Leaf morphological variability and intraspeci fic taxonomic units for [629554]

Leaf morphological variability and intraspeci fic taxonomic units for
pedunculate oak and grayish oak (genus Quercus L., series Pedunculatae
Schwz.) in Southern Carpathian Region (Romania)
Ecaterina Nicoleta Apostola,b,⁎, Alexandru Lucian Curtub, Liviu Mihai Daiac, Bogdan Apostola,
Cristiana Georgeta Dinua,N e c u l a e Șofleteab
aNational Institute for Research and Development in Forestry (INCDS) “Marin Dr ăcea”,E r o i l o rN o .1 2 8 ,V o l u n t a r i ,I l f o v ,R o m a n i a
b“Transilvania ”University of Bra șov, Bra șov, Romania
cNational Forest Administration –ROMSILVA, Petricani Street, 9A, Bucharest, Romania
HIGHLIGHTS
•Pedunculate oak and grayish oak taxa
have different adaptability.
•Morphological variability of Q.robur and
Q.pedunculi floraat inter-and intraspe-
cific levels were evaluated.
•Q.robur and Q.pedunculi florapower of
differentiation is con firmed by forming
two obvious clouds of points.GRAPHICAL ABSTRACT
Q.robur leaf traits: pubescence intensity (PU), lamina length (LL), petiole length (PL), lobe width (LW), sinus width (SW),
length of lamina at largest width (WP).
abstract article info
Article history:
Received 24 February 2017Received in revised form 30 May 2017
Accepted 30 May 2017
Available online 26 July 2017
Editor: Elena PAOLETTIEven though pedunculate oak ( Quercus robur L.) and grayish oak ( Quercus pedunculi floraK. Koch) have different
ecological requirements, they have been considered as having low differentiation at the level of morphological
traits and genetic variation. The leaf morphology for 862 trees has been assessed in 16 natural populations,seven of Q.robur ,e i g h to f Q.pedunculi floraand a mixed forest were both taxa coexist. In total, fifteen descriptors
have been analysed by using discriminant analysis, while it was found that with only four out of the fifteen leaf
traits (abaxial pubescence, abaxial colour of the leaf, petiole length and basal shape of lamina) the two taxa could
be clearly differentiated. A dendrogram has been constructed on the basis of these traits, where the populationsof each taxon have been clustered together. PU and CL traits of Q.pedunculi florawere discussed for their adaptive
value for drought resistance in the steppe habitats occupied by this taxon. Using the leaves' morphological de-
scriptors and data from the literature, intra-taxonomic units (varieties, forms and sub-forms) have been identi-fied in all analysed populations. Eight intraspeci fic units for Q.robur and six for Q.pedunculi florahave been
identi fied in the investigated area. An analysis of spatial distribution of the two taxa and of their intraspeci fic
units has been performed using maps of ecoregions for the study area.
© 2017 Elsevier B.V. All rights reserved.Keywords:
Quercus robur
Quercus pedunculi flora
Series PedunculataeTaxonomyLeaf descriptorsIntraspeci ficu n i t sScience of the Total Environment 609 (2017) 497 –505
⁎Corresponding author at: National Institute for Research and Development in Forestry (INCDS) “Marin Dr ăcea”, Eroilor No. 128, Voluntari, Ilfov, Romania.
E-mail addresses: [anonimizat] ,[anonimizat] (E.N. Apostol).
http://dx.doi.org/10.1016/j.scitotenv.2017.05.274
0048-9697/© 2017 Elsevier B.V. All rights reserved.
Contents lists available at ScienceDirect
Science of the Total Environment
journal homepage: www.elsevier.com/locate/scitotenv

1. Introduction
As one of the most important economically and ecologically tree
genera, widespread in the Northern Hemisphere, the genus Quercus
(oaks) includes about 350 –500 species ( Kubitzki 1993, Nixon 1993 ).
From the European white oaks ( i.e.Quercus robur L.,Q.petraea (Matt.)
Liebl., Q.pubescens Willd., Q.frainetto Ten., Q.cerris L.,Q.faginea Lam.,
Q.macranthera Fisch. & C.A.Mey, Q.pyrenaica Willd., Q.canariensis
Willd., Q.alnifolia Poech, Q.infectoria Oliv., Q.coccifera L.), in Romania,
the genus Quercus is represented by 5 –9 species (the firstfive stated, in-
cluding Q.virgiliana Ten., Q.pedunculi floraK. Koch), according to several
taxonomical classi fications ( Georgescu & Morariu 1948, St ănescu et al.
1997, Șofletea & Curtu 2007 ). Oaks are the second most important
broadleaved species, following beech, covering about 16% of the forest
area of the country (Forest National Inventory 20121).
In the context of climate change, with increasing temperatures and
decreasing rainfall, in most of the cases, the white oak species were reg-istered highly increasing periodicity of fructi fication, even to up to
10 years ( Curtu et al. 2015 ). Thus, to assess oak's genetic variability in
order to preserve its gene pool and the “valuable gene funds character-
ized by increased genetic diversity, particularly bioaccumulation capac-
ity, exceptional wood quality and remarkable adaptive traits ”(Șofletea
2005 ) is currently of outmost importance.
Pedunculate oak ( Quercus robur L.) and grayish oak ( Quercus
pedunculi floraK. Koch) belong to sub genus Lepidobalanus (Endl.)
Oerst, section Robur Schb., series Pedunculatae Schwz. The two taxa
have different adaptations: the first one is a mesophilic to
mesohydrophyllic and mesothermic element ( Becker & Levy 1990 ),
and the second one is thermophilic and xero fitic (Curtu et al. 2011,
Enescu 1993, Tomescu 2000 ). These adaptive differences should be con-
sidered in the future management of forests in the distribution area of
the two taxa.
Quercus robur L. (pedunculate oak) is considered crux botanicorum
by taxonomists ( Gömöry et al. 2001 ) and one of the broadleaves with
the highest intraspeci fic variability ( Masarovi čová 1991 ), especially in
terms of the leave's size and shape as well as the leave's colour, the
type and number of lobes (Willkomm 1875 –1887 in Kleinschmit
1993 ). This tree species is also valuable for its longevity and its spectac-
ular habit; furthermore, from a biodiversity point of view, it represents a
host to many organisms ( Lefort et al. 1998 ).
Quercus pedunculi floraK. Koch (grayish oak) is the most important
forest tree taxon of the Romanian wood steppe ( Enescu 1993 ). It has
been considered as a distinct species in several studies from Romania
(Doni țăet al. 2005, Georgescu & Cretzoiu 1941, Georgescu & Morariu
1948, Sanda et al. 2008 ), Greece ( Athanasiadis et al. 2000 ), Italy
(Carella 2013 ), Serbia ( Vuckovic 1984 ) as well as in Flora Europaea
(Schwarz in Tutin et al. 1964 ), while it is considered sometimes as an in-
traspeci fic unit of
Q.robur (Bordács et al. 2002 ), being identi fied as Q.
robur ssp. pedunculi flora(Broshtilov 2006, Davis 1965 –1988, Govaerts
& Frodin 1998, Mehrnia et al. 2013, Ozturk et al. 2004 )o rQ.pedunculata
var.pedunculi flora,Q.pedunculata ssp.pedunculi floraorQuercus haas2in
the Balkan Peninsula ( Moraru 1944 ).
Since 1936, when grayish oak has been identi fied for the first time in
Romania by the famous botanist Alexandru Borza ( Borza 1936 ), the
identi fication of the taxon has been initiated in the country's forests in
order to plot its areas of distribution ( Georgescu et al. 1942 ). Thereby,
the corresponding phytocoenosis of the grayish oak has been delimited
from the pedunculate oak areas in the forest steppe sites from Oltenia
and Muntenia (southern Romania), as well as those from south-eastern
Moldavia and Dobrogea. However, there are areas of interference be-
tween Q.robur and Q.pedunculi flora, especially in the xerophyte oakforests and in the floodplain forests with Populus spp., Salix spp., Alnus
glutinosa , where, because of their morphological similarities, their dis-
crimination is very dif ficult. There are many studies regarding the mor-
phological variability of European white oaks, ( Aas 1993, Bruschi et al.
2000, Dupouey & Badeau 1993, Kleinschmit et al. 1996 ), but the differ-
entiation between Q. robur and Q. pedunculi florawas only rarely
analysed. The two taxa were also genetically very similar as revealedbyCrăciunesc et al. (2015) , who studied the differentiation of five
white oak species based on the frequency of three alleles of a dehydryn
gene, and reported that the lowest level of differentiation among the
species studied was detected between Q.robur and Q.pedunculi flora
(F
ST= 0.020). Moreover, in the overlapping areas, the two taxa may hy-
bridize ( Chesnoiu et al. 2009, Curtu et al. 2011 ).
The goal of the present study was to evaluate the morphological var-
iability of Q.robur and Q.pedunculi floraat the inter- and intraspeci fic
level, in order to assess the phenotypic variability and to identify new
and relevant discriminatory traits between the two taxa by using multi-
variate statistical methods. A high number of morphological traits were
used, so that, several traits that differentiate the two species could be
identi fied. In addition to a previous study on morphological and iso-
zyme genetic variation between both taxa Curtu et al. (2011) that
could not clarify the morphological and genetic differences (Fst
0.039) between the two taxa, in the present study the sample size in
terms of leaves/individual tree and number of populations were in-
creased and, a new trait, the abaxial colour of the leaves was assessed.
This trait is mentioned also in literature as a morphological descriptor
for the leaves colour of the taxon, but was never evaluated before.
Also, for the first time, the variability of the two taxa at the level of intra-
specific taxonomic units was evaluated.
2. Materials and methods
Across natural range of the species in Romania, samples were col-
lected from 16 natural populations established as Forest Genetic Re-
sources (7 of Q.robur ,8o f Q.pedunculi floraand one in a mixed forest
where both taxa coexist) and have been used in the current study
(Table 1 andFig. 5 ). From each population, 49 –53 trees were sampled.
The distance among individual trees was approximatelly 50 m, in
order to minimize the possibility of sampling related individuals
(Franji ćet al. 2006, Toader et al. 2009 ). From each tree, five mature
leaves were collected in the mid- to upper crown of the trees, from
the inner side of the crown ( Borazan & Babac 2003, Curtu et al. 2011,
Gailing 2008, Kremer et al. 2002, Masato 2009 ) and from the current
years' growth ( Dupouey & Badeau 1993 ). Part of the collected plant ma-
terial (only 3 leaves per individual tree and 1 population less) was
analysed by Curtu et al. (2011) , including for molecular genetic analy-
ses. In total, 862 trees and 4310 leaves have been measured for their
morphological traits and were then subjected to statistical analysis.
For each leaf, fifteen variables have been evaluated: observed morpho-
logical variables : abaxial colour of the leaf (CL), scored from 1 to 3 (1 –
dark green, 2 –light green, 3 –bluish green), the basal shape of lamina
(BS), scored from 1 to 9 ( Kremer et al. 2002 ) and, using a ×30 stereomi-
croscope, the pubescence intensity (PU), scored from 1 to 6 (1 – no hair
and 6 – densely hairiness), ( Kissling 1997 );counted variables :n u m b e ro f
lobes (NL) and number of intercalary veins (NV). For the counted vari-
ables, the number of pairs of lobes (NL/2) and the number of pairs of in-
tercalary veins (NV/2) were also calculated ( Wigston 1975 ). Using
WinFOLIA software (Regent instrument 2007), dimensional variables
have been measured: lamina length (LL), petiole length (PL), lobe
width (LW), sinus width (SW) and length of lamina at largest width
(WP) ( Kremer et al. 2002 ). Based on counted and dimensional parame-
ters,fivetransformed variables ( Kremer et al. 2002, Viscosi et al. 2009b )
have been calculated: lamina shape (LS = 100 ∗WP/LL), petiole ratio
[PR = 100 ∗PL / (LL + PL)], lobe depth ratio (LDR = 100 ∗(LW−
SW) / LW), percentage venation (PV = 100 ∗NV / NL) and lobe width
ratio (LWR = 100 ∗LW / LL).1Forest National Inventory, 2012 (source: http://www.mmediu.ro/app/webroot/
uploads/ files/2016-06-08_Rezultate_IFN.pdf ).
2Quercus haas : synonym denomination for Quersus robur ssp. robur (source: http://
www.theplantlist.org/tpl/record/kew-173012 ).498 E.N. Apostol et al. / Science of the Total Environment 609 (2017) 497 –505

According to intraspeci fic units characteristics of the taxa described
in the literature ( Beldie 1952, 1977, Dumitriu-T ătăranu 1960,
Georgescu & Cretzoiu 1941, Georgescu & Morariu 1948, St ănescu et al.
1997, Șofletea & Curtu 2007 ), using observed, counted and dimensional
variables, each tree has been allocated into an intraspeci fic correspond-
ing taxonomic unit. The mean values of each variable were estimated on
a population and taxon basis by using IBM SPSS Statistics software v20.0
(2011). To test the signi ficance of differences of the species mean values
for all the leaf variables the t-test was used. Levene's test was used to
test for the homogeneity of variances of the taxa. When variances of
the groups were unequal, the Welch t-test was applied. The correlations
between variables were calculated using the Corrgram package R 1.8
(Wright 2015 ) and R software version 3.2.4 ( RC o r eT e a m .2 0 1 6 ).
Three different multivariate procedures were used for analysing the
data: ( i) discriminant analysis (DA) of the 10 original variables (ob-
served, counted and measured), by using the tree as the classi fication
variable; ( ii) principal component analysis (PCA) and ( iii) cluster analy-
sis to investigate the inter-relationships between populations.
For the purpose of cross-validation of the discriminant functions we
have used the holdout method at the inter- and intraspeci ficl e v e l s(Viscosi et al. 2009a ). Thus, discriminant functions have been calculated
both at the inter- and intraspeci fic levels for all the populations, except
for the mixed one. Afterwards, the calibrated discriminant functions
have been validated using the mixed population, both at the inter-
and intraspeci fic levels.
3. Results
3.1. Morphological descriptors analysis
Morphological differences between Q.robur and Q.pedunculi flora
(Table 2 )w e r es i g n i ficant (p b0.05) for 12 out of the 15 variables, but
not signi ficant at counted variables level (NL, NV) and transformed var-
iable PV. Pubescence is almost absent in Q.robur , with just some unicel-
lular trichomes on the abaxial surface of the leaf; Q.pedunculi flora
shows densely pubescent unicellular and fasciculate trichomes. The ab-
axial colour of the Q.robur leaves is usually green, while for the Q.
pedunculi florais bluish green to bluish. Basal shape of lamina (BS) is
from weak to strong auriculate for both taxa, but more prominently au-
riculate at Q.robur . The average number of pairs of lobes (NL/2) and theTable 1
Geographic coordinates of the studied populations and the Romanian ecoregions they fall into.
Population name and abbreviation Latitude N Longitude E Altitude m Temperature0C Relative humidity % Ecoregiona
Q.robur L.
Dacia (RUP) 45°58 ′53.01 ″N 25°06 ′19.18 ″E 540 8.0 78.96 1B
Cenu șa (POD) 47°03 ′29.13 ″N 27°13 ′10.02 ″E 300 9.6 76.59 1B
Vânju Mare (VJM) 44°26 ′08.06 ″N 22°50 ′04.04 ″E 89 11.0 73.15 1B
Reșca (RES) 44°10 ′34.79 ″N 24°25 ′22.48 ″E 75 11.3 75.49 3A
Noroieni (NOR) 47°51 ′24.38 ″N 22°54 ′43.02 ″E 120 9.8 77.64 1B
Bazo ș(TIM) 45°44 ′15.37 ″N 21°33 ′59.68 ″E 98 10.8 76.60 1B
Păunoaia (PAU) 44°45 ′10.01 ″N 25°57 ′49.46 ″E 150 10.6 76.88 1B
Q.pedunculi floraK. Koch
Viișoara (VIS) 44°52 ′25.48 ″N 27°39 ′54.96 ″E 30 10.9 76.19 3B
Pogoanele (POG) 44°53 ′46.03 ″N 26°55 ′09.09 ″E 80 11.0 74.85 3A
Băneasa (BAN) 44°05 ′41.59 ″N 27°49 ′37.75 ″E 110 11.5 76.17 3A
Ciornuleasa (MIT) 44°13 ′15.98 ″N 26°45 ′14.02 ″E 60 11.1 76.34 3A
Snagov (SNA) 44°37 ′52.76 ″N 26°21 ′31.28 ″E 90 11.1 76.11 3A
Urziceni (URZ) 44°34 ′46.75 ″N 26°28 ′33.04 ″E 60 10.9 76.40 3A
Brani ștea Catârilor (BRA) 43°53 ′46.75 ″N 24°14 ′42.38 ″E 65 11.2 76.15 3A
Punghina (PUN) 44°15 ′15.33 ″N 22°50 ′4.34 ″E 50 11.4 72.95 4B
Mix pedunculate and grayish oak forest
Letea (LET) 45°19 ′57.75 ″N 29°30 ′57.23 ″E 5 11.5 80.3 4A
a1B – forests with Quercus robur ,Q.cerris ,Q.frainetto and other species, on low hills and plains; 3A – xerophyte oak forests in wood steppe; 3B –steppe; 4A – floodplain forests with
Populus spp .,Salix spp .,Alnus glutinosa andQuercus robur ; 4B – high floodplain forests with Quercus robur andFraxinus spp .(Gancz & P ătrășcoiu 2000 ).
Table 2
Coefficients of variation, mean values for the 15 leaf traits of the two taxa and signi ficance of their differences.
Leaf descriptors Q.robur Q .pedunculi flora t -value (t & Welch t-test) p-Value
Mean SD CV% Mean SD CV%
PU 1.03 ±0.25 24.3 4.67 ±0.76 16.3 96.45 0.00+
CL 1.12 ±0.34 30.4 2.42 ±0.69 28.5 35.92 0.00+
BS 8.23 ±0.68 8.3 7.89 ±0.83 10.5 6.73 0.00+
NL 10.12 ±1.57 15.5 10.09 ±1.48 14.7 0.26 0.80
NV 4.37 ±1.44 33.0 4.35 ±1.15 26.4 0.28 0.78+
LL (mm) 112.08 ±17.39 15.5 118.16 ±17.13 14.5 5.17 0.00
PL (mm) 6.29 ±2.01 32.0 8.21 ±2.19 26.7 13.38 0.00+
LW (mm) 36.23 ±6.38 17.6 39.15 ±6.20 15.8 6.80 0.00
SW (mm) 13.49 ±3.77 27.9 12.81 ±4.16 32.5 2.51 0.01+
WP (mm) 66.39 ±11.60 17.5 68.62 ±12.40 18.1 2.72 0.01
LS 59.20 ±5.59 9.4 57.92 ±5.25 9.1 3.47 0.00
PR 5.42 ±1.70 31.4 6.57 ±1.69 25.7 9.95 0.00
LDR 62.00 ±9.62 15.5 66.52 ±10.39 15.6 6.6 0.00
PV 44.79 ±16.20 36.2 44.29 ±12.42 28.0 0.50 0.62+
LWR 32.42 ±3.48 10.7 33.24 ±3.24 9.7 3.57 0.00
Statistical signi ficant p-value (p b0.05) of the morphological leaf descriptors [pubescence intensity (PU), abaxial colour of the leaf (CL), basal shape of lamina (BS), number of lobes ( NL),
number of intercalary veins (NV), lamina length (LL), petiole length (PL), lobe width (LW), sinus width (SW), length of lamina at largest width (WP), l amina shape (LS), petiole ratio (PR),
lobe depth ratio (LDR), percentage venation (PV) and lobe width ratio (LWR)] among the two species ( Q.robur andQ.pedunculi flora) are given in bold; p-values calculated using Welch t-
test are indicated with“+”symbol.499 E.N. Apostol et al. / Science of the Total Environment 609 (2017) 497 –505

average number of pairs of intercalary veins (NV/2) are similar for both
taxa. Concerning dimensional traits, the leaves of grayish oak, compared
to those of pedunculate oak, are characterized by higher mean values
with 5.4% (LL), 30.5% (PL) and 8.0% (LW). For transformed variables,
the results indicate that the leaves of pedunculate oak are more obovate,
and that the proportion of the petiole to the leaf length is smaller than
the one estimated for Q.pedunculi flora. With regards to LDR and LWR
values, Q.pedunculi florahas deeper sinuses and narrower leaves than
Q.robur .
Compared to the study of Curtu et al. (2011) , where signi ficant dif-
ferences were obtained for 4 out of the 14 variables analysed, in the
present study, by increasing the number of evaluated leaves per tree
from 3 to 5, by introducing a new population of grayish oak, Pogoanele
and considering , as well as, for the first time in the literature, a new
trait, the abaxial colour of the leaf (CL), signi ficant differences were
found for 12 variables out of 15 analysed.
3.2. Discriminant analysis (DA)
A discriminant analysis was performed for the two groups of Q.robur
and Q.pedunculi florapure populations. This analysis showed that the
pubescence intensity (PU), the abaxial colour of the leaf (CL), the petiole
length (PL) and the basal shape of lamina (BS) are the variables with a
significant contribution (p b0.05 and Partial Lambda values between
0.21 and 0.99) to the discrimination of the two taxa. Therefore, the dis-
criminant analysis generated a 4 variable discriminant function:
ID¼−528 :835ț168 :591/C3PUț49 :364/C3CLț4:874/C3PL−12 :842
/C3BS
The function generated a negative value for Q.robur and a positive
one for Q.pedunculi flora. The cross-validation results (holdout method)
of the two taxa for the trees from the Letea mixed population show a
very high percentage of function classi fication (100% for Q.robur and
92% for Q.pedunculi flora).
Theprincipal component analyses (PCA) performed by using 15 vari-
ables for the two Q.robur and Q.pedunculi florapure populations,
showed that separation between the two taxa is mainly due to Factor
1 (21.19%), which explains a notable part of the total variation. LL
(23%), LW (13%) and WP (26%) are the variables that contribute the
most to the separation of the two groups ( Fig. 1 a). Performing the
same analysis for the two groups, but using only the four variables de-
rived from the discriminant function (PU, CL, BS and PL) a clear separa-
tion was revealed among the two groups ( Kelleher et al., 2005 ) and theseparation is due to Factor 2 (23.66%). Regarding the contribution of the
variables, in this case BS (86%) and PL (13%) were the variables that con-
tributed to the separation of the two distinct groups ( Fig. 1 b).
3.2.1. Cluster analysis
The populations of the two species were grouped into two main
clusters: one that includes populations from central-eastern and cen-
tral-western Romania, and another with populations from southern Ro-
mania ( Fig. 2 ). Although in the primary clusters, the grayish oak and the
pedunculate oak populations are mixed, in the clusters of the highest
order they are separated, showing a certain level of discrimination be-
tween the populations of the two species relative to the 13 characters
which differed signi ficantly (p b0.05). The second cluster analysis
(Fig. 3 ), which considers only the four traits derived from the discrimi-
nant analysis (PU, CL, BS and PL), resulted in separation of all popula-
tions of pedunculate oak and grayish oak; nevertheless, the analysis
did not reveal any intraspeci fic subgroups based on their geographical
origins.
3.3. Taxonomic intraspeci fica n a l y s i s
Eight intraspeci fic units of Q.robur and six units of Q.pedunculi flora
were identi fied in the investigated area ( Fig. 5 ). While for Q.robur both
varieties mentioned in Romanian literature ( Q.robur var.glabra , respec-
tively Q.robur var.puberula )w e r ei d e n t i fied, for Q.pedunculi floraonly
var. atrichoclados was identi fied. For pedunculate oak, 85% of the re-
search material falls in var. glabra , mostly the forms acutifolia (25.4%),
multilobata (22.7%) and vulgaris (22.1%) ( Fig. 4 a). At the taxon level,
theQ.pedunculi floravar. atrichoclados f.typica sub-form obtusiloba is
the most common (about 65%) and the most widespread. Shows signif-
icant weights and f. maxima sub-form simplex (14%) and f. maxima sub-
form frainettoides (12.7%) ( Fig. 4 b).
3.3.1. Discriminant analysis and spatial distribution of intraspeci ficu n i t s
ForQ.robur and Q .pedunculi floraintraspeci fic units, discriminant
functions were generated with greater frequency. Only the discriminant
functions with N85% correct classi fication percent were considered.
After testing the realized functions on intraspeci ficu n i t so f Q.robur
and Q. pedunculi flora, the cross-validation results show a very high per-
centage of correct classi fication ( Tables 3 & 4 ), of over 85%. The function
ID1:−764.953 + 3.376 ∗LL−12.084 ∗SW + 11.617 ∗LW + 12.305
∗NL produced a negative value for var. glabra f.vulgaris , the function
ID 2:−145.354 + 0.127 ∗LL + 26.525 ∗SW + 1.668 ∗LW−28.978
∗NL generated a negative value for var. glabra f.multilobata ,t h ef u n c t i o n
Fig. 1. PCA graph for the two taxa using different variables.500 E.N. Apostol et al. / Science of the Total Environment 609 (2017) 497 –505

ID 3: 87.802 –9.74∗LL−4.352 ∗SW + 25.731 ∗LW + 13.548 ∗NL gave a
negative value for var. glabra f.acutifolia and a positive value for var.
puberula f.rotundiloba , while the function ID4: −561.313 −1.653 ∗LL
+ 10.405 ∗SW−0.415 ∗LW + 60.935 ∗NL showed a negative value
for var. glabra f.heterophylla .
ForQuercus pedunculi floravar.atrichoclados the following discrimi-
nant functions have been generated:
ID1: 965.586 –2.835 ∗LL + 4.15 ∗SW−11.284 ∗LW−22.308 ∗NL
−0.901 ∗PL, gave positive value for f. typica , subf. Obtusiloba; ID2: −
88.049 + 5.258 ∗LL + 22.97 ∗SW−12.372 ∗LW-33.64 ∗NL−1.48
∗PL, gave negative value for f. typica , subf. goniolobula ,
ID3:−271.334 −8.613 ∗LL + 14.172 ∗SW + 14.527 ∗LW + 39.919
∗NL + 17.492 ∗PL gave negative value for f. maxima subf. stellatoides
and positive value for f. maxima subf. frainettoides and.
ID4:−374.677 + 2.16 ∗LL−4.733 ∗SW−8.711 ∗LW + 18.609
∗NL + 40.582 ∗PL gave negative value for f. maxima subf. stellatoides .
The testing of the realized functions on intraspeci ficu n i t so f Q.
pedunculi floracross-validation results shows a very high percentage of
correct classi fication. For intraspeci ficu n i t so f Q.pedunculi floraof higheroccurrence, discriminant functions have been generated, showing vali-
dation percentages of over 85%.
The interpretation of the spatial distribution of the two species in the
Romanian forest ecoregion map, in relation to the level of intraspeci fic
units ( Fig. 5 ) shows that pedunculate oak is mostly present in the forests
with Quercus robur ,Q.cerris ,Q.frainetto and other species, on low hills
and plains. However, among the high intra-population taxonomic vari-
ability, a high frequency of var. glabra f.acutifolia presence was regis-
tered in western and south-western Romania, and of var. glabra f.
multilobata in the north-west. Grayish oak is present mainly in the xero-
phyte oak forests, in silvosteppe ecoregion, where a high frequency was
registered for var. atrichoclados f.typica subf. Obtusiloba , in the south-
east of the natural range of Q.pedunculi flora, in the study area.
4. Discussion
Because in most of the studies Q.pedunculi florawas considered an
infrataxon of Q.robur (Broshtilov 2006, Davis 1965 –1988, Govaerts &
Frodin 1998, Mehrnia et al. 2013, Ozturk et al. 2004 ), while in others it
was considered as a separate species ( Athanasiadis et al. 2000, Carella
2013, Doni țăet al. 2005, Georgescu & Cretzoiu 1941, Georgescu &
Morariu 1948, Sanda et al. 2008, Schwarz in Tutin et al. 1964,
Vuckovic 1984 ), during the recent years there has been signi ficant re-
search into its taxonomic status ( Broshtilov 2006, Carella 2013, Curtu
et al. 2009, 2011 ). In the current study, an integrated morphological
analysis involving Q.robur and its closest relative Q.pedunculi florawas
carried out, by using a data set of morphological traits that included ob-
served, counted, measured and transformed variables.
Unlike the discriminant function developed by Curtu et al. (2011) ,
the one developed in this study has considered a new trait, the abaxial
colour of the leaves (CL), which together with PU, BS and PL showed a
greater differentiation for the two taxa, as shown in Fig. 1 b. The fact
that the two taxa also have common or similar traits is highlighted in
the PCA analysis for all 15 descriptors analysed ( Fig. 1 a), where a signif-
icant cloud overlapping area was recorded, denoting the overall pheno-
type of the analysed specimens.
At the same time, in the cluster analysis performed for the 13 de-
scriptors that generated signi ficant differences (p ˂0.05) between the
two taxa, the populations grouped in three subcategories differentiated
according to speci fic regions in the analysed area. Furthermore, within
these sub-clusters, the pedunculate oak populations have been separat-
ed from the grayish oak. These data validate the morphological
Fig. 2. Cluster analysis using means of 13 morphological traits (p b0.05). In this figure, R meaning Q.robur , P meaning Q.pedunculi flora, and the other three letters are the abbreviation of
the populations as in Table 1 .
Fig. 3. Cluster analysis using variables PU, CL, BS and PL. In this figure, R meaning Q.robur ,P
meaning Q.pedunculi flora, and the other three letters are the abbreviation of the
populations as in Table 1 .501 E.N. Apostol et al. / Science of the Total Environment 609 (2017) 497 –505

differentiation of the two taxa, but also indicate some of their regional
morphological particularities. The discriminant function constructed
on the basis of the above four descriptors provided a great validation
of the species differentiation, and for two (PU and CL) out of the four
traits, an adaptive value could be suggested. The presence of trichomes
may enhance leaf surface roughness and ultimately the hydrophobic
nature of its surface ( Brewer & Nuñez 2007, in Naz et al. 2010 ), a fact
proved to be true for many plant genera and species ( Hamed et al.
2009, Naz et al. 2010, Pugnaire et al. 1999 ). On the other hand, the glau-
cous leaf colour was considered to be an adaptive strategy for transpira-
tion reduction in plants ( Membrives et al. 2003 ). The above two traits
(PU and CL) characterize and clearly differentiate grayish oak from pe-
dunculate oak, explaining its adaptability to drought in the forest steppe
lands where it grows. Bluish-green colour has been identi fied on indi-
viduals from the Letea mixed forest and can be explained by the coexis-
tence of the two species in the same phytocoenosis. The highest
amplitude of variation for this trait was evident in this population,
which could be a possible result of hybridization between the two
taxa. The spatial distribution of the two taxa in the Letea mixed popula-
tion reveals their ecological speci ficity, as the grayish oak can be found
growing in sites characterized by harsher xerophytic conditions.
The values of pubescence estimated for Q.pedunculi floraranged be-
tween 4.2 and 5. The maximum value of PU was recorded for specimens
of the form rotundiloba , in the populations PUN and BRA, in which the
abaxial colour of the leaf is glaucous. In the present study, the value of
PU for Q.robur has been similar to those reported in a similar study in
France; no hair ( Dupouey et al. 1990 ) or with simple hairs as identi fied
in some others studies from England ( Wigston 1975 ), from France
(Bacilieri et al. 1996 ), where was recorded a value of 1.2 for PU, or
from Western France, where was recorded a value of 0.2 for PU
(Ponton et al. 2004 ). The abaxial colour of the leaves for Q.
pedunculi florawas glaucous to grayish, while that of Q.robur was green.
Petiole length (PL) is another important character discriminating the
two taxa. For this trait, the smallest amplitude of variation for Q.robur
(CV% = 32%) was estimated, a result which is in agreement with the
findings of a study carried out in Bulgaria ( Broshtilov 2006 ) in which
the CV% varied among 37.3% and 44.7%. In most of the populations,the average petiole length has been lower than that reported by
Boratynski et al. 2008 in Poland and Ponton et al. 2004 in France, but
similar to that reported by Bašićet al. 2007 in Bosnia. The average peti-
ole length (PL) for Q.pedunculi flora, being under 1 cm in all populations,
fits in the top of the variation recorded in the literature ( Negulescu &
Savulescu 1957, Șofletea & Curtu 2007 ). Values between 1.7 cm and
3.3 cm were registered in Bulgaria ( Broshtilov 2006 ).
ForQ.robur the average width of the lobe at the point of maximum
leaf width varied between 3.25 and 4.26 cm, showing slightly lower
values than those recorded in the studies from Poland ( Boratynski et
al. 2008 ) and Bosnia ( Bašićet al. 2007 ). The average value of grayish
oak (3.9 cm) is higher by about 8% when compared with that of pedun-
culate oak. Also, statistically signi ficant differences were recorded
among the two species for the leaf base form –BS (Kissling 1997 ),
which ranges, from strong auriculate for Q.robur (Wigston 1975 ) and
even re flected articulated (BS = 9) ( Stanescu et al. 1997 ), to weak au-
riculate for Q.pedunculi flora, and corresponds to the speci ficf o r mo f
the species described in the literature ( Dumitriu-T ătăranu 1960,
Șofletea & Curtu 2007 ).
The average number of the pairs of lobes is within the limits de-
scribed for Q.robur in the literature ( Negulescu & Savulescu 1957,
Dumitriu-T ătăranu 1960, Stanescu et al. 1997 ). Five pairs of lobes
were observed by Dupouey & Badeau 1993 in France, in the north-
east populations, also by Ponton et al. (2004) in the western France
ones and by Bacilieri et al. (1996) in Le Mans. In England ( Wigston
1975 ) the average number of the pairs of lobes was 3 –5(Becker &
Levy, 1990 ). A medium level of variability (CV = 16%) was estimat-
ed for this trait in the study area, which is similar to the one found
byBroshtilov (2006) in Bulgaria (12 –16.7%). For Q.pedunculi flora
the number of pairs of lobes ranged among 4 and 7, a result similar
to the ones found in the literature ( Dumitriu-T ătăranu 1960 ), but
the speci fic trait did not contribute to the discrimination of the
two taxa.
The number of intercalary veins (NV), which was proved to be a use-
ful descriptor to differentiate the pedunculate oak from the sessile oak
(Kremer et al. 2002 ), showed similar values in the two taxa analysed
in the current study [mean 4.4; between 32 ( Georgescu & Cretzoiu,
Fig. 4. Proportion of intraspeci fic units within taxa.
Table 3
Grayish oak discriminant function sign and the percentage of the correct classi fication.
Species Discriminant functions ID1 ID2 ID3 ID4 ID5
Intraspeci fic units Sign % of correct
classi ficationSign % of correct
classi ficationSign % of correct
classi ficationSign % of correct
classi ficationSign % of correct
classi fication
Quercus pedunculi flora
var. atrichocladosf.typica subf. obtusiloba + 100.00 + 22.73 + 77.27 − 72.73 + 40.91
subf. goniolobula + 50.00 − 100.00 − 33.33 + 83.33 − 50.00
f.maxima subf. stellatoides − 100 − 100 − 100 − 100 +5 0
subf. acutiloba − 0.00 + 33.33 − 16.67 + 50.00 + 66.67
subf. simplex − 28.57 + 57.14 − 28.57 − 85.71 − 28.57
subf. frainettoides − 42.86 − 100.00 + 100.00 + 57.14 − 14.29
Values in bold – % of correct classi fication according to which the discriminatory functions considered502 E.N. Apostol et al. / Science of the Total Environment 609 (2017) 497 –505

1941 ) % and 55%]. With respect to the above trait, the two taxa appear to
follow similar adaptation strategies, even though its role and involve-
ment in ecophysiological processes is highly stressed in the literature,
together with its evolutionary signi ficance ( Kull & Herbig 1994,
Scoffoni et al. 2015 ). On the other hand, it appears that leaves with larg-
er number of lobes tend to have lower values for PV and SW but, on the
other hand, LL does not in fluence the PV value (data presented in table
of correlation between characters, supplementary material). In Quercus
robur , the grand mean for NV (grand mean 4.4 for all analysed popula-
tions) is very close to the value of 4.9 found by Dupouey et al. (1990)
in France, but about two times higher than the values reported by
Ponton et al. (2004) andBacilieri et al. (1996) .The LL value for Q.robur falls within the values mentioned in the lit-
erature ( Bacilieri et al. 1996, Ba šićet al. 2007, Boratynski et al. 2008,
Ponton et al. 2004 ).
For many species, adaptation in xerophytic environments results in
reduced leaf sizes ( Ogaya & Peueñlas 2007, Yadollahi et al. 2011 )i n
order to control water loss by transpiration. In this context, the great
length of the grayish oak leaves (ranging between 7 and 19 cm), that
grows in the forest steppe, doesn't follow the abovementioned adapta-
tion strategy. There is a striking difference of grayish oak with Quercus
pubescens , a very drought tolerant oak ( Wellstein & Cianfaglione
2014 ), which is found in similar habitats, including the habitat of the
mixed stand included in the current study. Comparing to grayish oak,Table 4
Pedunculate oak discriminant function sign and the percentage of the correct classi fication.
Species Discriminant functions ID1 ID2 ID3 ID4
Intraspeci fic units Sign % of correct classi fication Sign % of correct classi fication Sign % of correct classi fication Sign % of correct classi fication
Q.robur var. glabra f.heterophylla − 66.67 + 50.00 − 0.00 − 100.00
f.multilobata + 46.15 − 92.31 + 92.31 + 38.46
− 57.14 − 57.14 − 85.71 + 57.14
f.acutifolia
f.macrophylla + − + −− −− −
f.vulgaris − 91.67 − 83.33 − 16.67 + 33.33
var. puberula f.acutiloba − 66.67 − 0.00 + 66.67 − 66.67
f.microphylla −− −− + −− −
f.rotundiloba − 75.00 + 50.00 + 87.50 + 25.00
Values in bold – % of correct classi fication according to which the discriminatory functions considered
Fig. 5. Q.robur andQ.pedunculi floraintraspeci fic units spatial distribution across Romania [base map –Romania Forest Ecoregion map ( Gancz & P ătrășcoiu 2000 )].503 E.N. Apostol et al. / Science of the Total Environment 609 (2017) 497 –505

pubescent oak leaves are smaller [(4,5 –8 (12) cm], present more dense-
ly pubescence with longer trichomes. The petiole is longer, usually 10 –
15 mm ( Rehder, 1960; Șofletea și Curtu, 2007; Doni ță, 2008; Bordács et
al., 2009 ), and occasionally 2 cm ( Trinajti ć, 2007 ). Undoubtedly, both
species are very well adapted for survival on drier sites
(Apostol-Chesnoiu et al. 2015, Curtu et al., 2011 ), especially in the
steppe zones.
Across Romanian ecoregions, pedunculate oak is mostly present in
forests with Q.cerris ,Q.frainetto and other species, on low hills and
plains, but the population RES, which is in another ecoregion (xero-
phyte oak forests in wood steppe), has the lowest intraspeci fic taxo-
nomic diversity. Grayish oak is present mainly in the xerophyte oak
forests in wood steppe ecoregion, only VIS population in steppe, and
PUN population in high floodplain forests with Quercus robur and
Fraxinus spp. ecoregion. Grayish oak shows a lower frequency to f. typica
subf. obtusiloba in populations in southern and southwestern Romania
(BRA and PUN), and SNA and BAN populations are poorer in intraspecif-
ic morphological units (only three); nevertheless, it presents the
highest frequency for grayish oak, predominantly infrataxon (f. typica
subf. obtusiloba ). On the other hand, in the Letea mixed stands, a signif-
icant percentage of trees (22%) are from Q.r.v a r puberula , which could
be potentially considered a putative hybrid between pedunculate oak
and grayish oak. This aspect requires further research for validation.
5. Conclusions
Twelve morphological traits give high signi ficance values that can be
used in order to discriminate the two taxa. Furthermore, the two enti-
ties, sometimes considered species, show characteristics with the
greatest power of differentiation such as abaxial pubescence, the abaxial
colour of the leaf, petiole length and basal shape of lamina. Their power
of differentiation is con firmed by forming two obvious clouds of points
and two separate clusters corresponding to the two taxa.
Q.robur and Q.pedunculi flora populations from Southern
Carpathians show higher phenotypic variability highlighted by leaves
morphological variability of the intraspeci fic units. The leaves traits of
Q.pedunculi florapresent a good plasticity and adaptability in the con-
text of global warming, which recommends this taxon to be promoted
within breeding programmes ( Bruschi et al. 2003 ) and conserved both
in situ andex situ .
Our study showed that the assessment of the abaxial colour of the
leaf, a new trait used for the first time in the literature, proved to be
very useful to distinguish Q. pedunculi florafrom Q. robur which is of
great help for both scientists and forest practitioners.
Acknowledgements
Ecaterina Nicoleta Apostol's (Chesnoiu) research was supported by
the Sectorial Operational Programme Human Resources Development
(SOP HRD), financed from the European Social Fund (2014-2020) and
by the Romanian Government under the project number POSDRU/
159/1.5/S/134378, and by the Nucleu Gedefor Programme (46N).
References
Aas, G., 1993. Taxonomical impact of morphological variation in Quercus robur and Q.
petraea : a contribution to the hybrid controversy. Ann. For. Sci. 50 (1), 107s –113s.
Apostol-Chesnoiu, E.N., Curtu, A.L., Șofletea, N., 2015. Structura taxonomic ăintraspeci fică
într-un complex de cvercinee din estul României, la contactul cu zona silvostepei ex-
terne. Revista de Silvicultura si Cinegetica 37, 47 –51.
Athanasiadis, N., Tonkov, S., Atanassova, J., Bozilova, E., 2000. Palynological study of Holo-
cene sediments from Lake Doirani in northern Greece. J. Paleolimnol. 24 (3),
331 –342.
Bacilieri, R., Ducousso, A., Petit, R.J., Kremer, A., 1996. Mating system and asymmetric hy-
bridization in a mix stand of European oaks. Evolution 50, 900 –908.
Bašić, N., Kapi ć, J., Ballian, D., 2007. Morfometrijska analiza varijabilnosti svojstava lista
hrasta lu žnjaka ( Quercus robur L.) na podru čju sjeverne Bosne. Rad. Šumar. inst.
Jastrebar 42 (1), 5 –18.Becker, M., Levy, G., 1990. Le point sur l'écologie comparée du chêne sessile et du chêne
pédonculé. Revue Forestière Française 42, 48 –54.
Beldie, Al, 1952. Genul Quercus . In: Nyarady, E. (Ed.), Flora R.P.R. Vol. I. Editura “Academiei
R.P.R, pp. 224 –261.
Beldie, A., 1977. Flora Ramâniei. Determinator ilustrat al plantelor vasculare. vol. I. Editura
Academiei R. S, România.
Boratynski, A., Marcysiak, K., Lewandowska, A., Jasinsk, A., Iszkulo, G., Burczyk, J., 2008.
Differences in leaf morphology between Quercus petraea and Q.robur adult and
young individuals. Silva Fennica 42 (1), 115 –124.
Borazan, A., Babac, M., 2003. Morphometric leaf variation in oaks ( Quercus )o fB o l u .T u r –
key. Ann. Bot. Fenn. 40, 233 –242.
Bordács, S., Popescu, F., Slade, D., Csaikl, U.M., Lesur, I., Borovics, A., Kézdy, P., König, A.O.,
Gömöry, D., Brewer, S., Burg, K., Petit, R.J., 2002. Chloroplast DNA variation of white
oaks in northern Balkans and in the Carpathian Basin. For. Ecol. Manag. 156, 197 –209.
Bordács, S., Zhelev, P., Schirone, B., 2009. EUFORGEN Technical Guidelines for Genetic
Conservation and Use for Pubescent oak ( Quercus pubescens Willd.). International
Plant Genetic Resources Intitute, Rome, Italy.
Borza, Al, 1936. Quercus pedunculi floraC. Koch, un stejar nou pentru România. Bul. Gr ăd.
Bot. Muz. Bot., Univ. Cluj, XVI. 1 –4p p .5 5 –62.
Brewer, C.A., Nuñez, C.I., 2007. Patterns of leaf wettability along an extreme moisture gra-
dient in western Patagonia, Argentina. Int. J. Plant Sci. 168, 555 –562.
Broshtilov, K., 2006. Quercus robur L. leaf variability in Bulgaria. Plant Genet. Resour.
Newsl. 147, 64 –71.
Bruschi, P., Vendramin, G.G., Bussotti, F., Grossoni, P., 2000. Morphological and molecular
differentiation between Quercus petraea (Matt.) Liebl. and Quercus pubescens Willd.
(Fagaceae ) in Northern and Central Italy. Ann. Bot. 85, 325 –333.
Bruschi, P., Vendramin, G.G., Bussotti, F., Grossoni, P., 2003. Morphological and molecular
diversity among Italian populations of Quercus petraea (Fagaceae ). Ann. Bot. 91,
707 –716.
Carella, R., 2013. First observation of Quercus pedunculi floraK. Koch in the Italian Peninsu-
la. J. For. Sci. 59 (3), 130 –135.
Chesnoiu, E.N., So fletea, N., Curtu, A.L., Toader, A., Radu, R., Enescu, M., 2009. Bud burst
andflowering phenology in a mixed oak forest from Eastern Romania. Ann For.
Res. 52, 199 –206.
Crăciunesc, I., Vornam, B., Leinemann, L., Finkeldey, R., Șofletea, N., Curtu, A.L., 2015. High
genetic differentiation among European white oak species ( Quercus spp .) at a
dehydrin gene. Notulae Botanicae Horti Agrobotanici Cluj-Napoca 43 (2), 582 –588.
Curtu, A.L., Șofletea, N., Toader, A.V., Enescu, M., Moldovan, C., Chesnoiu, E.N., 2009.
Stejarul brum ăriu: specie sau unitate intraspeci ficăa stejarului pedunculat? Revista
Pădurilor 124 (5), 24 –30.
Curtu, A.L., Șofletea, N., Toader, A.V., Enescu, C.M., 2011. Leaf morphological and genetic
differentiation between Quercus robur L. and its closest relative, the drought tolerant
Q.pedunculi floraK Koch. Ann. For. Sci. 68, 1163 –1172.
Curtu, A.L., Craciunesc, I., Enescu, C.M., Vidalis, A., Șofletea, N., 2015. Fine-scale spatial ge-
netic structure in a multi-oak-species ( Quercus spp .) forest. iForest. Biogeosci. For. 8,
324 –332.
Davis, P.H., 1965 –1988. Flora of Turkey and the East Aegean Islands, Vol: 1 –10. Edinburgh
University Press, Edinburgh.
Doni ță, N., 2008. Quercus virgiliana Ten., un arbore de interes pentru silvicultura din
zonele aride. Revista P ădurilor 4, 18 –19.
Doni ță, N., Popescu, A., Pauc ă-Com ănescu, M., Mih ăilescu, S., Biri ș, I.A., 2005. Habitatele
din România. Editura Tehnic ăSilvic ă,B u c u r e ști.
Dumitriu-T ătăranu, I., 1960. Arbori și arbu ști forestieri și ornamentali cultiva ți în R.P.R.
Editura Agro-Silvic ă.
Dupouey, J.L., Badeau, V., 1993. Morphological variability of oaks ( Quercus robur L .Quercus
petraea (Matt) Liebl. Quercus pubescens Willd) in northeastern France: preliminary
results. Ann. For. Sci. 50 (1), 35s –40s.
Dupouey, J.L., Fougère, V., Kremer, A., 1990. Variabilité génétique des Chênes sessile et
pédonculé estimée à l'aide de marqueurs morphologiques et moléculaires. Rev. For.Fr. XLII-2, 198 –204.
Enescu, V., 1993. A test of half – sib progenies of greyish oak, Quercus pedunculi floraK
Koch. Ann. For. Sci. 50, 439s –4443 (No. Supplement).
Franji ć,J . ,L i b e r ,Z . , Škvorc, Ž., Idžojtić,M . , Šoštarić, R., Stan čić, Z., 2006. Morphological and
molecular differentiation of the Croatian populations of Quercus pubescens Willd.
(Fagaceae ). Acta Soc. Bot. Pol. 75 (2), 123 –130.
Gailing, O., 2008. QTL analysis of leaf morphological characters in a Quercus robur full-sib
family ( Q.robur ×Q.robur ssp.slavonica ). Plant Biol. 10, 624 –634.
Gancz, V., P ătrășcoiu, N., 2000. Cartogra fierea ecosistemelor forestiere din România prin
mijloace GIS și de teledetec ție. Revista P ădurilor 2, 35 –40.
Georgescu, C.C., Cretzoiu, P., 1941. Considera țiuni sistematice asupra speciei Quercus
pedunculi floraK. Koch în România. VII. Analele ICEF. I, pp. 3 –37.
Georgescu, C.C., Morariu, I., 1948. Monogra fia stejarilor din România. Universul, Bucure ști.
Georgescu, C.C., Lupe, L., Cretzoiu, P., 1942. Răspândirea stejarului brum ăriu ( Quercus
pedunculi floraK. Koch). [Natural distribution of Grayish oak ( Quercus pedunculi flora
C. Koch).]. Analele Icas 8, 165 –172.
Gömöry, D., Yakovlev, I., Zhelev, P., Jedináková, J., Paule, L., 2001. Genetic differentiation of
oak populations within the Quercus robur /Quercus petraea complex in Central and
Eastern Europe. Heredity 86, 557 –563.
Govaerts, R., Frodin, D.G., 1998. World Checklist and Bibliography of Fagales . Royal Botanic
Gardens, Kew, Kew.
Hameed, M., Ashraf, M., Naz, N., 2009. Anatomical adaptations to salinity in cogon grass
[Imperata cylindrica (L.) Raeuschel] from the Salt Range. Pakistan. Plant Soil 322,
229 –238.
Kelleher, C., Hodkinson, T., Douglas, G., Kelly, D., 2005. Species distinction in Irish popula-
tions of Quercus petraea andQ.robur : morphological versus molecular analyses. Ann.
Bot. 96, 1237 –1246.504 E.N. Apostol et al. / Science of the Total Environment 609 (2017) 497 –505

Kissling, P., 1997. Les poils des quatre espèces de chênes du Jura ( Quercus pubescens .Q.
petraea .Q.robur etQ.cerris ). Bericht der Schweizerischen Botanischen Gesellschaft
87, 1 –18.
Kleinschmit, J., 1993. Intraspeci fic variation of growth and adaptive traits in European oak
species. Ann. For. Sci. 50, 166 –185.
Kleinschmit, J., Elsner, G., Schlums, K., 1996. Interspeci fic variation between Quercus robur
and Quercus petraea for leaf morphological traits. In: Kremer, A., Muhs, A.J. (Eds.),
Inter – and Intra – Speci fic Variation in European Oaks: Evolutionary Implications
and Practical Consequences – Conference Proceedings. EC Directorate – General XII
Science. Research and Development, pp. 3 –16 EUR 16717 EN.
Kremer, A., Dupouey, J.L., Deans, J.D., Cottrel, J., Csaikl, U.M., Finkeldey, R., Espinel, S.,
Jensen, J.S., Kleinschmit, J., Van Dam, B., Ducousso, A., Forrest, I., De Herdia, U.L.,
Lowe Andew, J., Tutkova, M., Minro, R.C., Badeau, V., 2002. Leaf morphological differ-
entiation between Quercus robur and Quercus petraea is stable across western Euro-
pean mixed stand. Ann. For. Sci. 59, 777 –787.
Kubitzki, K., 1993. Fagaceae. In: Kubitzki, K., Rohwer, J.G., Bittrich, V. (Eds.), The Families
and Genera of Vascular Plants. Spinger, Berlin, pp. 301 –309.
Kull, U., Herbig, A., 1994. Leaf venation patterns and principles of evolution. 9.
Mitteilungen des SFB 230. Heft, Stuttgart, pp. 167 –175.
Lefort, F., Lally, M., Thompson, D., Douglas, G.C., 1998. Morphological traits, microsatellite
fingerprinting and genetic relatedness of elite oaks ( Q.robur L.) at Tullynally, Ireland.
Silvae Genetica 47 (5 –6), 257 –262.
Masarovi čová, E., 1991. Leaf shape, stomata density and photosynthetic rate of the com-
mon oak. Biol. Plant. 33 (6), 495 –500.
Masato, I., 2009. Variation in leaf morphology of Quercus crispula and Quercus denata as-
semblages among contact zones: a method for detection of probable hybridization.
J. For. Res. 14, 240 –244.
Mehrnia, M., Nejadsattari, T., Assadi, M., Mehregan, I., 2013. Taxonomic Study of the
Genus Quercus L. Sect. Quercus in the Zagros forests of Iran. J. Bot. 19 (1), 62 –74.
Membrives, N., Pedrola-Monfort, J., Caujape-Castells, J., 2003. Correlations between mor-
phological-anatomical leaf characteristics and environmental traits in Southwest Af-
rican species of Androcymbium (Colchicaceae ). Botanica Macaronesica 24, 73 –85.
Moraru, I., 1944. Asupra ecologiei și sociologiei lui Quercus pedunculi floraC. Koch, Revista
Pădurilor 10 (12), 257 –267.
Naz, N., Hameed, M., Ashraf, M., Al-Qurainy, F., Arshad, M., 2010. Relationships between
gas-exchange characteristics and stomatal structural modi fications in some desert
grasses under high salinity. Photosynthetica 48 (3), 446 –456.
Negulescu, E., S ăvulescu, A.L., 1957. Dendrologie. Bucure ști, Editura Agro-Silvic ăde Stat.
Nixon, Kevin, C., 1993. Infrageneric classi fication of Quercus (Fagaceae ) and typi fication of
sectional names. Ann. For. Sci. 50 (1), 25s –34s.
Ogaya, R., Peñuelas, J., 2007. Leaf mass per area ratio in Quercus ilex leaves under a wide
range of climatic conditions. The importance of low temperatures. Acta Oecol. 31 (2),168 –173.
Ozturk, M., Ozcelik, H., Sakcali, S., Guvensen, A., 2004. Land degradation problems in Eu-
phrates Basin, Turkey. 10(3). EnviroNews, Newsletter of ISEB India, pp. 7 –9.
Ponton, S., Dupouey, J.L., Dreyer, E., 2004. Leaf morphology as species indicator in seed-
lings of Quercus robur L. and Q.petraea (Matt.) Liebl.: modulation by irradiance and
growth flush. Ann. For. Sci. 61 (1):73 –80.http://dx.doi.org/10.1051/forest:2003086 .
Pugnaire, F.I., Serrano, L., Pardos, J., 1999. Constraints by water stress on plant growth.
Handbook of plant and crop stress. 2, pp. 271 –283.
R Core Team, 2016. R: A Language and Environment for Statistical Computing. R Founda-
tion for Statistical Computing, Vienna. Austria URL. https://www.R-project.org/ .
Rehder, A., 1960. Manual of Cultivated Trees and Shrubs. second edition. The Macmillan
Company, New York Edit.Sanda, V., Ollerer, K., Burescu, P., 2008. Fitocenozele din România – Sintaxonomie,
Structur ăși Evolu ție. Editura Ars Docendi, Universitatea din Bucure ști.
Schwarz, O., 1964. Quercus L. In: Tutin, T.G., Heywood, V.H., Burges, N.A., Valentine, D.H.,
Walters, S.M., Webb, D.A. (Eds.), Flora Europaea, vol. 1: Lycopodiaceae to Platanaceae.
Cambridge University Press, Cambridge, pp. 61 –64.
Scoffoni, C., Kunkle, J., Pasquet-Kok, J., Vuong, C., Patel, A.J., Montgomery, R.A., Givnish, T.J.,
Sack, L., 2015. Light-induced plasticity in leaf hydraulics, venation, anatomy, and gas
exchange in ecologically diverse Hawaiian lobeliads. New Phytol. 207 (1), 43 –58.
Șofletea, N., 2005. Genetic ăși ameliorarea arborilor. Editura “Pentru Via ță”,B r a șov.
Șofletea, N., Curtu, A.L., 2007. Dendrologie. Editura Universit ății Transilvania, Bra șov.
Stănescu, V., Șofletea, N., Popescu, O., 1997. Flora forestier ălemnoas ăa României. Editura
Ceres, Bucure ști.
Toader, A., Șofletea, N., Curtu, A.L., 2009. Varia ția genetic ăizoenzimatic ăas t e j a r u l u i
pedunculat ( Quercus robur L.)și stejarului brum ăriu ( Quercus pedunculi floraK.
Koch.) din România. Proceedings of the Biennial International Symposium Forest
and Sustainable Development. Editura Universit ății Transilvania din Bra șov, Bra șov,
pp. 1 –8.
Tomescu, A.M.F., 2000. Evaluation of Holocene pollen records from the Romanian Plain.
Rev. Palaeobot. Palynol. 109 (3), 219 –233.
Trinajsti ć,I . ,2 0 0 7 . About the problem of differentiation between the oaks Quercus
pubescens Willd. and Quercus virgiliana Ten., Pregledni Članci –Rewievs UDK 630
∗164. Šumarski list br. 1 –2. CXXXI pp. 57 –60.
Viscosi, V., Fortini, P., Slice, D.E., Loy, A., Blasi, C., 2009a. Geometric morphometric analyses
of leaf variation in four oak species of the subgenus Quercus (Fagaceae ). Plant Biosys.
143 –575.
Viscosi, V., Lepais, O., Gerber, S., Fortini, P., 2009b. Leaf morphological analyses in four Eu-
ropean oak species ( Quercus ) and their hybrids: a comparison of traditional and geo-
metric morphometric methods. Plant Biosys. 143 (3), 564 –574.
Vuckovic, B., 1984. A new habitat of Quercus pedunculi floraC. Koch in Serbia (Yugoslavia).
Zbornik radova Instituta za sumarstvo i drvnu industriju. 22 –23, pp. 111 –113.
Wellstein, C., Cianfaglione, K., 2014. Impact of extreme drought and warming on survival
and growth characteristics of different provenences of juvenile quercus pubescens
willd. Folia Geobotanica 49 (1), 31 –47.
Wigston, D.L., 1975. The distribution of Quercus robur L,Q petraea (Matt) Liebl and their
hybrids in south-western England. 1. The Assessment of the Taxonomic Status of
Populations From Leaf Characters. 10, pp. 345 –369 Watsonia.
Wright, K., 2015. Corrgram: Plot a Correlogram. R package version. 1:8. https://CRAN.R-
project.org/package=corrgram .
Yadollahi, A., Arzani, K., Ebadi, A., Wirthensohn, M., Karimi, S., 2011. The response of dif-
ferent almond genotypes to moderate and severe water stress in order to screen for
drought tolerance. Sci. Hortic. 129 (3), 403 –413.
Further reading
***2012. Forest National Inventory of Romania.
***Herbarul Facult ății de Silvicultur ășiE x p l o a t ări Forestiere Bra șov (BVS).
***Herbarul Institutului Na țional de Cercetare Dezvoltare în Silvicultur ă“Marin Dr ăcea”
Bucure ști (BUCF).
***IBMSPSS software.
***2008. STATISTICA 8. StatSoft Inc., Tulsa. OK. USA.
***2007. WinFOLIA. Regent Instruments INC.505 E.N. Apostol et al. / Science of the Total Environment 609 (2017) 497 –505