomic value
and provide support to the existing classification of the
genus. The leaf anatomical and surface features analyzed
may facilitate a proper identification of the species and
higher taxa of Ficus if considered together in a complex.
Handling editor: Hiroshi Tobe.
Y. Sosnovsky (&)
Botanical Garden of Ivan Franko National University of Lviv, 44
Cheremshyna str, Lviv 79014, Ukraine
e-mail: eugene_biol@i.ua
Keywords Diagnostic value
Epidermal features
Ficus
infrageneric taxa
Leaf anatomy
Quantitative parameters
Introduction
Ficus L. (Moraceae) is one of the largest angiosperm
genera comprising ca 750 species distributed in the tropics
and subtropics throughout the world. The genus is featured
in a closed inflorescence called ‘‘syconium’’ and the pol-
lination syndrome based on the species-specific mutualism
with insects from the family Agaonidae (Hymenoptera)
(Berg 1989; Berg and Wiebes 1992; Berg and Corner
2005). Many classifications of Ficus have been suggested
since its first subdivision by Thunberg (Berg 2003a). The
last one, proposed by Berg (1986) and Berg and Corner
(2005), includes six subgenera, 19 sections, and 26 sub-
sections and is thought to be most comprehensive.
A great morphological diversity in Ficus combines with
high variability of its leaf anatomical features (Berg and
Corner 2005). Numerous studies evidence for the taxo-
nomic and diagnostic value of the leaf structural traits in
the genus, though this question remains arguable. Renner
(1907) was the first to study the leaf anatomy in Ficus and
revealed a number of the leaf traits distinguishing its in-
frageneric taxa recognized at that time. Philpott (1953)
mainly focused on the tissue volumes and spacing in Ficus
leaves as well as functional interpretation of the leaf ana-
tomical features. Corner (1965), in his classification of
Ficus, used several leaf parameters, such as the shape and
venation pattern of lamina, the features of indumentum,
and the distribution of cystoliths. Van Greuning et al.
(1984) and Chantarasuwan et al. (2014) indicated the tax-
onomic importance of leaf anatomy at the species level of
Ficus, whereas Berg and Corner (2005) suggested the leaf
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microscopic structure to be of value for the higher infra-
generic taxa. Certain authors have contributed to the
knowledge of Ficus leaf architecture by focusing their
attention on particular leaf traits, such as stomata (Gram-
bast 1954), hydathodes (Chen and Chen 2005, 2006, 2007),
and crystal inclusions (Kuo-Huang et al. 2002; Sharawy
2004).
A growing number of papers on Ficus leaf architecture
reveal some discrepancy as to the diagnostic value of the
leaf traits, especially of the epidermal features. In recent
works, for instance, they were found useful for distin-
guishing Ficus infrageneric taxa (Ogunkunle and Oladele
2008; Ogunkunle 2013). Other studies showed their diag-
nostic value for the species, but not higher taxa of Ficus
(van Greuning et al. 1984; Loutfy et al. 2005; Khan et al.
2011). In contrast, Sonibare et al. (2004, 2005, 2006)
concluded on the epidermal characters to be useless for
Ficus identification and indicated the importance of only
the leaf anatomy and macroscopic traits.
The need of reliable diagnostic criteria of Ficus based
on the plant sterile material especially concerns the
greenhouse-cultivated specimens since they rarely produce
flowers, which are the main features for distinguishing
Ficus taxa. Nevertheless, most of the studies on Ficus leaf
structure dealt with herbarium or living plant material
collected in the nature, and in only some of them the plants
grown ex situ were considered (e.g., Philpott 1953; Klimko
and Truchan 2006; Khan et al. 2011). The latter demon-
strated that the leaf microscopic traits are useful for dis-
tinguishing Ficus species, but not its higher taxa.
The objective of the present work was to perform a
detailed study of the leaf microscopic structure in Ficus
cultivated in greenhouses of botanical gardens and to check
the suitability of the leaf characters for the delimitation of
Ficus infrageneric taxa.
Materials and methods
Plant material collection
Plant material was collected at four sites––greenhouses of
botanical gardens in Ukraine: (1) Botanical Garden of Ivan
Franko National University of Lviv, Lviv city; (2) M.M.
Gryshko National Botanical Garden of the National
Academy of Sciences of Ukraine, Kyiv city; (3) O.V. Fo-
min Botanical Garden of Taras Shevchenko National
University of Kyiv, Kyiv city; and (4) Donetsk Botanical
Garden of the National Academy of Sciences of Ukraine,
Donetsk city.
In total, 33 Ficus species were examined, which were
unevenly distributed between the areal sites (Table 1). The
species were classified according to the recent
123
Y. Sosnovsky
classification of the genus (Berg 1986, 2003a, b, c, d, e,
2004a, b; Berg and Corner 2005). The synonymy and
position of the species within the classification were
checked using electronic sources (Global Biodiversity
Information Facility 2014; van Noort and Rasplus 2014;
USDA Agricultural Research Service 2014) as well as
available publications (van Greuning 1990; Berg and
Wiebes 1992; Erhardt et al. 2002; Zhou and Gilbert 2003;
Berg and Corner 2005; van Noort et al. 2007; Kumar et al.
2011; Chaudhary et al. 2012; Ibarra-Manrı́quez et al. 2012;
Pelissari and Neto 2013).
The leaves were sampled from all the individual plants
(from one to three) of each Ficus species available in the
greenhouse collections. For anatomical studies, up to three
mature leaves were sampled from the different parts of
crown (inside and on the periphery) of each individual
plant and fixed in 70 % ethanol. The old and young leaves
were avoided and the plant cultivars were not examined.
The examined plants varied in height from one to 10 m
(except for climbers).
Microscopic studies and statistical data analysis
For microscopic study, temporary preparations of freehand
paradermal and transverse sections of the leaf blades were
made in glycerin. The sections were taken from different
parts of each leaf blade––near the midrib, in the middle,
and near the edge. Transverse sections of the veins of a
particular order were made at their base, in the middle, and
near the tip. To reveal the presence of lignified tissue, a
part of the vein sections was placed in 0.5 % phloroglu-
cinol and then in concentrated hydrochloric acid (for 30 s
each), after which mounted in glycerin (Metcalfe and
Chalk 1979).
The size and density of leaf anatomical structures were
measured under light microscope. Also, scanning electron
microscope Jeol JSM-T220A was used to observe the leaf
surface. SEM photographs were taken with the help of
camera Olympus C8080WZ. Types of anatomical struc-
tures were identified using works of Aneli (1975), Bara-
nova (1985), Barthlott et al. (1998) and Berg and Corner
(2005).
To estimate the importance of leaf architecture for dis-
tinguishing the species of Ficus, one-way factorial
ANOVA was performed on quantitative leaf traits, using
plant species as a factor. Also, t test was used to compare
Ficus subgenera with respect to the leaf traits; mean values
of the latter were previously calculated for each subgenus.
Principal component analysis (PCA) was applied for the
classification of species based on the leaf parameters. The
leaf traits used for the PCA were the same as for ANOVA.
Statistical analyses were performed using Microsoft Office
Excel 2007 as well as Statistica 8.0 software.
Ficus leaf architecture

Table 1 Ficus species Section Subsection Species Site of data

examined in the study collectinga

Subgenus Ficus L.
Ficus Ficus 1 F. carica L. 1, 2, 3, 4
Frutescentiae Sata 2 F. erecta Thunb. 1, 3, 4
3 F. formosana Maxim. 2
Subgenus Synoecia (Miq.) Miq.
Kissosycea Miq. – 4 F. hederacea Roxb. 2, 4
Rhizocladus Plagiostigma (Miq.) 5 F. pumila L. 1, 2, 3, 4
Endl. C.C.Berg
Punctulifoliae Sata 6 F. sagittata J.Koenig ex Vahl 1, 2
7 F. villosa Blume 2
Subgenus Sycidium (Miq.) Mildbr. & Burret
Sycidium Miq. – 8 F. montana Burm.f. 2, 3
9 F. ulmifolia Lam. 3
Palaeomorphe – 10 F. tinctoria G.Forst. subsp. 1, 4
King gibbosa (Blume) Corner
Subgenus Sycomorus (Gasp.) Miq.
Sycomorus Sycomorus 11 F. mucuso Ficalho 2
12 F. sycomorus L. 2, 3, 4
13 F. vallis-choudae Delile 4
Sycocarpus Sycocarpus 14 F. septica Burm.f. 2
Miq.
Subgenus Pharmacosycea (Miq.) Miq.
Oreosycea Pedunculatae Sata 15 F. callosa Willd. 1
(Miq.) Miq.
Subgenus Urostigma (Gasp.) Miq.
Americanae – 16 F. crocata (Miq.) Miq. 3
Miq. 17 F. luschnathiana (Miq.) Miq. 2, 3
18 F. petiolaris Kunth 1, 3
Urostigma Urostigma (Gasp.) C.C.Berg 19 F. lacor Buch.-Ham. 3
(Gasp.) Endl. 20 F. religiosa L. 1, 2, 3, 4
21 F. virens Aiton 1
Conosycea (Miq.) C.C.Berg 22 F. benjamina L. 1, 2, 3, 4
23 F. drupacea Thunb. 1, 2, 3, 4
24 F. microcarpa L.f. 2, 4
Stilpnophyllum Stilpnophyllum (Endl.) 25 F. elastica Roxb. ex Hornem. 1, 2, 3, 4
Endl. C.C.Berg
Malvanthera (Corner) 26 F. macrophylla Desf. ex Pers. 1, 2, 4
a C.C.Berg 27 F. platypoda A.Cunn. ex Miq. 1
1, Botanical Garden of Ivan
Franko National University of Galoglychia Platyphyllae (Mildbr. & 28 F. vasta Forssk. 1
Lviv; 2 M.M. Gryshko National (Gasp.) Endl. Burret) C.C.Berg
Botanical Garden of the Chlamydodorae (Mildbr. & 29 F. craterostoma Mildbr. & Burret 2, 3, 4
National Academy of Sciences Burret) C.C.Berg 30 F. lingua De Wild. & T.Durand 2
of Ukraine; 3, O.V. Fomin
subsp. lingua De Wild.
Botanical Garden of Taras
Shevchenko National University 31 F. natalensis Hochst. subsp. 1, 2, 3, 4
of Kyiv; 4, Donetsk Botanical leprieurii (Miq.) C.C.Berg
Garden of the National Cyathistipulae (Mildbr. & 32 F. cyathistipula Warb. 2, 4
Academy of Sciences of Burret) C.C.Berg 33 F. lyrata Warb. 1, 2, 3, 4
Ukraine

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Results
Survey of leaf structural traits
The following leaf characters were analyzed for their
diagnostic value in the genus Ficus.
Primary epidermal cells
Leaf epidermis was found quite variable between Ficus
species. Adaxial epidermal cells were irregularly polygonal
with straight or arched walls; on the veins, they were
slightly elongate or rectangular. The number of epidermal
layers ranged from one to five (Table 2); within an indi-
vidual leaf, it usually increased near the midrib. In F. pu-
mila having leaves of two types in our study, the adaxial
epidermis of so-called leaves––‘‘bathyphylls’’ was single-
layered, whereas that of leaves––‘‘acrophylls’’ was multi-
layered (Table 2). The quantitative leaf traits of F. pumila
(Tables 3, 4) and the results of their analyses are presented
for the bathyphylls only.
Abaxial epidermal cells located on the veins were highly
elongate with pointed or truncated tips, sometimes irregu-
larly polygonal or rectangular. The cells between veins had
undulate walls, except for the species of Ficus subgen.
Urostigma having mostly straight or slightly undulate cell
walls. Abaxial epidermis was single-layered in all the
species except those of Ficus subgen. Urostigma sect.
Stilpnophyllum as well as Ficus subsect. Conosycea and
subsect. Cyathistipulae (see Tables 1, 2).
The cells of a single-layered epidermis were quite large,
though smaller on the abaxial side. In the multilayered
epidermis, the cell size gradually increased towards the
deeper layers. Only in F. hederacea the cells of both
adaxial epidermal layers were similar in size. The cells of
the innermost adaxial layer were sometimes vertically
elongate and, therefore, resembled a hypodermis; this was
also observed abaxially in F. macrophylla.
Cuticle and wax depositions
The topography of the leaf surface greatly differed between
the species studied, which was partly caused by the
roughness of cuticle and the morphology of wax layer
deposited on it. In the examined Ficus species, leaf cuticle
roughness was formed by folds or ridges, according to
which, three types of cuticle were recognized: (1) smooth
cuticle with small ridges radiating sometimes from stomata
and lithocysts; (2) slightly folded cuticle forming small
ridges (almost) throughout the leaf surface; (3) folded
cuticle, which makes the leaf surface highly rugose. Upper
and lower leaf surfaces were often different with respect to
the cuticle morphology (Table 2; Fig. 1a–d).
123
Y. Sosnovsky
Epicuticular wax layer formed a smooth film or a rough
crust on the leaves of most of the studied species; waxy
flakes and spherical granules were often present as well.
Other types of wax deposits, which were of rare occur-
rence, include plates, platelets, and threads; the latter
covered the leaf surface with a fine cobweb (Table 2;
Fig. 1a–f).
Trichomes
Leaf indumentum of the studied Ficus species consisted of
non-glandular and glandular trichomes. The general den-
sity of trichomes was observed to be higher on the abaxial
leaf surface, while their length was not related to the leaf
side or was higher adaxially (Table 3). Non-glandular
trichomes were either unicellular or uniseriate; in some
species (F. carica, F. montana), they had a papillate sur-
face. The basal cell of trichomes was either located at the
level of adjacent epidermal cells or (in case of multilayered
epidermis and on the veins) was sunken, so that the tri-
chome appeared to arise from a socket of epidermis
(Fig. 2a, b). In a single-layered epidermis, such a socket
was sometimes formed by the additional epidermal cells
surrounding the base of trichome (Fig. 2a). Several types of
non-glandular trichomes were recognized on the leaves of
the studied species:
1. Straight—stiff filiform trichomes, unicellular and un-
iseriate. Occurred in species of all the subgenera
except Ficus subgen. Synoecia and subgen. Pharma-
cosycea (Table 2; Fig. 2a).
2. Tortuous—weak ferruginous filiform trichomes, uni-
cellular (in F. drupacea) or uniseriate (in F. platy-
poda). In the multilayered epidermis of F. drupacea,
trichomes arose from the outermost epidermal layer,
not forming sockets. In F. platypoda, trichomes of the
adaxial and abaxial leaf side were different in shape
(Fig. 2b).
3. Prickle-like—either recumbent or suberect stiff tric-
homes with an enlarged base, unicellular. Occurred in
all the subgenera except Ficus subgen. Pharmacosycea
and subgen. Urostigma, mostly on the veins (Table 2).
In F. ulmifolia, large prickle-like trichomes of the
adaxial leaf side contained cystoliths.
4. Papilla-like—short unicellular trichomes with fully
enlarged bases containing cystoliths––crystals of cal-
cium carbonate. They were found only in Ficus
subgen. Synoecia, subgen. Sycidium, and subgen.
Sycomorus (Table 2; Fig. 2c). In the other species
studied, lithocysts did not produce trichome-resem-
bling structures. Also, on the abaxial side of the
leaves––acrophylls of F. pumila, distinct ribbed tric-
homes arose from every epidermal cell without a
 Table 2 Some leaf structural traits in Ficus species examined
Species Type of cuticle Wax layer Number of cell layersa Trichomes Hydathodes
Ad Ab Ad Ab Ad Ab PM Ad-Ngl Ab-Ngl Ad-Gl Ab-Gl Ad Ab

1 F. carica SF Sm F, G F 1 1 1-2 Pr S – Ca – –
2 F. erecta SF Sm C, G C, G 1-(2) 1 1 – S – – Pt St
Ficus leaf architecture

3 F. formosana Sm Sm C, G C, G 1-(2) 1 (1)-2 S S – – Pt St

4 F. hederacea Fo SF F, G F, G 1-2 1 1 Pa, Pr, S Pa, Pr Ca Ca Pt St
5 F. pumila SF SF F, G F, G 1-2-(3) 1 1-2-(3) Pa, Pr, S Pa, Pr, S Ca Ca Pt St
6 F. sagittata Sm Sm F, G F, Th 2 1 2 S S Fa Fa Pt St
7 F. villosa Sm Sm F, G F (1)-2-(4) 1 2 – – Fa Fa Pt St
8 F. montana Sm Sm F, G F, G, Th 1 1 1 Pa Pa Ca Ca Pt St
9 F. ulmifolia Sm Sm F, G F 1 1 1 Pr Pa, S Ca Ca W St
10 F. tinctoria subsp. gibbosa Sm Sm C, G C, G 2 1 1 – Pa, Pr – Ca Pt St
11 F. mucuso Sm Sm F, G F, G 1 1 (1)-2 Pa, Pr, S Pa, Pr, S Ca Ca Pt St
12 F. sycomorus Sm Sm F, G F, G 1-(2) 1 (1)-2 Pa, Pr, S Pa, Pr, S Ca Ca Pt St
13 F. vallis-choudae Sm SF C, G F, G, Th (1)-2 1 2-(3) Pa Pa, Pr Ca Ca Pt St
14 F. septica Sm SF F, G F 1 1 2 – – – Ca Pt St
15 F. callosa Sm Sm C, G F 1 1 1-2 – – Ca Ca Pt St
16 F. crocata SF SF F, G F, G (1)-2 1 1-2 ? (1) S S – E Pt St
17 F. luschnathiana SF Fo F, G F, G (1)-2-(3) 1 2-(3) ? (1) – – – E Pt Pt, St
18 F. petiolaris SF Fo F F 1-2 1-(2) (1)-2 S S E – Pt St
19 F. lacor Sm Sm C, G C, G 1 1 1 – – E – Pt St
20 F. religiosa Sm Sm F, Pl F, Pl 1 1 1-2 ? (1) – – – – Pt St
21 F. virens Sm Sm F F 1 1 (1)-2 ? (1) S S Ca Ca W St
22 F. benjamina Sm Sm C, G C 2 2 1-(2) ? (1) – – – E W St
23 F. drupacea Sm Sm C, G C 2-3 2 2 ? (1) T T – E Pt St
24 F. microcarpa Sm Sm C, G C 2-3 2 2?1 – – – – – St
25 F. elastica Sm Sm C, G F, G 3-4 3 2?1 – – – E Pt St
26 F. macrophylla Sm Sm C, G C 3-4-(5) (2)-3 2 – B – – – St
27 F. platypoda Sm Sm F, G F, G 3-4-(5) 3-(4) 2?1 T S, T – – Pt St
28 F. vasta SF SF F F 1-2 1 2 S S Ca Ca W St
29 F. craterostoma Sm Sm C C, G 2-(3) 1 2 – – – – Pt St
30 F. lingua subsp. lingua Sm Fo C P 2 1 2 – – – E Pt St
31 F. natalensis subsp. leprieurii Sm Sm C, P C 2 1 2 – – – E – St
32 F. cyathistipula Sm Sm C C 2 2 1-2 ? (1) – – E E – St
33 F. lyrata Sm Sm C, G, Th P 2 2 2 – – E E – St

Ab abaxial epidermis/surface, Ad adaxial epidermis/surface, B branched, C crust, Ca capitate, E elongate, F film, Fa fan-shaped, Fo folded, G granules, Gl glandular, Ngl non-glandular,
P plates, Pa papilla-like, Pl platelets, PM palisade mesophyll, Pr prickle-like, Pt pits, SF slightly folded, Sm smooth, S straight, St stomata, T tortuous, Th threads, W warts
a
The values in parentheses mean that the feature is of rare occurrence or underdeveloped

123
 Table 3 Trichome length (lm) and density (per 1 mm2) on the leaves of Ficus species examined (mean ± SE)
Species Non-glandular trichome length Non-glandular trichome density Glandular trichome length Glandular trichome density

123
Adaxial side Abaxial side Adaxial side Abaxial side Adaxial side Abaxial side Adaxial side Abaxial side

1 F. carica 142.06 ± 7.7 154.36 ± 6.98 1.82 ± 0.18 17.39 ± 0.99 0 34.7 ± 1.05 0 0.22 ± 0.08
2 F. erecta 0 228.47 ± 18.07 0 0.32 ± 0.12 0 0 0 0
3 F. formosana 350.0 ± 24.58 320.25 ± 25.22 0.03 ± 0.02 0.18 ± 0.06 0 0 0 0
4 F. hederacea 180.99 ± 22.93 20.23 ± 3.66 0.28 ± 0.09 11.52 ± 1.59 46.1 ± 2.77 36.84 ± 1.66 1.78 ± 0.27 31.68 ± 2.8
5 F. pumila 289.83 ± 27.11 59.11 ± 8.15 0.3 ± 0.1 0.75 ± 0.17 43.82 ± 1.53 38.58 ± 1.75 1.89 ± 0.25 27.73 ± 2.6
6 F. sagittata 453.95 ± 42.6 218.1 ± 52.78 0.15 ± 0.06 0.22 ± 0.07 37.0 ± 5.32 30.48 ± 1.03 0.25 ± 0.12 26.86 ± 1.86
7 F. villosa 0 0 0 0 29.31 ± 0.38 31.01 ± 0.38 12.6 ± 1.09 28.16 ± 1.37
8 F. montana 10.31 ± 1.23 23.13 ± 3.52 68.0 ± 4.12 93.8 ± 4.24 35.61 ± 1.91 35.33 ± 0.77 9.8 ± 1.26 52.4 ± 2.93
9 F. ulmifolia 102.9 ± 21.49 92.37 ± 5.02 4.99 ± 0.35 42.6 ± 2.73 37.23 ± 2.06 32.22 ± 1.24 3.77 ± 1.35 29.4 ± 2.29
10 F. tinctoria subsp. gibbosa 0 25.42 ± 3.56 0 2.24 ± 1.21 0 40.48 ± 1.59 0 3.2 ± 1.02
11 F. mucuso 216.76 ± 38.36 131.59 ± 9.89 5.08 ± 0.25 9.77 ± 0.78 41.23 ± 4.39 45.34 ± 0.91 0.58 ± 0.13 6.69 ± 0.67
12 F. sycomorus 156.71 ± 17.59 235.06 ± 45.74 0.26 ± 0.09 0.8 ± 0.35 57.14 ± 4.82 58.07 ± 1.56 0.37 ± 0.1 1.12 ± 0.41
13 F. vallis-choudae 57.25 ± 4.46 41.62 ± 3.5 6.56 ± 1.39 12.64 ± 1.44 48.0 ± 1.42 41.18 ± 1.38 13.46 ± 1.33 59.2 ± 3.35
14 F. septica 0 0 0 0 0 48.42 ± 1.21 0 10.77 ± 0.84
15 F. callosa 0 0 0 0 50.66 ± 5.31 42.88 ± 0.94 0.12 ± 0.06 32.0 ± 1.94
16 F. crocata 192.23 ± 14.09 348.85 ± 15.06 19.87 ± 1.02 19.03 ± 0.5 0 70.52 ± 1.98 0 4.5 ± 0.4
17 F. luschnathiana 0 0 0 0 0 73.23 ± 1.08 0 1.11 ± 0.17
18 F. petiolaris 186.3 ± 22.83 158.91 ± 12.01 0.95 ± 0.15 3.54 ± 0.31 53.55 ± 4.07 0 0.25 ± 0.08 0
19 F. lacor 0 0 0 0 79.07 ± 9.3 0 0.07 ± 0.06 0
20 F. religiosa 0 0 0 0 0 0 0 0
21 F. virens 266.83 ± 18.12 234.1 ± 19.79 5.15 ± 1.31 48.49 ± 3.62 43.92 ± 0.92 41.4 ± 1.78 6.19 ± 0.84 17.6 ± 2.57
22 F. benjamina 0 0 0 0 0 61.66 ± 2.55 0 0.07 ± 0.04
23 F. drupacea 855.02 ± 57.18 961.21 ± 99.22 1.64 ± 0.45 20.49 ± 2.85 0 87.0 ± 3.48 0 0.3 ± 0.17
24 F. microcarpa 0 0 0 0 0 0 0 0
25 F. elastica 0 0 0 0 0 50.95 ± 9.05 0 4.64 ± 0.89
26 F. macrophylla 0 238.0 ± 31.16 0 239.73 ± 10.81 0 0 0 0
27 F. platypoda 207.85 ± 14.27 213.05 ± 10.92 14.95 ± 2.0 90.92 ± 5.48 0 0 0 0
28 F. vasta 278.99 ± 21.55 413.96 ± 29.65 6.98 ± 0.58 15.83 ± 0.6 48.72 ± 3.75 45.73 ± 0.94 0.22 ± 0.08 8.48 ± 1.08
29 F. craterostoma 0 0 0 0 0 0 0 0
30 F. lingua subsp. lingua 0 0 0 0 0 78.96 ± 3.32 0 1.12 ± 0.41
31 F. natalensis subsp. leprieurii 0 0 0 0 0 53.05 ± 1.39 0 3.04 ± 0.63
32 F. cyathistipula 0 0 0 0 52.1 ± 3.39 53.8 ± 1.14 0.66 ± 0.16 1.33 ± 0.58
33 F. lyrata 0 0 0 0 72.5 ± 2.5 65.63 ± 1.36 0.28 ± 0.09 21.44 ± 1.61
Y. Sosnovsky
Ficus leaf architecture

Table 4 Some quantitative leaf traits of Ficus (mean ± SE)

Species Stomatal Stomatal Stomatal Lamina Adaxial Abaxial Spongy/
length (lm) width (lm) density (per thickness (lm) epidermis epidermis palisade
1 mm2) thickness (lm) thickness (lm) mesophyll
ratio

1 F. carica 20.12 ± 0.89 13.89 ± 0.45 79.52 ± 5.52 78.33 ± 2.8 18.5 ± 1.67 10.78 ± 0.69 1.5 ± 0.1
2 F. erecta 21.38 ± 0.67 17.97 ± 0.36 107.52 ± 8.6 148.95 ± 7.3 31.28 ± 1.68 21.0 ± 1.68 3.19 ± 0.43
3 F. formosana 22.19 ± 0.66 15.03 ± 0.29 127.6 ± 8.74 146.7 ± 10.15 31.35 ± 2.5 19.3 ± 1.32 2.32 ± 0.19
4 F. hederacea 17.37 ± 0.23 16.61 ± 0.15 155.52 ± 13.99 100.25 ± 2.13 34.52 ± 1.54 12.22 ± 0.49 1.16 ± 0.07
5 F. pumila 15.96 ± 0.31 14.13 ± 0.21 212.53 ± 16.2 100.27 ± 2.92 28.71 ± 0.98 13.81 ± 0.51 1.42 ± 0.07
6 F. sagittata 17.29 ± 0.35 14.68 ± 0.29 271.98 ± 30.74 236.81 ± 3.97 63.43 ± 1.79 15.41 ± 0.94 2.3 ± 0.11
7 F. villosa 19.51 ± 0.31 13.76 ± 0.17 145.12 ± 15.06 207.39 ± 7.04 44.2 ± 3.86 26.22 ± 2.34 2.01 ± 0.13
8 F. montana 17.84 ± 0.41 13.92 ± 0.22 226.9 ± 18.07 116.57 ± 2.64 26.96 ± 0.88 16.77 ± 0.99 2.3 ± 0.11
9 F. ulmifolia 15.75 ± 0.32 11.62 ± 0.33 254.1 ± 14.7 97.13 ± 3.7 24.83 ± 1.6 12.28 ± 0.87 1.51 ± 0.11
10 F. tinctoria 16.63 ± 0.41 13.44 ± 0.35 195.52 ± 23.43 175.25 ± 11.88 32.83 ± 2.1 16.43 ± 0.74 5.12 ± 0.5
subsp.
gibbosa
11 F. mucuso 19.21 ± 0.41 12.02 ± 0.39 180.0 ± 8.01 115.16 ± 2.36 27.5 ± 0.65 12.74 ± 0.4 2.25 ± 0.07
12 F. sycomorus 20.45 ± 0.46 12.28 ± 0.46 211.52 ± 11.08 209.49 ± 8.13 44.05 ± 2.34 13.52 ± 0.93 2.12 ± 0.17
13 F. vallis- 19.75 ± 0.45 13.86 ± 0.35 215.36 ± 14.72 240.48 ± 21.32 44.08 ± 3.82 12.57 ± 0.62 1.9 ± 0.16
choudae
14 F. septica 21.92 ± 0.47 13.75 ± 0.22 242.87 ± 17.05 193.58 ± 16.76 31.6 ± 1.23 17.65 ± 1.46 3.1 ± 0.31
15 F. callosa 17.72 ± 0.32 14.14 ± 0.35 364.96 ± 21.06 241.93 ± 10.29 43.9 ± 2.4 15.4 ± 0.99 5.95 ± 0.41
16 F. crocata 32.39 ± 0.55 22.53 ± 0.29 85.04 ± 2.56 205.85 ± 3.4 50.07 ± 0.77 27.4 ± 0.99 2.46 ± 0.12
17 F. 31.06 ± 0.77 23.63 ± 0.33 113.77 ± 4.29 294.79 ± 15.33 63.82 ± 3.01 31.43 ± 1.72 2.39 ± 0.13
luschnathiana
18 F. petiolaris 29.84 ± 0.58 23.55 ± 0.5 67.4 ± 3.08 260.21 ± 16.05 52.33 ± 2.84 26.18 ± 2.14 2.59 ± 0.23
19 F. lacor 26.46 ± 0.5 19.49 ± 0.29 64.64 ± 3.32 137.26 ± 2.88 26.61 ± 0.55 22.38 ± 0.72 3.62 ± 0.12
20 F. religiosa 26.41 ± 0.73 20.59 ± 0.54 130.21 ± 3.89 187.18 ± 18.77 37.5 ± 3.51 22.41 ± 1.66 1.87 ± 0.39
21 F. virens 17.23 ± 0.5 13.7 ± 0.25 189.2 ± 19.4 106.05 ± 4.23 25.6 ± 1.19 10.79 ± 0.6 1.07 ± 0.1
22 F. benjamina 27.12 ± 0.24 29.13 ± 0.42 137.76 ± 6.28 212.62 ± 15.57 48.5 ± 2.93 26.17 ± 1.27 3.43 ± 0.25
23 F. drupacea 36.72 ± 0.66 33.34 ± 0.83 114.72 ± 7.04 335.68 ± 11.83 63.12 ± 3.0 32.64 ± 0.94 3.07 ± 0.11
24 F. microcarpa 32.14 ± 0.43 32.16 ± 0.39 118.48 ± 3.53 297.86 ± 15.25 62.67 ± 2.68 34.09 ± 1.41 2.11 ± 0.3
25 F. elastica 44.14 ± 1.05 41.4 ± 1.0 107.36 ± 5.02 642.47 ± 53.47 174.85 ± 12.8 65.99 ± 2.52 4.08 ± 0.32
26 F. macrophylla 30.49 ± 0.53 25.35 ± 0.28 110.67 ± 11.28 435.55 ± 5.88 91.41 ± 2.25 61.08 ± 1.26 2.4 ± 0.07
27 F. platypoda 33.79 ± 0.56 29.52 ± 0.42 88.8 ± 7.23 341.31 ± 11.71 82.65 ± 2.2 48.17 ± 1.3 1.94 ± 0.09
28 F. vasta 22.72 ± 0.58 18.81 ± 0.46 80.72 ± 7.12 179.56 ± 11.41 29.58 ± 2.62 20.03 ± 1.52 2.35 ± 0.19
29 F. 22.15 ± 0.46 17.59 ± 0.31 118.24 ± 8.27 379.12 ± 18.06 77.99 ± 4.72 22.63 ± 1.01 3.69 ± 0.33
craterostoma
30 F. lingua 22.88 ± 0.61 16.5 ± 0.29 186.24 ± 12.25 282.72 ± 14.39 51.3 ± 2.39 17.45 ± 0.87 3.19 ± 0.14
subsp. lingua
31 F. natalensis 20.71 ± 0.5 14.52 ± 0.27 224.32 ± 12.95 564.06 ± 20.35 68.53 ± 2.85 21.66 ± 0.95 7.82 ± 0.44
subsp.
leprieurii
32 F. cyathistipula 24.0 ± 0.42 20.38 ± 0.27 155.35 ± 14.38 493.22 ± 28.54 55.06 ± 1.64 38.47 ± 2.45 8.65 ± 0.47
33 F. lyrata 22.84 ± 0.33 21.41 ± 0.35 155.52 ± 16.84 269.51 ± 7.03 42.16 ± 1.04 33.55 ± 0.78 2.92 ± 0.14

cystolith (Fig. 2d). The longest ones (up to 24–26 lm)
were located together with stomata within deep
depressions of the leaf surface between veins, whereas
on the large veins such trichomes were very short or
almost absent.
5. Branched—weak, ribbon-like ferruginous trichomes
with a scyphiform to cylindrical base, which occurred
in F. macrophylla (Table 2; Fig. 2e). Trichome
branches were not observed to be separated by septa.
These trichomes were densely interwoven and covered

123

the abaxial leaf surface within slight depressions
between veins where stomata were located.
Glandular trichomes consisted of a one-celled stalk and
a 1–9-celled head; the basal cell was sometimes sunken
into the epidermis similar to that of non-glandular tric-
homes. Several forms of glandular trichomes were found:
1. Capitate, spherical to elliptic, with 2–8-celled head.
Occurred in the species of all the subgenera, but rarely
in Ficus subgen. Urostigma where they were mostly
elliptic (Table 2; Fig. 3a).
2. Elongate or cylindrical, with 1–8-celled head.
Occurred in the species of Ficus subgen. Urostigma
(Table 2; Fig. 3b), sometimes pairwise in F. luschna-
thiana and F. lyrata.
3. Fan-shaped, with the head consisting of up to nine cells
which radiate from the stalk in one longitudinal plane,
often at right angle to the stalk. They were found only
in Ficus subgen. Synoecia sect. Rhizocladus subsect.
Punctulifoliae (Table 2; Fig. 3c).
Stomata
The leaves of the studied species were hypostomatic, except
for Ficus petiolaris having stomata on both leaf sides; the
adaxial stomata were larger (36.61 ± 0.74 lm in length)
and much less dense (0.22 ± 0.09 per 1 mm2) compared to
the abaxial ones (Table 4; Fig. 4a). The stomata were of
anomocytic type with mostly 4–6 subsidiary cells. In Ficus
sect. Stilpnophyllum and sect. Galoglychia as well as Ficus
subsect. Conosycea, encyclocytic-like stomata were present
on the same leaves with the anomocytic ones.
In the studied species having single-layered abaxial
epidermis (see Table 2), stomata were located at the level
of primary epidermal cells or slightly protruded in Ficus
sect. Americanae, which was seen on the transverse leaf
sections. In the other species, stomata were sunken and had
a peristomal rim (Fig. 4b). Stomata were mostly distributed
throughout between veins, but in some species (F. crater-
ostoma, F. cyathistipula, F. lyrata, F. platypoda, and F.
villosa) they were aggregated into more or less large
groups (Fig. 4c).
Hydathodes
In the studied species, three types of laminar hydathodes
were identified: in the form of pits, warts, and stomata
(Table 2; Fig. 4d–f). Pits and warts occurred on the upper
side of the leaf lamina, either nearer to its margin (in Ficus
benjamina, F. elastica, F. formosana, F. pumila), or
throughout between large veins. They were often accom-
panied by a single non-glandular trichome and/or a group
123
Y. Sosnovsky
of glandular ones. In some species (F. craterostoma, F.
drupacea, F. lingua subsp. lingua, F. platypoda), such
hydathodes were sparse and present only on some leaves,
whereas in others (F. mucuso, F. religiosa, F. septica, F.
sycomorus) they were dense––up to four hydathodes per
1 cm2 of the lamina. In F. luschnathiana, pits were also
found on the lower leaf side. Stomata-resembling hydath-
odes were confined to the lower side of leaves and usually
occurred singly on the veins.
Mesophyll
In the examined species, several types of mesophyll were
distinguished largely based on the structure of spongy
parenchyma.
1. Spongy tissue was of typical structure, more or less
dense, with irregular shaped or isodiametric cells. The
palisade was mostly 1–2-layered. Besides, additional
palisade layer on the abaxial side (marked with ‘‘?’’ in
Table 2) was often present in Ficus subgen. Urostig-
ma, though better distinguished near the vascular
bundles and rarely extended along the whole abaxial
epidermis. In some species (F. luschnathiana, F.
religiosa, F. vallis-choudae, F. virens, and in the
leaves––acrophylls of F. pumila), the tendency was
observed as to formation of a third adaxial palisade
layer, which was usually more developed near large
bundles.
2. The whole mesophyll tissue resembled the palisade,
largely 3-layered. It was a characteristic of the thin
blades of F. pumila and F. virens in our study.
However, the portions of inner and abaxial palisade
layers often alternated with the typical but dense
spongy parenchyma.
3. Aerenchymatous (diffuse) spongy parenchyma occu-
pied a large volume in the leaves; the cells were thin
and elongate and the intercellular spaces were large.
The features of palisade parenchyma were the same as
in the typical mesophyll. Several species studied had
this type of mesophyll, namely F. callosa, F. crater-
ostoma, F. cyathistipula, F. drupacea, F. lingua subsp.
lingua, F. macrophylla, and F. natalensis subsp.
leprieurii.
Vein anatomy
The leaf veins contained collateral vascular bundles and the
inverted bundles were additionally present in large veins,
so that the vascular system consisted of two opposite
arcs––abaxial (main bundle) and adaxial (inverted bundle).
Based on the presence and location of the bundles, several
types of the vascular system were recognized in the studied
Ficus leaf architecture
Fig. 1 SEM photographs of the leaf surface in Ficus showing the
morphology of cuticle and wax depositions. a F. vallis-choudae, slightly
folded cuticle and waxy threads (Bar 20 lm). b F. carica, slightly folded
cuticle and glandular wax (Bar 10 lm). c F. luschnathiana, folded
species: (1) abaxial arc is only present; (2) besides the main
arc, only phloem strands are present adaxially (Fig. 5a); (3)
both arcs are clearly distinguished, though the adaxial one
may be separated into several bundles (Fig. 5b); (4) the
arcs are partially or fully merged to form a more or less
closed ring; (5) phloem strands or vascular bundles are
additionally present in the center (Fig. 5b). These types
were confined to the veins of a particular order and
appeared stable within the species studied (Table 5). The
above types were observed through the whole length of
particular veins, but certain transitions occurred within the
cuticle (Bar 50 lm). d F. erecta, smooth cuticle and waxy crust (Bar
10 lm). e F. lyrata, waxy plates (Bar 10 lm). f F. religiosa, waxy
platelets (Bar 20 lm)
primary (midrib or vein I) and secondary veins. Namely,
the third type often changed into the fourth one or vice
versa closer to the vein base; in this case, the both types
featuring the veins of a particular order are presented in
Table 5.
Several forms of the bundle sheath were present in the
leaf veins of the studied plants: (1) the sheath fibers, sur-
rounding the bundle, extend to both adaxial and abaxial
epidermal layers, and from the adaxial side the sheath is
usually much narrower (Fig. 5a–c); (2) the bundle sheath
extends to the abaxial epidermis only (Fig. 5d); (3) the
123
 Y. Sosnovsky

Fig. 2 Non-glandular
trichomes in the cross section of
Ficus leaves. a Uniseriate (F.
crocata) and unicellular (F.
platypoda) straight trichomes
(Bar 40 lm). b Weak filiform
trichomes of F. platypoda (Bar
40 lm). c Papilla-like trichomes
with cystoliths of F. montana
(Bar 30 lm). d A row of ribbed
trichomes of F. pumila (Bar
30 lm). e Branched trichome,
surface view (Bar 30 lm)

bundle sheath is closed, that is not extending to the epi-
dermis (Fig. 5e). The adaxial bundle sheath extensions
were collenchymatous (Fig. 5a, c), whereas the abaxial
ones were either collenchymatous (Fig. 5c) or parenchy-
matous (Fig. 5d). The bundle sheath types of particular
veins were stable within each Ficus species (Table 5). In
some species, the bundle sheath was partially separated
from the epidermis or the extensions were very thin, con-
sisting of 1–2 strands of fibers, and were difficult to notice.
In such cases, both of the bundle sheath types, which were
hard to distinguish, are presented in Table 5 for the veins
of a particular order.
Also, laticifers were found to be mainly associated with
the bundle sheath and the vascular tissue in the leaves of the
studied species (Fig. 5c–e), although they often occurred in
the mesophyll as well. In our study, the laticifers appeared
to have no diagnostic or taxonomic value in Ficus.
Crystal inclusions
In the leaves of the examined Ficus species, crystals of
calcium carbonate (cystoliths) and calcium oxalate were
found. Several forms of calcium oxalate crystals were
present, namely druses, prismatic crystals, and crystal sand.
Prismatic crystals were of two types: large prismatic
crystals mainly confined to bundle sheath cells (especially
dense in minor veins of F. formosana, F. hederacea, and F.
pumila) and small rod-like crystals. The occurrence of
crystal types in particular leaf tissues of the examined
species is presented in Table 5.

123
Ficus leaf architecture
Fig. 3 Glandular trichomes in
the surface view. a Capitate and
elliptic trichomes with three-,
four-, and two-celled head (Bar
20 lm). b Elongate trichomes
(Bar 20 lm). c Fan-shaped
trichome of Ficus villosa (Bar
20 lm)
The cystoliths were mostly present within the enlarged
cells––lithocysts in the adaxial and/or abaxial epidermis
(Table 5); the adjacent epidermal cells radiated from the
lithocysts in the surface view. In the adaxial epidermis of
F. religiosa, the cystoliths occurred in ordinary cells of
normal size. They were usually found in a number of
adjacent cells and so aggregated into groups in contrast to
the normal lithocysts being distributed more or less evenly
in the epidermis.
Results of statistical data analyses
The results of ANOVA showed that many quantitative
parameters of leaf architecture greatly depend on the spe-
cies of Ficus (Table 6) and, therefore, are of importance for
the species recognition. Nevertheless, due to the effect of
uncounted factors, the intraspecific variation of several leaf
parameters exceeded their interspecific variation, namely
of the adaxial non-glandular trichome length and the den-
sity of stomata and calcium oxalate crystals in the epi-
dermis. This can be seen based on the values of sum of
squares (SS), which were low for the interspecific variation
of the mentioned leaf parameters (Table 6). In contrast, the
species factor explained around 80–90 % of the variation
in stomatal length and width, abaxial non-glandular tri-
chome density, abaxial glandular trichome length, and the
thickness of abaxial epidermis and palisade mesophyll
(Table 6).
The results of t test showed significant differences
between Ficus subgenera with regard to some quantitative
leaf traits (Table 7). However, each of the latter distin-
guished only some of the subgenera, and only the features
of stomata and abaxial glandular trichomes separated most
of these taxa. The thickness of lamina and its inner layers
distinguished only Ficus subgen. Pharmacosycea and
subgen. Urostigma from other subgenera. The leaf traits,
which did not differ significantly between subgenera, were
not included in Table 7 (the whole list of quantitative leaf
traits considered is given in Table 6). The latter especially
concerns the densities of trichomes and crystal inclusions,
because they were quite dense in some species and absent
in others even in the same subgenera and sections.
Five factors of PCA analysis of the leaf traits explained
93.19 % of the total variance (Table 8). Factor 1 showed
123

Fig. 4 SEM photographs of stomata and hydathodes on Ficus leaves.
a F. petiolaris, stomata on the adaxial leaf side (Bar 20 lm). b F.
lyrata, sunken stomata (Bar 10 lm). c F. cyathistipula, groups of
significant values (higher than 0.6) of 16 species, most of
which belong to Ficus subgen. Urostigma and were gen-
erally featured in having thick glabrous laminas. Factor 2
showed high values of six species of Ficus subgen. Uro-
stigma, all the studied species of Ficus subgen. Ficus, two
species of Ficus subgen. Sycomorus, and one species of
Ficus subgen. Synoecia. All these species were similar to
have long non-glandular trichomes, which were either
scarce or dense. The lamina was thin to average thick and
the density of stomata was generally lower than in the
species of the previous group; the calcium oxalate crystals
123
Y. Sosnovsky
stomata (Bar 300 lm). d F. lyrata, stoma-like hydathode (Bar
10 lm). e F. elastica, pit-like hydathode (Bar 200 lm). f F. vasta,
wart-like hydathode, note the base of trichome (arrow) (Bar 50 lm)
were either absent or very rare in the abaxial epidermis.
Finally, this group included all the species (except F. val-
lis-choudae) with the additional central phloem strands or
vascular bundles in secondary veins. Factor 3 showed high
values of two species of Ficus subgen. Synoecia and F.
ulmifolia from Ficus subgen. Sycidium. These species were
featured in a thin leaf lamina, a relatively large volume of
the adaxial epidermis, and small and dense stomata. Also,
the adaxial non-glandular trichomes were long and sparse,
whereas the abaxial ones were short and dense. High values
of F. montana and F. septica fell into another separate
Ficus leaf architecture

Fig. 5 Cross sections of veins in the leaves of Ficus. a F. religiosa c F. lacor vein III, note the laticifers (arrow) (Bar 50 lm). d F. lingua
vein II, note the additional phloem strands above (arrow) surrounded subsp. lingua vein II (Bar 50 lm). e F. natalensis subsp. leprieurii
by lignified tissue (Bar 200 lm). b F. religiosa vein I (Bar 500 lm). vein IV (Bar 100 lm)

factors (Table 8). The first species was distinct to have very Discussion
dense and short papilla-like trichomes with cystoliths on
both leaf sides and the second one represented a separate Results of the study show that many qualitative and
section in our study. quantitative leaf structural traits are good for diagnosing

123
 Y. Sosnovsky

Table 5 Anatomical features of the veins of I–V orders and morphological types of crystal inclusions in the leaves of Ficus species examined
Species Type of vascular systema Type of bundle sheathb Type of crystal inclusion
I II III IV V I II III IV V Ad Ab M BS

1 F. carica 4, 5 2 1 1 1 1 1 1 1 1 Dr Cy Dr –
2 F. erecta 3-4 1 1 1 1 1-2 2 2 3 3 Cy Cy Dr Dr, Pr
3 F. formosana 3-4 1 1 1 1 1-2 2 2 3 3 Cy, S Cy, Dr, R, S Dr Dr, Pr
4 F. hederacea 1 1 1 1 1 1 1 1 1 2 – Cy Dr Pr
5 F. pumila 2 1 1 1 1 1 1 1 1 1 Cy, Dr Cy Dr Pr
6 F. sagittata 4, 5 3 1 1 1 1 1 1 1 1 Cy, Dr Cy Dr –
7 F. villosa 4, 5 3 1 1 1 1 2 2 2 2 Cy, Dr Cy, Dr Dr Dr
8 F. montana 4, 5 2 1 1 1 1 1 1 1 1 Cy Cy Dr Dr
9 F. ulmifolia 3-4, 5 4 1 1 1 1 1 1 1 1 Cy Cy, Dr Dr Dr
10 F. tinctoria subsp. gibbosa 3 1 1 1 1 1 1 1-2 1-2 2 Cy, Dr Cy, Dr Dr Dr
11 F. mucuso 3, 5 3, 5 1 1 1 1 1 1 1 1 Cy, Dr, Pr, R Cy Dr Dr, Pr
12 F. sycomorus 4, 5 4 1 1 1 1 1 1 1 1 Dr, Pr, R Cy Dr Dr, Pr
13 F. vallis-choudae 4, 5 3-4, 5 2 1 1 1 1 1 1 1 Cy Cy Dr Dr, Pr
14 F. septica 3, 5 3, 5 1 1 1 1 1 1 1 1 Dr, Pr, R Cy Dr Dr, Pr
15 F. callosa 4 3 1 1 1 1 1 1 1 2 Cy Cy – Dr, Pr
16 F. crocata 4, 5 3, 5 1 1 1 1 1 1 1 2 Cy Cy Dr Dr, Pr
17 F. luschnathiana 3, 5 3 1 1 1 1 1 1 1 1 Cy, Dr Cy, Dr, S Dr Dr, Pr
18 F. petiolaris 4, 5 1 1 1 1 1 1 1 1 1 Cy, Dr Cy Dr Dr, Pr
19 F. lacor 4, 5 1 1 1 1 1 1 1 1 1 – Cy, Pr, S – Pr
20 F. religiosa 4, 5 2 1 1 1 1 1 1 1 1 Cy Cy, Dr Dr Pr
21 F. virens 3-4, 5 3, 5 1 1 1 1 1 1 1 1 Dr Cy Dr Dr
22 F. benjamina 3, 5 1 1 1 1 1 1 1 1 3 Cy, Dr Cy, Dr Dr Dr
23 F. drupacea 3, 5 3, 5 3 3 2 1 1 1 1 1-3 Cy, Dr Cy, Dr – Dr, Pr
24 F. microcarpa 3-4, 5 1 1 1 1 1 2 2 3 3 Cy, Dr Cy Dr –
25 F. elastica 3, 5 1 1 1 1 1 2 3 3 3 Cy, R Cy, R – Dr, Pr
26 F. macrophylla 3-4, 5 3-4 1 1 1 1 1 1 1 1 Cy, Dr Cy Dr Dr
27 F. platypoda 3, 5 1 1 1 1 1 1 1 1 1 Cy Cy, Dr Dr Pr
28 F. vasta 4, 5 3-4, 5 1 1 1 1 1 1 1 1 Cy, Dr Cy, Dr Dr Dr, Pr
29 F. craterostoma 3-4, 5 1 1 1 1 1 2 3 3 3 Cy Cy, Dr – Pr
30 F. lingua subsp. lingua 3-4 1 1 1 1 1 2 3 3 3 Cy Cy, Dr Dr Pr
31 F. natalensis subsp. leprieurii 4, 5 1 1 1 1 2 2 2 3 3 Cy Cy, Dr, S Dr Dr
32 F. cyathistipula 4, 5 4 1 1 1 2 2 2 2 2 Cy, Dr, Pr Cy, Dr, Pr Dr –
33 F. lyrata 4, 5 3 1 1 1 1 1 1 1 1 Cy Cy, Dr Dr Pr
Ab abaxial epidermis, Ad adaxial epidermis, BS bundle sheath and midrib parenchyma, Cy cystoliths, Dr druses, M mesophyll, Pr prismatic
crystals, R rod-like crystals, S crystal sand
a
1, abaxial arc is only present; 2, besides the main arc, only phloem strands are present adaxially; 3, adaxial and abaxial arcs are clearly
distinguished; 4, the arcs are partially or fully merged to form a closed ring; 5, phloem strands or vascular bundles are additionally present in the
center
b
1, bundle sheath extends to both adaxial and abaxial epidermal layers; 2, bundle sheath extends to the abaxial epidermis only; 3, bundle sheath
is closed

Ficus taxa if considered in a complex. Statistical analyses
also demonstrate that the abaxial leaf side in Ficus provides
more traits of diagnostic value than the adaxial one, which
confirms previously published results (Klimko and Truchan
2006). Meanwhile, many of leaf traits are known to depend
on the environmental conditions, e.g., the stomatal density,
the development of leaf indumentum and palisade
mesophyll, etc. (Fahn 1990; Berg and Corner 2005; Klimko
and Truchan 2006; Glukhov and Strelnikov 2011; Chant-
arasuwan et al. 2014). As a result, not all of the leaf
characters are equally useful for the species recognition
and each of them will be further discussed.
Epidermis Many studies evidence for the taxonomic
importance of the leaf epidermis in Ficus (van Greuning

123
Ficus leaf architecture

Table 6 ANOVA results for quantitative leaf structural variables between Ficus species (P \ 0.001)
Source SS df MS F

Lamina thickness
Between species 20,979,470.04 32 655,608.44 107.51
Error 7,208,156.98 1,182 6,098.27
Adaxial epidermis thickness
Between species 930,122.75 32 29,066.34 104.67
Error 327,958.5 1,181 277.7
Abaxial epidermis thickness
Between species 231,548.68 32 7,235.9 133.39
Error 64,067.22 1,181 54.25
Mesophyll thickness
Between species 11,818,841.42 32 369,338.79 85.56
Error 5,097,940.49 1,181 4316.63
Palisade mesophyll thickness
Between species 426,074.79 32 13,314.84 134.57
Error 116,855.53 1,181 98.95
Spongy mesophyll thickness
Between species 8,935,926.49 32 279,247.7 73.52
Error 4,485,681.7 1,181 3,798.21
Spongy/palisade mesophyll ratio
Between species 3,168.51 32 99.02 59.08
Error 1,979.44 1,181 1.68
Adaxial non-glandular trichome length
Between species 18,117,556.46 15 1,207,837.1 34.32
Error 28,299,699.29 804 35,198.63
Adaxial non-glandular trichome density
Between species 402,065.46 32 12,564.55 206.52
Error 186,898.77 3,072 60.84
Adaxial glandular trichome length
Between species 14,908.26 15 993.88 12.56
Error 14,483.4 183 79.14
Adaxial glandular trichome density
Between species 26,986.2 32 843.32 98.93
Error 24,379.55 2,860 8.52
Abaxial non-glandular trichome length
Between species 31,460,533.23 18 1,747,807.4 62.11
Error 30,475,333.29 1,083 28,139.74
Abaxial non-glandular trichome density
Between species 4,673,242.17 32 146,038.82 420.97
Error 980,709.35 2,827 346.91
Abaxial glandular trichome length
Between species 111,746.75 23 4,858.55 87.15
Error 32,056.25 575 55.75
Abaxial glandular trichome density
Between species 719,421.54 32 22,481.92 159.16
Error 390,994.34 2,768 141.26
Stomatal length
Between species 44,539.17 32 1,391.85 155.93
Error 8,631.46 967 8.93

123
 Y. Sosnovsky

Table 6 continued
Source SS df MS F

Stomatal width
Between species 49,827.48 32 1,557.11 318.88
Error 4,721.96 967 4.88
Stomatal density
Between species 14,362,428.76 32 448,825.9 30.83
Error 43,187,681.93 2,967 14,556.01
Cystolith density in the adaxial epidermis
Between species 2,168,493.12 32 67,765.41 196.94
Error 952,095.25 2,767 344.09
Cystolith density in the abaxial epidermis
Between species 1,082,495.62 32 33,827.99 190.04
Error 471,173.87 2,647 178.0
Oxalate crystals density in the adaxial epidermis
Between species 17,763,487.9 32 555,108.99 77.25
Error 20,099,098.51 2,797 7,185.95
Oxalate crystals density in the abaxial epidermis
Between species 32,234,956.27 32 1,007,342.38 73.67
Error 40,160,867.0 2,937 13,674.11

et al. 1984; Sonibare et al. 2005; Klimko and Truchan
2006; Chantarasuwan et al. 2014). We found that the shape
of epidermal cell wall and the number of epidermal layers
appear to be of diagnostic value for the species and higher
taxa of Ficus. The multilayered adaxial epidermis is typical
for Ficus subgen. Synoecia and subgen. Urostigma, except
Ficus subsect. Urostigma (Table 2). According to Chant-
arasuwan et al. (2014), some species of this subsection also
have the multiple epidermis. Among the examined species
of Ficus subgen. Synoecia, F. pumila is an exception,
because its leaves––bathyphylls have single-layered adax-
ial epidermis, unlike to the acrophylls. Berg (2003a) and
Berg and Corner (2005) described these types of leaves for
the subgenus as a whole, although an individual plant may
have either two or one leaf type. The multiple abaxial
epidermis features only Ficus sect. Stilpnophyllum, sub-
sect. Conosycea, and subsect. Cyathistipulae. The straight
or arched walls of the abaxial epidermal cells feature Ficus
subgen. Urostigma, while in the other subgenera they are
undulate.
Cuticle and epicuticular wax layer The type of cuticle
and wax depositions was found to be of taxonomic value,
although at the level of species only. The presence of
folded cuticle and waxy plates is especially important since
they feature only several Ficus species out of all the ones
studied (Table 2). However, in certain species (F. cyathi-
stipula, F. microcarpa, F. natalensis subsp. leprieurii, and
F. religiosa), the wax layer type appears different from that
reported by Klimko and Truchan (2006) for the same
species. Barthlott et al. (1998) reported about the system-
atic significance of the wax layer in plants. By contrast,
certain authors indicated that the wax structure mainly
depends on the environmental conditions (Klimko and
Truchan 2006), which makes it useless for diagnosing plant
taxa.
Trichomes In general, the distribution of trichome types
among Ficus taxa in our study is similar to that reported by
Berg and Corner (2005). The studied plants of Ficus sub-
gen. Urostigma and subgen. Pharmacosycea are featured in
having glabrous leaves compared to the other taxa, except
for F. drupacea, F. macrophylla, F. platypoda, and F.
vasta with quite dense and long hairs. Certain trichome
types are typical for particular taxa, e.g., fan-shaped glan-
dular trichomes are typical for the species of Ficus subsect.
Punctulifoliae, elongate glandular and weak ferruginous
non-glandular trichomes occur only in Ficus subgen.
Urostigma, which is consistent with Berg and Corner
(2005). However, we have not found any published data
concerning the branched trichomes in Ficus. Dixon (2001),
when describing the leaf indumentum in F. macrophylla,
reported about similar hairs, but their branching was not
mentioned. Berg and Corner (2005) indicated moniliform
trichomes in F. macrophylla and F. rubiginosa Vent. In our
study, the moniliform type can be assigned only for the
trichomes in F. platypoda (Fig. 2b), which is closely
related to the former. Certain species have distinct com-
binations of non-glandular trichomes on the leaves
(Table 2). Namely, in F. hederacea, F. mucuso, F. pumila,

123
Ficus leaf architecture

Table 7 Student coefficient values between Ficus subgenera compared for some quantitative leaf traits
Ficus taxa Fic Syn Sycid Sycom Pharm Urost

Lamina thickness
Fic 0
Syn 0.86 0
Sycid 0.15 0.74 0
Sycom 1.84 0.64 1.69 0
Pharm 4.63*** 2.18* 4.38*** 1.83 0
Urost 4.54*** 3.06** 4.4*** 2.83* 1.97 0
Adaxial epidermis thickness
Fic 0
Syn 1.8 0
Sycid 0.24 1.82 0
Sycom 1.62 0.68 1.76 0
Pharm 3.44*** 0.15 4.63*** 1.45 0
Urost 3.78** 1.69 3.97*** 2.71* 2.09* 0
Abaxial epidermis thickness
Fic 0
Syn 0.03 0
Sycid 0.54 0.5 0
Sycom 0.86 0.83 0.56 0
Pharm 0.49 0.46 0.14 0.83 0
Urost 3.06** 3.08** 4.34*** 4.73*** 4.45*** 0
Mesophyll thickness
Fic 0
Syn 0.67 0
Sycid 0.22 0.45 0
Sycom 2.09 1.05 1.72 0
Pharm 5.62*** 2.86** 4.36*** 1.78 0
Urost 4.54*** 3.15** 4.03*** 2.32* 1.34 0
Palisade mesophyll thickness
Fic 0
Syn 1.65 0
Sycid 0.59 2.03 0
Sycom 2.05 0.65 2.33 0
Pharm 1.29 1.12 3.05** 1.65 0
Urost 5.72*** 2.1* 7.16*** 0.93 5.46*** 0
Spongy mesophyll thickness
Fic 0
Syn 0.38 0
Sycid 0.29 0.07 0
Sycom 1.93 1.18 1.24 0
Pharm 6.03*** 3.99*** 3.97*** 3.34*** 0
Urost 3.99*** 3.22** 3.24** 2.52* 0.54 0
Spongy/palisade mesophyll ratio
Fic 0
Syn 1.1 0
Sycid 0.54 1.11 0
Sycom 0.02 1.67 0.56 0
Pharm 5.69*** 8.71*** 2.54* 7.44*** 0
Urost 1.47 3.01** 0.29 1.84 4.3*** 0

123
 Y. Sosnovsky

Table 7 continued
Ficus taxa Fic Syn Sycid Sycom Pharm Urost

Abaxial glandular trichome length

Fic 0
Syn 0.21 0
Sycid 0.5 0.56 0
Sycom 3.62** 3.39* 2.83* 0
Pharm 5.82*** 3.85*** 2.66** 1.45 0
Urost 6.1*** 5.77*** 5.22*** 2.44* 4.29*** 0
Abaxial glandular trichome density
Fic 0
Syn 26.9*** 0
Sycid 1.99 0.02 0
Sycom 1.45 0.68 0.46 0
Pharm 16.42*** 1.53 0.26 0.93 0
Urost 2.34* 13.78*** 1.74 1.18 11.65*** 0
Stomatal length
Fic 0
Syn 3.9* 0
Sycid 5.26** 0.83 0
Sycom 1.06 2.98* 4.26** 0
Pharm 5.17*** 0.23 1.43 3.91*** 0
Urost 4.05*** 6.09*** 6.75*** 4.6*** 6.48*** 0
Stomatal width
Fic 0
Syn 0.61 0
Sycid 1.88 1.91 0
Sycom 2.03 2.29 0.02 0
Pharm 1.17 0.91 1.46 1.95 0
Urost 3.78** 4.81*** 5.73*** 5.97*** 5.41*** 0
Stomatal density
Fic 0
Syn 2.82* 0
Sycid 5.5** 0.86 0
Sycom 5.67*** 0.51 0.62 0
Pharm 10.3*** 4.68*** 5.16*** 6.18*** 0
Urost 1.16 2.3* 5.08*** 5.31*** 10.24*** 0
Abbreviations designate subgenera: Fic, Ficus subgen. Ficus; Syn, Ficus subgen. Synoecia; Sycid, Ficus subgen. Sycidium; Sycom, Ficus subgen.
Sycomorus; Pharm, Ficus subgen. Pharmacosycea; Urost, Ficus subgen. Urostigma
Significance level: *** P \ 0.001, ** P \ 0.01, * P \ 0.05

and F. sycomorus trichomes are most diverse––papilla-,
prickle-like and straight hairs occur together on both leaf
sides. In the three studied species of Ficus subgen. Syci-
dium as well as in F. vallis-choudae, papilla-like trichomes
are predominant and mainly combined with the prickle-like
ones. The distinct feature of F. platypoda is a combination
of straight and tortuous trichomes. Trichome length and
density seem useless for the identification of Ficus taxa.
Although the trichome length is generally higher in Ficus
subgen. Urostigma compared to the other groups (Table 3),
statistical analyses indicate that the quantitative features of
only glandular abaxial trichomes are of diagnostic value for
higher taxa of Ficus (Table 7). Also, according to Berg and
Corner (2005), many Ficus species may have glabrous and
pilose leaves, which impedes using hair density as a
diagnostic feature.
Stomata The quantitative features of stomata are argu-
able as to their taxonomic value, both in general and in the

123
Ficus leaf architecture

Table 8 Factor loadings from the PCA of Ficus quantitative leaf Ficus subgen. Urostigma, the smallest ones are for Ficus
traits subgen. Sycidium. However, the stomatal width signifi-
Species Factor Factor Factor Factor Factor cantly separates only Ficus subgen. Urostigma from the
1 2 3 4 5 other taxa, while it greatly depends on the species of Ficus.
The densest stomata feature Ficus subgen. Pharmacosycea
1 F. carica 0.06 0.94 0.29 0.03 0.08
and subgen. Sycidium, the least dense ones occur in Ficus
2 F. erecta 0.46 0.71 -0.08 0.05 0.32
subgen. Ficus (see Tables 1, 4). Meanwhile, the diagnostic
3 F. formosana 0.01 0.94 0.29 -0.02 -0.1
value of stomatal density at the species level appears low
4 F. hederacea 0.22 0.49 0.77 0.13 -0.01
due to its high intraspecific variation. The separation of
5 F. pumila 0.08 0.59 0.76 0.13 -0.05
Ficus subgen. Pharmacosycea based on any leaf traits
6 F. sagittata 0.18 0.79 0.54 0.09 -0.06
seems doubtful, because it is represented by only one
7 F. villosa 0.77 0.05 0.15 0.61 -0.01
species in our study. Sunken stomata are found diagnostic
8 F. montana 0.47 -0.04 0.55 0.06 0.63
for Ficus sect. Stilpnophyllum, subsect. Conosycea, and
9 F. ulmifolia 0.35 0.36 0.75 0.05 0.07
subsect. Cyathistipulae, because they are associated with
10 F. tinctoria 0.69 -0.06 0.38 -0.08 0.05 multiple abaxial epidermis. Also, the three studied species
subsp.
gibbosa of Ficus sect. Americanae are featured in quite small sto-
11 F. mucuso 0.12 0.74 0.54 0.37 -0.01 matal guard cells compared to the size of primary epider-
12 F. sycomorus 0.3 0.69 0.21 0.59 0.04 mal cells if seen in a transverse section, and the stomata are
13 F. vallis- 0.78 0.31 0.42 0.18 0.22
often slightly protruded from the epidermis. Certain
choudae authors (Ogunkunle and Oladele 2008; Ogunkunle 2013)
14 F. septica 0.42 -0.01 0.15 0.88 0.03 reported about the diagnostic value of the number of sub-
15 F. callosa 0.73 0.08 0.52 0.23 0.3 sidiary cells in Ficus, which is not confirmed in our study.
16 F. crocata 0.2 0.96 -0.00 -0.00 0.08 Likewise to our results, in many studies, the stomatal type
17 F. 0.95 0.14 0.06 0.13 0.01 in Ficus is defined as anomocytic (Grambast 1954; van
luschnathiana Greuning et al. 1984; Klimko and Truchan 2006; Araújo
18 F. petiolaris 0.5 0.8 0.06 0.19 -0.14 2012). However, some evidence occur in the literature as
19 F. lacor 0.85 0.02 0.19 -0.06 -0.31 for actinocytic, anisocytic, diacytic, paracytic, and poly-
20 F. religiosa 0.91 0.15 0.24 0.15 0.2 cytic types (Sonibare et al. 2005; Ogunkunle and Oladele
21 F. virens -0.01 0.87 0.48 0.06 0.1 2008; Glukhov and Strelnikov 2011; Khan et al. 2011;
22 F. benjamina 0.93 0.08 0.22 0.17 -0.02 Ogunkunle 2013; Chantarasuwan et al. 2014). Certain
23 F. drupacea -0.07 0.99 0.09 -0.04 -0.07 authors indicate the presence of several stomatal types in
24 F. microcarpa 0.94 0.19 0.05 0.11 0.09 one Ficus species or even on the same leaves (Ogunkunle
25 F. elastica 0.92 0.16 -0.13 0.22 -0.18 and Oladele 2008; Glukhov and Strelnikov 2011). Sup-
26 F. macrophylla 0.71 0.5 -0.24 0.08 0.24 posedly, this may have resulted from the ambiguity of
27 F. platypoda 0.54 0.8 0.02 0.00 0.02 interpretations of the stomatal patterns because of their
28 F. vasta 0.05 0.98 0.06 -0.03 0.01 morphological resemblance and partly overlapped defini-
29 F. craterostoma 0.95 0.21 0.00 0.09 0.01 tions proposed (e.g., Prabhakar 2004).
30 F. lingua subsp. 0.92 0.15 0.23 0.15 0.11
Hydathodes No diagnostic value has been previously
lingua reported for laminar hydathodes in Ficus, and only few
31 F. natalensis 0.95 0.18 0.06 0.09 -0.02 works have dealt with their structure (Chen and Chen 2005,
subsp. 2006, 2007). Klimko and Truchan (2006) described the
leprieurii types and general occurrence of the hydathodes in selected
32 F. cyathistipula 0.77 -0.05 0.07 0.02 -0.58 species of Ficus, without referring to the relation between
33 F. lyrata 0.91 0.14 0.19 0.15 0.12 hydathode type and Ficus taxa. Meanwhile, the laminar
Percent of total 55.1 22.97 7.67 3.86 3.59 hydathodes similar to those in Ficus appear taxonomically
variance explained
specific since they are known from only several plant
The values higher than 0.6 are marked with italics families (Lersten and Curtis 1991). In our study, the den-
sity of pit- and wart-like adaxial hydathodes and their
position on the leaf lamina are constant in some species
genus Ficus in particular (Chantarasuwan et al. 2014). We (indicated in the Results section) and variable in others.
found them to be of taxonomic and diagnostic value in the The hydathode type appears to be the most stable param-
genus since they appeared quite stable within particular eter at the species level (Table 2). This indicates that
taxa (Tables 6, 7). The largest stomata are characteristic for hydathodes may contribute to the separation of Ficus

123

species. However, the diagnostic value of the adaxial
hydathodes is limited by the fact that only one of their
types occurs in most of the examined species. Hence,
additional studies are needed to reveal a potential taxo-
nomic importance of these structures in the genus Ficus. It
should be also noted that Klimko and Truchan (2006)
reported about the absence of hydathodes on the upper leaf
side of F. benjamina, F. elastica, and F. sagittata, which is
different from our results (Table 2). Meanwhile, the
absence of hydathodes in F. carica observed by those
authors is confirmed in our study. Also, F. elastica was
noted to have hydathodes only on the sapling leaves (Berg
and Corner 2005), while in our study they occurred on the
mature leaves of all the individual plants including the old
ones reaching up to 10 m in height.
Mesophyll Van Greuning et al. (1984) indicated the
taxonomic importance of spongy mesophyll structure in
Ficus, while Sonibare et al. (2006) reported the features of
the palisade to be of diagnostic value. Our results show that
the presence of either single- or multilayered palisade
mesophyll, continuous abaxial palisade layer, and diffuse
spongy parenchyma are of particular value for distin-
guishing Ficus species and higher taxa. In the examined
species of Ficus subgen. Sycidium, the palisade is single-
layered, as well as in F. erecta, F. hederacea, F. lacor, and
leaves––bathyphylls of F. pumila. In Ficus sect. Stilpno-
phyllum, sect. Galoglychia, and subsect. Conosycea
(except F. benjamina), two dense palisade layers are
clearly recognized. The continuous abaxial palisade layer
features F. elastica, F. microcarpa, and F. platypoda. The
diffuse spongy parenchyma is found in F. callosa and six
species of Ficus subgen. Urostigma indicated in the Results
section. The all-palisade mesophyll seems to be of less
diagnostic value since it often alternates with the typical
mesophyll in the same leaves. This indicates the inner and
abaxial palisade layers to be a palisade-like spongy
parenchyma described by certain authors in Ficus (Philpott
1953; van Greuning et al. 1984). Also, Ficus subgen.
Pharmacosycea and subgen. Urostigma are generally fea-
tured in thick mesophyll layers, which separates them well
from the other taxa of Ficus. However, the feature of Ficus
subsect. Urostigma is a relatively thin mesophyll layer.
Vascular bundles Diagnostic value of the vascular sys-
tem in Ficus leaves was reported by Sonibare et al. (2006)
and confirmed in our study. The type of vascular system
which is most different between the species is that of pri-
mary and secondary veins (Table 5). Among the species
examined, only F. hederacea is featured in total absence of
additional bundles and phloem strands, whereas F. drup-
acea is distinct to have additional phloem strands adaxially
even in small veins. The central phloem strands in sec-
ondary veins are only found in the species of Ficus subgen.
Sycomorus and subgen. Urostigma. Also, in the midrib
123
Y. Sosnovsky
base of some species (F. crocata, F. drupacea, F. lusc-
hnathiana, F. natalensis subsp. leprieurii, F. vallis-chou-
dae), the vascular system distinctively consists of a number
of separate bundles, which suggests the potential diagnostic
value of the petiole anatomy. The latter was demonstrated
by Carvajal and Shabes (2005) for the American species of
Ficus.
Bundle sheath The bundle sheath structure is often
considered to be of diagnostic value in the genus Ficus
(Berg and Corner 2005; Sonibare et al. 2006; Chantarasu-
wan et al. 2014). However, in the mentioned works, the
type of bundle sheath was described when it extends to the
adaxial epidermis only, which was not found in our study.
On the other hand, the authors did not report about the type
when it only extends abaxially, except for some earlier
works on Ficus (Philpott 1953; van Greuning et al. 1984).
Our results show that the bundle sheath type which is most
different between the species is that of the vein V
(Table 5). The species of Ficus subsect. Frutescentiae and
subsect. Chlamydodorae and certain other species (F. cy-
athistipula, F. elastica, and F. microcarpa) are featured in
early separation of the closed bundle sheath (i.e., in the
veins III–IV and following). Chantarasuwan et al. (2014)
reported about the closed (or circular) bundle sheath in the
tertiary veins of all the species of Ficus subsect. Urostigma
studied. This is in contrast to our results showing the pre-
sence of both bundle sheath extensions in the same veins of
species of this subsection. However, the authors considered
the minor extensions inessential, while in our study they
are thin but clear (except for some cases indicated in the
Results section) and so we take them into account.
Lithocysts and calcium crystals Crystal inclusions in the
leaves are shown useful for the species recognition. The
presence and absence of cystoliths in the epidermis of the
studied species conforms well with that reported by Berg
and Corner (2005) for particular taxa of Ficus. Chantar-
asuwan et al. (2014) suggested the taxonomic importance
of cystolith presence in ordinary adaxial epidermal cells of
some species of Ficus subgen. Urostigma. However, we
found such cystoliths only in one species, namely F. reli-
giosa, for which they were already reported previously
(Chantarasuwan et al. 2014). The occurrence of lithocysts
forming prickle- or papilla-like trichomes also appears
diagnostic, which is discussed above. Certain authors
(Kuo-Huang et al. 2002) indicate that the presence of either
hair-like or papillate lithocysts is related to the number of
epidermal layers in the family Moraceae, which is not
confirmed in our study. The combinations of calcium
oxalate crystals types in the epidermis and bundle sheath
are found stable in each Ficus species studied (Table 5),
which seems useful for the species identification. Among
the higher taxa, the feature of Ficus subgen. Sycomorus is
the presence of prismatic and rod-like crystals in the
Ficus leaf architecture
adaxial epidermis of most of its examined species. The
taxonomic importance of the crystal type distribution in
Ficus leaves was also shown by Sharawy (2004). However,
the density of both cystoliths and oxalate crystals in the
epidermis of the species studied is very variable and,
therefore, not useful as diagnostic feature. The latter can be
also supported by the fact that the formation and dissolu-
tion of calcium crystals in plants was reported to depend on
the availability of calcium ions (e.g., Wu et al. 2006).
From the foregoing, the new data on Ficus leaf anat-
omy presented provide some strength to the existing
classification of the genus. The considered qualitative leaf
traits allow to identify species as well as some subsections
and sections of Ficus. However, most of such characters
are very variable and, therefore, of less diagnostic value at
the subgenus level. By contrast, the quantitative leaf
parameters separate well most of Ficus subgenera. This
all suggests that many leaf characters of Ficus are
genetically determined and so may potentially reflect the
phylogenetic relationships between the taxa. Indeed, early
molecular studies on Ficus largely supported its current
classification (Berg and Corner 2005). Later on, Rønsted
et al. (2005, 2008a, b) evidenced for paraphyly of all the
subgenera except Ficus subgen. Sycidium, although most
of the sections were recognized as monophyletic.
Recently, Chantarasuwan et al. (2014) suggested that
additional studies are needed to resolve some intrasec-
tional discrepancies in Ficus. Results of the present study
do not precisely fit the mentioned above groupings of
Ficus, but they do demonstrate that the species placed
closely in the current classification of the genus show a
degree of affinity based on the microscopic leaf structure.
Hence, the considered leaf traits are evidently of notable
value for the taxonomy of Ficus and may be used to
identify its infrageneric taxa. Furthermore, conformity of
the presented results with Ficus taxonomy largely based
on plant material obtained in the wild indicates that the
greenhouse conditions do not cause considerable changes
in leaf anatomy of cultivated plants exceeding the reaction
norm of the species. This evidences for greenhouse
material to be equally useful for plant taxonomy as wild-
growing specimens.
Given below is a dichotomous diagnostic key of the
species examined based on the most recognizable leaf traits
among those analyzed in this paper. The key enables to
identify not only the species of Ficus, but also most of its
subsections and some sections studied. If a specific leaf
trait or a complex of traits features all the examined species
of a particular supra-specific taxon, the latter is given
parenthetically in the key.
Tentative key to Ficus species examined based on leaf
anatomy
1a. Adaxial epidermis multilayered... 2
1b. Adaxial epidermis mostly single-layered... 22
2a. Abaxial epidermis multilayered... 3
2b. Abaxial epidermis single-layered... 10
3a. Abaxial epidermis 2-layered... 4
3b. Abaxial epidermis 3-layered... (Ficus sect. Stilpno-
phyllum) 8
4a. Leaves covered with tortuous ferruginous hairs;
hydathodes pit-like adaxially; central phloem strands
present in secondary veins additionally to main bundles;
vascular system of veins III–V consists of either abaxial
and adaxial arcs or abaxial arc and only phloem strands
adaxially... F. drupacea
4b. Leaves glabrous; hydathodes wart-like or absent
adaxially; central phloem strands absent in secondary
veins; vascular system of veins III–V consists of only
abaxial arc... 5
5a. Hydathodes present adaxially... F. benjamina
5b. Hydathodes absent adaxially... 6
6a. Glandular hairs present; stomata aggregated into
large groups; abaxial palisade mesophyll layer underde-
veloped or absent; vascular system of secondary veins
consists of abaxial and adaxial arcs, which may be
merged to form a closed ring; bundle sheath extends to
adaxial and/or abaxial epidermis in veins IV–V... (Ficus
subsect. Cyathistipulae) 7
6b. Glandular hairs absent; stomata distributed evenly
between large veins; abaxial palisade mesophyll layer
clearly distinguished; vascular system of secondary
veins consists of only abaxial arc; bundle sheath closed
in veins IV–V... F. microcarpa
7a. Epicuticular wax layer does not form plates; lamina
thick (450–500 lm); bundle sheath extends to only
abaxial epidermis in veins I–V; calcium oxalate forms
druses and prismatic crystals in the epidermis above
midrib; calcium crystals absent in the bundle sheath... F.
cyathistipula
7b. Epicuticular wax layer forms plates abaxially;
lamina of average thickness (200–300 lm); bundle
sheath extends to both abaxial and adaxial epidermal
layers; calcium oxalate forms only druses in the
epidermis and prismatic crystals in the bundle sheath...
F. lyrata
8a. Leaves covered with weak ferruginous hairs adax-
ially and/or abaxially; bundle sheath extends to both
abaxial and adaxial epidermal layers in veins I–V;
calcium oxalate forms druses in the epidermis and either
druses or prismatic crystals in the bundle sheath... (Ficus
subsect. Malvanthera) 9
8b. Leaves glabrous; bundle sheath closed in veins III
and following; calcium oxalate forms rod-like crystals in
the epidermis as well as both druses and prismatic
123

crystals in the bundle sheath... (Ficus subsect. Stilpno-
phyllum) F. elastica
9a. Hairs not branched; hydathodes present adaxially;
the cells of the innermost abaxial epidermal layer not
vertically elongate; abaxial palisade mesophyll layer
present; spongy mesophyll not diffuse, the cells are
irregularly shaped or rounded; vascular system of
secondary veins consists of only abaxial arc; calcium
oxalate forms prismatic crystals in the bundle sheath...
F. platypoda
9b. Hairs branched; hydathodes absent adaxially; the
cells of the innermost abaxial epidermal layer often
vertically elongate; abaxial palisade mesophyll layer
absent; spongy mesophyll diffuse, the cells are often
elongate; vascular system of secondary veins consists of
both abaxial and adaxial arcs; calcium oxalate forms
druses in the bundle sheath... F. macrophylla
10a. Bundle sheath closed in veins III(IV)-V... (Ficus
subsect. Chlamydodorae) 11
10b. Bundle sheath extends adaxially and/or abaxially in
veins III-V... 13
11a. Cuticle folded abaxially; epicuticular wax layer
forms plates abaxially; central phloem strands absent in
midrib... F. lingua subsp. lingua
11b. Cuticle smooth; epicuticular wax layer does not
form plates abaxially; central phloem strands present in
midrib... 12
12a. Epicuticular wax layer forms plates adaxially;
hydathodes absent adaxially; stomata distributed evenly
between veins; bundle sheath extends only abaxially in
veins I–III; calcium oxalate forms druses in the bundle
sheath... F. natalensis subsp. leprieurii
12b. Epicuticular wax layer does not form plates
adaxially; hydathodes present adaxially; stomata aggre-
gated in groups of 3–10; bundle sheath extends both
abaxially and adaxially in vein I, only abaxially in vein
II, and is closed in vein III; calcium oxalate forms
prismatic crystals in the bundle sheath... F. craterostoma
13a. Cuticle (partly) folded adaxially... 14
13b. Cuticle smooth adaxially... 19
14a. Hairs filiform straight, papilla- and prickle-like on
the same leaves; central phloem strands absent in midrib;
calcium oxalate forms dense prismatic crystals in the
bundle sheath... 15
14b. Hairs only filiform straight or absent; central
phloem strands present in midrib; calcium oxalate forms
both druses and prismatic crystals in the bundle sheath...
16
15a. Abaxial surface smooth; the cells of two adaxial
epidermal layers are (almost) equal in size; palisade
mesophyll single-layered; vascular system of midrib
consists of only abaxial arc; cystoliths absent adaxially...
(Ficus sect. Kissosycea) F. hederacea
123
Y. Sosnovsky
15b. Abaxial surface covered with papillae raised from
each epidermal cell; the cell size of adaxial epidermis
sharply increases towards the deeper layers; palisade
mesophyll mostly 2-layered; vascular system of midrib
consists of abaxial arc and additional phloem strands
adaxially; cystoliths present adaxially... (Ficus subsect.
Plagiostigma) F. pumila
16a. Hydathodes pit-like adaxially; glandular hairs
elongate or absent; stomata slightly protruded from the
epidermis if seen in transverse section... (Ficus sect.
Americanae) 17
16b. Hydathodes wart-like adaxially; glandular hairs
capitate; stomata on the level with epidermis... (Ficus
subsect. Platyphyllae) F. vasta
17a. Cuticle slightly folded abaxially; central phloem
strands present in secondary veins additionally to main
bundles... F. crocata
17b. Cuticle heavily folded abaxially; central phloem
strands absent in secondary veins... 18
18a. Straight hairs present; pit-like hydathodes absent
abaxially; vascular system of secondary veins consists of
only abaxial arc... F. petiolaris
18b. Straight hairs absent; pit-like hydathodes present
abaxially; vascular system of secondary veins consists of
both abaxial and adaxial arcs... F. luschnathiana
19a. Non-glandular hairs filiform straight or absent;
glandular hairs fan-shaped... (Ficus subsect. Punctulifo-
liae) 20
19b. Non-glandular hairs papilla- and prickle-like;
glandular hairs capitate... 21
20a. Bundle sheath extends only abaxially in veins II-
V... F. villosa
20b. Bundle sheath extends both abaxially and adaxial-
ly... F. sagittata
21a. Cuticle smooth abaxially; palisade mesophyll
single-layered; vascular system consists of abaxial and
adaxial arcs in midrib and only abaxial arc in secondary
veins; calcium oxalate forms only druses in the bundle
sheath... F. tinctoria subsp. gibbosa
21b. Cuticle slightly folded abaxially; palisade meso-
phyll 2-layered; vascular system consists of both arcs
and additional central phloem strands in midrib and
secondary veins; calcium oxalate forms druses and
prismatic crystals in the bundle sheath... F. vallis-
choudae
22a. Bundle sheath extends only abaxially in secondary
and tertiary veins... (Ficus subsect. Frutescentiae) 23
22b. Bundle sheath extends both abaxially and adaxial-
ly... 24
23a. Cuticle smooth adaxially; calcium oxalate crystals
present in the epidermis and are multiform... F.
formosana
Ficus leaf architecture
23b. Cuticle slightly folded adaxially; calcium oxalate
crystals absent in the epidermis... F. erecta
24a. Cystoliths absent adaxially... 25
24b. Cystoliths present... 29
25a. Central phloem strands present in secondary veins
additionally to main bundles... 26
25b. Central phloem strands absent in secondary veins...
27
26a. Hydathodes pit-like adaxially; spongy mesophyll
2.5–3.5 times thicker than palisade; calcium oxalate
forms druses and prismatic crystals in adaxial epidermis
and bundle sheath... (Ficus subsect. Sycocarpus) F.
septica
26b. Hydathodes wart-like adaxially; spongy mesophyll
equal to palisade or thinner; calcium oxalate forms only
druses... F. virens
27a. Cuticle slightly folded adaxially; hairs with papil-
late surface abaxially; hydathodes absent... (Ficus sub-
sect. Ficus) F. carica
27b. Cuticle smooth; hairs with smooth surface or
absent; hydathodes present... 28
28a. Non-glandular hairs absent; glandular hairs elon-
gate or absent; palisade mesophyll single-layered; vas-
cular system of secondary veins consists of only abaxial
arc... F. lacor
28b. Non-glandular hairs present; glandular hairs capi-
tate; palisade mesophyll 2-layered; vascular system of
secondary veins consists of both abaxial and adaxial arcs
partially or fully merged to form a closed ring... F.
sycomorus
29a. Cuticle slightly folded; vascular system of second-
ary veins consists of only abaxial arc... (Ficus subsect.
Plagiostigma) F. pumila
29b. Cuticle smooth; vascular system of secondary veins
consists of either both abaxial and adaxial arcs or abaxial
arc and only phloem strands adaxially... 30
30a. Palisade mesophyll single-layered... (Ficus sect.
Sycidium) 31
30b. Palisade mesophyll mostly 2-layered... 32
31a. Hairs papilla-like with papillate surface; hydathodes
pit-like adaxially; vascular system of secondary veins
consists of abaxial arc and phloem strands adaxially... F.
montana
31b. Hairs filiform, papilla- and prickle-like with smooth
surface; hydathodes wart-like adaxially; vascular system
of secondary veins consists of both abaxial and adaxial
arcs partially or fully merged to form a closed ring... F.
ulmifolia
32a. Central phloem strands present in midrib addition-
ally to main bundles... 33
32b. Central phloem strands absent... (Ficus subsect.
Pedunculatae) F. callosa
33a. Hairs present; epicuticular wax does not form
platelets; vascular system of secondary veins consists of
abaxial and adaxial arcs and additional central phloem
strands; calcium oxalate forms druses, prismatic and rod-
like crystals in the epidermis... F. mucuso
33b. Hairs absent; epicuticular wax forms platelets;
vascular system of secondary veins consists of abaxial
arc and phloem strands adaxially; calcium oxalate forms
only druses in the epidermis... F. religiosa
Acknowledgments I am pleased to thank Dr. Andriy Prokopiv for
reviewing this manuscript, constructive discussions and comments.
Thanks to curators of greenhouse plant collections––Dr. Lyudmyla
Buyun, Dr. Vira Nikitina, Dr. Olha Usoltseva, and M.Sc. Nadiya
Lutsyshyn for providing permissions to work in greenhouses of the
botanical gardens. Laboratory assistant Yuryi Datsyuk provided tech-
nical assistance in the use of scanning electron microscope. Thanks to
Dr. Mariya Dyka for the help in statistical data analysis. The anonymous
reviewer’s helpful comments are gratefully acknowledged.
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