Morphological and genetic diversity within Pilosella hoppeana aggr. (Asteraceae) in Italy and taxonomic implications

Abstract

Morphological variation, ploidy level and genetic diversity have been studied on 10 populations of the Pilosella hoppeana aggr. from the Alps, Abruzzo, Calabria and Sicily. Chromosome counts showed that the plants from Abruzzo and those from Sicily are tetraploid (2n = 36); they are assigned to P. hoppeana subsp. macrantha. The plants from the Alps (P. hoppeana subsp. hoppeana) and those from Calabria are diploid. The Calabrian populations, previously included in P. hoppeana subsp. macrantha, are shown to belong to a separate species, P. leucopsilon. The principal component analysis, based on 25 morphological characters, allowed distinguishing clearly four groups. An allozyme study using 10 enzyme systems revealed 7 polymorphic loci with a total of 20 alleles, some of them exclusive at regional level, others shared between populations showing similar morphological features. The genetic differentiation between populations was relatively high. The obtained dendrogram supports recognition of the morphologically defined taxa.

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Morphological and genetic diversity within Pilosella
hoppeana aggr. (Asteraceae) in Italy and taxonomic
implications
E. Di Gristinaa, G. Dominab, G. Gottschlichc, P. Mazzolab & A. Geracia
a Dipartimento Scienze e Tecnologie Biologiche, Chimiche e Farmaceutiche, Università degli
Studi di Palermo, Palermo, Italy
b Dipartimento Scienze Agrarie e Forestali, Università degli Studi di Palermo, Palermo, Italy
c Hermann-Kurz-Straße 35, 72074, Tübingen, Germany
Published online: 20 Aug 2013.
To cite this article: Plant Biosystems - An International Journal Dealing with all Aspects of Plant Biology (2013):
Morphological and genetic diversity within Pilosella hoppeana aggr. (Asteraceae) in Italy and taxonomic implications, Plant
Biosystems - An International Journal Dealing with all Aspects of Plant Biology: Official Journal of the Societa Botanica
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Morphological and genetic diversity within Pilosella hoppeana aggr.
(Asteraceae) in Italy and taxonomic implications
E. DI GRISTINA
1
, G. DOMINA
2
, G. GOTTSCHLICH
3
, P. MAZZOLA
2
, & A. GERACI
1
1
Dipartimento Scienze e Tecnologie Biologiche, Chimiche e Farmaceutiche, Universita
`degli Studi di Palermo, Palermo, Italy
and
2
Dipartimento Scienze Agrarie e Forestali, Universita
`degli Studi di Palermo, Palermo, Italy and
3
Hermann-Kurz-Straße
35, 72074 Tu
¨bingen, Germany
Abstract
Morphological variation, ploidy level and genetic diversity have been studied on 10 populations of the Pilosella hoppeana aggr.
from the Alps, Abruzzo, Calabria and Sicily. Chromosome counts showed that the plants from Abruzzo and those from Sicily
are tetraploid (2n¼36); they are assigned to P. hoppeana subsp. macrantha. The plants from the Alps (P. hoppeana
subsp. hoppeana) and those from Calabria are diploid. The Calabrian populations, previously included in P. hoppeana
subsp. macrantha, are shown to belong to a separate species, P. leucopsilon. The principal component analysis, based on 25
morphological characters, allowed distinguishing clearly four groups. An allozyme study using 10 enzyme systems revealed 7
polymorphic loci with a total of 20 alleles, some of them exclusive at regional level, others shared between populations
showing similar morphological features. The genetic differentiation between populations was relatively high. The obtained
dendrogram supports recognition of the morphologically defined taxa.
Keywords: Allozymes, genetic variability, Italy, morphology, Pilosella, taxonomic relationships
Introduction
Pilosella was formerly included, as a subgenus, in
Hieracium L., but is now generally treated as a
separate genus, based on a whole range of
morphological, biochemical, cytological and geneti-
cal characteristics (Brau
¨tigam & Greuter 2007).
According to Zahn (1923, under Hieracium), Pilosella
comprises 181 “basic” species or species aggregates
distributed from Eurasia to north-western Africa
(Zahn 1923; Sell & West 1975). Widespread
polyploidy, various modes of reproduction (sexuality,
obligate and facultative apomixis of aposporous type,
haploid parthenogenesis and vegetative propagation)
and inter- and intra-specific hybridisation within and
across ploidy levels are the most important processes
involved in microevolution of this genus (Krahulcova
´
et al. 2000).
The Pilosella hoppeana aggregate (Hieracium sect.
Pilosellina Zahn) is very polymorphic. Zahn (1923)
recognised 25 subspecies P. hoppeana, distributed from
central and southern Europe to the Caucasus. These
taxa are hemicryptophyte rosulate, flowering between
May and early August, and they differ in size, shape,
colour and indumentum of bracts (Gottschlich 2009).
According to Pignatti (1982) and Greuter (2008), in
Italy, this group consists of P. h o p p e a n a (Schult.) F. W.
Schultz & Schp. Bip. subsp. hoppeana and P. hoppeana
subsp. macrantha (Ten.) S. Bra¨ut. & Greuter. As
pointed out in Gottschlich (2009, 2011), the name
macranthum was often used for other taxa of the
P. hoppeana aggregate, especially those occurring in
south-east Europe that must be named P. leucopsilon
(Arv.-Touv.) Gottschl. [Syn.: H. leucopsilon Arv.-Touv.,
H. macranthum subsp. testimoniale (Peter) Gottschl.
and P. hoppeana subsp. testimonialis (Peter) P. D. Sell &
C. West].
Pilosella hoppeana subsp. macrantha was described
for the first time by Tenore (1835 – 1838) as
Hieracium macranthum. The original specimens
were collected on Mt Velino and Mt Majella
(Abruzzo, central Italy). The name has been
employed for polymorphic Italian mountain popu-
lations distributed in Abruzzo, Basilicata, Calabria
q2013 Societa
`Botanica Italiana
Correspondence: E. Di Gristina, Dipartimento Scienze e Tecnologie Biologiche, Chimiche e Farmaceutiche, Universita
`degli Studi di Palermo, via Archirafi 38,
90123 Palermo, Italy. Tel: þ39 091 23891262. Email: emilio.digristina@unipa.it
Plant Biosystems, 2013
http://dx.doi.org/10.1080/11263504.2013.829880
Downloaded by [Universita di Palermo] at 01:45 02 September 2013

and Sicily (Zahn 1923; Pignatti 1982). In Sicily, they
are limited to the Madonie (Gussone 1844; Strobl
1878; Lojacono Pojero 1903) and Nebrodi moun-
tains (Gussone 1844; Lojacono Pojero 1903). The
plants of the Madonie (north-central Sicily), growing
on calcareous and quartzarenitic stony pastures and
slopes between 1400 and 1850 m a.s.l., are very
variable, especially with respect to the proportion of
simple and glandular hairs on the involucral bracts.
Individuals with dense simple and sparse or absent
glandular hairs on the bracts are found growing
together with others that have dense glandular and
sparse or absent simple hairs. Indeed, Lojacono
Pojero (1903) referred the individuals with dense
simple hairs to H. macranthum and those with dense
glandular hairs to H. hoppeanum s. str. However,
Zahn (1923) and the latest Floras (Pignatti 1982;
Greuter 2008) consider that a single taxon is
present in Sicily corresponding to P. h o p p e a n a subsp.
macrantha.
Until now, the knowledge on the distribution and
the taxonomy of the taxa belonging to the group of P.
hoppeana in Italy has been rather uncertain and
confused because of the poor field collections, the
low number of specimens in the herbaria and, above
all, the lack of genetic surveys. Indeed, chromosome
counts on south-east European and Italian materials
have demonstrated the presence of both diploid
(2n¼2x¼18: Buttler 1991; Rotreklova
´et al. 2005)
and tetraploid plants (2n¼4x¼36: Raimondo et al.
1983; Brullo et al. 1994, 2004). Moreover, the
high variability observed call for further investigation
of the Sicilian populations of P. h o p p e a n a subsp.
macrantha.
The present study, integrating the knowledge on
the Loci classici of the studied taxa (cf. Domina et al.
2012), proposes to answer the following questions,
for Italy: (1) How are the levels and the distribution
of genetic variability within and among populations
of P. hoppeana aggregate? (2) What is the population
genetic differentiation? (3) Is there a correlation
among morphological features, geographic distri-
bution and genetic variability?
Materials and methods
Ten populations representing the whole range of
variation of the P. hoppeana aggr. in Italy were
examined. In each population, 20 or 25 randomly
selected individuals separated by at least 5 m from
each other were sampled (Table I and Figure 1). In
addition, the morphology of specimens kept in BRIX
(now BOZ), FI, NAP, CLU and PAL were studied.
The sampled individuals were pressed and dried
prior to morphological study. Altogether, 25 char-
acters were observed and quantified, as detailed in
Table II. Similar to Fung Boix et al. (2011) and
Giuliani and Maleci Bini (2011), a number of simple,
glandular and stellate hairs of different organs were
counted per surface unit of 4 mm
2
, then reduced to
number per mm
2
and grouped into four frequency
classes as defined in Table II. Principal component
analysis (PCA) was performed using PAST software
(Hammer et al. 2001).
For karyological analyses, achenes collected in
the wild from the same populations (Table I) were
germinated at 258C on moist filter paper in Petri
dishes. Actively growing root tips of 5– 10 mm in
length were excised from the germinating seeds and
pre-treated in 0.5% colchicine in saturated paradi-
chlorobenzene solution for 1 h at room temperature
and fixed with acetic alcohol (1:3) for 12– 18 h. Then
Table I. Details of sampled Italian populations of the Pilosella hoppeana aggr.
Population code Individuals sampled Area Locality Altitude (m a.s.l.)
Alps s 25 Alps
Trentino Alto Adige: Val di Peder (BZ),
46829042.49200N, 10841015.4100 E 2203
Alps g 25 Alps
Trentino Alto Adige: Passo San Pellegrino (TN),
46822059.5400N, 11846059.7500 E 2002
Vel 20 C-Apennines
Abruzzo: Monte Velino (AQ), 42808050.9500 N,
13822004.9800E 1835
Maj 20 C-Apennines
Abruzzo: Blockhaus, Majella (CH), 42808055.0300N,
14806053.1100E 2050
Car 20 S-Apennines
Calabria: Monte Caramolo (CS), 39848016.2500N,
16805025.4900E 1568
Stilo 20 S-Apennines
Calabria: Bosco di Stilo (RC), 38830047.0100N,
16821035.5800E 1180
Neb s 20 Nebrodi Mts.
Sicily: Monte Campanito (ME), 37849045.8700N,
14823009.9400E 1390
Neb g 20 Nebrodi Mts.
Sicily: Monte Campanito (ME), 37849038.1200N,
14823014.6600E 1422
Mad g 25 Madonie Mts.
Sicily: Monte Scalone (PA), 37850027.8300N,
14801006.0200E 1510
Mad s 25 Madonie Mts. Sicily: Quacella (PA), 37850043.3200N, 14800052.7600E 1352
E. Di Gristina et al.2
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the root tips were rinsed in water, hydrolysed with
1 N HCl for 6 –8 min at 608C, and again rinsed in
water for a minimum of 3 –5 min. Feulgen staining of
root tips was carried out for 1 – 2 h. Finally, squash
preparations were made in 1% aceto-orcein. At least
20 somatic metaphase plates, ideally suited for
chromosome counting, were studied to determine
chromosome numbers. Slides were examined under
a Leica DM 3000 LB photomicroscope, and
photographs were taken with the same microscope.
For allozyme analysis, fresh young leaves of each of
the 220 individuals (Table I), sampled in the wild and
transferred to the laboratory, were crushed in a 200-ml
buffer containing Tris – HCl, pH 7.5 and 1% reduced
glutathione. Crude extracts were adsorbed onto What-
man3MMpaperwicksandstoredat2808Cuntil
electrophoresis. Electrophoresis was performed under
constant voltage at 48C on 11% starch gel following the
general protocols of Wendel and Weeden (1989)
according to Kephart (1990). Ten enzyme systems
were tested for activity and consistent banding patterns
in two different electrode buffers following the staining
recipes of Soltis et al. (1983). Only five of them gave
enough consistent resolution in Tris citrate,
pH 7.0 (Meizel & Markert 1967): PGI ¼phosphoglu-
coisomerase (E.C.5.3.1.9) and PGM ¼phosphoglu-
comutase (E.C.2.7.5.1.); and in morpholine citrate, pH
6.1 (Clayton & Tretiak 1972): IDH ¼isocitrate
dehydrogenase (E.C.1.1.1.42), MDH ¼malate dehy-
drogenase (E.C.1.1.1.37) and 6PGD ¼6-phosphoglu-
conate dehydrogenase (E.C.1.1.1.44). For the
complete methodology, see Geraci et al. (2004, 2009)
and Bancheva et al. (2006, 2011). After migration, the
gel slices were incubated in a staining solution specific
for each enzyme according to Wendel and Stuber
(1984) and Vallejos (1983). Zymograms, then, were
interpretedintermsoflociandalleles,whichwere
labelled starting from the most anodally migrating
band.
The allozyme frequencies, the mean number of
alleles per locus (A), the mean percentage of
polymorphic loci (P), the observed (Ho) and
expected (He) heterozygosity (according to the
Hardy– Weinberg law) were calculated using
BIOSYS-2 (Swofford & Selander 2000). Wright’s
(1951) fixation index (F) was calculated as
F¼12Ho/He. A
x
2
test was used to evaluate the
significance of the deviation from the Hardy –
Weinberg’s equilibrium. The amount of genetic
diversity at the gene pool level was estimated using
Wright’s (1965) F-statistics: F
IT
,F
IS
and F
ST
.F
IT
and
F
IS
coefficients measure the excess of homozygotes
or heterozygotes relative to the panmictic expec-
tations within the entire sample and within popu-
lations, respectively. The F
ST
coefficient estimates
the relative population differentiation. Gene flow
(Nm) among populations was estimated indirectly
from the population genetic structure using Wright’s
(1951) equation as modified by Crow and Aoki
(1984): Nm ¼[(1/F
ST
)1]/4a, where a¼[n/(n21)]
2 and nis the number of populations.
Nei’s (1978) genetic distance and identity
measures were calculated for all pairs of populations
to estimate genetic divergence among populations. A
cluster analysis based on Nei’s genetic distance
values by unweighted pairwise groups method using
arithmetic average (UPGMA) was generated to
examine genetic associations among populations.
Results
Morphological analysis
For those characters that, according to the literature
(Gottschlich 2009), have high diagnostic value (see
Table IX), our results are summarised in Table III.
The populations from Alps show blackish or green-
ish-grey, ovate, rounded, 1.8– 3.3-mm wide bracts
with dense to sparse or absent simple hairs, dense to
sparse or absent glandular hairs and dense to
numerous stellate hairs. Those from Abruzzo have
greyish or greenish-white, ovate, rounded, 1.7 – 3.2-
mm wide bracts with dense to numerous simple
hairs, no or few glandular hairs and dense stellate
hairs. Calabrian populations possess whitish or
greenish-grey, ovate, rounded or acute, 1.3 – 2.1-
mm wide bracts with sparse or no simple hairs,
Figure 1. Localisation of the sampled populations (see also
Table I).
Diversity in Pilosella hoppeana aggr. in Italy 3
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Table II. Characters, and their states or unit, used in morphometric analysis.
Organ Character States or unit
Stolons Length cm
Rosette leaves Number
Length cm
Width cm
Shape 1: Oblanceolate-spathulate 2: Lanceolate-obovate 3: Obovate 4: Oblanceolate
id., adaxially Simple hairs (mm
22
)–:0 (þ): 1–4 þ:5–10 þþ:.10
Glandular hairs (mm
22
)–:0 (þ): 1–4 þ:5–10 þþ:.10
Stellate hairs (mm
22
)–:0 (þ): 1 – 14 þ:15–30 þþ:.30
id., abaxially Simple hairs (mm
22
)–:0 (þ): 1–4 þ:5–10 þþ:.10
Glandular hairs (mm
22
)–:0 (þ): 1–4 þ:5–10 þþ:.10
Stellate hairs (mm
22
)–:0 (þ): 1 – 14 þ:15–30 þþ:.30
Cauline leaves Number
Stem Height cm
id., proximally Simple hairs (mm
22
)–:0 (þ): 1–4 þ:5–10 þþ:.10
Glandular hairs (mm
22
)–:0 (þ): 1–4 þ:5–10 þþ:.10
Stellate hairs (mm
22
)–:0 (þ): 1 – 14 þ:15–30 þþ:.30
id., distally Simple hairs (mm
22
)–:0 (þ): 1–4 þ:5–10 þþ:.10
Glandular hairs (mm
22
)–:0 (þ): 1–4 þ:5–10 þþ:.10
Stellate hairs (mm
22
)–:0 (þ): 1 – 14 þ:15–30 þþ:.30
Bracts Colour 1: Blackish to greenish-grey 2: Greyish to greenish-white 3: Whitish to greenish-grey
Number
Width mm
id., apex Apex 1: Rounded 2: Acute
Simple hairs (mm
22
)–:0 (þ): 1–4 þ:5–10 þþ:.10
Glandular hairs (mm
22
)–:0 (þ): 1–4 þ:5–10 þþ:.10
Stellate hairs (mm
22
)–:0 (þ): 1 – 14 þ:15–30 þþ:.30
E. Di Gristina et al.4
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numerous glandular hairs and dense stellate hairs.
Last, populations from Sicily present greyish or
greenish-white, ovate, rounded, 1.8 – 3.4-mm wide
bracts with dense to sparse or no simple hairs, dense
to sparse or no glandular hairs and dense to
numerous stellate hairs.
For comparison, the comprehensive morphologi-
cal character set (Table II) was statistically analysed
(PCA) for all the populations. All eigenvalues were
positive (one very close to zero), which means that
the input data matrix is well represented in a
Euclidean distance space. The scatter diagram
(Figure 3) reveals four consistent groups: (1) the
Alpine populations; (2) the Sicilian and Abruzzese
populations with prevailing simple hairs; (3) the
Sicilian populations with mainly glandular hairs and
(4) the Calabrian populations.
Chromosome counts
The results are given in Table III. They include the
first records of the chromosome number for Abruzzo
(2n¼4x¼36), Calabria (2n¼4x¼18) and
Nebrodi (2n¼4x¼36).
Isozyme analysis
Six of the seven scored loci (85.7%) were poly-
morphic at least in one population. Only Mdh-1 was
monomorphic across all the tested populations.
Overall, 20 different alleles were detected in the
polymorphic loci (Table IV). Two alleles occurred
only in Mdh-1 and Idh-2 loci, three alleles in Idh-1,
Mdh-2,6Pgd-1 and Pgm-1; the most polymorphic
locus was Pgi-1, where four alleles were detected.
Table III. Chromosome numbers and involucral characters of the 10 studied populations of the P. hoppeana group.
Phyllaries Hairs on involucres
Population
code
Chromosome
number (2n) Colour Apex Width (mm) Simple Glandular Stellate
Alps s 18 Blackish to greenish-grey Rounded 1.8–3.1 þ/þþ 2/(þ)þ/þþ
Alps g 18 Blackish to greenish-grey Rounded 1.9–3.3 2/(þ)þ/þþ þ/þþ
Vel 36 Greyish to greenish-white Rounded 1.7–3 þ/þþ 2/(þ)þþ
Maj 36 Greyish to greenish-white Rounded 1.8– 3.2 þ/þþ 2/(þ)þþ
Car 18 Whitish to greenish-grey Rounded to acute 1.3– 2 2/(þ)þþþ
Stilo 18 Whitish to greenish-grey Rounded to acute 1.4 –2.1 2/(þ)þþþ
Neb s 36 Greyish to greenish-white Rounded 1.8– 3.2 þ/þþ 2/(þ)þ/þþ
Neb g 36 Greyish to greenish-black Rounded 1.9–3.4 2/(þ)þ/þþ þ/þþ
Mad s 36 Greyish to greenish-white Rounded 1.8–3.3 þ/þþ 2/(þ)þ/þþ
Mad g 36 Greyish to greenish-black Rounded 1.8 – 3.2 2/(þ)þ/þþ þ/þþ
Table IV. Allele frequencies of seven allozyme loci for 10 populations of the Pilosella taxa studied.
System Locus Allele Alps g Alps s Mad s Mad g Neb S Neb g Vel Maj Stilo Car
IDH Idh-1 a 0.083 0.500 0.250 0.167 0.350 0.273 0.250 0.333 0.708 0.792
b 0.917 0.500 0.750 0.583 0.650 0.500 0.750 0.667 0.292 0.208
c 0.250 0.227
Idh-2 a 0.583 0.540 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000
b 0.417 0.460
MDH Mdh-1 a 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500
b 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500
Mdh-2 a 0.636 0.538
b 1.000 1.000 0.667 1.000 0.917 1.000 0.833 0.917 0.364 0.462
c 0.333 0.083 0.167 0.083
6-PGD 6Pgd-1 a 1.000 0.708 0.917 0.538 0.917 0.500 1.000 1.000 0.667 0.577
b 0.292 0.083 0.154 0.083 0.292 0.083 0.192
c 0.308 0.208 0.250 0.231
PGI Pgi-1 a 0.292 0.154 0.600 0.364 0.700 0.500 0.864 0.750 0.650 0.682
b 0.250 0.385 0.200 0.455 0.150 0.350 0.136 0.250 0.250 0.136
c 0.250 0.385 0.200 0.182 0.150 0.150 0.100 0.182
d 0.208 0.077
PGM Pgm-1 a 0.583 0.577 0.091 0.300 0.136 0.350 0.167 0.125 0.333 0.389
b 0.417 0.423 0.773 0.650 0.773 0.600 0.833 0.875 0.667 0.611
c 0.136 0.050 0.091 0.050
Diversity in Pilosella hoppeana aggr. in Italy 5
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Idh-2 was polymorphic only for Alpine popu-
lations, consequently Idh-2-b was an exclusive allele
of these populations, as was Pgi-1-d. At Mdh-2 locus,
allele “a” was found to be limited to Calabrian
populations, while at Idh-1 allele “c” was unique to
Sicilian populations characterised by glandular hairs.
The allele “c” of Pgm-1 locus was found in all the
Sicilian populations, but with a rather low frequency.
6-Pgd-1-c occurred in the Sicilian populations
characterised by glandular hairs and in the Calabrian
populations.
The mean number of alleles per locus (A) was
2.05 (range: 1.70 – 2.30). The percentage of poly-
morphic loci (P) was between 71.4 and 85.7. The
mean expected population heterozygosity (He) was
0.368, ranging from 0.249 to 0.456. The mean
observed heterozygosity (Ho) was 0.367, ranging
from 0.217 to 0.521 (Table V).
The inbreeding coefficient (F), calculated for
each population, was greatly positive only for one
Alpine population (0.226), while the index values
were significantly negative for the Calabrian popu-
lations (20.237 and 20.208). The other values were
positive or slightly negative. The
x
2
test showed that
all populations were not in equilibrium for the Mdh-1
locus showing a fixed heterozygosity, and six of them
indicated a significant (P,0.001) departure from
Hardy– Weinberg equilibrium by an excess of
homozygosity in some other loci (Table V).
Genetic structure
F-Statistic was calculated for each polymorphic locus
(Table VI). F
IT
indicated an overall deficiency of
heterozygote at five of six polymorphic loci (mean
value 0.438). High values of F
IS
were also scored in
Idh-2,6Pgd-1 and Pgi-1 loci showing a large excess of
homozygous pattern, while a negative value was
found for Pgm-1. The mean value was 0.302 in F
IS
,
showing an excess of homozygotes.
The resulted F
ST
values, which represent a
measure of differentiation among all populations,
ranged between 0.124 and 0.368, the mean value
being 0.224. The loci mainly implicated in the
differentiation among these populations are Idh-2
and Mdh-2.
F
ST
was also calculated between pairs of popu-
lations, between pairs of geographical territories and
between morphological groups (Tables VII and VIII).
The values ranged from 0.012 to 0.239. The highest
values were found between Alpine and Calabrian
populations. Considering the pairs of territories, the
lowest value was found between Sicilian and
Abruzzese populations (0.108); an even lower value
(0.039) resulted when only populations few or no
glandular hairs on the bracts were compared.
The number of migrants per generation (Nm)
was 0.908, if we consider all populations, while the
values range between 0.666 (Alpine and Calabrian
populations) and 1.607 (Abruzzese and Sicilian
populations), when pairs of territories are con-
sidered. This value becomes very high when
calculated for populations with prevailing simple
hairs within Sicily and Abruzzo (3.605). Between the
Sicilian populations with mainly simple hairs and
those with mainly glandular hairs, Nm was 1.450
(F
ST
¼0.097).
Table VI. Summary of F-statistics at polymorphic loci.
Locus F
IS
F
ST
F
IT
Idh-1 0.156 0.193 0.319
Idh-2 1.000 0.363 1.000
Mdh-2 0.162 0.368 0.470
6Pgd-1 0.326 0.175 0.444
Pgi-1 0.288 0.125 0.378
Pgm-1 20.118 0.124 0.020
Mean 0.302 0.224 0.438
Notes: F
IS
, fixation index, F
ST
, inbreeding coefficient and F
IT
,
genetic differentiation between each population compared with all
other populations.
Table V. Parameters of genetic variability.
Heterozygosity
Population A P Ho He F Unbalanced loci
Alps g 2.0 71.4 0.274 0.354 0.226 Mdh-1, Idh-2, Pgm-1
Alps s 2.1 85.7 0.461 0.456 20.011 Mdh-1, Idh-2
Mad s 2.1 85.7 0.303 0.360 0.158 Mdh-1, Mdh-2
Mad g 2.3 71.4 0.410 0.414 0.010 Mdh-1
Neb s 2.1 85.7 0.263 0.315 0.165 Mdh-1, 6Pgd-1, Pgi-1
Neb g 2.3 71.4 0.448 0.429 20.044 Mdh-1
Vel 1.7 71.4 0.253 0.249 20.016 Mdh-1, Mdh-2
Maj 1.7 71.4 0.217 0.253 0.142 Mdh-1, Mdh-2
Stilo 2.1 85.7 0.521 0.421 20.237 Mdh-1
Car 2.1 85.7 0.517 0.428 20.208 Mdh-1
Notes: A, number of alleles per locus; F, fixation index; He, expected heterozygosity; Ho, observed heterozygosity; P, polymorphism 95%.
E. Di Gristina et al.6
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Genetic distances
Table VI shows the matrix of genetic distances.
Values of genetic distance range between 0.001
(between Nebrodi and Madonie populations with
predominant simple hairs) and 0.305 (between
Alpine and Calabrian populations). Nevertheless,
high values (higher than 0.1) occur between
Calabrian populations and all others, and also
between Alpine populations and the others except
the Sicilian populations with prevailing glandular
hairs. The values of distance between pairs of
glandular-haired and simple-haired populations
from Madonie, Nebrodi and Alps were moderately
low, ranging from 0.037 (between Nebrodi popu-
lations) to 0.060 (between Madonie populations).
The dendrogram (Figure 3) reveals four consist-
ent groups embracing: (1) the Alpine populations;
(2) the Sicilian and Abruzzese populations having
simple hairs; (3) the Sicilian populations character-
ised by glandular hairs and (4) the Calabrian
populations.
Discussion
Morphological analysis
According to the literature data (Gottschlich 2009),
only the shape and indumentum of the involucral
bracts are useful for the discrimination of taxa within
the Pilosella hoppeana group (Table IX). However, the
indumentum features observed in our population
sample do not support this assumption. Whereas in
the plants from Abruzzo (the ditio classica of
P. hoppeana subsp. macrantha), simple hairs do
indeed prevail over glandular hairs and the same
holds true for some of the populations from the Alps
and for both Sicilian mountain massifs, while other
populations from the same areas show the prevalence
of glandular hairs over simple hairs allegedly
characterising P. hoppeana subsp. hoppeana.
Even though indumentum characters account for
60% of all characters employed in our statistical
analysis, the results of PCA (Figure 3) do not support
the grouping together of all glandular plants in a single
taxon. Sicilian populations with prevailing simple hairs
group with those from Abruzzo, but those with
predominant glands are far apart from both popu-
lations from the Alps. All other populations are well
separated from the Calabrian populations.
Chromosome numbers
The populations of Pilosella hoppeana
subsp. macrantha from the ditio classica (Abruzzo:
Table VII. Matrix with Nei’s distances (above the diagonal) and F
ST
(below the diagonal) between pairs of populations.
Alps g Alps s Mad s Mad g Neb s Neb g Vel Maj Stilo Car
Alps g – 0.040 0.112 0.104 0.098 0.115 0.114 0.110 0.295 0.305
Alps s 0.081 – 0.151 0.084 0.117 0.071 0.175 0.145 0.220 0.193
Mad s 0.137 0.134 – 0.060 0.001 0.057 0.003 0.003 0.124 0.139
Mad g 0.131 0.097 0.085 – 0.041 0.002 0.073 0.053 0.152 0.152
Neb s 0.129 0.125 0.027 0.076 – 0.037 0.003 0.003 0.108 0.109
Neb g 0.134 0.093 0.083 0.091 0.069 – 0.061 0.045 0.124 0.108
Vel 0.154 0.174 0.038 0.114 0.018 0.098 – 0.001 0.126 0.139
Maj 0.150 0.158 0.041 0.097 0.019 0.099 0.012 – 0.115 0.127
Stilo 0.199 0.153 0.127 0.129 0.127 0.108 0.162 0.154 – 0.002
Car 0.239 0.161 0.158 0.131 0.139 0.098 0.167 0.159 0.084 –
Table VIII. Matrix with pairwise values of gene flow (Nm: above
the diagonal) and Wright’s F
ST
coefficient (below the diagonal)
between regions.
Alps Sicily Abruzzo Calabria
Alps – 0.997 0.769 0.666
Sicily 0.174 – 1.607 1.142
Abruzzo 0.183 0.108 – 0.890
Calabria 0.211 0.152 0.158 –
Table IX. Comparison of involucral bract characters for Pilosella hoppeana subsp. hoppeana,macrantha and P. leucopsilon.
Taxon Colour Shape Width (mm) Simple hairs Glandular hairs Stellate hairs
P. hoppeana subsp.
hoppeana Blackish to greenish-grey Rounded-ovate (1.5 – ) 2 – 4 2/(þ)(þ)/þ/þþ þ/þþ
P. hoppeana subsp.
macrantha Greyish to greenish-white Rounded-ovate (1.5 – ) 2 – 4 þ/þþ 2/(þ)þ/þþ
P. leucopsilon Whitish to greenish-grey
Rounded-ovate
to ovate-acute 1.5–2 2/(þ)/þ2/(þ)/þþþ
Source: Modified from Gottschlich (2009).
Diversity in Pilosella hoppeana aggr. in Italy 7
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Velino and Majella Mts) are tetraploid. Their
chromosome number (2n¼36) agrees with that
reported for a population from the north side of Mt
Pollino in Lucania (Brullo et al. 1994) and previous
counts from the Madonie Mts in Sicily (Raimondo
et al. 1983; Brullo et al. 2004), but not with the
diploid number (2n¼18) reported for the same
taxon by Rotreklova
´et al. (2005) for Slovakia.
Furthermore, the Nebrodi populations are also
tetraploid.
Calabrian populations that were generally
ascribed to Pilosella hoppeana subsp. macrantha, but
differ significantly in bract width from typical
populations of that taxon, resulted to be diploid
(2n¼18). This number coincides with that given for
P. testimonialis from the Balkan Peninsula (Buttler
1991, as Hieracium hoppeanum subsp. testimoniale).
For the Alps, the diploid chromosome number
here observed confirms the literature data for Pilosella
hoppeana subsp. hoppeana (Favarger 1965; Gadella
1987).
Genetic diversity
Isozymes are codominant, neutral or nearly neutral
markers and have been used to assess plant genetic
diversity, population structure and plant conserva-
tion biology. Despite the development of a big range
of DNA markers (random amplified polymorphic
DNAs, inter-simple sequence repeat and amplified
fragment length polymorphisms), allozymes con-
tinue to be an important and reliable tool for the
study of genetic variation and evolutionary processes
in plant populations (Hamrick 1990; Wang et al.
2004; Liu et al. 2006; Levsen et al. 2008, Zaouali &
Boussaid 2008; Ben El Hadj Ali et al. 2012; Marino
et al. 2012; Troia et al. 2012; Yang et al. 2012). Our
analysis revealed an interesting allele distribution in
the examined populations. Some alleles are exclusive
at the regional level: the Alpine populations of
Pilosella hoppeana subsp. hoppeana showed two
unique alleles (Idh-2a and Pgi-1d); also Calabrian
and Sicilian populations referred to P. hoppeana
subsp. macrantha presented an exclusive allele, Mdh-
2a and Pgm-1c, respectively. Some other alleles are
shared between populations that show similar
morphological features: Mdh-2cwasfoundin
populations of P. hoppeana subsp. macrantha from
the ditio classica (Abruzzo) and in Sicilian popu-
lations with prevailing simple hairs on the bracts,
whereas 6-Pgd-1c allele was found in Calabrian
populations and in the Sicilian populations with
predominant glandular hairs on the bracts.
It was found that in the Pilosella hoppeana aggr.,
the values of genetic variation measures (A, P, He;
see Table V) are higher than the average values for
sexual plants (Hamrick & Godt 1989). Such a great
variability was also reported by S
ˇingliarova
´et al.
(2011) for populations of the P. alpicola group and by
Tyler (2005) who studied several populations of
Nordic Pilosella. Our results suggest that an efficient
gene flow within the populations is detectable. The
highest values were obtained in diploid populations
from the Alps and Calabria, where the principal
mode of reproduction may be sexual; the other
populations also show a high level of genetic
diversity: apart from gene flow and breeding system,
polyploidy could also affect the level of neutral
variation because a greater number of allelic forms
may be found in polyploid individuals or taxa. In
fact, in our study two polyploid populations showed
the biggest number of alleles per locus (A¼2.3).
F-values resulted very negative in diploid Calab-
rian populations, while, according to Fstatistics, the
mean values of F
IS
and F
IT
calculated for the
polymorphic loci revealed a high level of inbreeding.
Pilosella populations retain a significant degree of
sexuality, and hybridisation involving all species is a
frequent, recurrent phenomenon. Taxa range from
fully sexual, both in diploid and polyploid species, to
almost totally apomictic, occurring as widespread,
morphologically stable clones (Fehrer et al. 2007b).
Similar studies conducted on Hieracium, a genus
closely related to Pilosella (Chrtek et al. 2007),
showed a low level of variability. In Hieracium almost
all taxa are apomicts that rarely hybridise, and
whenever they do this gives rise to new, stable
apomictic lines that are customarily given taxonomic
recognition as species or subspecies (Brau
¨tigam &
Greuter 2007).
The UPGMA dendrogram shows significant
genetic distances between populations of Pilosella
hoppeana subsp. hoppeana from the Alps and all the
other Italian populations. The plants from Calabria
were also very distant from those from Abruzzo and
Sicily. Surprisingly in view of their geographic
separation, the highest genetic affinity was observed
between populations from Abruzzo and Sicily.
This trend is confirmed by F
ST
values. It averages
0.224 (Table VI), but shows great variation when
calculated between pairs of populations (from 0.239
to 0.012: Table VII). Considering groups of
populations, the lowest values were found between
Abruzzese and Sicilian populations.
Nm, which on the whole resulted ,1.000
(0.908), was very low between populations from
the Alps and Calabria and all the others, but very
high (3.605), when calculated between Abruzzese
and Sicilian populations with the same type of bract
indumentum (prevailing simple hairs). This result
highlights the fact that the latter populations possess
a very similar allelic composition. Moreover, our
results show the strong isozyme characterisation of
these taxa, whereas in some other studies (Tyler
E. Di Gristina et al.8
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2005; S
ˇingliarova
´et al. 2011) it was not possible to
use isozyme data to discriminate the morphologically
defined populations, because the majority of the
alleles were present in most of them. Neither do
chloroplast DNA data, especially within Pilosella,
show evident correlation with morphology; these
data are indicative of the complex evolutionary
mechanisms and reticulate connections prevailing in
the group from its very origin, rather than of
morphologically expressed phylogenetic affinities
(Fehrer et al. 2007a).
Conclusions: taxonomic implications
The combined morphological, karyological and
isoenzyme approach, here described, confirms the
presence in Italy of Pilosella hoppeana subsp. hoppeana
(Alps) and P. hoppeana subsp. macrantha (Peninsula
and Sicily). The former is a diploid, whereas
P. hoppeana subsp. macrantha is a tetraploid taxon,
probably an Italian endemic, restricted to the central
and southern Apennines and Sicily. However, the
Calabrian populations clearly differ by their narrower
bracts from P. hoppeana subsp. macrantha, to which
they had been assigned so far, and instead are to be
included in P. leucopsilon. Their diploid chromosome
number (2n¼18), agreeing with data obtained for
P. leucopsilon by Grau and Erben (1988), Buttler
(1991), Schuhwerk and Lippert (1997), Suda et al.
(2007), Krahulcova
´et al. (2009) and Ilnicki and
Szelag (2011), supports this attribution. P. leucopsilon
is a very polymorphic taxon, widespread in central
and south-east Europe, distributed in Italy, besides
Calabria, along the southern periphery of the eastern
Alps (Gottschlich & Pujatti 2002).
Our analyses showed that allozymes can be useful
as taxonomic markers, as was reported in several
previously studies (Samuel et al. 1990; Ho¨ randl
2004; Ruiz et al. 2004; Be
´jaoui et al. 2010; Go¨mo¨ry
et al. 2013). In fact, the obtained results are
congruent with detected morphological variations
of the Italian Pilosella taxa here investigated. The
allelic distribution confirms the morphologically
defined taxa. Alpine populations belonging to
P. hoppeana subsp. hoppeana are genetically differ-
entiated. The great isozyme affinity between Abruzz-
ese and Sicilian populations, in particular those with
mainly simple hairs on the bracts, mirrors their
overall morphological similarity (Figure 2) and
suggests that both can be referred to P. hoppeana
subsp. macrantha. The fact that Sicilian populations
characterised by mainly glandular hairs on the bracts
show genetic dissimilarities with the mainly simple-
haired Sicilian populations (Figures 2 and 3) calls for
their critical reappraisal and possible distinction at a
subordinate taxonomic rank. Finally, the strong
genetic differentiation between populations from
Calabria and all the others confirms that they belong
to a separate species.
Specimina visa
Pilosella hoppeana subsp. hoppeana:O
¨sterreich,
Tirol-Osttirol, Schobergruppe, Lienz, Thurneralpe,
03.08.1868, H. Gander, sub: H. pilosellaeforme
Hoppe, rev. K. H. Zahn sub: H. hoppeanum Schult.
var. subnigrum N.P., rev. G. Gottschlich sub: H.
hoppeanum Schult. ssp. hoppeanum, BRIX-6317;
O
¨sterreich, Tirol-Osttirol, Matrei, auf der Alpe
Figure 2. Cluster analysis using UPGMA of 10 populations of the
Pilosella hoppeana aggr. based on Nei’s genetic distance.
Figure 3. Scatter diagram of Pilosella hoppeana aggr. taxa in the first
and second principal components.
Diversity in Pilosella hoppeana aggr. in Italy 9
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Bergernitzen, 08.07.1868, H. Gander, sub: H.
pilosellaeforme Hoppe, rev. K. H. Zahn sub: (a) f.
genuinum, (b) zu subnigrum N.P., rev. G. Gottschlich
sub: H. hoppeanum Schult. ssp. hoppeanum, BRIX-
6168; O
¨sterreich, Tirol-Osttirol, Matrei, Alpe no¨ rdl.
der ?itteldorf, 03.07.1861, H. Gander, sub: H.
pilosellaeforme Hoppe, rev. K. H. Zahn sub: f.
subnigrum N.P., rev. G. Gottschlich sub: H.
hoppeanum Schult. ssp. hoppeanum, BRIX-6176;
O
¨sterreich, Tirol-Osttirol, Schobergruppe, Lienz,
Alpe Zetterfeld, 30.06.1868, H. Gander, sub: H.
pilosellaeforme Hoppe, rev. G. Gottschlich sub: H.
hoppeanum Schult. ssp. hoppeanum, BRIX-6166;
O
¨sterreich, Tirol-Osttirol, Schobergruppe, Lienz,
01.08.1872, in pratis alpinis montis Schleinitz,
6000– 70000, sol. schist., H. Gander (Huter: Exs.),
sub: H. pilosellaeforme Hoppe, rev. G. Gottschlich
sub: H. hoppeanum Schult. ssp. hoppeanum, BRIX-
6164; O
¨sterreich, Tirol-Osttirol, Lienzer Dolomiten,
Lienz, Waldesrand beim Kraitmayer ( ¼Kreithof ?),
07.1870, H. Gander, sub: H. pilosellaeforme Hoppe,
rev. G. Gottschlich sub: H. hoppeanum Schult.
ssp. hoppeanum, BRIX-6170; O
¨sterreich, Tirol-
Osttirol, Lienzer Dolomiten, Lienz, Wiesen beim
Kraitmayer, 30.06.1870, solo calc., H. Gander, sub:
H. pilosellaeforme Hoppe, rev. G. Gottschlich sub: H.
hoppeanum Schult. ssp. hoppeanum, BRIX-6167;
O
¨sterreich, Tirol-Osttirol, Karnische Alpen, prope
Hollbruck, 07.1893, in pratis montanis, sol. schist,
1800 –2000 m., A. Goller (Huter: Exs.), sub: H.
pilosellaeforme Hoppe, rev. G. Gottschlich sub: H.
hoppeanum Schult. ssp. hoppeanum, BRIX-6165;
Italy, Trentino, ex parte Tyrol. austr., Moena, in
alpe Pellegrino in Fiemme, F. Facchini, det. C. H.
Schultz sub: P. hoppeana Sz. Sz., rev. G. Gottschlich
sub: H. hoppeanum Schult. ssp. hoppeanum,
BRIX-6160; Italy, Trentino-Sudtirolo, Trento,
Moena, in Alpe Pellegrino in Fiemme, F. Facchini,
det. C. H. Schultz sub: P. hoppeana Sz. Sz., rev.
G. Gottschlich sub: H. hoppeanum Schult.
ssp. hoppeanum, BRIX-6162; Italy, Trentino-Sudtir-
olo, Trento, Judikarien, M. Lamiada, 07.1888, in
pascuis, sol. cal., 1000– 2000 m., P. Porta, sub: H.
pilosella L., rev. R. Huter sub: H. hoppeanum Schult.,
rev. G. Gottschlich sub: H. hoppeanum Schult.
ssp. hoppeanum, BRIX-6163; Italy, Trentino Alto
Adige, Bolzano, Val di Peder, 08.08.2010, 2203m s.l.
m., 46829042.49200N, 10841015.4100E, E. Di Gristina,
PAL; Italy, Trentino Alto Adige, Trento, Moena, Passo
San Pellegrino, 09.08.2010, 2002 m s.l.m.,
46822059.5400N, 11846059.7500 E, E. Di Gristina, PAL.
Pilosella hoppeana subsp. macrantha: Italy,
Abruzzo, L’Aquila, M. Majella, Guado di Coccia,
08.07.1899, in pascuis saxosis, 1200– 1500 m.,
G. Rigo (Iter Italicum quintum anni 1899, Nr.
167), sub: H. macranthum Ten., rev. G. Gottschlich
sub: H. hoppeanum Schult. ssp. hoppeanum (Gotts-
chlich 2009, H. hoppeanum subsp. macranthum
(Ten.) Na¨geli & Peter), BRIX-6149; Italy, Abruzzo,
Pescara, M. Morrone, 11.07.1899, pascuis alpinis
rupestribus, 2000 – 2400 m, G. Rigo (Iter Italicum
quintum anni 1899, Nr. 56), sub: H. hoppeanum f.
oolepium N.P., rev. G. Gottschlich sub: H. hoppeanum
Schult. ssp. hoppeanum (Gottschlich 2009, H.
hoppeanum subsp. macranthum (Ten.) Na¨geli &
Peter), BRIX-6155/6156/6157/6158; Italy, Abruzzo,
L’Aquila, M. Velino, Al Passo del Vorticchio,
09.08.1875, E. Levier, sub: H. macranthum Ten.,
rev. ? sub: H. hoppeanum f. viridisquamum N.P., rev.
G. Gottschlich sub: H. hoppeanum Schult.
ssp. virentisquamum Na¨geli & Peter (Gottschlich
2009, H. hoppeanum subsp. macranthum (Ten.)
Na¨geli & Peter), BRIX-6159; Italy, Abruzzo,
L’Aquila, M. Velino, Passo del Vorticchio, in alpinis
editioribus, 2200– 2300 m., E. Levier, sub: H.
macranthum Ten., rev. S. Belli sub: H. hoppeanum
melanotrichum, FI; Italy, Abruzzo, L’Aquila,
M. Velino, Cafornia, in rupestribus subalpinis,
20.07.1876, E. Levier, sub: H. hoppeanum, FI;
Italy, Abruzzo, L’Aquila, Majella alli trocchi,
M. Tenore, sub: H. macranthum Te n . , r e v.
C. Arvet-Touvet sub: H. pilosellaeforme Hoppe,
NAP; Italy, Abruzzo, L’Aquila, M. Velino,
22.07.2010, 1835 m s.l.m., 42808050.9500N,
13822004.9800E, E. Di Gristina, PAL; Italy, Abruzzo,
Chieti, M. Majella, Blockhaus, 23.07.2010, 2050 m
s.l.m., 42808055.0300N, 14806053.1100E, E. Di Gris-
tina, PAL; Italy, Calabria, Cosenza, Morano, ad
Montem Dirupata, 10.07.1877, pascuis aridis, sol.
calcar., 1000 m., R. Huter, P. Porta, G. Rigo (Huter,
Porta, Rigo, ex itinere Italico III, Nr. 662), sub: H.
pilosella L., rev. R. Huter sub: H. hoppanum Schult.,
rev. G. Gottschlich sub: H. macranthum (Ten.) Ten.
subsp. macranthum, BRIX-6161; Sicily, Messina,
Monti Nebrodi, Monte di Mistretta, 19 Giugno,
Gussone, sub: H. macranthum Ten., NAP; Sicily,
Messina, Monti Nebrodi, Monte Campanito,
28.06.2009, 37849045.8700N, 14823009.9400E, E. Di
Gristina, PAL; Sicily, Palermo, Madonie, Junio, in
elatioribus calcareis montosis, Todaro (Todaro Flora
Sicula Exsiccata, Nr. 1472), sub: H. macranthum Ten . ,
PAL-11587/11588/11589/11590/11591; Sicily,
Palermo, Madonie, 19.05.1830, sub: H. macranthum
Ten., PAL-84851; Sicily, Palermo, Madonie, 06.1859,
sub: H. macranthum Ten., PAL-85297; Sicily,
Palermo, Madonie, 07.1874, Citarda, sub: H.
macranthum Ten., PAL-85298; Sicily, Palermo,
Madonie, 18?1, Mina
`, sub: H. macranthum Te n . ,
PAL-85299; Sicily, Palermo, Madonie, 06.1873,
Bonafede, sub: H. macranthum Ten., PAL-85300;
Sicily, Palermo, Madonie, Vallone del Passo della
Botte, 17.06.1847, sub: H. macranthum Te n . , PA L -
85302; Sicily, Palermo, Madonie, Quacella,
37850043.3200N, 14800052.7600 E, E. Di Gristina, PAL.
E. Di Gristina et al.10
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Pilosella leucopsilon: Deutschland, Bayern,
Mu
¨nchen, Garchinger Heide, 29.06.1855,
J. Grabmayr, sub: H. pilosellaeforme Hoppe, rev.
R. Huter sub: f. testimoniale N.P., rev. G. Gottschlich
sub: H. macranthum subsp. testimoniale (Na¨geli ex
Peter) Gottschl., BRIX-6150; Italy, Trentino, Trento,
u
¨ber Povo, 05.07.1900, auf sumpfigen Boden, 700 m.,
J. Murr, sub: H. hoppeanum. ssp. testimoniale Naeg., rev.
G. Gottschlich sub: H. macranthum subsp. testimoniale
(Na¨geli ex Peter) Gottschl., BRIX-6151/6152/6153;
Italy, Trentino, Valsugana, S. Cristoforo, 06.1906,
Bahndamm, 500 m., J. Murr, sub: H. hypeuryum N.
P.,rev.G.Gottschlichsub:H. macranthum
subsp. testimoniale (Na¨geli ex Peter) Gottschl., BRIX-
6384/6385. Italy, Calabria, Saracena (CS), Monte
Caramolo, versante occidentale del Massiccio del
Pollino, 12.08.1992, prato sassoso su calcare, 1700–
1827 m s.l.m., UTM 33S WE 93.06, L. Bernardo,
Cesca G., sub: H. cfr. pilosella,rev.G.Gottschlichsub:
H. macranthum (Ten.) Ten., CLU-3818.
Acknowledgements
The authors are grateful to Prof. F. M. Raimondo
and Prof. W. Greuter for their useful and con-
structive suggestions and for a critical revision of the
text. Thanks are due also to Prof. F. Pedrotti from
University of Camerino for his help in the collection
of plants from Alps, to the Museo Scienze Naturali
Alto Adige, the Riserva Naturale Orientata “Monte
Velino”, Dr B. Petriccione (Ufficio Territoriale per la
Biodiversita
`di Castel di Sangro-AQ), the Comando
Forestale di Magliano dei Marsi (AQ), the Parco
Nazionale della Majella, Dr G. Ciaschetti, Dr
L. Bernardo from University of Calabria and to
F. Cannizzaro for English translation of the text.
Financial support by the International Foundation
pro Herbario Mediterraneo and by Universita
`degli
Studi di Palermo (Fondi di Ateneo per la Ricerca) are
acknowledged.
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