Morphological Characters Useful for the Delimitation of Taxa Within Viola Subsect. Viola (Violaceae): A Morphometric Study from the West Carpathians

Abstract

Forty-nine morphological characters were scored or measured on 44 populations (376 individuals) of Viola subsect. Viola from the West Carpathians and adjacent areas (Slovakia, Czech Republic, Austria and Hungary). The presence of six species,
namely V. alba (represented by subsp. alba), V. ambigua, V. collina, V. hirta, V. odorata and V. suavis s.l. was revealed based on pollen fertility, cytological and morphometric analyses. The morphological characters traditionally
used to delimit taxa within the subsection and those revealed by our study as most reliable are widely discussed. A key for
identifying the taxa and most common hybrids of subsection Viola occurring in the West Carpathians is presented. Chromosome counting and flow cytometry were used to determine the ploidy
levels of the populations studied. All individuals of V. alba subsp. alba, V. collina, V. hirta and V. odorata were tetraploid, while those of V. ambigua and V. suavis s.l. were octoploid.

Full text

Morphological Characters Useful for the Delimitation
of Taxa Within Viola Subsect. Viola (Violaceae):
A Morphometric Study from the West Carpathians
Iva Hodálová & Pavol MereďaJr.&
Pavol Mártonfi & Lenka Mártonfiová &
Jiří Danihelka
#
Institute of Botany, Academy of Sciences of the Czech Republic 2008
Abstract Forty-nine morphological characters were scored or measured on 44 populations (376
individuals) of Viola subsect. Viola from the West Carpathians and adjacent areas (Slovakia, Czech
Republic, Austria and Hungary). The presence of six species, namely V. alba (represented by subsp. alba),
V. ambigua, V. collina, V. hirta, V. odorata and V. suavis s.l. was revealed based on pollen fertility,
cytological and morphometric analyses. The morphological characters traditionally used to delimit taxa
within the subsection and those revealed by our study as most reliable are widely discussed. A key for
identifying the taxa and most common hybrids of subsection Viola occurring in the West Carpathians is
presented. Chromosome counting and flow cytometry were used to determine the ploidy levels of the
populations studied. All individuals of V. alba subsp. alba, V. collina, V. hirta and V. odorata were
tetraploid, while those of V. ambigua and V. suavis s.l. were octoploid.
Folia Geobot (2008) 43:83–117
DOI 10.1007/s12224-008-9005-x
I. Hodálová (*)
:
P. MereďaJr.
Institute of Botany, Slovak Academy of Sciences, Dúbravská cesta 14, 845 23 Bratislava, Slovakia
e-mail: iva.hodalova@savba.sk
P. Mártonfi
Institute of Biology & Ecology, Faculty of Science, P. J. Šafárik University, Mánesova 23,
041 54 Košice, Slovakia
e-mail: pavol.martonfi@upjs.sk
L. Mártonfiová
Botanical Garden, P. J. Šafárik University, Mánesova 23, 043 52 Košice, Slovakia
e-mail: lenka.martonfiova@upjs.sk
J. Danihelka
Institute of Botany and Zoology, Faculty of Science, Masaryk University, Kotlářská 2,
611 37 Brno, Czech Republic
e-mail: danihel@sci.muni.cz
Department of Ecology Brno, Institute of Botany, Academy of Sciences of the Czech Republic,
Poříčí 3b, 603 00 Brno, Czech Republic
; e-mail: pavol.mereda@savba.sk

Keywords Chromosome numbers
.
Flow cytometry
.
Multivariate morphometrics
.
Taxonomy
Introduction
The genus Viola L., the largest of the Violaceae family, comprises 525–600 species
distributed throughout most parts of the world (Ballard et al. 1999; Yockteng et al.
2003). It is divided into ca. 14 sections and many infrasectional groups (for a review of
infrageneric classification see Ballard et al. 1999). The genus probably originated in
South America but recent centers of morphological and taxonomic diversity are found
mainly in the Northern Hemisphere (Ballard et al. 1999; Yockteng et al. 2003). The
section Viola, one of the largest infrageneric groups of violets in Europe, is traditionally
divided into five subsections: Viola, Rostratae Kupffer (including Repentes (Kupffer)
W. Becker), Stolonosae Kupffer, Adnatae W. Becker, and Boreali-Americanae W.
Becker (Valentine et al. 1968; Marcussen et al. 2007), with the last one represented in
Europe only by the alien species V. sororia Willd., native to North America.
The subsection Viola, members of which are treated here, includes approximately
25 species native to the temperate zones of Eurasia and adjacent parts of North
Africa (Marcussen and Borgen 2000; Marcussen 2006). The only actual autapo-
morphy of this subsection is the unique capsule morphology (capsules globose,
inexplosive, on decumbent peduncles). Nevertheless, it can also be delimited from
other subsections within V. sect. Viola by combinations of the following characters:
absence of stem, presence of short and stout rhizome, rooting stol ons (which may be
reduced or absent), stipules free, sepals almost rounded, obtuse or obtusely acute at
apex, style beaked at apex, ovate seeds with conspicuous elaiosome adapted to
myrmecochory (Valentine 1962; Valentine et al. 1968; Kirschn er and Skalický 1990;
Okamoto et al. 1993; Mereďa et al. 2008).
The subsection Viola has traditionally been divided into two series, Viola (the
species occurring in the West Carpathians include V. alba Besser, V. odorata L., and
V. suavis M. Bieb.) and Eflagellatae Kitt. (in the West Carpathians represented by V.
ambigua Waldst. & Kit., V. collina
Besser, and V. hirta L.), based on the presence or
absence of stolons (cf. Becker 1925; Gams 1925; Marcussen and Borgen 2000; Dinç
et al. 2003). This classification, however, may be artificial, not reflecting true
phylogenetic relationships within this group (Marcussen and Borgen 2000). For
example, V. collina occasionally produces short stolons (Gams 1925; Marcussen and
Nordal 1998). A study of allozyme markers has also shown that e.g., the stoloniferous
V. suavis in its allozymic pattern is more similar to the non-stoloniferous V. pyrenaica
Ramond than to any stoloniferous subsection member (Marcussen and Borgen 2000).
In the subsection Viola two cytotypes, 2n=20 (the more common cytotype) and
2n=40 (found in V. ambigua and V. suavis), have been reported (for numerous
references see Mereďaetal.2006 ). Traditionally, the base chromosome number of V.
sect. Viola was believed to be x=10; however, Marcussen and Nordal (1998) and
Marcussen and Borgen (2000), interpreting isoenzyme phenotypes, suggested that
the base chromosome number within this subsection is x=5. So true diploids are not
known in this subsection, and plants with 2n=20 should be considered as
palaeotetraploid, and those with 2n=40 as palaeooctoploid.
84 I. Hodálová et al.

Species of the subsection Viola are notorious for their taxonomic complexity, and
their delimitation has been the topic of many studies (for references see Marcussen
and Borgen 2000). In general , problems in taxonomy of this group arise from the
facts that (1) there are only a few morp hological characters used to delimit taxa, with
most of them overlapping across recognized species, (2) some taxa exhibit strong
phenotypic plasticity, and (3) interspecific hybridization is frequent (e.g. Schmidt
1961;Kuta1981; Marcussen and Borgen 2000; Marcussen et al. 2001). A
combination of karyological, morphological and molecular approaches, such as
DNA sequences (Ballard et al. 1999; Ballard and Sytsma 2000; Nadot et al. 2000;
Yockteng et al. 2003) and allozyme markers (Marcussen and Nordal 1998;
Marcussen and Borgen 2000; Marcussen et al. 2001, 2005; Marcussen 2003,
2006), has substantially contributed to the elucidation of phylogenetic relationships
within the genus and within sect. Viola, as well as to the understanding of
intraspecific variation in a number of related species.
Six species of Viola subsect. Viola have been reported from the West Carpathians,
of which V. alba, V. ambigua, V. collina, and V. hirta are considered native, and V.
odorata and V. suavis are naturalized (for more details see Mereďa et al. 2008).
Viola alba (2n=20) is well known for its infraspecific variation. As shown by
Marcussen (2003) and Marcussen et al. (2005), it comprises three ± vicarious
subspecies: (1) V. alba subsp. alba (including two colour morphotypes: alba and
scotophylla), occurring from the Caucasus and the Middle East westwards to Central
Europe and northern Spain, (2) V. alba subsp.
dehnhardtii (Ten.) W. Becker, growing in
the Mediterranean region from T urkey westwards to the Iberian Peninsula and
Morocco, and (3) V. alba subsp. cretica (Boiss. & Heldr.) Marcussen, endemic to Crete.
In the West Carpathians V. alba reaches the northern limit of its native distribution
range. It occurs rather rarely from planar to submontane belt. Its main habitats are
colline oak-hornbeam and beech woods and shrubberies, mainly on basic substrata.
Viola ambigua (2n=40) is distributed from the Caucasus westwards to Central
Europe. The northwestern limit of its distribution range runs through southern
Moravia (Czech Republic). Isolated occurrences exist in northern Bohemia and
central Germany (Danihelka and Čeřovský 1999). In Central Europe it is known
only from a few localities in the Czech Republic, eastern Austria, southern Slovakia,
and northern Hungary. It grows in open, dry and sunny places from the planar to the
colline belt and prefers calcareous substrata.
Viola collina (2n=20) is distributed in most of the temperate parts of Eurasia
(Marcussen et al. 2001), but morphologically it is rather uniform (Marcussen and
Borgen 2000). It is common in the West Carpathians, growing mainly in their central
part from the colline to the montane belt. It occupies sunny pastures and open places
in beech and coniferous woods, mainly on basic substrata.
Viola hirta (2n=20) is widespread from the Iberian Peninsula and British Isles in
the west to Lake Baikal in the east (Marcussen et al. 2001). It is closely related to V.
ambigua in its allozymic pattern (Marcussen and Borgen 2000) but both species are
morphologically well differentiated. In the West Carpathians V. ambigua and V. hirta
can be found growing together in dry grasslands; however, V. hirta has a much
broader ecological niche including also moderately shaded places, open wood s,
shrubberies and forest edges on different substrata. It also has the widest distribution
Morphological characters of Viola subsect. Viola (Violaceae) 85

in the West Carpathians of the species considered here and is common throughout
the area studied.
Viola odorata (2n=20) is morphologically rather uniform and is commonly
distributed in most parts of Europe and adjacent parts of Asia and North Africa. As
the only member of subsection Viola, it is naturalized in North America. According to
Marcussen (2006), V. odorata is native only to the Mediterranean region south of the
Alps and to some parts of western Europe. The species is frequently cultivated, and
numerous ornamental cultivars of V. odorata can be found in temperate zones throughout
all continents (Marcussen 2006). In the West Carpathians, V. odorata is naturalized and
common from the planar to the submontane belt on different substrata. It occurs in man-
made and man-influenced habitats, such as parks and cemeteries, as well as in natural
and semi-natural open dry grasslands and shrubberies or in shaded alluvial woods.
Viola suavis (2n=40) represents a taxonomically critical species with a series of
morphologically and geographically defined races treated on different taxonomic
levels (for further details see e.g. Becker 1910;Schmidt1961; Marcussen and Nordal
1998). It is distributed in the Mediterranean region from Morocco eastwards to the
Middle East but due to cultivation its distribution area expanded also to Central and
northern Europe (Marcussen and Nordal 1998). Though not native and with limited
distribution, V. suavis is morphologically the most variable species of the subsection
Viola in the West Carpathians. It is common in the southern part of the West
Carpathians from the planar to the submontane belt, whereas only a few isolated
localities exist in northern Slovakia (Mereďaetal.2008). Viola suavis,likeV. odorata,
occurs in man-made habitats, such as gardens, parks and old cemeteries, as well as in
natural and semi-natural habitats close to settlements, including shaded parts of dry
grasslands, open deciduous woods, shrubberies and forest edges on different substrata.
Despite various biosystematic studies of Viola subsect. Viola, species boundaries
and relationships among species are not always clear. In addition, no statistically
based morphological study including the West-Carpathian (or Central European)
populations of the subsection Viola has yet been done. Thus, we present a co mbined
cytological and morphometric study focusing on populations in this part of Europe.
The main objectives of this study were (1) to reconsider the value of morphological
characters used to delimit the taxa within the subsection, and (2) to explore their
chromosome number variation in the West Carpathians.
In the present study we intended to exclude hybrid individuals as much as
possible and to focus only on variation of non-hybrid specimens. The identification
of non-hybrid and hybrid specimens was relied on assessing their ploidy levels and
pollen fertility. Nevertheless, we are aware that only heteroploid hybridization events
(between tetraploid and octoploid parental species) and primary hybrids (mostly F
1
)
in the case of homoploid hybridizations may have been safely identified using this
approach. It was documented that backcrossed-hybrid plants (introgressants) derived
from homoploid interspecific crosses can restore pollen fertility in subsequent
generations (cf. Kuta 1990; Krahulcová et al. 1996; Marcussen and Borgen 2000 ). A
certain percentage of the individuals investigated here, therefore, might be a result of
such backcrosses. A detailed morphometric analysis of hybrid populations of V.
subsect. Viola will be published separately, based on more extensive sampling.
86 I. Hodálová et al.

Material and Methods
Field Sampling
A total of 465 individuals from 57 popula tions of Viola subsection Viola were
collected in the West Carpathians and its adjacent areas in 2003–2005 (see
Appendix, Fig. 1). The intra-populational homogeneity known among species
within subsection Viola (Marcussen and Nordal 1998) allowed us to collect only one
to two plants with spring chasmogamous flowers for cytological and 10 (1–12)
plants for morphometric analyses at each site.
Plants for cytological analyses were cultivated in the experimental garden of the
Institute of Botany, Slovak Academy of Sciences, Bratislava, Slovakia. For morpho-
metric analyses all vegetative (stolons, leaves, stipules) and floral parts (peduncles,
calyx, corolla) per individual were attached to paper using adhesive tape, dried, and
preserved as herbarium specimens. Voucher specimens for all analyses are deposited in
the herbarium in the Institute of Botany, Slovak Academy of Sciences, Bratislava (SAV).
Pollen Fertility
To eliminate hybrid individuals, a step necessary for the reconsideration of
diagnostic morphological characters of taxa within the subsection, pollen fertility
Fig. 1 Map showing sample sites of the studied populations of Viola sect. Viola subsect. Viola from the West
Carpathians and adjacent areas. Viola alba subsp. alba (including alba morphotype (asterisk)andscotophylla
morphotype (triangle)), V. ambigua (square ), V. collina (star), V. hirta (plus sign), V. odorata (circle), V.
suavis (including violet-flowered morphotype (spade) and white-flowered morphotype (trefoil)), hybrid
populations of V. alba × V. hirta, V. alba × V. odorata, V. ambigua × V. odorata, V. hirta × V. odorata and V.
odorata × V. suavis s.str. (all represented by the symbol “×”)(formoredetailsseeAppendix)
Morphological characters of Viola subsect. Viola (Violaceae) 87

was analyzed in all tetraploid and octoploid specimens studied (in total 460 plants;
see Appendix). Pollen fertility analyses were not performed on hexaploid
individuals (results of heteroploid hybridization events, five plants).
Pollen fertility, indicated by pollen stainability, was examined in one chasmog-
amous flower of each plant studied. Pollen grains were removed from anthers of
open flowers or flower buds and mounted on slides in aceto-carmine jelly (Radford
et al. 1974). One hundred pollen grains per individual were observed. The unstained
grains, usually shrunk and empty, were considered as sterile and well-stained and
regularly developed grains were considered as fertile.
Cytological Analyses
Chromosome numbers and/or ploidy level estimations were determined using
chromosome counts in root tips and flow cytometry in all 57 populations studied.
Chromosome numbers
Root-tip meristems of potted plants were used for chromosome counts. They were pre-
treated in 0.1% water solution of colchicine for about 3 h, fixed in 98% acetic acid:96%
ethanol mixture (ratio 1:3) for 1–24 h, washed in distilled water, macerated in 1 N HCl
at the temperature 60°C for 5–6 min and washed in distilled water. The squashes were
made using a cellophane square (Murín 1960), stained in the 10% solution of Giemsa
stock dye in Sörensen phosphate buffer for about 1 h, washed, dried, and observed.
Flow cytometry
Samples were prepared from the fresh tissues of petals and/or flower peduncles and/or
young leaf petioles. Probably because of pronounced slime production, no FCM signal
was detected in the samples prepared from leaf laminas. A two-step procedure (Otto 1990;
Doležel and Göhde 1995) was used for sample preparation. Fresh material was chopped
with a sharp razor blade in a glass Petri dish containing 0.5 ml of ice-cold Otto I buffer
(0.1 M citric acid, 0.5% Tween 20). The nuclei suspension was filtered through a
42 μm nylon mesh and centrifuged at 150 g for 5 min. The supernatant was removed
and nuclei were resuspended in 100 μl of fresh ice-cold Otto I buffer and incubated for
20 min at room temperature with occasional shaking. For DNA staining a solution of
1 ml of Otto II buffer (0.4 M Na
2
HPO
4
·12H
2
O) supplemented with 50 μg/ml
propidium iodide and 50 μg/ml RNAse was added and analyzed after 10 min. Relative
DNA content was estimated with a Becton Dickinson FACSCalibur flow cytometer
using BD Cellquest Pro Software. A spontaneous plant of V. reichenbachiana Jord. with
2n=20 (counted by L. Mártonfiová and P. Mártonfi) from the decorative area of the
Botanical Garden in Košice, Slovakia, 223 m, 48°44′06″ N, 21°14′18″ E(17Apr2003
P. Mártonfi 2685 KO s.n.; plant cult. no. M23 in Botanical Garden, P. J. Šafárik
University in Košice) was used as the reference plant (standard). Internal standardiza-
tion (the nuclei of the standard were isolated, stained and analyzed simultaneously with
the nuclei of a sample), or , in some cases, pseudo-internal standardization (the nuclei of
the standard and a sample were isolated and stained separately, but they were mixed
and analyzed together) were employed (Noirot et al. 2005;Greilhuberetal.2007).
88 I. Hodálová et al.

Morphometric Analyses
Apparent hybrid individuals identified by significantly decreased pollen fertility or
hexaploid level (see Results) were excluded from the morphomet ric analyses. Forty-
one characters (24 vegetative and 17 floral) were measured or scored on 376
flowering plants from 44 populations collected in spring in the field, and eight ratios
were derived from them (together 49 characters, see Table 1, Fig. 2). Most characters
were quantitative, nine were binary, and two semi-quantitative. Characters StN,
StUL, StAL, StW, StP, LP, SFI, KP, CO, CEN, CP, CSS, CSP, CPSP, and CaVN
were scored immediately in the field on fresh plants, vegetative and floral parts were
subsequently attached to paper using adhesive tape, and dried. The remaining
characters were evaluated on such dried herbarium specimens. Characters on lamina,
petioles and stipules were scored on three (if present) well-developed leaves of each
plant; characters on peduncle, calyx and corolla on two (if pr esent) largest
chasmogamous flowers of each plant. Average values were then entered into the
data matrix. Out of the 49 characters scored or measured, only 37 were used in
morphometric analyses (Table 1). Altogether 14 characters were excluded from the
morphometric analyses: 11 were used solely for calculating ratios, one character
(CaVN) was const ant across all populations and taxa, and one character (StN) was
not included because the information expressed was already given by the characters
StAL, StUL and StW. Character CP was excluded from the canonical discriminant
analyses, because it was constant within most of the groups.
Characters in the morp hometric analyses included those traditionally used for the
delimitation of taxa within the subsection Viola (e.g. Kirschner and Skalický 1990;
Fischer et al. 2005), or used in modern taxonomic treatments and morphometric
papers (e.g. Marcussen and Nordal 1998; Marcussen et al. 2001; Marcussen 2003 ),
as well as those found useful in our preliminary screening of Carpathian populations.
Spearman correlation coefficients (Sneath and Sokal 1973; Krzanowski 1990)
were computed for the matrix including all plants studied to eliminate pairs of highly
correlated characters from further analyses.
Cluster analysis (CA; UPGMA – average clustering; Everitt 1986) based on popu-
lations (characterized by average values of characters) as operational taxonomic units
(OTUs) was performed to generate a hypothesis on population groupings. The charac-
ters in the primary matrix were standardized by zero mean and unit standard deviation,
and the Euclidean coefficient was used to compute the secondary distance matrix.
Principal component analyses (PCA; Sneath and Sokal 1973) based on populations
as OTUs and a correlation matrix between the characters were performed on subsets
of (I) tetraploids and (II) octoploids. Principal component analyses were used to
determine non-hierarchical structure within both tetraploid and octoploid populations.
Canonical discriminant analyses (CDA; Klecka 1980) based on individual plants
as OTUs were performed to test the results from cluster and p rincipal component
analyses that were based on population averages. Six groups, resolved by UPGMA
and PCA (see Results), were defined as groups for CDA 1–8 (see Results).
Mean, standard deviation, minimum, maximum, 10 and 90 percentiles were
computed for all quantitative characters.
Morphological characters of Viola subsect. Viola (Violaceae) 89

Table 1 Characters scored and measured for morphological analyses (see Fig. 2)
Character Character explanation
Stolons
StN (Aboveground or underground) stolons 0 absent; 1 present
StAL
a,b
Maximum length of aboveground stolon (cm)
StUL
a,b
Maximum length of underground stolon (cm)
StW
a
Width of the most robust stolon (mm)
StP
a
Violet pigmentation of stolons 0 absent; 1 present
Laminas and petioles
LHL
a,c
Maximum hair length (on petiole) (mm)
LL
d
Lamina length (cm)
LW Lamina width (cm)
LL1
e
Lamina length from the base to
maximum width
(cm)
LSL Lamina sinus depth (cm)
LSW Lamina sinus width (cm)
LSA
a
Lamina sinus angle (degree)
LCN
a
Number of crenulae along both lamina
margins (=lamina dentations)
–
LAA
a
Lamina apex angle (degree)
LP
a
Violet pigmentation of lamina 0 absent; 1 present
LL/LW
a
Lamina length/lamina width –
LW/LSW
a
Lamina width/lamina sinus width –
LL1/LL
a
Lamina length from the base to maximum
width/lamina length
–
LSL/LL
a
Lamina sinus depth/lamina length –
Stipules
f
SL Stipule length (mm)
SW Stipule width (mm)
SFN
a
Number of fimbriae (=glandular fimbriae,
non-glandular fimbriae and sessile glandule)
along both stipule margins
–
SFL
a
Maximum fimbriae length on stipule (mm)
SGN Number of glandular fimbriae along both
stipule margins
–
SFI
a
Indumenta of stipule and fimbriae margin 0 glabrous; 1 hairy
SYGN
a
Yellow or yellowish-brown glandular fimbriae
on stipule (including yellow or
yellowish-brown sessile glandule)
0 absent; 1 present
SBGN
a
Blackish glandular fimbriae on stipule
(including blackish sessile glandule)
0 absent; 1 present
SL/SW
a
Stipule length/stipule width –
SGN/SFN
a
Number of glandular fimbriae along both
stipule margins/number of fimbriae along
both stipule margins
–
Peduncles
PL Peduncle length (cm)
PL1 Peduncle length below bracteoles (cm)
PL1/PL
a
Peduncle length below bracteoles/peduncle
length
–
Calyx (sepals)
KAL
a
Anterior sepal length (mm)
KAW
a
Anterior sepal width (mm)
KP
a
Violet pigmentation of sepals 0 absence; 1 presence
90 I. Hodálová et al.

Analyses were performed using the SAS version 9.1 statistical package (SAS
Institute 2000) and the SYN-TAX 2000 package (Podani 2001).
Results
Pollen Fertility
Pollen stainability of individuals attributed to putatively pure V. alba subsp. alba
(including alba and scotophylla morphotypes) was 93–100%, to those of V. ambigua
95–100%, V. collina 94–1 00%, V. hirta 92–100%, V. odorata 95–100%, and of V.
suavis 91–100%. In addition, the following hybrid populations showing low fertility
were found: V. alba × V. hirta with 0–17% fertility, V. alba × V. odorata with 0–20%,
and V. hirta × V. odorata with 10–60%.
Individuals displaying low pollen stainability (less than 70% fertility, altogether
84 individuals, 10 populations) were considered as primary (F
1
) hybrids and they
were excluded from the morphometric analyses.
Table 1 (continued)
Character Character explanation
Corolla (petals)
CO
a
Corolla odour 0 scentless; 1 scented
CEN
a
Number of emarginated petals per corolla –
CPL
a
Posterior petal length (mm)
CPW
a
Posterior petal width (mm)
CPL1 Posterior petal length from the base to
the first maximum width
g
(mm)
CLL
a
Lateral petal length (mm)
CLW
a
Lateral petal width (mm)
CAL
a
Anterior petal length (including spur)
(=length of flower)
(mm)
CP
a
Corolla colour (excluding spur) 0 white; 1 violet
CSL
a
Spur length (mm)
CSS
a
Spur shape 0 straight or curved up at full length;
1 hook-shaped at apex
CSP
a
Spur colour 0 white; 1 pale blue or (bluish-)violet;
2 deep violet
CPSP
a
Pigmentation of corolla in contrast to
pigmentation of spur
0 spur paler than corolla; 1 spur the
same colour as corolla; 2 spur darker
than corolla
CPL1/CPL
a
Posterior petal length from the base to
the first maximum width/posterior
petal length
–
Capsule
CaVN Number of veins on one valve of capsule –
a
Characters marked with a superscripted “a” were used in multivariate analyses
b
Stolon length was measured as distance between two (rooting) leaf rosettes
c
Measured on young summer leaves
d
Measured from basal lobes to apex
e
Measured from basal lobes to maximal width
f
Outer stipules of main rosette-leaves
g
See Fig. 2
Morphological characters of Viola subsect. Viola (Violaceae) 91

Cytological Analyses
Chromosome numbers and DNA ploidy levels of 57 populations studied are listed in
Appendix. Viola alba, V. collina, V. hirta, V. odorata and their hybrids (V. alba × V.
hirta, V. alba × V. odorata, V. hirta × V. odorata) were found to be tetraploid with
2n∼ 4x∼ 20; V. ambigua and V. suavis s.l. were octoploid with 2n∼ 8x∼ 40. These
counts agree with previously published chromosome numbers (for further references
see Mereďa et al. 2006).
The hexaploid chrom osome number (2n∼ 6x∼ 30) was found in five plants (two
populations) growing in contact zones of tetraploid and octoploid species V. odorata
and V. ambigua or V. odorata and V. suavis s.l. Four of them, determined on the basis
of their morpholog y and vicinity of pure populations, represented the parental
combination V. odorata × V. suavis
s.l., and one represented V. ambigua × V.
odorata.
Morphometric Analyses
Based on the results of pollen fertility and cytological analyses, from the total of 465
individuals (57 populations) only 376 individuals (44 populations) were considered
as non-hybrid and were used for the mul tivariate morphomet ric analyses.
Fig. 2 Morphological characters scored and measured for morphometric analyses. For character
explanations see Table 1. Drawings by P. MereďaJr.
92 I. Hodálová et al.

Spearman Correlation Coefficients
Spearman correlation coefficients of any pair of characters in the whole dataset, used
in morphometric analyses, did not exceed the value of 0.9. Thus, all characters were
used in subsequent morphometric analyses.
Cluster Analysis
Cluster analysis of the complete dataset indicated that all 44 non-hybrid populations
can be divided into six main clusters (Fig. 3). The first cluster includes two
populations, which can be classified as V. alba subsp. alba (alba morphotype ). The
second cluster contains five populations that can be classified as V. alba subsp. alba
(scotophylla morphotype). The third cluster includes four population samples
traditionally recognized as V. collina. The fourth cluster is composed of two groups:
the first group (A) is formed of eight populations generally corresponding to V. hirta,
and the second group (B) includes four populations classifiable as V. ambigua. The
fifth cluster contains seven populations of V. odorata. The sixth cluster is formed by
14 populations that correspond to two colour morphotypes of V. suavis s.l. occurring
in the Carpathians: populations with blue to (bluish-) violet corollas (further referred
Fig. 3 Cluster analysis (UPGMA) based on 37 morphological characters and all 44 studied populations
from the West Carpathians and its adjacent areas: ALB – Viola alba subsp. alba (alba morphotype), SCO – V.
alba subsp. alba (scotophylla morphotype), AMB – V. ambigua, COL – V. collina, HIR – V. hirta, ODO – V.
odorata, SUB – V. suavis s.str ., SUW – white-flowered morphotype of V. suavis; abbreviations of taxa names
are followed by population numbers (see Appendix) and ploidy levels
Morphological characters of Viola subsect. Viola (Violaceae) 93

to as V. suavis s.str.), and populations with white corollas (further referred to as
white-flowered morphotype of V. suavis).
Principal Compone nt Analyses
The studied populations were divi ded into two gro ups ac cording t o their
chromosome numbers (tetraploids and octoploids), and these two groups were
separately subjected to PCA. To get a better resolution within the group of
tetraploids, another PCA based on a subset of tetraploid populations was performed.
Thus, three different data subsets (matrices) were assembled and analyzed separately
(in all cases only two-dimensional ordination graphs are shown, because the third
axis did not contribute to further differentiation):
Matrix A (PCA 1) – all 26 tetraploid populations (clusters 1, 2, 3, 4A, 5 in Fig. 3)
Matrix B (PCA 2) – 18 tetraploid populations of V. alba, V. collina and V.
odorata (clusters 1, 2, 3, 5 in Fig. 3)
Matrix C (PCA 3) – all 18 octoploid populations (clusters 4B, 6 in Fig. 3).
Fig. 4 Principal component analysis (PCA 1) based on 37 morphological characters and 26 tetraploid
populations: Viola alba subsp. alba, including alba morphotype (asterisk) and scotophylla morphotype
(triangle), V. collina (star), V. hirta (plus sign) and V. odorata (circle). The first two axes explain 30.35%
and 21.59% of variation among OTUs
94 I. Hodálová et al.

Principal Compone nt Analyses of Tetraploids
PCA 1, based on matrix A, resulted in two distinct groups (Fig. 4): (1) on the left
side of the ordination graph there is a group corresponding to populations
traditionally understood as V. hirta, (2) on the right side of the diagram populations
corresponding to V. alba subsp. alba (including alba and scotophylla morphotypes),
V. collina and V. odorata are found intermingled. Characters most correlated with the
first axis are (with the values of eigenvectors in brackets): CO (0.267), LAA (0.262),
SBGN (0.261), StW (0.260), LL/LW (−0.256), LHL (− 0.251), LL1/LL (0.249) and
LSL/LL (0.242), while characters CAL (0.328), CLL (0.320), CPL (0.315) and SFI
(−0.300) are most strongly correlated with the second axis. In an effort to portray
differences among V. alba subsp. alba (including alba and scotophylla morpho-
types), V. collina and V. odorata, a separate PCA (PCA 2; based on matrix B) was
performed. PCA 2 (Fig. 5) showed three clear groups corresponding to the species V.
alba subsp. alba (including alba and scotophylla morphotypes), V. collina and V.
odorata. On the contrary to cluster analysis (cf. Fig. 3) the scatter plot of PCA did
not show any tendency towards a further separation of V. alba subsp. alba into alba
Fig. 5 Principal component analysis (PCA 2) based on 37 morphological characters and 18 tetraploid
populations: Viola alba subsp. alba, including alba morphotype (asterisk) and scotophylla morphotype
(triangle), V. collina (star) and V. odorata (circle). The first two axes explain 34.89% and 18.47% of
variation among OTUs
Morphological characters of Viola subsect. Viola (Violaceae) 95

and scotophylla morp hotypes; both morphotypes were grouped together without any
discontinuity. The clusters are delimited by SFI (−0.258), CO (0.236), CPL (0.234),
CLL (0.234), LHL (−0.230), CAL (0.227) and LAA (0.223) (with the highest
eigenvector values for the first axis) and by CPSP (0.318), CPW (0.306), CLW
(0.300), CP (−0.293), CPL1/CPL (−0.275) and SL/SW (−0.253) (with the highest
values for the second axis).
Principal Compone nt Analysis of Octoploids
The result of PCA 3, based on matrix C, is shown in Fig. 6. Two groups separated
along the first principal component can be seen: a group on the left side of the
diagram corresponding to plants traditionally understood as V. ambigua, and a group
on the right side corresponding to V. suavis s.l. The scatter plot showed some
tendency towards a further, but incomplete separation of V. suavis s.l. into two
subgroups along the second principal component: (1) plants with blue to (bluish-)
violet corollas (V. suavis s.str.) are located in the upper right corner of the diagram
and (2) plants with white corollas (white-flowered morphotype of V. suavis) in the
lower right corner. The first axis, contributing most to the separation of the groups,
was highly correlated (eigenvecto r values) with the characters LAA (0.252), LSA
Fig. 6 Principal component analysis (PCA 3) based on 37 morphological characters and 18 octoploid
populations: Viola ambigua (square), V. suavis s.str. (spade) and the white-flowered morphotype of V.
suavis (trefoil). The first two axes explain 36.77% and 14.45% of variation among OTUs
96 I. Hodálová et al.

(0.251), LSL/LL (0.246), LL/LW (−0.246), CAL (0.230), SFN (0.224), PL1/PL
(−0.208) and StAL (0.201); the second axis, expressing a slight shift of the V. suavis
s.l. specimens, was highly correlated with SL/SW (−0.328), CP (0.319), LP (0.318),
SGN/SFN (0.245) and CSP (0.242).
Canonical Discriminant Analyses
Several CDA were run to reveal the differentiation among the groups resolved by
cluster analysis and PCA. On the contrary to the latter analyses, CDA were based on
individual plants as OTUs. CDA 1 was performed on the whole dataset, with six
groups (taxa) pre-defined, as they were delimited by the cluster analysis and
principal component analyses (PCA 1–3): V. alba subsp. alba (including alba and
scotophylla morphotypes), V. ambigu a , V. collina, V. hirta , V. odorata and V. suavis
s.l. (includin g V. suavis s.str. and white-flowered morphotype of V. suavis). The
scatter plot affirms the phenetic distinctness of the taxa studied, although they partly
overlap morphologically. The ordination diagram of CDA 1 (Fig. 7) showed
relatively distinct clusters of V. alba subsp. alba (including alba and scotophylla
morphotypes), V. ambigua, V. hirta and V. odorata individuals, whi le there was an
Fig. 7 Results of CDA 1 including 36 morphological characters and 376 individuals as OTUs. The six
groups defined on the basis of UPGMA and PCA represent: Viola alba subsp. alba (triangle), V. ambigua
(square), V. collina (star), V. hirta (plus), V. odorata (circle) and V. suavis s.l. (spade). The first two axes
explain 12.6% and 8.66% of variation among OTUs
Morphological characters of Viola subsect. Viola (Violaceae) 97

overlap between the clusters of V. collina and V. suavis s.l. (includin g V. suavis s.str.
and white-flowered morphotype of V. suavis). Correlation values of the characters
(total canonical structure) with the first and second axis are presented in Table 2.
To reveal differences between V. collina and V. suavis s.l., a separate CDA
(CDA 2) with two groups corresponding to the two taxa was run. The histogram of
CDA 2 showed clear separation between the species ( Fig. 8). They differ in a
Table 2 Total canonical structure expressing correlation of characters with canonical axes; those
exceeding the arbitary level of 0.5 are printed in italics
Character CDA 1 CDA 2
CAN1 CAN2 CAN1
StAL −0.393 0.123 0.317
StUL −0.234 0.179 0.256
StW −0.544 0.247 0.490
StP −0.119 0.391 0.144
LHL 0.820 0.190 −0.534
LSA 0.356 −0.504 0.207
LCN 0.007 −0.399 0.002
LAA −0.772 0.186 0.508
LP 0.126 0.413 −0.403
LL/LW 0.670 −0.414 −0.347
LW/LSW −0.347 −0.084 0.228
LL1/LL −0.535 0.166 0.220
LSL/LL −0.406 0.568 0.014
SFN −0.454 −0.125 −0.209
SFL −0.251 0.211 0.322
SFI −0.043 0.587 −0.125
SYGN 0.338 −0.079 0.551
SBGN −0.340 0.267 −0.566
SL/SW 0.264 0.107 0.342
SGN/SFN 0.021 0.013 −0.337
PL1/PL −0.202 −0.063 0.348
KAL −0.361 0.054 0.386
KAW 0.053 −0.184 −0.390
KP 0.032 −
0.180 0.640
CO −0.671 −0.062 0.073
CEN 0.563 0.057 0.771
CPL 0.005 −0.377 −0.809
CPW 0.014 0.127 0.606
CLL −0.031 −0.379 0.771
CLW −0.066 0.127 0.593
CAL −0.123 −0.318 0.711
CSL −0.288 −0.023 0.465
CSS 0.481 −0.304 −0.329
CSP −0.513 0.104 0.814
CPSP −0.110 0.573 0.482
CPL1/CPL 0.171 −0.213 −0.043
The values were retrieved from canonical discriminant analyses performed on individual samples as
OTUs: CDA 1 with six groups pre-defined, corresponding to Viola alba subsp. alba (including alba and
scotophylla morphotypes), V. ambigua, V. collina, V. hirta, V. odorata, and V. suavis s.l. (see Fig. 7); CDA
2 with two groups pre-defined, representing V. collina and V
. suavis s.l. (see Fig. 8).
98 I. Hodálová et al.

number of characters; correlation values with the discriminant axis are presented in
Table 2.
A series of six CDA (diagrams not shown) were further performed to identify
morphological characters most suitable for the recognition of a particular species
from the other species studied. They were computed using datasets with specimens
divided into two groups: one including specimens of a particular species and the
other including specimens of all the remaining species studied (e.g. CDA 3 was
computed using individuals of V. alba subsp. alba as group 1 and individuals of V.
ambigua, V. collina, V. hirta, V. odorata and V. suavis s.l. as group 2, etc.). The
characters pigmentation of corolla in contrast to pigmentation of spur (CPSP),
lamina dentations (LCN) and length of petals (CLL, CAL, CPL) were identified to
be the best characters for the definition of V. alba subsp. alba from the remaining
species studied; shape of lamina (LS A, LL/LW, LSL/LL) for the recognition of V.
ambigua; pigmentation of corolla in contrast to pigmentat ion of spur (CPSP) and
size of flower (CPL, CPW, CLL, CLW, CAL) for the recognition of V. collina;
maximum length of petiole hairs (LHL), odour of flower (CO), shape of lamina
(LAA, LL/LW), and shape of spur (CSS) for the recognition of V. hirta; lamina apex
angle (LAA) and indument of stipule (SBGN, SFI) for the recognition of V. odorata;
and fimbriation and indument of stipule (SFL, SFI), insertion of bracteoles on
peduncle (PL1/PL), and length of anterior sepals (KAL) for the recognition of V.
suavis s.l.
Fig. 8 Results of CDA 2 in-
cluding 36 morphological char-
acters and 165 individuals as
OTUs of Viola collina (white)
and Viola suavis s.l. (black)
Morphological characters of Viola subsect. Viola (Violaceae) 99

Table 3 Mean (x) ±standard deviation (s.d.), minimum (min.), maximum (max.) and percentiles (10%, 90%) of quantitative characters used in morphometric analyses in Viola
alba subsp. alba (including alba and scotophylla morphotypes), V. ambigua, V. collina, V. hirta, V. odorata and V. suavis s.l. from the West Carpathians and adjacent areas
Character V. alba subsp. alba V. ambigua V. collina V. hirta V. odorata V. suavis s.l.
n=51 n=27 n=38 n=68 n=65 n=127
(n
L
=152; n
F
=78) (n
L
=87; n
F
=34) (n
L
=112; n
F
=54) (n
L
=199; n
F
=125) (n
L
=178; n
F
=107) (n
L
=376; n
F
=206)
Min.
10%
x ± s.d. 90%
max.
Min.
10%
x ± s.d. 90%
max.
Min.
10%
x ± s.d. 90%
max.
Min.
10%
x ± s.d. 90%
max.
Min.
10%
x ± s.d. 90%
max.
Min.
10%
x ± s.d. 90%
max.
StAL (cm) 3 8.26 11 stolons absent 1 1.7 2.84 stolons absent 2 11.42 19.37 1 6.05 10.12
6.1 2.72 16.5 1 1.21 3.5 5.88 5.49 23 1.77 4.14 18.5
StUL (cm) 5 10.72 17.3 stolons absent Undeground
stolons absent
stolons absent 3 12.77 24.36 2 9.64 15.55
6.35 4.41 19 4.1 7.65 28.5 4.17 5.57 28
StW (mm) 0.7 1.19 1.5 stolons absent 1.6 1.95 2.24 stolons absent 0.9 1.47 1.9 1 1.91 2.7
0.9 0.28 1.9 1.66 0.31 2.3 1 0.33 2.5 1.42 0.51 3.7
LHL (mm) 0.5 1 1.4 0.15 0.3 0.4 0.2 0.64 1.1 0.7 1.21 1.5 0.1 0.28 0.42 0.1 0.39 0.66
0.7 0.3 1.8 0.2 0.1 0.5 0.4 0.24 1.2 0.9 0.24 1.7 0.15 0.11 0.5 0.13 0.22 1.1
LSA (°) −38 57.94 100 80 145.33 180 10 59.23 86.8 30 99.31 142 −35 53.62 90 −20 70.97 105.25
18.2 32.40 125 120 21.98 190 30 22.94 115 65 31.98 180 9.4 31.45 143 28.95 31.39 155
LCN (number) 20 30.82 37 22 34.66 45 28 38.93 46 31 41.87 49.2 22 39.18 44 20 38.16 47
25 5.27 50 28 6.24 52 33 5.54 53 36 5.62 58 33 4.74 50 32 6.17 60
LAA (°) 60 93.24 120 50 78.98 95 45 89.32 120 40 71.15 90 80 120.64 140 65 106.34 130
70 19.66 170 60 16.34 135 65 19.52 135 55 13.82 135 96.6 18.15 180 87.5 17.05 180
LL/LW 0.71 1.13 1.37 0.91 1.47 1.72 0.85 1.21 1.45 0.82 1.43 1.72 0.6 1.01 1.15 0.62 1.11 1.29
0.91 0.18 1.58 1.16 0.22 1.91 0.96 0.19 1.73 1.16 0.25 2.21 0.81 0.14 1.44 0.93 0.16 1.71
LW/LSW 1.36 1.9 2.3 1.43 1.76 2 1.24 2.07 2.4 1.27 2 2.33 1.33 2.35 3 1.17 2.26 2.95
1.58 0.37 3.5 1.57 0.19 2.5 1.78 0.27 3.3 1.67 0.31 3.67 1.79 0.52 4.5 1.77 0.49 4.29
LL1/LL 0.18 0.31 0.38 0.13 0.28 0.36 0.17 0.32 0.39 0.1 0.27 0.33 0.05 0.36 0.44 0.24 0.36 0.43
0.25 0.05 0.46 0.2 0.11 0.8 0.26 0.05 0.43 0.2 0.06 0.52 0.28 0.07 0.59 0.28 0.07 0.58
LSL/LL 0.06 0.19 0.26 0 0.04 0.07 0.07 0.14 0.19 0 0.08 0.12 0.05 0.18 0.25 0.04 0.14 0.23
0.11 0.06 0.32 0.003 0.03 0.11 0.09 0.04 0.23 0.04 0.03 0.21 0.11 0.06 0.41 0.09 0.05 0.34
SFN (number) 3 15.35 22 8 15.87 22 13 23.99 31 3 19.73 29 10 26.1 33.6 7 22.26 29
9 5.08 28 10 4.56 26 16.3 5.78 41 10 6.82 35 18.4 6.13 43 14.5 5.81 40
100 I. Hodálová et al.

SFL (mm) 0.2 0.8 1.1 0.2 0.84 1.53 0.6 1.34 1.8 0.2 0.67 1 0.2 0.59 0.9 0.2 1.64 2.2
0.4 0.3 2.3 0.34 0.49 2.3 0.9 0.35 2.2 0.34 0.28 1.5 0.3 0.25 1.6 1.1 0.46 3.7
SL/SW 3.05 4.88 6.28 2 5.38 7.88 2.07 3.51 4.13 1.88 3.53 5 1.6 2.72 3.29 1.34 4.18 5.51
3.75 1.12 10 3.14 1.77 9.54 2.81 0.61 5.33 2.41 1.11 7.5 2.09 0.55 5.07 3.12 0.99 6.88
SGN/SFN 0 0.56 0.88 0 0.77 1 0 0.33 0.63 0 0.64 1 0.21 0.83 1 0 0.59 0.91
0.25 0.24 1 0.27 0.31 1 0.07 0.21 0.83 0.29 0.25 1 0.48 0.21 1 0.18 0.28 1
PL1/PL 0.2 0.42 0.5 0.25 0.42 0.52 0.3 0.46 0.55 0.1 0.27 0.36 0.15 0.45 0.61 0.05 0.22 0.31
0.28 0.09 0.59 0.3 0.09 0.56 0.37 0.08 0.62 0.18 0.08 0.49 0.33 0.11 0.71 0.11 0.05 0.51
KAL (mm) 3.7 6.01 7.07 4.4 6.05 7.15 4.3 6.17 7.5 4.5 5.79 6.6 5 6.47 7.4 4.8 7 8.2
4.9 0.86 7.8 5.05 0.91 8.8 5.06 0.91 8.2 4.95 0.68 8 5.4 0.76 8.4 5.81 0.94 10
KAW (mm) 1.6 2.12 2.57 1.5 2.27 2.75 1.3 2.07 2.68 1.7 2.52 3 1.5 2.31 2.8 1.5 2.49 3.15
1.8 0.3 2.8 1.8 0.43 3.2 1.52 0.46 3 2.1 0.35 3.4 1.8 0.4 3.5 2 0.45 4
CEN (number) 0 3.37 5 1 3.31 5 0 2.02 5 0 4.42 5 0 1.2 3 1 2.29 4
1 1.45 5 2 1.2 5 0 1.64 5 3 0.91 5 0 1.01 5 0.5 1.31 5
CPL (mm) 7.2 11.1 13 9 12.62 14.88 7.3 9.92 11.68 10 13.76 16 10 12.89 15 9.5 13.44 15.2
8.5 1.61 13.7 11 1.57 15.8 8.54 1.18 12.5 11.08 1.81 18 11 1.53 17.2 11.5 1.45 17
CPW (mm) 4.2 7.15 9.1 4 5.72 6.74 2.7 4.66 6.28 4.4 6.42 7.5 4 6.86 9 4 6.33 7.5
5.3 1.41 10.5 4.86 1.02 9 3.42 1.03 6.9 5 0.91 9.2 5 1.46 11 5 1 9
CLL (mm) 6.7 10.97 12.7 10 12.8 14.76 7.2 10.16 12 10 13.63 15.8 9 12.8 15 9.8 13.58 15.5
8.2 1.73 14 11.12 1.27 15.3 8.7 1.25 12.5 11.14 1.67 17.8 11 1.3 15 11.75 1.34 17
CLW (mm) 4.5 6.89 8.16 3 5.9 7.2 3.5 5.02 6.92 4 6.31 7.53 4 6.7 8 4.5 6.72 8
5.44 1.14 10 4 1.18 8.3 3.8 1.11 7.5 5 0.88 8 5.4 1.09 9 5.25 1.05 11
CAL (mm) 8.5 14.7 16.9 12.5 15.78 17.58 9.7 13.75 16.24 13.7 17.06 19 13 17.02 19 13.3 17.34 19.1
11.4 2.21 19.1 14 1.61 20 11.64 1.93 18 15 1.69 21.4 15 1.68 22 15.5 1.54 21
CSL (mm) 2 4.2 5.06 3 4.13 5.12 2.7 3.89 4.78 2.7 4.17 5 3.3 4.7 5.62 3 4.77 5.85
3 0.76 5.7 3.3 0.68 5.3 3.3 0.6 5.8 3.3 0.71 6.7 3.8 0.7 6.6 3.75 0.8 6.9
CPL1/CPL 0.35 0.51 0.59 0.33 0.57 0.67 0.37 0.56 0.66 0.44 0.61 0.69 0.4 0.53 0.62 0.37 0.59 0.68
0.44 0.06 0.66 0.46 0.09 0.75 0.46 0.07 0.69 0.53 0.06 0.75 0.45 0.07 0.73 0.5 0.08 0.77
CaVN (number) 3–53–53–53–53–53–53–53–53–53–53–53–53–53–53–53–53–53–5
Character CaVN was not used in multivariate analyses because it was constant for all studied individuals
For character explanations see Table 1
n Number of individuals studied, n
L
number of measured leaves, n
F
number of measured flowers
Morphological characters of Viola subsect. Viola (Violaceae) 101

Table 4 Character state frequencies of qualitative characters used in morphometric analyses in Viola alba subsp. alba (both alba and scotophylla morphotypes), V. ambigua, V.
collina, V. hirta, V. odorata, V. suavis s.l. from the West Carpathians and adjacent areas
Character V. alba subsp. alba V. ambigua V. collina V. hirta V. odorata V. suavis s.l.
n=51 n=27 n=38 n=68 n=65 n=127
(n
L
=152; n
F
=78) (n
L
=87; n
F
=34) (n
L
=112; n
F
=54) (n
L
=199; n
F
=125) (n
L
=178; n
F
=107) (n
L
=376; n
F
=206)
0 (%) 1 (%) 2 (%) 0 (%) 1 (%) 2 (%) 0 (%) 1 (%) 2 (%) 0 (%) 1 (%) 2 (%) 0 (%) 1 (%) 2 (%) 0 (%) 1 (%) 2 (%)
StN 37.25 62.75 (27.45+
23.53 +
11.77)
– stolons absent – 89.47 10.53 (10.53+
0+0)
– stolons absent – 7.69 92.31
(55.39 +
7.69 +
29.23)
– 33.08 66.92
(37.27+
9.31+
20.34)
–
StP 9.38
a
90.62 – stolons absent – 50 50 – stolons absent – 65 35 – 75.64 24.36 –
LP 17.65
a
82.35 – 66.67 33.33 – 26.31 73.69 – 82.35 17.65 – 89.23 10.77 – 66.76 33.24 –
SFI 18.42 81.58 – 94.74 5.26 – 0 100 – 83.77 16.23 – 98.87 1.13 – 22.15 77.85 –
SYGN 23.68 76.32 – 2.3 97.7 – 42.98 57.02 – 1.54 98.46 – 51.63 48.37 – 5.38 94.62 –
SBGN 44.08 55.92 – 100 0 – 44.74 55.26 – 96.44 3.56 – 28.81 71.19 – 88.97 11.03 –
KP 17.65
a
82.35 – 0 100 – 0 100 – 10.29 89.71 – 0 100 – 41.23 58.77 –
CO 28.21 71.79 – 0 100 – 22.22 77.78 – 100 0 – 0 100 – 11.66 88.34 –
CSS 93.59 6.41 – 64.71 35.29 – 72.22 27.78 – 19.2 80.8 – 89.71 10.29 – 94.18 5.82 –
CP 100 0 – 0 100 – 0 100 – 0 100 – 0 100 – 50 50 –
CSP 17.65
a
82.35 0 0 55.88 44.12 5.56 94.44 0 0 100 0 0 0 100 1.46
b
66.98 31.56
CPSP 0 17.65
a
82.35 34.48 65.52 0 92.59 7.41 0 36.8 63.2 0 0 100 0 0 51.46 48.54
Character StN was not used in multivariate analyses because the same information is expressed by the characters StAL, StUL and StW (cf. Table 3). In stoloniferous taxa the
percentage of plants bearing: only aboveground stolons + together aboveground and underground stolons + only underground stolons is indicated in brackets (cf. character StN).
For character explanations see Table 1
n Number of individuals studied, n
L
number of measured leaves, n
F
number of measured flowers
a
Values belong solely to alba morphotype
b
Three flowers (=1.46%) of the white-flowered morphotype of V. suavis had a white spur
102 I. Hodálová et al.

Exploratory Data Analysis
Means, standard deviations, minima, maxima, 10 and 90 percentiles of quantitative
characters are presented in Table 3, and frequencies of qualitative characters in
Table 4. Although there is a more or less continuous variation across the whole
dataset in many characters, the combination of characters allows unambiguous
species identification.
Discussion
Our morphological analyses support the existence of six well-delimited and morpholog-
ically distinct taxa within Viola subsect. Viola in the West Carpathians: V. alba subsp.
alba (including alba and scotophylla morphotypes), V. ambigua, V. collina, V. hirta, V.
odorata and V. suavis s.l. Each of the above-mentioned taxa can be clearly distinguished
by a unique set of morphological features (see identification key below, and Fig. 9).
In most identification keys to the West-Carpathian or Central-European violets,
the presence or absence of stolons is considered a crucial character (e.g. Valentine et
al. 1968; Dostál 1989; Kirschner and Skalický 1990; Suda 2002; Fischer et al.
2005), and the speci es of this subsection are described either as non-stoloniferou s (V.
ambigua, V. collina and V. hirta; series Eflagellatae) or as stol oniferous (V. alba, V.
odorata and V. suavis; series Viola). However, V. collina can also form stolons up to
3.5 cm long, and in contrary, stolons can sometimes be lacking in V. alba, V.
odorata
and V. suavis, as already pointed out e.g. by Gams (1925), Marcussen and Nordal
Fig. 9 Shape of leaf laminas, hairs on leaf petiole and outer stipules of main rosette-leaves in Viola sect.
Viola subsect. Viola: a V. alba subsp. alba, b V. ambigua, c V. collina, d V. hirta, e V. odorata, f V. suavis s.
l. Drawings by P. MereďaJr.
Morphological characters of Viola subsect. Viola (Violaceae) 103

(1998), and Marcussen (2003). In the present study, four plants (10%) of V. collina
were observed having short stolons, whereas stolons were lacking in 8% of V.
odorata specimens studied, in 33% of V. suavis s.l., and in 37% of V. alba subsp.
alba specimens (cf. character StN, Table 4).
In Central Europe the morphological separation of taxa within subsection Viola
has also commonly relied on the presence or absence of glands on stipules. Many
previous author s stated that stipules of V. alba (cf. Gams 1925; Dostál 1989;
Kirschner and Skalický 1990); V. collina, V. hirta (e.g. Kirschner and Skalický 1990)
and V. suavis (e.g. Dostál 1989) are eglandular, whereas stipules of V. ambigua and
V. odorata are glandular. Our study demonstrated a large variation in this character
and gave reason to doubt its importance in the infrasubsectional classification. All
members of subsection Viola studied by us possessed sparsely or densely glandular
stipules (cf. characters SFN and SGN/SFN, Table 3), with a proportion of glandular
fimbriae to the total number of fimbriae in V. alba subsp. alba (0–)25–88(–100)%, in
V. ambigua (0–)27–100%, in V. collina (0–)7–63(–
83)%, in V. hirta (0–)29–100%, V.
odorata (21–)48–100% and in V. suavis s.l. (0–)18–91(–100)%.
Although the insertion of bracteoles on peduncles is a relevant diagnostic
character, it may vary much more than given in most identification keys and floras.
According to many authors bracteoles in V. alba, V. collina, V. odorata (e.g. Gams
1925; Valentine et al. 1968; Dostál 1989; Kirschner and Skalický 1990; Marcussen
and Nordal 1998; Suda 2002; Fischer et al. 2005), and in V. ambigua (e.g. Gams
1925; Dostál 1989) should be inserted at or above the middle of the peduncl e. In
fact, the insertion place varied considerably in the plants studied, and bracteoles of
these species were relatively often inserted below the middle of the peduncle (cf.
character PL1/PL, Table 3).
Viola alba subsp. alba The concept of two subspecies in V. alba = subsp. alba and
subsp. scotophylla (Jord.) Gremli – has been traditionally accepted in Central Europe
(e.g. Dostál 1989). Division of the species into these races was based mainly on
pigmentation of plants: subsp. scotophylla possesses strongly pigmented stolons,
leaves, peduncles, sepals and capsules and its spur is purplish; all organs of subsp.
alba are without anthocyan pigmentation and its spur is white with a yellowish-
green apex.
The re sults of our morpho logical ana lyses (cluster a nalysis and princ ipal
component analyses, PCA 1–2) are quite discordant for V. alba. The specimens
identified as alba and scotophylla morphotypes formed two distinct clusters in the
CA, however, they did not show any tendency towards a separation in the PCA 1–2.
The most important characters influencing the position of alba and scotophylla
morphotypes in the CA are connected with the pigmentation of stolons, lamina,
sepals, corolla and spur (StP, LP, KP, CP, CSP, CPSP). Nevertheless, we have not
observed any other significant differences between morphological characters of these
two morphotypes, and their morphological and ecological ranges broadly overlap.
This result is fully in agreement with the morphometric and allozymic studies by
Marcussen and Borgen (2000) and Marcussen (2003). They demonstrated that the
above-mentioned characters are taxonomically rather unimportant, and the popula-
tions of “subsp. scotophylla” should be included into the nominate subspecies. There
104 I. Hodálová et al.

is a note by Marcu ssen (2003) concerning differences in colour: “the genetic basis
for such polymorphism is probably simple and may be explained by bi-allelic
variation in two loci, one coding for anthocyan production, and another for its
expression in the corolla (thus, giving rise to three possible morphotypes: the alba
morphotype, and the white- and lilac-flowered scotophylla morphotype)”. In the
West Carpathians mixed populations of both morphotypes occur and morphological
differences between them are likely to break down. Thus, following the results of
Marcussen (2003) we included “subsp. scotophylla” in the synonymy of subsp. alba.
In some parts of the distributio n range of V. alba subsp. alba,ascotophylla
morphotype with completely violet corollas (classified by Gams (1925)asV. alba
var. scotophylla f. violacea Wiesb.) is rather common. Mainly in southern parts of
the distribution range of V. alba subsp. alba, violet-flow ered individuals may even
prevail in compa rison to the white-flowered individuals (cf. Marcussen 2003;
Hodálová and Mereďa Jr., unpublished). The violet-flowered morphotype occurs in
the West Carpathians as well (Hodálová and Mereďa Jr., unpublished), but we did
not include specimens with violet flowers in the present analyses because of their
rarity.
According to numero us prev ious studies (e.g. Gams 1925; Kirschner and
Skalický 1990; Suda 2002; Marcussen et al. 2005), V. alba subsp. alba should
possess only aboveground stolons. However, our study showed that V. alba subsp.
alba develops both aboveground and underground stolons (in proportion of ca. 2:1;
cf. character StN, Table 4). So we consider this character unsuitable for its
delimitation from other (mostly stoloniferous) members of subsection Viola.
The results of our morphometric study are not in full agreem ent with those of
Marcussen (2003) and Marcussen et al. (2005) regarding limits of some
morphological traits within V. alba subsp. alba
. Discrepancies concern mainly
lamina dentations and maximum petiole hair length: both characters are shown in
our study as most important for the separation of V. alba within subsection Viola.
Marcussen (2003) also uses these characters for the delimitation of subspecies within
V. alba, i.e., subsp. alba, subsp. cretica and subsp. dehnhardtii. However, Marcussen
(2003) reported only interquartile ranges as follows: maximum hair length on
laminas 0.5–1 mm in subsp. alba,1–1.3 mm in subsp. cretica, and 0.4–0.6 mm in
subsp. dehnhardtii; and number of crenulae along one leaf margin 19–26 in subsp.
alba,13–17 in subsp. cretica, and 14–18 in subsp. dehnhardtii. In our material,
maximum hair length on petiole in V. alba subsp. alba was (0.5–)0.7–1.4(–1.8) mm
and number of crenulae along one lamina margin was (10–)13–19(–25) (cf.
characters LHL and LCN, Table 3).
Viola ambigua Dostál (1989), Kirschner and Skalický (1990), and Suda (2002)
considered the numbe r of veins on capsule valves as one of the most imp ortant
diagnostic characters for the delimitation of V. ambigua from the other species of
subsection Viola: according to them, V. ambigua should have three veins on each
valve, whereas the other species of subsection Viola should have only one vein.
However, in fact, all individuals of this subsection from the West Carpathians that
we had the opportunity to study had 3–5-veined capsule valves (cf. character CaVN,
Table 3
).
Morphological characters of Viola subsect. Viola (Violaceae) 105

Viola collina and V. hirta The morphological separation of V. collina and V. hirta is
often based on the number of emarginated petals per corolla. Many authors (e.g.
Gams 1925 ; Dostál 1989; Kirschner and Skalický 1990; Suda 2002) reported that V.
collina tends to have only one emarginated petal (the anterior one), wher eas in V.
hirta all five petals are usually emarginated. However, much greater variation in this
character has been observed on our material and V. collina partly shared the number
of emarginated petal with V. hirta. Among the plants of V. collina studied, 11% of
the flowers possessed two emarginated petals, 15% three emarginated petals, 7%
four emarginated petals, and 13% five emarginated petals. In contrary, 39% of
flowers of V. hirta possess four or less emarginated petals (cf. character CEN,
Table 3). Thus, it is clear that the numbers of emarginated petals per corolla partly
overlap, and the species cannot be unambiguously distinguished by this trait.
Viola odorata and V. suavis s.l. The morphological separation of V. odo rata and V.
suavis is, in addition to other characters, commonly based on the presence or
absence of aboveground and underground stolons: V. odorata should have only
aboveground stolons (e.g. Gams 1925; Dostál 1989), whereas V. suavis only
underground ones (e.g. Dostál 1989; Fischer et al. 2005). Based on results of this
study, it is apparent that there are only slight differences between those two in this
respect, and both species possess aboveground as well as underground stolons (in
proportion of 2:1; cf. character StN, Table 4).
Viola suavis s.l. It represents a taxonomically critical species, and its morphological
variation has been repeatedly discussed (e.g. Becker 1910; Gams 1925, Marcussen
and Nordal 1998 ). Recently, many European authors have expressed doubts as to the
further subdivision of V. suavis because of a lack of reliable characters, but they have
emphasized that further investigations of infraspeci fic variation of this taxon are
necessary (cf. Marcussen and Nordal 1998). According to Marcussen and Borgen
(2000) V. suavis is enzymatically highly variable but geographic patterns are not
seen. It was hypothesized that V. suavis originated recurrently from V. pyrenaica
(distributed from the Atlas and Pyrenees to the Caucasus) and other unidentified
tetraploids (Marcussen and Borgen 2000).
According to our study, V. suavis s.l. is morphologically the most variable species
of subsection Viola also in the West Carpathians. Like in V. alba subsp. a lba,we
found in V. suavis s.l. two colour morphotypes: (1) blue to (bluish-) violet-flowered
plants (V. suavis s.st r.) and (2) white- flowered plants (white-flower ed morphotype of
V. suavis). These morphotypes can be unambiguously distinguished by flower
colour: V. suavis s.str. has blue to (bluish-) violet petals (excluding spur) with a large
conspicuous white throat at base (reaching 1/3 – 1/2 of the length of lateral and
anterior petals) and a pale blue to deep (bluish-) violet spur; the white-flowered
morphotype of V. suavis has white petals with a pale to deep (bluish-) violet spur. In
addition to this character, stolons, laminas, and sepals are, on average, more
intensively anthocyanine-tinted in V. suavis s.str. than in the white-flowered
morphotype. On the basis of our observ ation, V. suavis s.str. and its white-flowered
morphotype differ (apart from characters connected with pigmentation of vegetative
and generative parts) also in other morphological characters. Generally, the white-
flowered morphotype of V. suavis has more narrow stipules, longer fimbriae on
106 I. Hodálová et al.

stipules, and bracteoles are inserted in lower parts of peduncles. The populations of
the white-flowered morphotype of V. suavis are commonly cultivated in the gardens
and parks, but in contrast to V. suavis s.str. they are not often estab lished in natural
and semi-natural habitats. Up to now the white-flowered morphotype of V. suavis has
never been report ed from Central Europe.
The taxonomic structure of V. suavis s.l. in the West Carpathians might be much
more complicated as mentioned above and the taxonomy of this species should be
studied within the whole species range. For this reason the taxonomic status and
origin of the white- flowered morphotype of V. suavis should be further studied,
based on more plant material.
Key to the Species of Viola Sect. Viola Subsect. Viola Occurring in the West
Carpathians
Notes: When identifying species of the subsection Viola it is necessary to remember
that many importan t diagnostic characters are changing after the period of flowering
in the course of late spring and summer (e.g. leaf shape, lamina consistence, and the
length of hairs on petioles) or they disappear totally (characters in stipules and
flowers). Therefore most plants can be reliably identifi ed only in spring when
chasmogamous flowers are present. The chasmogamous flowers appearing some-
times in summer or autumn (reflorescence) are usually smaller, poorly developed
and lack some attributes. For these reasons, the descriptions of leaves in the key refer
to vernal flowering plants, and the descriptions of flowers refer exclusively to open
(chasmogamous) vernal flowers.
Because in most species the first leaves appearing in early spring have glabrous or
subglabrous petioles, the indument should be observed on petioles of younger, more
hairy and still developing “summer” leaves. At that time, the longest hairs can be
sometimes found also on the petioles of over-wintering leaves, developed in summer
or autumn of the previous year.
The stipule shape described in the key refers to outer stipules of the main leaf
rosette. Towards the middle of the main rosette and in filial leaf rosettes stipules get
narrower and are less characteristically fimbriate or glandular and lack characters
typical of different species.
Flower length is measured from the apex of the spur to the apex of the anterior
petal (character CAL, Fig. 2).
The fragrance of flowers should be inspected when the weather is warm and
sunny and the flowers are in full bloom. On cold or rainy days flowers partly or
completely lose their odour.
In the key the main diagnostic characters (i.e., those sufficient for the safe
identification of a particular species) are separated by the symbol • from the supple-
mentary ones (which are less reliable and have no complementary statements con-
sistently given in the other branch of the key). Main characters are arranged according
to their importance, the order of supplementary characters corresponds to the usual
arrangement of morphological descriptions. In the key, corolla colour is described in
more detail than in the coded descriptions used in morphometric studies; so besides
violet colour also its tints that could not be reliably coded are given in the key.
The most common hybrids are described in notes.
Morphological characters of Viola subsect. Viola (Violaceae) 107

Character values given in the key represent 10 and 90 percentiles, those in
brackets minima and maxima.
1a Plants without stolons (but often with a thick many-headed rhizome), or with
aboveground or underground stolons up to 3.5 cm long……………………… 2
1b Plants at least with one aboveground or underground stolon more than 3.5 cm
long…………………………………………………………………………….8
2a Lamina truncate to shallowly cordate at base, with sinus angle (80–)120–180
(–190)°, lamina sinus depth reaching (0–)0.3–7(–11)% of lamina length…….3
2b Lamina shallowly to deeply cordate at base, with sinus angle [(−38)–]9–110
(–155)°, lamina sinus depth reaching (3–)8–26(–41)% of lamina length…… 4
3a Petioles with short hairs, the longest hairs (0.15–)0.2–0.4(–0.5) mm long.
Flowers strongly fragrant. • Lamina fleshy. Bracteoles inserted in (25–)30–52
(–56)% of peduncle length from the base. Petals (including spur) usually deep
(bluish-) violet, flowers with (1–)2–5 emarginated petals. Spur ± straight or
rarely hook-shaped, pointing upwards ……………………………… V. ambigua
Note: Plants with shallowly cordate laminas at base, the longest hairs on petioles
0.3–1 mm long, and pale (bluish-)violet corollas may be of hybrid origin between V.
ambigua and V. hirta. However, such plants can often represent only extreme
variants of V. ambigua or V. hirta and cannot usually be exactly identified without a
cytological analysis.
3b Petioles with long hairs, the longest hairs (0.7–)0.9–1 .5(–1.7) mm long.
Flowers non-fragrant. • Lamina not fleshy……………… V. hirta (see also 4a)
4a Flowers non-fragrant. Stipules elongated to narrowly triangular, 2–5(–7.5)×
longer than wide, short-fimbriate or entire, the longest fimbriae (0.2–)0.3–1
(–1.5) mm long, ± glabr ous or near apex of the stipule sparsely ciliate, most of
glandular fimbriae yellow or yellowish-brown. The longest hairs on petioles
(0.7–)0.9–1.5(–1.7) mm long. Lamina shallowly to deeply cordate, rarely
truncate at base, with sinus angle (30–)65–140(–180)° and sinus depth reaching
(0–)4–12(–21)% of lamina length. Spur hook-shaped at apex, pointing upwards,
rarely ± straight, pinkish-violet, rarely whitish. • Lamina (transversely)
rotundate-ovate to elongate-trian gular, (0.8–)1.2–1.7(–2.2)× longer than wide.
Bracteoles inserted in (10–)18–36(–49)% of peduncle length from the base.
Petals pale (bluish-) violet with a pink tint, rarely pink or white, flowers with
(0–)3–5 emarginated petals………………………………………………V. hirta
Note: Plants forming stout tufts, occasionally with short stolons; the longest hairs
on petioles 0.25–1 mm long, laminas ovate-lanceolate to rotundate-ovate, 0.7– 1.8×
longer than wide, stipules often ovate-lanceolate, flowers sometimes fragrant and
spur deep (bluish-) violet belong to hybrid V. hirta × V. odorata. It is the most
frequent hybrid among the species of subsection Viola, which occurs almost
anywhere where parental species grow together.
4b Flowers gently or strongly fragrant, rarely non-fragrant. Stipules either (1) ovate
to lanceolate, 1.5–3.5(–5)× longer than wide, short-fimbriate, the longest
fimbriae (0.2–)0.3–0.9(–1.6) mm long, ± glabrous, most of glandular fimbriae
blackish ( V. odorata
); or (2) stipules ovate-lanceolate to narrowly triangular, 3–
108 I. Hodálová et al.

6(–10)× longer than wide, long-fimbriate, the longest fimbriae (0.2–)0.4–2.5
(–3.7) mm long, stipules (and fimbriae) along the whole margin or at least near
apex ciliate, glandular fimbriae yellow, yellowish-brown or blackish. The
longest hairs on petioles (0.1–)0.15–1.4(–1.8) mm long. Lamina deeply cordate,
with sinus ang le [(−38)–]9–110(–155)°, and sinus depth reaching (3–)8–26
(–41)% of lamina length. Spur ± straight, evenly curved up at full length or
rarely hook-shaped at apex, either (1) pinkish-violet (V. collina) or (2) pale to
deep (bluish-)violet, or yellowish-green……………………………………… 5
5a Stipules short-fimbriate, the longest fimbriae (0.2–)0.3–0.9(–1.6) mm long,
(0.02–)0.06–0.2(–0.33)× as long as the width of the undivided part of stipule,
stipules and fimbriae ± glabrous. The longest hairs on petioles (0.1–)0.15–0.4
(–0.5) mm long………… …….…………………………V. odorata (see also 8b)
5b Stipules long-fimbriate, the longest fimbriae (0.2–)0.4–2.5(–3.7) mm long,
(0.06–)0.19–0.78(–1.31)× as long as the width of the undivided part of stipule;
stipules in the upper half (including fimbriae) usually sparsely ciliate. The
longest hairs on petioles 0.1–1.8 mm long…………………………………… 6
6a Stipules ovate-lanceolate to narrowly lanceolate, (1.9–)2.4–3.7(–4) mm wide,
long-fimbriate, the longest fimbriae (0.6–)0.9–
1.8(–2.2) mm long. Bracteoles
inserted in (30–)37–55(–62)% of peduncle length from the base. • The longest
hairs on petioles (0.2–)0.4–1.2 mm long. Stipules and fimbriae ciliate along the
whole margin or at least near apex. Lamina (transversely) rotundate-ovate to
ovate, (0.8–)1–1.5(–1.8)× longer than wide, deeply cordate at base. Flowers
small, (9.5–)11.5–16.5(–18) mm long. Petals (excluding spur) pale pinkish-
violet. Spur whitish- to pale pinkish-violet, ± paler than petals……… V. collina
Note: Plants with short- to long-fimbriate stipules and ±ovate lamina are hybrids
V. collina × V. hirta.
6b Stipules either (1) narrowly tria ngular, (0.9–)1.6–2.9(–4.5) mm wide, the
longest fimbriae (0.2–)0.4–1.1(–2.3) mm long and bracteoles inserted like in 6a
(V. alba subsp. alba); or (2) stipule shape like in 6a, (1.6–)2.6–4.7(–6.2) mm
wide, the longest fimbriae (0.2–)0.7–2.5(–3.7) mm long and bracteoles inserted
in (5–)9–38(–51)% of peduncle length from the base (V. suavis s.l.)………… 7
7a The longest hairs on petioles (0.5–)0.7–1.4(–1.8) mm long. Stipu les narrowly
triangular, (0.9–)1.6–2.9(
–4.5) mm wide; the longest fimbriae (0.2–)0.4–1.1
(–2.3) mm long. Bracteoles inserted in (20–)28–50(–59)% of peduncle length
from the base. • Stolons slender, (3–)6–17(–19) cm long and (0.7–)0.9–1.5
(–1.9) mm thick. Lamina apex obtusely acute to obtuse, with apex angle
(60–)70–120(–170)°. Glandular fimbriae on stipules yellow, yellowish-brown
or blackish. Flowers (8.5–)11.4–16.9(–19.1) mm long, posterior petals usually
markedly asymmetric, 1.34–1.8(–2)× longer than wide, petals (excluding spur)
white or yellowish-white, rarely pale to deep (blui sh-)violet or violet. Spur
white with yellowish-green colour at apex, or purplish… V. alba subsp. alba
(including alba and scotophylla morphotypes)
Note: Plants with short stolons, laminas elongate-triangular, stipules 2–4mm
wide, spur usually purplish, and petals white, usually with purpl ish spots (mainly on
Morphological characters of Viola subsect. Viola (Violaceae) 109

the outside of upper petals) and/or violet venati on near the base of the anterior petal
belong to hybrid V. alba × V. hirta.
Note: Plants with numerous and very long stolons, lamina ±rounded or triangular-
ovate, petiole hairs 0.4–1 mm long, stipules ovate to narrowly lanceolate (2–4×
longer than wide), 2.5–5 mm wide, petals inside paler (pinkish) than outside
(purplish) with violet venation near the base of the anterior petal and spur pinkish-
violet belong to hybrid V. alba × V. odorata.
7b Longest hairs on petioles 0.1–0.7(–1.1) mm long. Stipules ovate to narrowly
lanceolate, (1.6 – )2.6–4.7(–6.2) mm wide; the longest fimbriae (0.2–)1.1 –2.5
(–3.7) mm long. Bracteoles inser ted in (5–)11–31(–51)% of peduncle length
from the base. • Stolons stout, (1–)2–19(–28) cm long and (1–)1.4–2.7(–3.7) mm
thick. Lamina apex obtusely acute to obtuse, with apex angle (65–)85–130
(–180)°. Glandular fimbriae on stipules yellow or yellowish-brown. Calycine
appendages appressed to the peduncle. Flowers (13–)15.5–19(–21) mm long,
petals (excluding spur) either blue to (bluish-)violet, with large conspicuous
white throat (reaching to ±1/3 of petal length from the base) or entirely white.
Spur pale blue to deep (bluish-)violet……………………………… V. suavis s.l.
Note: Plant s with short-fimbriate sti pules, bracteoles inserted in 1/3 to 1/2 of
peduncle and calycine appendages slightly patulous from peduncle represent hybrids
V. odorata × V. suavis s.str. This hybrid can be very difficult to recognize, especially
from some individuals of V. suavis s.str. representing extreme variation, and
cytological analysis is necessary for their safe identification.
8a Stipules long-fimbriate, the longest fimbriae (0.2–)0.4–2.3(–3.7) mm long,
(0.06–)0.19
–0.78(–1.31)× as long as the width of the undivided part of stipule,
stipules and fimbriae ciliate along the whole margin or least near apex. The
longest hairs on petioles 0.1–1.8 mm long…………………………………… 7
8b Stipules short-fimbria te, the longest fimbriae (0.2–)0.3–0.9(–1.6) mm long,
(0.02–)0.06–0.2(–0.33)× as long as the width of the undivided part of stipule,
stipules and fimbriae ± not ciliate. The longest hairs on petioles (0.1–)0.15–0.4
(–0.5) mm long. • Stolons slender, (2–)4–19(–28.5) cm long and (0.9–)1–1.9
(–2.5) mm thick. Lamina apex obtuse to rounded, rarely obtusely acute, with
apex angle (80–)95–140(–180)°. Bracteoles inserted in (15–)33–61(–71)% of
peduncle length. Stipules ovate to narrowly triangular, (1.5–)2–3.5(–5)× longer
than wide, (3–)3.6–5.2(–7) mm wide, most of glandular fimbriae blackish.
Calycine appendages straight, not appressed to the peduncle. Petals (including
spur) (bluish-)violet, deep-violet, rarely whitish or (in cultivars) pink, yellow
etc. wi th small white throat at base (reaching up to ±1/4–1/5 of petal length
from the base)………………………….……………………………… V. odorata
Acknowledgements The authors wish to express their thanks to K. Marhold for valuable discussions
and to three anonymous reviewers for their useful comments on the manuscript. We are much obliged to P.
Mereďa sen. for his help with plant collecting and during our study. We are thankful to V. Kolarčik and Ľ.
Majeský for help with flow cytometric analyses, V. Polakovičová and J. Kučera for technical help, and to
H. Šípošová and D. Dítě for their assistance in the field. This study was supported by the Grant Agency of
110 I. Hodálová et al.

Ministry of Education of the Slovak Republic and Slovak Academy of Sciences VEGA (grant no. 6054)
and by Research and Development Support Agency of Slovak Republic (grant no. 6404). The
participation of J. D. was supported by the Ministry of Education, Youth and Sports of the Czech
Republic, project no. MSM 0021622416, and by the long-term research plan no. AV0Z60050516 of the
Institute of Botany, Czech Academy of Sciences.
Appendix
List of the studied populations of Viola sect. Viola subsect. Viola. Each record is
given as follows: population number, country, locality description, geographic
coordinates (WGS84), altitude, date of collection, name of collector(s); mitotic
chromosome numbe r and/or DNA ploidy level, name(s) of the author(s) of the
chromosome count(s) or author(s) of the measurements of DNA ploidy level; in
parenthesis the total number of plants studied: number of plants studied for pollen
fertility/for chromosome numbers/in flow cytometr y/in morp hometric analyses.
Phytogeographical division of the Czech Republic follows Skali cký (1988), that of
Slovakia Futák (1984) and that of Hungary Soó (1964). Abbreviations of collectors,
authors of the chromosome counts and authors of the measurements of DNA ploidy
level: JD – J. Danihelka, DD – D. Dítě,IH– I. Hodálová, PMA – P. Mártonfi, LM –
L. Mártonfiová, PM
J – P. Mereďa Jr., PMs – P. Mereďa sen., HŠ – H. Šípošová.
Viola alba Besser subsp. alba (incl. subsp. scotophylla (Jord.) Gremli)
109A – Slovakia, Devínska Kobyla Mts., city quarter of Bratislava-Dúbravka, E of
the elevation point 406, near the red-marked tourist path, 48°11′15″ N, 17°00′25″ E,
390 m, 14 Apr 2003, coll. PM
J;2n=20, det. IH & PMJ (1:1/1/0/1); alba morphotype.
109B – Slovakia, Devínska Kobyla Mts., city quarter of Bratislava-Dúbravka, E
of the elevation point 406, near the red-m arked tourist path, 48°11′15″ N, 17°00′25″
E, 390 m, 14 Apr 2003, coll. PM
J;2n=20, det. IH & PMJ (8:8/1/0/8); scotophylla
morphotype.
6 – Slovakia, Podunajská nížina Lowl ands, SW of the settlement of Čenkov, 47°
45′51″ N, 18°31′39″ E, 110 m, 2 Apr 2003, coll. IH & PM
J;2n=20, det. IH & PMJ
(10:10/1/0/10); scoto phylla morphotype.
111A – Slovakia, Strážovské and Súľovské vrchy Mts., village of Om šenie,
0.5 km NW of the top of Omšenská Baba Hill, near the red-marked tourist path, 48°
54′56″ N, 18°13′56″ E, 530 m, 19 Apr 2003, coll. PM
J &PMS;2n=20, det. IH &
PM
J (8:8/1/0/8); alba morphotype.
111B – Slovakia, Strážovské and Súľovské vrchy Mts., village of Omšenie,
0.5 km NW of the top of Omšenská Baba Hill, near the red-marked tourist path,
48°54′56″ N, 18°13′56″ E, 530 m, 19 Apr 2003, coll. PM
J & PMs; 2n∼ 4x∼ 20, det.
PMA, IH & PMJ (4:4/0/1/4); scotophylla morphotype.
112 – Slovakia, Strážovské and Súľovské vrchy Mts., town od Nová Dubnica,
Markovica Hi ll, 0.5 km SW of the summit, 48°55′25″ N, 18°10′43″ E, 450 m, 21
Apr 2003, coll. PM
J;2n∼ 4x∼ 20, det. PMA, IH & PMJ (10:10/0/1/10); scotophylla
morphotype.
123 – Hungary, Sokoró Mts., SE of village of Györújbarát, near the camp
Ifjuságy, 47°35′12″ N, 17°39′09″ E, 242 m, 1 Apr 2004, coll. PM
J;2n=20, det. IH
&PMJ (10:10/1/0/10); scotophylla morphotype.
Morphological characters of Viola subsect. Viola (Violaceae) 111

Viola ambigua Waldst. & Kit.
9 – Czech Republic, Moravia, Pavlovské kopce Mts., Pálava Hill, SE facing slope
N of limestone quarry, 48°51′26″ N, 16°38′37″ E, 400 m, 4 Apr 2003, coll. IH &
PM
J;2n=40, det. LM (8:8/2/0/8).
21 – Slovakia, Devínska Kobyla Mts., city quarter of Bratislava-Devín, 1.2 km
SW of the top of Devínska Kobyla Hill, 48°10′52″ N, 16°59′ 12 ″ E, 250 m, 8 Apr
2003, coll. IH & PM
J;2n=40, det. IH & PMJ (2:2/1/0/2).
151 – Hungary, Gerecse Mts., village of Csolnok, Magos-hegy Hill (elevation
point 317 m), 47°41′20″ N, 18°42′10″ E, 314 m, 14 Apr 2005, coll. IH & PMj;
2n=40, det. IH & PMj (8:8/1/0/8).
149 – Hungary, Gerecse Mts., village of Dág (SSE of town of Dorog), SE slope of
Kecske-hegy Hill, 47°40′31″ N, 18°42′36″ E, 207 m, 14 Apr 2005, coll. IH & PM
J;
2n=40, det. IH & PM
J (9:9/1/0/9).
Viola collina Besser
30 – Austria, Lower Austria, Eastern Alps, town of Baden Ali Wien, slope of
Rauheneck Castle Hill, 48°00′ 34 ″ N, 16°12′25″ E, 350 m, 15 Apr 2003, coll. IH;
2n=20, det. LM (10:10/2/0/10).
128 – Slovakia, Strážovské and Sú ľovské vrchy Mts., village of Omš enie, 0.5 km
NW of the top of Omšenská Baba Hill, W facing slope, 48°54′40″ N, 18°14′07″ E,
580 m, 11 Apr 2004, coll. PM
J &PMS;2n∼ 4x∼ 20, det. PMA, IH & PMJ (8:8/0/
1/8).
127 – Slovakia, Strážovské and Súľovské vrchy Mts., town of Nová Dubnica,
Markovica Hi ll, 0.5 km SW of the summit, 48°55′30″ N, 18°10′40″ E, 425 m, 10
Apr 2004, coll. PM
J;2n=20, det. IH & PMJ (10:1 0/1/0/10).
130 – Slovakia, Nízke Tatry Mts., village of Kráľova Lehota, near the settlement
of Hlboké, 49°02′06″ N, 19°47 ′15″ E, 605 m, 15 Apr 2004, coll. IH & PM
J;2n=20,
det. IH & PMJ (10:10/1/0/10).
Viola hirta L.
15 – Czech Republic, Moravia, Pavlovské kopce Mts., town of Mikulov, Svatý
kopeček Hill, 48°48′25″ N, 16°38′56″ E, 330 m, 4 Apr 2003, coll. JD, IH & PM
J;
2n∼ 4x∼ 20, det. PMA, IH & PM
J (10:10/0/1/10).
14 – Czech Republic, Moravia, Pavlovské kopce Mts., town of Mikulov, Svatý
kopeček Hill, 48°48′26″ N, 16°38′53″ E, 260 m, 4 Apr 2003, coll. JD, IH & PM
J;2n
=20, det. IH & PM
J (3:3/1/0/3).
24 – Slovakia, Slovenský kras Karst, village of Silica, 1 km W of the edge of the
village, 48°33′20″ N, 20°30′10″ E, 530 m, 9 Apr 2003, coll. IH; 2n=20, det. LM
(5:5/2/0/5).
103 – Slovakia, Devínska Kobyla Mts., city quarter of Bratislava-Devín, 0. 8 km
SWS of the top of the Devínska Kobyla Hill, near the red-marked tourist path,
48°10′58″ N, 16°59′34″ E, 344 m, 5 Apr 2003, coll. PM
J;2n∼ 4x∼ 20, det. PMA, IH
&PM
J (10:10/0/1/10).
116 – Slovakia, Biele Karpaty (southern part) Mts., village of Chocholná-Velčice,
Chocholničianska dolina valley, 0.5 km NWW of Urbanová Hill, 48°53′48 ″ N,
17°54′57″ E, 460 m, 23 Apr 2003, coll. PM
J;2n∼ 4x∼ 20, det. PMA, IH & PMJ
(10:10/0/1/10).
112 I. Hodálová et al.

113 – Slovakia, Strážovské and Súľovské vrchy Mts., town of Nová Dubnica,
valley of Veľkokolačanský potok stream, 0.7 km SWW of the top of Markovica Hill,
48°55′25″ N, 18°10′35″ E, 415 m, 21 Apr 2003, coll. PM
J;2n∼ 4x∼ 20, det. PMA,
IH & PM
J (10:10/0/1/10).
114 – Slovakia, Strážovské and Súľovské vrchy Mts., town of Nová Dubnica,
upper part of the city quarter of Veľký Kola č ín, 48°55′55″ N, 18°10 ′13″ E, 285 m,
22 Apr 2003, coll. PM
J;2n=20, det. IH & PMJ (10:1 0/1/0/10).
23 – Slovakia, Spišské kotliny Basin, settlement of Primovce, near E edge of
settlement, 49°00′49″ N, 20°23′07″ E, 620 m, 9 Apr 2003, coll. IH & DD; 2n=20,
det. LM (10:10/1/0/10).
Viola odorata L.
31 – Austria, Lower Austria, Eastern Alps, town of Baden Ali Wien, alluvium of
the river below the Rauheneck Castle Hill, 48°00′34″ N, 16°12′25″ E, 250 m, 15 Apr
2003, coll. IH; 2 n=20, det. IH & PM
J (9:9/1/0/9).
8 – Czech Republic, Moravia, Pavlovské kopce Mts., town of Mikulov, Pálava
Hill, SE slope north of limestone quarry, 48°51′28″ N, 16°38′42″ E, 380 m, 4 Apr
2003, coll. IH & PM
J;2n∼ 4x∼ 20, det. PMA, IH & PMJ (10:10/0/1/10).
3 – Slovakia, Burda Mts., settlement of Kováčov, in vicinity of railway station,
47°49′25″ N, 18°46′51″ E, 110 m, 2 Apr 2003, coll. IH & PM
J;2n=20, det. IH &
PM
J;2n∼ 4x∼ 20, det. PMA, IH & PMJ (9:9/1/1/9).
26 – Slovakia, Košická kotlina Basin, town of Turňa nad Bodvou, S foot of
Turniansky hradný vrch Castle Hill, 48°36′23″ N, 20°52′19″ E, 200 m, 10 Apr 2003,
coll. IH; 2n∼ 4 x∼ 20, det. PMA, IH & PM
J (8:8/0/1/8).
203 – Slovakia, Slanské vrchy Mts., village of Kalš a, 48°36′25″ N, 21°31′13″ E,
225 m, 8 Apr 2004, coll. PMA; 2n=20, det. LM; 2n∼ 4x∼ 20, det. PMA, IH & PM
J
(10:10/1/1/10).
204 – Slovakia, Slanské vrchy Mts., village of Kalša, 48°36′23″ N, 21°31′11″ E,
236 m, 8 Apr 2004, coll. PMA; 2n=20, det. LM; 2n∼ 4x∼ 20, det. PMA, IH & PM
J
(9:9/1/1/9).
120 – Hungary, Sokoró Mts., SE of village of Györújbarát, near the camp
Ifjuságy, 47°35′11″ N, 17°39′12″ E, 246 m, 1 Apr 2004, coll. PM
J;2n∼ 4x∼ 20, det.
PMA, IH & PM
J (10:10/0/1/10).
Viola suavis M. Bieb. s.l.
11 – Czech Republic, Moravia, Pavlovské kopce Mts., town of Mikulov, Pálava
Hill, 48°51′26″ N, 16°38′37″ E, 400 m, 4 Apr 2003, coll. IH & PM
J;2n=40, det.
LM (8:8/1/0/8).
4 – Slovakia, Burda Mts., settlement of Kováčov, in vicinity of railway station,
47°49′25″ N, 18°46′51″2 E, 110 m, 6 Apr 2004, coll . IH & PM
J;2n=40, det. LM
(10:10/1/0/10); viol et-flowered morphotype.
16 – Slovakia, Devínska Kobyla Mts., city quarter of Bratislava-Devín, 1 km
SWS of the top of Devínska Koby la Hill, 48°10′51″ N, 16°59′32″ E, 290 m, 6 Apr
2003, coll. IH; 2 n=40, det. IH & PM
J (7:7/1/0/7); violet-flowered morphotype.
7 – Slovakia, Devínska Kobyla Mts., city quarter of Bratislava-Dúbravka, Brižite
Hill, 48°11′49″ N, 17°01′29″ E, 240 m, 1 Apr 2003, coll. IH; 2n∼ 8x∼ 40, det. PMA,
IH & PM
J (10:10/0/1/10); white-flowered morp hotype.
Morphological characters of Viola subsect. Viola (Violaceae) 113

18 – Slovakia, Podunajská nížina Lowlands, city quarter of Bratislava-Petržalka,
Dudova street, 48°06′59″ N, 17°07′09″ E, 135 m, 4 Apr 2003, coll. HŠ & IH; 2n=
40, det. LM (4:4/2/0/4); white-flowered morphotype.
129 – Slovakia, Podunajská nížina Lowlands, town of Nitra, Šibeni čný vrch Hill, edge
of forest with Pinus nigra, Urbánkova street, 48°18′16″ N, 18°04′30″ E, 180 m, 13 Apr
2004, coll. PM
J;2n=40, det. IH & PMJ (10:10/1/0/10); violet-flowered morphotype.
106 – Slovakia, Podunajská nížina Lowlands, town of Nitra, Kalvária Hill, Pod
Borinou street, 48°17′52″ N, 18°05′22″ E, 175 m, 13 Apr 2003, coll. PM
J;2n=40,
det. LM (12:12/2/0/12); white-flowered morphotype.
25 – Slovakia, Košická kotlina Basin, town of Turňa nad Bodvou, S foot of
Turniansky hradný vrch Castle Hill, 48°36′23″ N, 20°52′22″ E, 205 m, 10 Apr 2003,
coll. IH; 2n=40, det. LM (10:10/2/0/10) ; white-flowered morphotype.
201 – Slovakia, Košická kotlina Basin, city of Košice, Botanical garden of P. J.
Šafárik University–Faculty of Science, Mánesova street, 48°44′05″ N, 21°14′18″ E,
227 m, 17 Apr 2003, coll. PMA & LM; 2n=40, det. LM (10:10/1/ 0/10); white-
flowered morphotype.
202 – Slovakia, Košická kotlina Basin, city of Košice, Humenská street, lawn in
kindergarten, 48°42′23″ N, 21°14′16″ E, 249 m, 17 Apr 2003, coll. PMA & LM; 2n
=40, det. LM (10:10/1/0/10); white-flowered morp hotype.
28 – Slovakia, Východoslovenská nížina Lowlands, village of Hrušov, near the
church, 48
o
26′10″ N, 21
o
51′41″ E, 105 m, 10 Apr 2003, coll. IH; 2n=40, det. LM
(10:10/2/0/10); viol et-flowered morphotype.
22 – Slovakia, Liptovská kotlina Basin, town of Ružomberok, E of railway
station, on the foot of Mních Hill, 49°05′00″ N, 19°18′37″ E, 490 m, 9 Apr 2003,
coll. DD & IH; 2n=40, det. LM (6:6/2/0/6); white-flowered morphotype.
125 – Hungary, Pilis Mts., town of Esztergom, 0.4 km NW of the top of Vaskapu
Hill, near the red-marked tourist path, 47°47′21″ N, 18°46′11″ E, 340 m, 6 Apr
2004, coll. IH & PM
J;2n=ca. 40, det. IH & PMJ;2n∼ 8x∼ 40, det. PMA, IH & PMJ
(10:10/1/1/10); viol et-flowered morphotype.
122 – Hungary, Sokoró Mts., SE of village of Györújbarát, near the camp
Ifjuságy, 47°35′12″ N, 17°39′09″ E, 242 m, 1 Apr 2004, coll. PM
J;2n∼ 8x∼ 40, det.
PMA, IH & PM
J (10:10/0/1/10); violet-flowered morphotype.
Viola alba × V. hirta (V.×adulterina Godr.)
17 – Slovakia, Devínska Kobyla Mts., city quarter of Bratislava-Devín, 1.3 km SW
of the top of Devínska Kobyla Hill, near the educational path, 48°10′48″ N, 16°59′
08″ E, 220 m, 6 Apr 2003, coll. IH; 2n∼ 4x∼ 20, det. PMA, IH & PM
J (10:10/0/1/0).
101 – Slovakia, Devínska Kobyla Mts., city quarter of Bratislava-Devín, 1 km
SWS of the top of Devínska Koby la Hill, 48°10′57″ N, 16°59′20″ E, 300 m, 5 Apr
2003, coll. PM
J;2n∼ 4x∼ 20, det. PMA , IH & PMJ (10:10/1/0/0).
Viola alba × V. odorata (V.×pluricaulis Borbás)
102 – Slovakia, Devínska Kobyla Mts., city quarter of Bratislava-Devín, 1 km
SWS of the top of Devínska Koby la Hill, 48°10′57″ N, 16°59′22″ E, 300 m, 5 Apr
2003, coll. PM
J;2n=20, det. IH & PMJ (6:6/1/0/0).
105 – Slovakia, Devínska Kobyla Mts., city quarter of Bratislava-Devín, 0. 8 km
SES of the top of Devínska Kobyla Hill, near the yellow- marked tourist path, 48°10′
114 I. Hodálová et al.

56″ N, 16°59′56″ E, 350 m, 6 Apr 2003, coll. PMJ;2n∼ 4x∼ 20, det. PMA, IH &
PM
J (5:5/0/1/0).
110 – Slovakia, Devínska Kobyla Mts., city quarter of Bratislava-Dúbravka, E of
elevation point 406 m, near the red-marked tourist path, 48°11′15″ N, 17°00′20″ E,
390 m, 14 Apr 2003, coll. PM
J;2n∼ 4x∼ 20, det. PMA, IH & PM J (8:8/0/1/0).
121 – Hungary, Sokoró Mts., SE of village of Györújbarát, near the camp
Ifjuságy, 47°35′12″ N, 17°39′09″ E, 242 m, 1 Apr 2004, coll. PM
J;2n=20, det. IH
&PM
J (8:8/1/0/0).
Viola ambigua × V. odorata (V.×hungarica Degen & Sabr.)
13 – Czech Republic, Moravia, Pavlovské kopce Mts., town of Mikulov, Svatý
kopeček Hill, 48°48′26″ N, 16°38 ′53″ E, 4 Apr 2003, coll. JD, IH & PM
J;2n=30,
det. LM (1:0/1/0/0).
Viola hirta × V. odorata (V.×scabra F. Braun)
12 – Czech Republic, Moravia, Pavlovské kopce Mts., saddle Nad Soutěskou,
48°52′07″ N, 16°38′18″ E, 4 Apr 2003, coll. IH & PM
J;2n=20, det. LM (9:9/2/
0/0).
104 – Slovakia, Devínska Kobyla Mts., city quarter of Bratislava-Devín, 0. 8 km
SW of the top of the Devínska Kobyla Hill, near the red-marked tourist path,
48°10′58″ N, 16°59′40″ E, 320 m, 5 Apr 2003, coll. PM
J;2n∼ 4x∼ 20, det. PMA, IH
&PM
J (8:8/0/1/0).
117 – Slovakia, Biele Karpaty (southern part) Mts., village of Chocholná-Velčice,
near N edge of part of Malá Chocholná, 48°52′37 ″ N, 17°56′44″ E, 270 m, 23 Apr
2003, coll. PM
J;2n=20, det. IH & PMJ (10:10/1/0/0).
115 – Slovakia, Strážovské and Súľovské vrchy Mts., town of Nová Dubnica,
lower part of the city quarter of Veľký Kolačín, 48°56′22″ N, 18°09′38″ E, 255 m,
22 Apr 2003, coll. PM
J;2n=ca. 20, det. IH & PMJ (10:10/1/0/0).
Viola odorata × V. suavis s.str. (V.×vindobonensis Wiesb.)
118 – Czech Republic, Moravia, Pavlovské kopce Mts., town of Mikulov, Svatý
kopeček Hill, 48°48′26″ N, 16°38′53″ E, 340 m, 4 Apr 2003, coll. JD, IH & PM
J;2n
=30, det. IH & PM
J;2n∼ 6x∼ 30, det. PMA , IH & PMJ (2:0/1/1/0).
124 – Hungary, Pilis Mts., town of Esztergom, 0.4 km NW of the top of Vaskapu
Hill, near the red-marked tourist path, 47°47′21″ N, 18°46′11″ E, 340 m, 6 Apr
2004, coll. IH & PM
J;2n∼ 6x∼ 30, det. PMA, IH & PMJ (2:0/0/2/0).
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Received: 29 January 2007 / Revised: 28 August 2007 / Accepted: 29 October 2007 /
Published online: 21 May 2008
Morphological characters of Viola subsect. Viola (Violaceae) 117