ORIGINAL ARTICLE

Differentiation and hybridization of Quercus frainetto, Q. petraea,and Q. pubescens (Fagaceae): insights from macro-morphologicalleaf traits and molecular data

Paola Fortini • Piera Di Marzio • Romeo Di Pietro

Received: 2 January 2014 / Accepted: 7 May 2014

� Springer-Verlag Wien 2014

Abstract Macro-morphological leaf traits and genetic

assignments were combined to study the differentiation and

hybridization of three sympatric and inter-fertile white oak

species (Quercus frainetto, Q. petraea, and Q. pubescens).

The sampling was performed in a single forest stand of

central Italy (Mount Vairano) in which the cover percent-

ages of each of these three oak species were almost equal.

The individuals classified as pure species and the hybrid

individuals were divided into two subsets (A and B) which

were subsequently statistically analysed. The results

regarding the subset of pure individuals showed a clear

separation between the three species on the basis of dif-

ferences observed in the following leaf traits: basal leaf

shape, petiole ratio, petiole length, number of intercalary

veins, pubescence of the petiole, leaf area, number of lobes,

lamina length, and percentage of venation. Regarding the

subset of hybrid individuals, as expected, a wider pattern of

leaf traits compared to that exhibited by the pure individ-

uals was observed. The leaf traits of the pure species that

had provided the greater genetic contribution in the

hybridization process were easily identifiable. Quercus

pubescens and its hybrids exhibited a higher degree of leaf

traits variability when compared with those observed for Q.

petraea and Q. frainetto.

Keywords Hybridization � Molecular data �Morphological leaf traits � Quercus � Statistical analysis �Taxonomy

Introduction

The majority of the forest landscape of Europe and western

Asia is dominated by species belonging to Fagaceae. This

family is widespread in the northern hemisphere in both the

temperate and the Mediterranean bioclimatic regions.

Hybridization and introgression are well-documented

phenomena inside Fagaceae and are important sources of

genetic and morphological variability. This latter is often

the cause of the taxonomic confusion which characterizes

the classification of various Fagaceae genera (especially

Fagus and Quercus). In fact the most of the studies on this

family published in the last decade emphasized the

importance of using both genetic and morphological data

for taxonomical classification purposes. For example, on

the basis of morphological traits and gene markers, the

variation patterns of beech in SE-Europe were described as

introgression between the beech subspecies sylvatica and

orientalis where the presumed ‘‘third’’ European beech

species, Fagus mohesiaca, was regarded as just an hybrid

swarm (Denk 1999; Gomory et al. 1999; Denk 2003; Pa-

pageorgiou et al. 2008). The taxonomical problems con-

cerning introgression and hybridization turn out to be

significantly amplified and complicated when the research

topic is Quercus L., by far the most widespread and

physiognomically important genus of trees in the whole

central and southern Europe. The low barriers to gene flow

among oak species cause the production of several hybrid

individuals, especially at the section level, which can lead

to enhanced diversity (Rushton 1983; Williams et al. 2001;

P. Fortini (&) � P. Di Marzio

Dipartimento di Bioscienze e Territorio, Universita degli Studi

del Molise, C.da Fonte Lappone, 86090 Pesche, IS, Italy

e-mail: fortini@unimol.it

R. Di Pietro

Dipartimento PDTA, Sezione Ambiente e Paesaggio, Universita

‘‘La Sapienza’’, Via Flaminia 72, 00196 Rome, Italy

e-mail: romeo.dipietro@uniroma1.it

123

Plant Syst Evol

DOI 10.1007/s00606-014-1080-2

Gonzalez-Rodrıguez et al. 2004; Tovar-Sanchez and Oy-

ama 2004). Crosses frequently occur in mixed forests,

where multiple oak taxa coexist, and the resulting vari-

ability is increased by the high phenotypic plasticity which

currently characterizes this genus. As a consequence, the

identification of species and subspecies can very often be

wrong or misleading (Jensen 1988; Jensen et al. 1993;

Ponton et al. 2004). From a taxonomical viewpoint, the

genus Quercus has always been considered an intricate

case among botanists and the debate concerning the species

concept or the role of hybridism is still open. One per-

sisting issue is to establish whether the common pheno-

typical differences among pure oak individuals are to be

attributed to environmental factors, hybridity, or both

(Govaerts and Frodin 1998). Various methods for delim-

iting species have been proposed (Henderson 2006; Zapata

and Jimenez 2012), and nowadays taxonomists advocate

the use of as many characters as possible for delimiting

groups. As a consequence, the traditional keys based

exclusively on the plant morphology have apparently

become marginalized in favour of an ‘‘omics’’ study

(Schonenberger and von Balthazar 2012). Despite these

recommendations, the majority of oak species are still

identified on the basis of their patterns of morphological

variation (Luckow 1995; Wiens and Servedio 2000) and

even when other kinds of data are available (environmen-

tal, geographical, molecular, biochemical, etc.), the mor-

phological traits continue to be considered the principal

ones for species identification (Bond and Stockman 2008).

The leaves have always been considered the basic

structure for identifying oak species since they are easy to

observe and compare. Regarding morphological oak leaf

variation, investigations have usually been performed on

the basis of traditional macro-morphometrics (Kremer et al.

2002; Borazan and Babac 2003; Skvorc et al. 2005), geo-

metric morphometrics (Viscosi and Fortini 2011), or

micro-morphological leaf traits (Scareli-Santos et al. 2007;

Fortini et al. 2009; Panahi et al. 2012; Scareli-Santos et al.

2013). Other studies (Bacilieri et al. 1995; Yucedag 2013;

Bruschi et al. 2000; Gomory and Schmidtova 2007) ana-

lysed the relationships between genetic assessments and

leaf morphological traits comparing couples of white oak

species (e.g. Q. petraea vs. Q. robur; Q. petraea vs. Q.

pubescens). In the present study, the role of the macro-

morphological leaf traits in delimiting three white oak

species was related to the genetic assessment of pure and

hybrid individuals in a geographical area where the pre-

sence of these latter had previously been documented. The

main aim of this paper was to identify which were the

macro-morphological characters which played the greater

taxonomical role among a sample of S-European white

oaks. In order to achieve this objective, three European

deciduous species, Quercus frainetto Ten., Q. petraea

(Matt.) Liebl, and Q. pubescens Willd. (subgen. Quercus

Sect. Quercus), which grow sympatrically in a large natural

forest, were considered.

Materials and methods

The study area, Mount Vairano, is a natural forest of about

700 hectares located in the Molise region (central Italy)

characterized by a mixed wood of Q. frainetto, Q. petraea,

and Q. pubescens developed at altitudes ranging between

710 and 935 m a.s.l. (latitude 41.55, longitude 14.60). The

parent material consists of limestone and sandstone

embedded in a matrix of flysch. The bioclimate is tem-

perate with upper hill thermotype and humid/subhumid

ombrotype (Blasi and Michetti 2007).

Collections of mature leaves were made randomly in

nine stands (from 27 to 30 individuals per stand) during the

month of July 2009. A total of 265 trees were analysed.

These trees had already been genotyped and micro-mor-

phologically analysed in two previous papers. In Viscosi

et al. (2012), pure genotypes of Q. frainetto (55 individu-

als), Q. petraea (100), and Q. pubescens (55), together with

mixed genotypes of frainettoxpubescens (9), frai-

nettoxpetraea (3), petraeaxpubescens (19), petraeaxfrai-

netto (5), pubescensxfrainetto (9), and pubescensxpetraea

(10), were identified. The assignment of individuals as

species or hybrids was performed using the estimated

membership coefficient (Q) for each individual in each

cluster (Q C 0.90 pure genotype). In Fortini et al. (2013),

the micro-morphological leaf traits were examined, and the

capability of stomata and trichomes type in discriminating

among species was established.

In the present paper, the macro-morphological leaf

traits of the 265 individuals analysed were measured in

order to complete the genetic/morphological comparison

among the three Quercus species occurring in the Mount

Vairano study area. The individuals which were already

identified as ‘‘genetically pure’’ (210 individuals) in Vis-

cosi et al. (2012) were selected from the whole data set to

form subset ‘‘A’’, while the 55 individuals identified as

‘‘hybrids’’ were assigned to subset ‘‘B’’. Ten leaves were

gathered randomly from the entire crown for each indi-

vidual. These leaves were subsequently pressed, dried,

and scanned (with the abaxial surface facing upwards)

using an Epson GT-15000 scanner with a resolution of

300 dpi and finally measured through ImajeTool program

(Rasband 1997–2007). In total, 15 macro-morphological

leaf variables were assessed following Kissling (1977):

five dimensional (leaf area, lamina length, petiole length,

lobe width, and sinus width); two counted (number of

lobes, number of intercalary veins); two observed (basal

shape of the lamina, pubescence of the petiole); six

P. Fortini et al.

123

transformed (leaf compactness, obversity, lamina/petiole

ratio, lobe depth ratio, percentage of venation, and lobe

width ratio); (Table 1; Fig. 1). The data sets were sub-

sequently treated with univariate and multivariate analysis

procedures using XLSTAT 2013.5.06 (Copyright Addin-

soft 1995–2013).

Fifteen box-plot diagrams were performed on the 210

pure individuals (subset ‘‘A’’) in order to identify their

range of leaf variation in Q. frainetto, Q. petraea, and Q.

pubescens. The discriminant role of those traits showing

both a normal distribution according to the Shapiro–Wilk’s

test (p [ 0.05) and an homogeneity of variance according

to a Levene’s test (p [ 0.05) was measured through a one-

way ANOVA, while the discriminant role of those traits

showing a homogeneity of variance only was measured

through a Kruskal–Wallis test.

Using discriminant function analysis (DFA), the best

discriminating combinations of variables among the three

oak species were identified. Box’s tests (chi-square

asymptotic approximation and Fisher’s asymptotic

approximation) were performed in order to select what

DFA type had to be used.

Finally, the canonical discriminant function coefficients

obtained from the previous DFA were used to calculate the

coordinates of the hybrid individuals (subset B). These

coordinates were plotted in order to observe the position of

hybrids and pure individuals in the DFA diagram space.

Results of analysis

Analysis on subset ‘‘A’’ (pure species)

The box-plots (Fig. 2) displayed a wide range of variability

in the leaf traits of the three species analysed. Two leaf

traits only, pubescence of the petiole and basal leaf shape,

did not exhibit overlaps in their values. All the other traits

exhibited a certain degree of overlapping on two groups, at

least.

On the basis of one-way ANOVA and Kruskal–Wallis

test (Table 2), the following leaf traits are to be considered

as discriminant among the three species: leaf area, leaf

length, petiole length, number of lobes, number of inter-

calary veins, pubescence of the petiole, basal leaf shape,

obversity, lobe depth ratio and percentage of venation.

Regarding the leaf traits of lobe width and petiole ratio, Q.

frainetto differed from the other two species, which

appeared more similar to each other. Instead, regarding

compactness and lobe width ratio, Q. frainetto exhibited a

higher similarity to Q. pubescens. Among all the leaf traits

considered, the sinus width only exhibited values that were

similar for all the three species, and consequently it was

excluded from the subsequent analyses that were per-

formed on 14 traits. The DFA for the three pure species and

the projections of the individuals in the factorial plans are

shown in Table 3 and Figs. 3 and 4. The first two quadratic

discriminant functions, accounting, respectively, for 71.75

and 28.25 %, of the total variance, indicated a strong

separation between the three groups of individuals: Q.

Table 1 List of the leaf morphological traits recorded

Code Variable

A Leaf area

PL Petiole length

SW Sinus width

LW Lobe width

LL Lamina length

NL Number of lobes

NV Number of intercalary veins

Co Compactness: sqrt[(4/pi) * area]/major axis

OB Obversity (lamina shape): WP/LL * 100

PR Petiole ratio: PL/(LL ? PL) * 100

LDR Lobe depth ratio: (LW - SW)/LW * 100

PV Percentage of venation: NV/NL * 100

LWR Lobe width ratio: LW/LL * 100

PI_PU Petiole pubescence

BSL Basal shape of the lamina

Fig. 1 Graphical display of the morphological leaf traits measured.

LL Lamina length, LW lobe width, PL petiole length, WP length of

lamina from base to widest point, SW sinus width

Morphological leaf traits in subgenus Quercus

123

frainetto, Q. petraea, and Q. pubescens (only three indi-

viduals of Q. pubescens and one individual of Q. petraea

are misgrouped). The distribution of the individuals of the

three oak species observed along F1 was shown to be

related to the following leaf traits: basal leaf shape, petiole

ratio, petiole length, number of intercalary veins, and leaf

area. This same distribution observed along F2 was shown

to be related to the pubescence of the petiole, the number of

lobes, lamina length, and percentage of venation.

Analysis on subset ‘‘B’’ (hybrid individuals)

The canonical discriminant function coefficients (Table 4)

determined the distribution of the hybrid individuals in the

Fig. 2 Box-plot showing the minimum, first quartile, median (line),

mean (cross) third quartile, and maximum together with both limits

beyond which values are considered anomalous. Values that are

outside the [Q1 - 3 (Q3 - Q1); Q3 ? 3 (Q3 - Q1)] interval are

displayed with the asterisk symbol. Values that are in the [Q1 - 3

(Q3 - Q1); Q1 - 1.5 (Q3 - Q1)] or the [Q3 ? 1.5 (Q3 - Q1);

Q3 ? 3 (Q3 - Q1)] intervals are displayed with the ‘‘o’’ symbol.

Quercus frainetto = dark grey, Q. petraea = light grey, and Q.

pubescens = white

P. Fortini et al.

123

factorial space (Fig. 5). The diagram showed that the

individuals genetically identified as hybrids did not always

exhibit an intermediate position between their ‘‘pure’’

parents in respect of their morphological leaf traits. In fact,

the individuals genetically identified as petxpub (19) were

grouped around the Quercus petraea barycentre (four

individuals only were found in an intermediate position

between Q. petraea and Q. pubescens barycentres). All the

petxfra individuals (5) are grouped around the barycentre

of Q. petraea. Hybrids identified as pubxpet (10) formed a

more or less continuous cloud of points between the

barycentres of Q. pubescens and Q. petraea. Among the

hybrids identified as pubxfra (9), five individuals were

located near the Q. pubescens barycentre and four in an

intermediate position. The hybrids identified as fraxpub (9)

were grouped around the barycentre of Q. frainetto with

just a single individual falling within the Q. pubescens

cloud. Finally, two of the individuals fraxpet (3) exhibited

Table 2 Variables, means, and results of one-way ANOVA (F value, with degree of freedom in brackets, and p value) or Kruskal–Wallis test

(K and p value) for the leaf traits recorded on ten leaves of individuals of Quercus frainetto, Q. petraea, and Q. pubescens (n = 210 trees)

Variable Q. frainetto Q. petraea Q. pubescens F; p K; p

A 67.951 ± 4.725a 33.044 ± 1.845b 30.692 ± 3.256c – 108.06; \0.0001

PL** 0.56 ± 0.037a 1.509 ± 0.069b 1.279 ± 0.107c 32.439 (2, 11.343); 0000

SW 1.454 ± 0.104a 1.331 ± 0.05a 1.41 ± 0.072a – 3.07; 0.222

LW 4.17 ± 0.153a 2.839 ± 0.096b 2.778 ± 0.147b – 101.26; \0.0001

LL 14.351 ± 0.435a 10.421 ± 0.359b 9.346 ± 0.452c – 108.40; \0.0001

NL 17.013 ± 0.467a 12.393 ± 0.297b 11.08 ± 0.460c – 124.29; \0.0001

NV 6.012 ± 0.446a 2.862 ± 0.173b 4.831 ± 0.337c – 119.99; \0.0001

Co* 0.634 ± 0.008a 0.597 ± 0.006b 0.644 ± 0.009a 4.257 (2, 207); \00001

OB 61.137 ± 1.044a 49.229 ± 1.406b 53.15 ± 1.317c – 93.99; \0.0001

PR** 3.786 ± 0.253a 12.694 ± 0.443b 11.981 ± 0.752b 69.321 (2, 11.356); 0000

LDR* 64.932 ± 2.375a 52.435 ± 1.788b 48.708 ± 1.853c 5.594 (2, 207); \00001

PV 35.876 ± 3.131a 23.466 ± 1.621b 44.882 ± 3.98c – 90.47; \0.0001

LWR 29.077 ± 0.626a 27.474 ± 0.653b 29.788 ± 0.768a – 26.12; \0.0001

PI_PU 3.575 ± 0.234a 1.418 ± 0.096b 4.722 ± 0.239c – 157.33; \0.0001

BSL 6.312 ± 0.451a 2.039 ± 0.175b 4.295 ± 0.209c – 150.97; \0.0001

Means with the same letter are not significantly (p B 0.05) different among groups (Tukey’s HSD test, REGWQ test, and Steel–Dwass–

Critchlow–Fligner, respectively)

* ANOVA, ** Welch ANOVA

Table 3 Variables/factors

correlationsF1 F2

A 0.687 -0.463

PL -0.781 0.221

LW 0.670 -0.433

LL 0.581 -0.556

NL 0.631 -0.613

NV 0.765 0.116

Co 0.452 0.362

OB 0.660 -0.119

PR -0.828 0.400

LDR 0.454 -0.450

PV 0.442 0.517

LWR 0.253 0.237

PI_PU 0.677 0.685

BSL 0.883 0.041

Fig. 3 Correlation circle showing the relationship between the initial

variables and the discriminant factors

Morphological leaf traits in subgenus Quercus

123

an intermediate position, while the other one fell down in

the Q. frainetto cloud.

Discussion

A close look at the morphological leaf traits currently

reported in the European Floras diagnostic keys for the

identification of the white oaks (Hegi 1935; Matyas 1970;

Hedge and Yaltirik 1982; Pignatti 1982; Franco 1990;

Schwarz 1993; Christensen 1997) reveals that the number

of leaf traits actually used is, in fact, quite low. Moreover,

some of these traits are rather vague, and in some cases

based on qualitative characters only (glabrous/pubescent;

obovate/ovate/oblong…). This runs counter to the more

recent advances in Quercus taxonomy and biosystematics.

The results presented in this paper confirm that species

identification in the European white oaks is not straight-

forward and often requires the careful evaluation of mul-

tiple characters and molecular data (Gugerli et al. 2008).

The high rate of hybridization which characterizes the

various white oaks species of southern Europe, which for

long periods coexisted in restricted areas (refugia) where

they succeeded in surviving the severe climatic conditions

of the ice ages (Brewer et al. 2002), has resulted in the

splitting of the taxonomical frameworks. It has also per-

suaded botanists to create an impressive number of new

species, most of which, however, are now considered

doubtful (Tenore 1835–1836; Brullo et al. 1999; Di Pietro

et al. 2012). This high number of taxa was also responsible

of the proposal of misleading taxonomical keys based on

‘‘unstable’’ morphological diagnostic traits. According to

Muir and Schlotterer (2005), the current traits similarity

observed between some oak species is just a consequence

of a shared ancestry. Nevertheless, ambiguous morpho-

logical traits could also be considered as the phenotypic

expression of the greater or lesser contributions of the pure

oak species in the hybridization process. Recently Lepais

and Gerber (2011) proposed introgressive hybridization as

Fig. 4 Discriminant function analysis of the 210 pure individuals in

the space of the first two quadratic discriminant functions (canonical

correlations: F1 = 0.997, F2 = 0.935). The barycentre of the three

oak species is shown as grey circles

Table 4 Canonical

discriminant function

coefficients

F1 F2

Intercepts -7.357 -14.204

Variables

A 0.007 -0.018

PL -2.231 1.341

LW 0.502 -0.822

LL 0.157 0.079

NL 0.146 0.038

NV 0.156 -0.470

Co -6.308 14.134

OB 0.017 0.021

PR 0.096 0.054

LDR 0.002 -0.001

PV 0.020 0.075

LWR 0.052 0.036

PI_PU 0.598 0.963

BSL 0.500 -0.007

Fig. 5 Scatter plot with the 55 hybrid individuals in the same space

as Fig. 4. Individuals are identified by genetic assignment

(square = Quercus frainetto, triangle = Q. petraea, circle = Q.

pubescens). The barycentres of the three species are marked by the

larger symbols

P. Fortini et al.

123

a mechanism of the postglacial expansion of Q. pubescens

in central Europe and as a possible explanation of the

increasing northwards affinity of Q. pubescens with Q.

petraea. The preliminary genetic identification of pure oak

individuals and the subsequent identification of their

diagnostic morphological traits have represented a signifi-

cant improvement in defining the correct sequence of

analysis for the creation of realistic taxonomical frame-

works. It is precisely by following this methodological

approach that was possible to select a group of quantitative

and qualitative diagnostic macro-morphological traits

(basal leaf shape, petiole ratio, petiole length, number of

intercalary veins, leaf area, the pubescence of the petiole,

number of lobes lamina length and percentage of venation)

for Q. frainetto, Q. petraea, and Q. pubescens. These

macro-morphological leaf traits, together with the micro-

morphological traits previously selected on the same pure

individuals (Fortini et al. 2013), act as an unequivocal pool

of traits to be used for taxonomic identification (Fig. 6).

Obviously, the species-specific diagnostic power of these

separate traits becomes more powerful when they are used

in combination with each other (Hardin 1979; Bruschi et al.

2000). It is worth-noting, however, that the majority of the

morphological characters (both micro and macro) identi-

fied in this paper are not considered at all in the analytic

keys of the most important European Floras, while those

few that are considered are often restricted to a qualitative

description only (Table 5). As regards the identification of

the hybrids, the problem is still not completely solved. The

combined arrangement of pure individuals and hybrids in

the same DFA diagram enabled us to hypothesize about

hybrids behaviour in relation to their previous genetic

assessment. In the main, the hybrids exhibited leaf traits

linked to those pure species which had provided the greater

genetic contribution in the hybridization process. When

this higher genetic contribution is provided by Q. frainetto

or Q. petraea, the hybrids tend to phenotypically express

the morphological traits of Q. frainetto or Q. petraea, with

Fig. 6 Summary of the micro-morphological and macro-morphological diagnostic leaf traits of Quercus frainetto, Q. petraea, and Q. pubescens

as identified in a previous paper (Fortini et al. 2013) and in the present one

Morphological leaf traits in subgenus Quercus

123

very few individuals showing an intermediate position.

More or less similar results were already reported by Ishida

et al. (2003) for two Quercus species occurring in the cool

temperate forests of Japan. Instead, in the hybrids deriving

from a greater genetic contribution of the parental Q. pu-

bescens (pubxfra and pubxpet), the leaf morphology traits

were mixed. Such hybrid individuals exhibited a more

irregular distribution in the diagram (Fig. 5) with several

individuals placed in an intermediate position with respect

to the barycentres of the three pure species. Similar evi-

dence on the different behaviour of the hybrids of Q. pu-

bescens was reported in Curtu et al. (2007), who performed

a similar analysis in a mixed forest of Q. frainetto, Q.

petraea, Q. pubescens, and Q. robur in Romania, and in

Salvini et al. (2009), who studied a mixed oak forest

composed of Q, petraea and Q. pubescens. This broad

morphological pattern identified in the hybrids of Q. pu-

bescens perfectly matches the broad morphological pattern

recognizable in the systematic taxa conventionally called

Q. pubescens Willd and in the various other doubtful taxa

currently included in the Q. pubescens collective group. (Q.

amplifolia, Q. congesta, Q. dalechampii, Q. humilis, Q.

leptobalana, Q. virgiliana, etc.). Recent papers (Curtu et al.

2011) demonstrated that the wide phenotypic pattern of

Quercus pubescens (the highest among the white oaks)

originated from a genetic base. Hence, it could have been

precisely the contribution of a hypothetical pure form of Q.

pubescens in countless past hybridization events which has

led to the high morphological variability and taxonomical

entropy currently characterizing the Q. pubescens collec-

tive group.

Despite the aforementioned extensive hybridization and

possible introgression events, the three studied species, Q.

frainetto Q. petraea, and Q. pubescens, remain well iden-

tifiable, both morphologically and coenologically, at least

in the relatively small and circumscribed study area of

Mount Vairano. It is not to be excluded, however, that

different physical or ecological features characterizing

some other geographical areas might lead these three spe-

cies growing in such areas to exhibit a different leaf mor-

phological pattern from that observed in the Mount

Vairano forest. In fact, some papers have already demon-

strated that the influence of the environmental parameters

in combination with genetic processes may contribute to

the development of leaf morphological trends (Bruschi

et al. 2003; Lepais and Gerber 2011; Chybicki et al. 2012).

Unfortunately, the experimental work performed on Mount

Vairano would be difficult to replicate elsewhere in Italy,

since the Italian sites where Q. petraea, Q. pubescens, and

Q. frainetto grow together are extremely few in number

due to the almost opposite chorological features of Q.

petraea and Q. frainetto (Abbate et al. 1990; Scoppola

Table 5 Treatment of the leaf traits which have been selected as

‘‘taxonomically diagnostic’’ for the Quercus genus in the present

paper as reported in other European floras: black squares = reported

using quantitative parameters; white squares = reported using qual-

itative parameters only

Pignatti

(1982)

Flora

d’Italia

Christensen

(1997)

Flora

Hellenica

Franco

(1990)

Flora

Iberica

Hedge and

Yaltirik (1982)

Flora of

Turkey

Savulescu (1952)

Flora Republicii

Socialiste

Romania

Matyas (1970)

Taxa nova

Quercum

Hungariae

Schwarz

(1993)

Flora

Europaea

A Leaf area

PL Petiole length j j j j j j j

SW Sinus width h

LW Lobe width j

LL Lamina length j j j j j j

NL Number of lobes j j j j j j

NV Number of intercalary

veins

j j j

Co Compactness

OB Obversity (lamina

shape)

h h h h h h h

PR Petiole ratio

LDR Lobe depth ratio

PV Percentage of venation

LWR Lobe width ratio

PI_PU Petiole pubescence h

BSL Basal shape of the

lamina

h h h h h h

P. Fortini et al.

123

et al. 1990; Blasi et al. 2004; Di Pietro et al. 2010). Instead,

forest situation where these three oaks grow together is

quite common in the central Balkans, where Q. frainetto

exhibits its wider ecological amplitude and Q. petraea is

far more common than in the Italian Peninsula (Horvat

et al. 1974; Carni et al. 2009). So it would be interesting if

the same experimental design performed in Mount Vairano

were to be re-proposed in the Balkans in order to verify

whether, and to what extent, a different biogeographical

context might influence the diagnostic pattern of the leaf

morphological traits.

Conclusions

The extensive felling of forests over thousands of years,

together with some primarily recent reforestation activity, has

fostered contacts among oaks coming from different habitats

and enhanced the possibility of interbreeding. However,

despite the well-known extensive hybridization and intro-

gression which characterize all oaks, Q. frainetto Q. petraea,

and Q. pubescens remain sufficiently distinct for clear iden-

tification purposes, even in areas of sympatry. The findings

confirm the very good congruence between the genetic and

morphological classification in terms of pure species, the lack

of consistency in terms of hybrids (introgressive forms), and

the high variability of Q. pubescens compared to the co-

occurring closely related species. Among the wide range a leaf

macro-morphological characters that were measured and

observed, there were some (e.g. petiole pubescence) that were

not frequently used in oaks taxonomical literature, but were

proved to be useful for taxonomical purposes (especially when

used in combination with micro-morphological and genetic

data). Those descriptors found to be diagnostic were nicely

compared between different European floras and could be

adopted, in the next future, for the elaboration of new and

updated diagnostic keys.

In terms of conservation biology, it is the opinion of the

authors that the stable micro-/macro-morphological and

genetic traits identified in this paper could act as a useful

tool for easier taxonomical identification of the species in

the field, and consequently for establishing more focused

conservation strategies at both local and regional levels. At

the same time, projects aimed at preserving a high species

diversity in oak forests could benefit from monitoring of the

small-scale direction of hybridization. This could lead in the

future to the creation of a network of pilot areas where the

evolutionary processes involving oaks can be investigated.

Acknowledgments We thank Vincenzo Viscosi for his help during

sampling and technical support, Luisa Gilardi for the morphological

trait measurements, and the anonymous reviewer for useful comments

on this manuscript. This research was partially financed by funds from

Italian Miur—Ricerche di Ateneo—Prot. C26A12PJJZ (Resp. R. Di

Pietro).

References

Abbate G, Blasi C, Spada F, Scoppola A (1990) Analisi fitogeografica

e sintassonomica dei querceti a Quercus frainetto dell’Italia

centrale e meridionale. Not Fitosoc 23:63–84

Bacilieri R, Ducousso A, Kremer A (1995) Genetic, morphological,

ecological and phenological differentiation between Quercus

petraea (Matt) Liebl and Quercus robur L. in a mixed stand of

Northwest of France. Silvae Genet 44:1–10

Blasi C, Michetti L (2007) Biodiversity and climate. In: Blasi C et al.

(eds) Biodiversity in Italy. Contribution to the National Biodi-

versity Strategy. Palombi Editori, Rome, pp 57–66

Blasi C, Di Pietro R, Filesi L (2004) Syntaxonomical revision of

Quercetalia pubescenti-petraeae in the Italian Peninsula. Fito-

sociologia 41:87–164

Bond JE, Stockman AK (2008) An integrative method for delimiting

cohesion species: finding the population-species interface in a

group of Californian trapdoor spiders with extreme genetic

divergence and geographic structuring. Syst Biol 57:628–646

Borazan A, Babac MT (2003) Morphometric leaf variation in oaks

(Quercus) of Bulu, Turkey. Ann Bot Fenn 40:233–242

Brewer S, Cheddadi R, Beaulieu JL, Reille M (2002) The spread of

deciduous Quercus throughout Europe since the last glacial

period. For Ecol Manag 156:27–48

Brullo S, Guarino R, Siracusa G (1999) Revisione tassonomica delle

querce caducifoglie della Sicilia. Webbia 54:1–72

Bruschi P, Vendramin GG, Bussotti F, Grossoni P (2000) Morpho-

logical and molecular differentiation between Quercus petraea

(Matt.) Liebl. and Quercus pubescens Willd. (Fagaceae) in

Northern and Central Italy. Ann Bot-Lond 85:325–333

Bruschi P, Grossoni P, Bussotti F (2003) Within- and among-tree

variation in leaf morphology of Quercus petraea (Matt.) Liebl.

natural populations. Trees 17:164–172

Carni A, Kosir P, Karadzic B, Matevski V, Redzic S, Skvorc Z (2009)

Thermophilous deciduous forests in Southeastern Europe. Plant

Biosyst 143:1–13

Christensen KI (1997) Pinaceae, Cupressaceae, Taxaceae, Ephedra-

ceae, Salicaceae, Juglandaceae, Betulaceae, Fagaceae, Ulma-

ceae, Moraceae. In: Strid A, Tan K (eds) Flora Hellenica, vol. 1.

Konigstein: Koeltz, pp 1–17, 26–55

Chybicki IJ, Oleksa A, Kowalkowsk K, Burczyk J (2012) Genetic

evidence of reproductive isolation in a remote enclave of

Quercus pubescens in the presence of cross-fertile species. Plant

Syst Evol 298:1045–1056

Curtu AL, Gailing O, Leinemann L, Finkeldey R (2007) Genetic

variation and differentiation within a natural community of five

oak species (Quercus spp.). Plant Biol 9:116–126

Curtu AL, Moldovan IC, Enescu CM, Craciunesc I, Sofletea N (2011)

Genetic differentiation between Quercus frainetto Ten. and Q.

pubescens Willd. in Romania. Not Bot Horti Agrobo 39:275–282

Denk T (1999) The taxonomy of Fagus in western Eurasia, 2: Fagus

sylvatica subsp. sylvatica. Feddes Repert 110:381–412

Denk T (2003) Phylogeny of Fagus L. (Fagaceae) based on

morphological data. Plant Syst Evol 240:55–81

Di Pietro R, Azzella MM, Facioni L (2010) The forest vegetation of

Tolfa–Ceriti mountains (northern Latium—central Italy). Hac-

quetia 9:5–64

Di Pietro R, Viscosi V, Peruzzi L, Fortini P (2012) A review of the

application of the name Quercus dalechampii. Taxon 61:1311–

1316

Morphological leaf traits in subgenus Quercus

123

Fortini P, Viscosi V, Fineschi S, Vendramin GG (2009) Compar-

ative leaf surface morphology and molecular data of five oaks

of subgenus Quercus Oerst. (Fagaceae). Plant Biosyst 143:

543–554

Fortini P, Antonecchia G, Di Marzio P, Maiuro L, Viscosi V (2013)

Role of micromorphological leaf traits and molecular data in

taxonomy of three sympatric white oak species and their

hybrids (Quercus L.). Plant Biosyst. doi:10.1080/11263504.

2013.868374

Franco JA (1990) Quercus L. Flora Iberica, vol. II (Platanaceae–

Plumbaginaceae (partim)). In: Castroviejo S et al. (eds), Real

Jardin Botanico, CSIC, pp 15-36

Gomory D, Schmidtova J (2007) Extent of nuclear genome sharing

among white oak species (Quercus L. subgen. Lepidobalanus

(Endl.) Oerst.) in Slovakia estimated by allozymes. Plant Syst

Evol 266:253–264

Gomory D, Paule L, Brus R, Zhelev P, Tomoviae Z, Gracan J (1999)

Genetic differentiation and phylogeny of beech on the Balkan

peninsula. J Evol Biol 12:746–754

Gonzalez-Rodrıguez A, Arias DM, Valencia S, Oyama K (2004)

Morphological and RAPD analysis of hybridization between

Quercus affinis and Q. laurina (Fagaceae), two Mexican red

oaks. Am J Bot 91:401–409

Govaerts R, Frodin DG (1998) World checklist and bibliography of

Fagales (Betulaceae, Corylaceae, Fagaceae and Ticodendra-

ceae). Royal Botanic Gardens, Kew, Richmond, pp 201–394

Gugerli F, Brodbeck S, Holderegger R (2008) Utility of multilocus

genotypes for taxon assignment in stands of closely related

European White Oaks from Switzerland. Ann Bot-Lond

102:855–863

Hardin JW (1979) Patterns of variation in foliar trichomes of eastern

north American Quercus. Am J Bot 66:576–585

Hedge IC, Yaltirik F (1982) Quercus L. In: Davis PH (ed), Flora

of Turkey and the East Aegean Islands, 7. Edinburgh,

pp 659–683

Hegi G (1935) Illustrierte Flora von Mittel-Europa. J. F. Lehmanns

Verlag, Munchen. New edition from Hanser Verlag, Munchen

and later Verlag Paul Parey, Berlin

Henderson A (2006) Traditional morphometrics in plant systematics

and its role in palm systematics. Bot J Linn Soc 151:103–111

Horvat I, Glavac V, Ellemberg H (1974) Vegetation Sudosteuropas.

Fischer, Stuttgart

Ishida TA, Hattori K, Sato H, Kimura MT (2003) Differentiation and

hybridization between Quercus crispula and Q. dentata (Faga-

ceae): insights from morphological traits, amplified fragment

length polymorphism markers, and leafminer composition. Am J

Bot 90:769–776

Jensen RJ (1988) Assessing patterns of morphological variation of

Quercus spp. in mixed oak communities. Am Midl Nat 120:120–

135

Jensen RJ, Hokanson SC, Isebrands JG, Hancock JF (1993)

Morphometric variation in oaks of the Apostle Islands in

Wisconsin: evidence of hybridization between Quercus rubra

and Q. ellipsoidalis (Fagaceae). Am J Bot 80:1358–1366

Kissling P (1977) Les poils des quatre especes de chenes du Jura

(Quercus pubescens, Q. petraea, Q. robur et Q. cerris). Berichte

der Schweizerischen Botanischen Gesellschaft 87:1–18

Kremer A, Dupouey LJ, Deans JD et al (2002) Morphological

variation in mixed oak stands (Quercus robur and Quercus

petraea) is stable western European population. Ann For Sci

59:777–787

Lepais O, Gerber S (2011) Reproductive patterns shape introgression

dynamics and species succession within the European white oak

species complex. Evolution 65:156–170

Luckow M (1995) Species concepts: assumptions, methods and

applications. Syst Bot 20:589–605

Matyas V (1970) Taxa nova Quercum Hungariae. Acta Bot Hung

Budapest 16:329–361

Muir G, Schlotterer C (2005) Evidence for shared ancestry polymor-

phism rather than recurrent gene flow at microsatellite loci

differentiating two hybridizing oaks (Quercus spp.). Mol Ecol

14:549–561

Panahi P, Arisa Ziba J, Pourmajidian MR, Fallah A, Pourhas-

hemi M (2012) Foliar epidermis morphology in Quercus

(subgenus Quercus, section Quercus) in Iran. Acta Bot

Croat 71:95–113

Papageorgiou AC, Vidalis A, Gailing O, Tsiripidis I, Hatziskakis S,

Boutsios S, Galatsidas S, Finkeldey R (2008) Genetic variation

of beech (Fagus sylvatica L.) in Rodopi (N.E. Greece). Eur J For

Res 127:81–88

Pignatti S (1982) Flora d’Italia, vol 1. Edagricole, Bologna,

pp 113–120

Ponton S, Dupouey JL, Dreyer E (2004) Leaf morphology as species

indicator in seedlings of Quercus robur L. and Q. petraea (Matt.)

Liebl.: modulation by irradiance and growth flush. Ann For Sci

61:73–80

Rasband WS (1997–2007) ImageJ, US National Institutes of Health,

Bethesda, Maryland, USA. http://rsb.info.nih.gov/ij/

Rushton BS (1983) An analysis of variation of leaf characters in

Quercus robur L. and Quercus petraea (Matt.) Liebl. population

samples from Northern Ireland. Irish For 40:52–77

Salvini D, Bruschi P, Fineschi S, Grossoni P, Kjaer ED, Vendramin

GG (2009) Natural hybridisation between Quercus petraea

(Matt.) Liebl. and Quercus pubescens Willd. within an Italian

stand as revealed by microsatellite fingerprinting. Plant Biol

(Stuttg) 11:758–765

Savulescu T (1952) Flora Republicii Socialiste Romania,Vol 1 Ed.

Academiei Republicii Populare Romane, pp 224–232

Scareli-Santos C, Herrera-Arroyo ML, Sanchez-Mondragon ML,

Gonzalez-Rodriguez A, Bacon J, Oyama K (2007) Comparative

analysis of micromorphological characters in two distantly

related Mexican oaks, Quercus conzattii and Q. eduardii

(Fagaceae) and their hybrids. Brittonia 59:37–48

Scareli-Santos C, Sanchez-Mondragon ML, Gonzalez-Rodriguez A,

Oyama K (2013) Foliar micromorphology of Mexican oaks

(Quercus: Fagaceae). Acta Bot Mex 104:31–52

Schonenberger J, von Balthazar M (2012) Modern plant morpholog-

ical studies. Bot J Linn Soc 169:565–568

Schwarz O (1993) Quercus L. In: Tutin TG, Burges NA, Chater AO,

Edmondson JR, Heywood VH, Moore DM, Valentine DH,

Walters SM, Webb DA (eds) Flora Europaea. Cambridge

University Press, Cambridge, pp 72–76

Scoppola A, Blasi C, Spada F, Abbate G (1990) Sulle cenosi a

Quercus petraea dell’Italia centrale. Not Fitosoc 23:85–106

Skvorc Z, Franjic J, Idzojtic M (2005) Population structure of

Quercus pubescens Willd. (Fagaceae) in Croatia according to

morphology of leaves. Acta Bot Hung 47:193–206

Tenore M (1835–1836) Flora Napolitana, Vol 5. Stampa Reale,

pp 260–261

Tovar-Sanchez E, Oyama K (2004) Natural hybridization and hybrid

zones between Quercus crassifolia and Quercus crassipes

(Fagaceae) in Mexico: morphological and molecular evidence.

Am J Bot 91:1352–1363

Viscosi V, Fortini P (2011) Leaf shape variation and differentiation in

three sympatric white oak species revealed by elliptic Fourier

analysis. Nord J Bot 29:632–640

Viscosi V, Antonecchia G, Lepais O, Fortini P, Gerber S, Loy A

(2012) Leaf shape and size differentiation in white oaks:

P. Fortini et al.

123

assessment of allometric relationships among three sympatric

species and their hybrids. Int J Plant Sci 173:875–884

Wiens JJ, Servedio MR (2000) Species delimitation in systematics:

inferring diagnostic differences between species. P R Soc Lond

Ser B 267:631–636

Williams JH, Boecklen WJ, Howard DJ (2001) Reproductive

processes in two oak (Quercus) contact zones with different

levels of hybridization. Heredity 87:680–690

Yucedag C (2013) Morphological and genetic variation within and

among four Quercus petraea and Q. robur natural populations.

Turk J Bot 37:619–629

Zapata F, Jimenez I (2012) Species delimitation: inferring gaps in

morphology across geography. Syst Biol 61:179–194

Morphological leaf traits in subgenus Quercus

123