Mating System and Hybridization of the Cyanus triumfetti and C. montanus Groups (Asteraceae)

Abstract

Mode of reproduction and presence of reproductive barriers were studied in two closely related members of the genus Cyanus: the C. triumfetti (diploid 2n = 22) and C. montanus (tetraploid 2n = 44) groups. Based on results from isolation and emasculation experiments, taxa of these groups can be considered allogamous with a low selfing rate (0.07 %–0.21 % of achenes developed after selfing). Taxa of the C. triumfetti group hybridize easily and produce viable progeny. Differences in the percentage of well-developed achenes per capitulum obtained from interspecific crosses between members of the C. triumfetti group suggested different levels of reproductive isolation. The percentage of well-developed achenes of most homoploid crosses was 3.47 %–8.87 %. Higher percentages of well-developed achenes were obtained from crosses between Eastern Carpathian C. pinnatifidus and Alpine C. triumfetti s. str. (18.36 % ; 26.56 %) and between geographically close taxa in Central Europe (C. dominii, C. strictus and ‘intermediate morphotype’; 12.75 % –17.60 %), which indicate their overall close relatedness. Crossing geographically remote C. strictus and C. triumfetti s. str. yielded no or only few (0.99 %) well-developed achenes, indicating an increased degree of incompatibility in allopatry. The success of heteroploid crosses between plants belonging to different groups was reduced (2.96 %) and suggested reproductive incompatibilities between ploidy levels. The progeny of heteroploid crosses comprised 63 % of triploids of presumable hybrid origin on tetraploid as well as diploid maternal plants. Another 15.22 % of progeny had the maternal cytotype, probably resulting from selfing. Low viability of heteroploid hybrids supports the existence of post-zygotic mechanisms.

Full text

Mating System and Hybridization of the Cyanus triumfetti
and C.montanus Groups (Asteraceae)
Katarína Olšavská &Carsten J. Löser
#Institute of Botany, Academy of Sciences of the Czech Republic 2013
Abstract Mode of reproduction and presence of reproductive barriers were studied
in two closely related members of the genus Cyanus:theC.triumfetti (diploid
2n=22) and C.montanus (tetraploid 2n=44) groups. Based on results from isolation
and emasculation experiments, taxa of these groups can be considered allogamous with
a low selfing rate (0.07 %–0.21 % of achenes developed after selfing). Taxa of the C.
triumfetti group hybridize easily and produce viable progeny. Differences in the per-
centage of well-developed achenes per capitulum obtained from interspecific crosses
between members of the C.triumfetti group suggested different levels of reproductive
isolation. The percentage of well-developed achenes of most homoploid crosses was
3.47 %–8.87 %. Higher percentages of well-developed achenes were obtained from
crosses between Eastern Carpathian C.pinnatifidus and Alpine C.triumfetti s. str. (18.36 % ;
26.56 %) and between geographically close taxa in Central Europe (C.dominii,C.
strictus and ‘intermediate morphotype’;12.75%–17.60 %), which indicate their overall
close relatedness. Crossing geographically remote C.strictus and C.triumfetti s. str.
yieldednooronlyfew(0.99%)well-developedachenes,indicatinganincreaseddegree
of incompatibility in allopatry. The success of heteroploid crosses between plants be-
longing to different groups was reduced (2.96 %) and suggested reproductive incom-
patibilities between ploidy levels. The progeny of heteroploid crosses comprised 63 %
of triploids of presumable hybrid origin on tetraploid as well as diploid maternal plants.
Another 15.22 % of progeny had the maternal cytotype, probably resulting from selfing.
Low viability of heteroploid hybrids supports the existence of post-zygotic mechanisms.
Folia Geobot (2013) 48:537–554
DOI 10.1007/s12224-013-9155-3
Electronic supplementary material The online version of this article (doi:10.1007/s12224-013-9155-3)
contains supplementary material, which is available to authorized users.
K. Olšavská (*)
Institute of Botany, Slovak Academy of Sciences, Dúbravská cesta 9, 845 23 Bratislava,
Slovak Republic
e-mail: katarina.olsavska@savba.sk
C. J. Löser
Institute of Systematic Botany, Friedrich Schiller University, Philosophenweg 16, 07743 Jena,
Germany

Keywords Centaurea section Cyanus .Compositae .Heteroploid hybridization .
Homoploid hybridization .Reproductive barriers .Self-incompatibility
Introduction
Natural hybridization is a relatively common feature in vascular plants and plays an
important role in their evolution. Hybrid individuals can suffice as progenitors of new
lineages and may give rise to entirely new species (Ellstrand et al. 1996; Rieseberg
1997). Stabilization of new hybrid derivates could be accompanied by extinction or
merging of hybridizing lineages. Several hybridization-isolation cycles may result in
a highly reticulated pattern of phylogeny. The effective speciation process requires
fertile and viable hybrids and the establishment of at least partial isolation barriers
(Stebbins 1969; Chapman and Burke 2007). Reduced hybrid fitness together with
persistence of gene flow can result in a stable hybrid zone preventing merging of
lineages but allowing genetic exchange, e.g., via introgression (Barton and Hewitt
1989; Chapman and Burke 2007). Frequency of natural hybridization in plants
apparently varies with life history, pollination mechanisms, breeding system and
genetic predisposition (Grant 1981). Beside the data on spontaneous hybridization
in natural populations, experimental crosses could contribute to the identification of
species compatibility and isolation barriers (Mráz and Paule 2006).
In the present study, we examined the breeding system and the contribution of
post-mating prezygotic and postzygotic mechanisms to the strength of reproductive
isolation in perennials of the genus Cyanus Mill. (tribe Cardueae Cass., subtribe
Centaureinae Dumort.). The possibilities of gene flow in natural populations should
contribute to the knowledge about ancestral relationships of these species and clarify
the role of hybridization in their evolution. We focused on taxa occurring in the
Carpathians, Pannonia, Bohemian Massif, Western Alps and Dinarides that have
recently received intensive biosystematic scrutiny (cf. Olšavská et al. 2011,2012).
The diploid C.triumfetti group (2n=2x=22) is represented by C.adscendens (Bartl.)
Soják (Dinarides), C.axillaris auct. (hereinafter referred to as ‘C.axillaris’; Central
Europe), C.dominii (Dostál) Holub (including C.d. subsp. dominii,C.d. subsp.
slovenicus (Dostál) Olšavská, C.d. subsp. sokolensis (Pawł.) Olšavská; Western
Carpathians), C.pinnatifidus (Schur) Holub (Eastern Carpathians), C.strictus
(Waldst. & Kit.) Soják (Eastern Slovakia and Hungary), C.triumfetti (All.) Dostál
ex Á.Löve & D.Löve s. str. (Western Alps), and the intermediate morphotype
between C.axillaris and C.dominii (hereinafter referred to as ‘intermediate morpho-
type’; Western Carpathians). This complex of species forms a geographic continuum
ranging from the northern Iberian Peninsula, the Alps and Central Europe to the
Carpathians, Stara Planina, the Apennine and Balkan Peninsulas (Fig. 1a). The
tetraploid (2n=4x=44) C.montanus group is formed by C.mollis (Waldst. & Kit.)
J.Presl & C.Presl (Carpathians) and C.montanus (L.) Hill s. str. (Western Europe)
(Fig. 1b). Previously, little attention has been given to reproductive biology and
breeding behaviour of these species. The mode of reproduction and breeding system
of C.montanus s. str. was included in a study of genetic factors determining flavonoid
variationbyGonnet(1992,1996). Two experimental hybrids were reported by
Guinochet (1957a,b).
538 K. Olšavská, C.J. Löser

The C.triumfetti and C.montanus groups are well differentiated karyologically
(diploids vs tetraploids) and morphologically (branched root system and fimbria paler
than margins of involucral bracts vs creeping rhizomes and black or dark-brown
margin and fimbria/dents of involucral bracts). Both groups have overlapping
distributions in the Alps and Carpathians, and a few sympatric populations of C.
triumfetti s. str. and C.montanus s. str. have been reported from the Alps
Fig. 1 Geographical distribution of the studied taxa (lines) and origin of the material for cultivation
(symbols) of the Cyanus triumfetti (a) and C.montanus groups (b)
Mating System and Hybridization in Cyanus 539

(cf. Gonnet 1993;Olšavská et al. 2012). Taxa of both groups are herbaceous long-
lived perennials. In general, plants of the C.triumfetti group occupy south-exposed
dry meadows and rocky places, while plants from the C.montanus group grow
mostly in humid and shady mountain meadows and forests. Plants of the C.montanus
group reproduce also vegetatively by rhizomes (Gonnet 1993).
On one hand, the C.triumfetti and C.montanus groups are regarded as very closely
related in the context of autopolyploid origin, based on similarities in morphological
characters, flavonoid glycoside variation, morphology of chromosomes and base chro-
mosome numbers (Baksay 1957;Guinochet1957a,b; Gonnet 1993). Furthermore, no or
only negligible differences in monoploid genome size (Cx-value), found between
sympatric diploids of the C.triumfetti group and tetraploids of the C.montanus group
(Olšavská et al. 2012), can be regarded as an indicator of the autopolyploid origin of the
tetraploids (cf. Trávníček et al. 2010). Recent analyses of nuclear ribosomal DNA
sequences (Boršićet al. 2011), however, did not support such an autopolyploid rela-
tionship and thus all abovementioned similarities might likewise result from common
ancestry or from recurrent gene flow. Interspecific hybrids and introgressed forms of C.
triumfetti s. str. and C.montanus s. str. are rarely reported in natural populations, but no
triploid hybrids 2n=33havebeendetected(Gugler1907;Gonnet1993).
The C.triumfetti group is taxonomically critical due to high polymorphisms of
taxa and because clinal variation often obscures boundaries between taxa. Moreover,
intermediate morphotypes among some taxa are also reported (cf. Olšavská et al.
2011). The patterns of genetic diversity in the C.triumfetti group obtained by AFLP
markers (Olšavská et al. 2011) and nuclear DNA sequences (Löser, unpubl. data)
reflect its geographic distribution. The genetic data showed a separate position of C.
triumfetti s. str. from the Alps. Samples of ‘C.axillaris’were divided into two
genetically differentiated and allopatric groups, originating from the Czech
Republic and Austria vs Slovakia and Hungary, which may reflect different glacial
refuges and/or postglacial migration routes. The weak genetic differentiation revealed
for morphologically well-separated taxa from Slovakia and Hungary (‘C.axillaris’
and C.dominii,C.strictus) could indicate a very recent diversification or ongoing
gene flow (Olšavská et al. 2011).
Plants of the C.triumfetti and C.montanus groups are insect-pollinated, mainly by
honeybees, bumblebees, wasps and ants (Hymenoptera). They possess several adap-
tations to myrmecophily and myrmecochory. Ants are attracted by nectar drops at the
involucral bracts and therefore, they tend to build nests in proximity of Cyanus plants.
Ants are also important for the dispersal of the diaspores because the achenes have
developed lateral elaiosomes (cf. Bancheva and Stoyanov 2009). Flowers of the
genus Cyanus show proterandry and develop centripetally within the inflorescences
(Rendle 1975).
The following questions were addressed in the present study: i) What is the mode
of reproduction of the C.triumfetti and C.montanus groups? ii) Are taxa of the C.
triumfetti group sharing the same cytotype 2n= 22 reproductively isolated? How
easily can viable hybrids be formed by cross pollination within the C.triumfetti
group? Is the genetic gap between the Austrian-Czech and the Hungarian-Slovak
samples of ‘C.axillaris’(Olšavská et al. 2011) expressed in reproductive isolation?
iii) Is there a reproduction barrier between the C.triumfetti and C.montanus groups
across ploidy levels? Are heteroploid hybrids viable?
540 K. Olšavská, C.J. Löser

Material and Methods
Plant Material
Roots/rhizomes from mature plants were collected in the field from 2006 to 2009
in Central, Eastern and Western Europe (Fig. 1;seeTableS1 in Electronic
Supplementary Material for locality details) and were transferred and cultivated under
uniform conditions in an experimental garden at Višňové (49°09′40″N, 18°47′13″E,
470 m a.s.l., Žilina district, Slovakia). Results of our previous papers were employed
for taxonomic identification of plant material (Olšavská et al. 2011,2012).
Mating System and Hybridization Experiments
The study of mating system and artificial crossings in the C.triumfetti and C.
montanus groups was performed in the experimental garden from May to July in
2008–2010 (Table 1). Capitula of the studied plants were left without treating for
open pollination or enclosed in paper, cotton or nylon bags to perform experiments.
During experiments all unisolated inflorescences were regularly removed before
anthesis to minimize the risk of unwanted cross-pollinations.
To test seed production by apomixis, inflorescences were emasculated by cutting
the upper part of the capitulum using a razor blade (two or three times depending on
gradual development of florets) and bagged.
To test seed production by autogamy, inflorescences were isolated in bags
before and during anthesis to prevent cross-pollination. The lack of differences
between hand-pollinated and not hand-pollinated inflorescences determined by
Ortiz et al. (2006) indicates that human assistance is unnecessary to determine
self-incompatibility.
To study reproductive barriers, crossing experiments within and between diploid
taxa of the C.triumfetti and tetraploid taxa of the C.montanus groups were
performed. For artificial crosses, pairs of capitula at similar developmental stages
were enclosed together in bags prior to anthesis. Three types of crosses were carried
out according to the scheme outlined in Table 2: (a) within a taxon, (b) between taxa
of the C.triumfetti group (homoploid crosses), (c) between species from the two
different groups C.triumfetti and C.montanus (heteroploid crosses). At anthesis, two
capitula were gently brushed against each other 2–4 times during flowering (receptive
styles were presented).
After harvesting, capitula were dried in a warm place for two weeks. Subsequently,
seed set (number of undeveloped and well-developed achenes per capitulum) were
calculated. Inflorescences in penetrated bags at the time of collection (mainly by
wasps and ants attracted by nectar) and inflorescences with achenes damaged by
granivorous insects (mainly by larvae of Chaetorellia (Diptera, Tephritidae) –their
eggs are injected into young buds) were excluded. Well-developed achenes were
stored at 4–6°C for four months. Subsequently, the seed germination was evaluated to
study fitness of hybrids. The achenes were germinated on wet filter paper in Petri
dishes under standard conditions (20–22°C). Some of the progeny (those from
crosses between different species) were transferred into experimental plots and are
still cultivated to assess their morphological variation.
Mating System and Hybridization in Cyanus 541

The success of crosses was calculated as the percentage of well-developed
achenes per capitulum (Fig. 2) or as the mean percentage of well-developed
achenes for all capitula used in particular experiments. The results from crosses
obtained from more than five capitula and germination rates from more than
five achenes were considered as relevant (marked in bold in Tables 2and 3).
The results for the particular subspecies of C.dominii are shown in Tables 1,2
and 3, while those at the species-level are presented in the text and in Figs. 2
and 3. The figures displaying success of hybridizations (Figs. 3and 4)were
produced only for Central and Western European species of the C.triumfetti
group for which each type of crossing was realized. To test for a correlation
between reproductive distances (means of crossing success) and geographic
distances (distances of midpoints of the ranges) between taxa, a matrix corre-
lation test (Mantel 1967) was performed using the program NTSYSpc Version
2.11a (Rohlf 2002). The Mann-Whitney U-test was used to check for differ-
ences between directions of reciprocal crosses and between two geographical
groups of samples of ‘C.axillaris’. Furthermore, the percentage of well-
developed achenes obtained for each cross type within C.triumfetti group
represented at least by five capitula were compared using the Kruskal-Wallis
test.
Flow Cytometry
The ploidy level of progeny (embryos) from heteroploid crosses was estimated using
flow cytometry (DAPI fluorochrome) and Partec Cyflow ML instrument (Partec
GmbH, Munster, Germany) equipped with a HBO-100 mercury arc lamp.
Lycopersicum esculentum Mill. was used as an internal standard. The procedure is
described in details in Olšavská et al. (2011).
Results
Mating System
Under open pollination (176 non-isolated inflorescences) massive seed produc-
tion was observed during three years of experiments; average percentage of
well-developed achenes per capitulum of particular taxa varied from 42.5 % to
66.53 % (mean numbers of well-development achenes per capitulum ranged
from 10 to 24) (Table 1,Fig.2). The highest mean number of well-developed
achenes per capitulum (= number of inner florets per capitulum) was recorded
for C.triumfetti s. str., C.mollis and C.montanus s. str. Our data clearly
exclude the presence of apomixis in the studied taxa as no well-developed
achenes were formed in emasculated capitula (N=32). Allogamy was revealed
for all investigated taxa. In the isolation experiment (N=334 inflorescences) no
well-developed achenes were produced by C.strictus,C.triumfetti s. str. and
‘intermediate morphotype’, and only a few well-developed and mature achenes
(0.07 %–0.21 %) were produced by C.adscendens,‘C.axillaris’,C.dominii,
C.mollis and C. montanus s. str. (Table 1).
542 K. Olšavská, C.J. Löser

Table 1 Summarized results of open pollination, isolation and emasculation experiments of the Cyanus triumfetti and C.montanus groups. Abbreviations used: I –number of
inflorescences, IW –number of inflorescences yielding at least one well-developed achene, A –number of all achenes, AW –number of well-developed achenes, SS –mean total
number of achenes per capitulum (seed set), SSE –mean total number of well-developed achenes per capitulum (effective seed set), MON –C.montanus s. str., MOL –C.mollis,
TRI –C.triumfetti s.str., AXI –‘C.axillaris’, STR –C.strictus, DOM –C.dominii subsp. dominii, SLO –C.dominii subsp. slovenicus, SOK –C.dominii subsp. sokolensis,
INT –‘intermediate morphotype’, ADS –C.adscendens,PIN–C.pinnatifidus
Open pollination Isolation experiments Emasculation experiments
IW/I (%) AW/A (%) SS SSE IW/I (%) AW/A (%) IW/I AW/A
MOL 16/16 (100 %) 287/532 (53.95 %) 33.25 17.94 1/28 (3.57 %) 1/1058 (0.09 %) 0/4 0/115
MON 7/7 (100 %) 110/226 (48.67 %) 32.29 15.71 1/20 (5 %) 1/755 (0.13 %) 0/2 0/56
TRI 8/8 (100 %) 188/308 (61.04 %) 38.5 23.5 0/14 0/541 0/3 0/107
AXI 41/41 (100 %) 603/1115 (54.08 %) 27.19 14.70 3/84 (3.57 %) 3/2127 (0.14 %) 0/10 0/305
STR 16/16 (100 %) 316/475 (66.53 %) 29.68 19.75 0/53 0/1078 0/3 –
DOM 7/7 (100 %) 77/143 (53.85 %) 20.43 11.00 0/12 0/326 0/2 0/49
SLO 23/23 (100 %) 190/445 (42.7 %) 19.35 10.26 0/22 0/399 0/4 0/57
SOK 5/5 (100 %) 75/142 (52.82 %) 28.4 15 1/10 (10 %) 2/248 (0.81 %) ––
INT 19/19 (100 %) 299/524 (57.06 %) 27.58 15.74 0/16 0/421 0/2 –
ADS 12/12 (100 %) 169/318 (53.14 %) 26.5 14.08 1/48 (2.08 %) 1/1343 (0.07 %) ––
PIN 5/5 (100 %) 51/120 (42.5 %) 24.00 10.20 0/27 0/719 ––
Mating System and Hybridization in Cyanus 543

Table 2 Summarized results of crossing experiments of the Cyanus triumfetti and C.montanus groups. Abbreviations used: I –number of inflorescences, IW –number of inflorescences
yielding at least one well-developed achene, A –number of all achenes, AW –number of well-developed achenes; MON –C.montanus s. str., MOL –C.mollis,TRI–C.triumfetti s. str.,
AXI –‘C.axillaris’,STR–C.strictus, DOM –C.dominii subsp. dominii,SLO–C.dominii subsp. slovenicus,SOK–C.dominii subsp. sokolensis,INT–‘intermediate morphotype’,
ADS –C.adscendens,PIN–C.pinnatifidus. Intraspecies crosses are indicated in italic
Maternal plant
MON
MOL
TRI
AXI
STR
DOM
SLO
SOK
INT
ADS
PIN
IW/I
%
AW/A
%
IW/I
%
AW/A
%
IW/I %
AW/A
%
IW/I
%
AW/A %
IW/I
%
AW/A
%
IW/I
%
AW/A
%
IW/I
%
AW/A
%
IW/I
%
AW/A
%
IW/I
%
AW/A
%
IW/I
%
AW/A
%
IW/I
%
AW/A
%
Pollen donor
MON
4/4
100%
33/149
22.15%
3/4
75%
3/141
2.13%
4/10
40%
40/461
8.68%
1/7
14.29%
9/270
3.33%
1/3
33.33%
5/133
3.75%
_
_
_
_
1/4
25%
6/104
5.77%
_
_
_
_
_
_
MOL
0/4
0%
0/133
0%
_
_
1/5
20%
5/248
2.02%
1/10
10%
2/265
0.75%
1/2
50%
1/66
1.51%
0/1
0%
0/33
0%
0/1
0%
0/39
0%
1/1
100%
5/18
27.78%
_
_
0/5
0%
0/189
0%
_
_
TRI
3/7
42.86%
11/252
4.37%
1/5
20%
1/158
0.63%
6/7
85.71%
51/254
20.08%
5/12
41.67%
29/379
7.65%
0/5
0%
0/182
0%
1/4
25%
1/115
0.87%
_
_
2/5
40%
6/137
4.38%
0/1
0%
0/27
0%
3/3
100%
10/72
13.89%
6/7
85.71%
38/207
18.36%
AXI
1/6
16.66%
3/190
1.58%
1/9
11.11%
2/278
0.72%
6/12
50%
35/424
8.25%
54/64
84.38%
279/1890
14.76%
12/20
60%
54/609
8.87%
2/4
50%
6/91
6.59%
7/13
43.75%
29/324
8.95%
9/24
37.50%
45/585
7.69%
7/24
29.17%
37/651
5.68%
1/2
50%
2/45
4.44%
2/3
66.67%
19/78
24.35%
STR
1/4
25%
1/134
0.75%
1/2
50%
3/45
6.67%
1/5
20%
2/203
0.99%
11/21
52.38%
50/615
8.13%
11/13
84.62%
71/348
20.40%
0/1
0%
0/15
0%
4/5
80%
20/143
13.99%
1/2
50%
6/46
13.04%
5/6
83.33%
22/125
17.6%
_
_
_
_
DOM
_
_
0/1
0%
0/35
0%
2/4
50%
15/116
12.93%
0/4
0%
0/108
0%
0/1
0%
0/19
0%
5/6
83.3%
28/223
12.56%
2/2
100%
9/51
17.65%
0/2
0%
0/69
0%
0/2
0%
0/51
%
1/3
33.33%
4/69
5.8%
0/2
0%
0/67
0%
SLO
_
_
0/1
100%
0/22
0%
_
_
4/14
28.57%
23/427
5.39%
6/6
100%
22/160
13.75%
2/3
66.67%
22/92
23.91%
11/13
84.62%
67/375
17.87%
8/13
61.54%
49/348
14.08%
2/3
66.67%
5/74
6.76%
_
_
_
_
SOK
2/4
50%
3/121
2.48%
0/1
0%
0/31
0%
4/5
80%
17/182
9.34%
10/24
41.67%
54/685
7.88%
2/3
66.67%
14/97
14.43%
0/3
0%
0/82
0%
2/11
18.18%
2/276
0.72%
_
_
2/5
40%
16/140
11.43%
_
_
_
_
INT
_
_
_
_
0/1
0%
0/39
0%
7/22
31.81%
34/530
6.42%
1/4
25%
20/115
17.39%
1/1
100%
1/24
4.17%
1/3
33.33%
11/87
12.64%
1/5
20%
11/137
8.03%
_
_
_
_
_
_
ADS
_
_
0/2
0%
0/70
0%
3/3
100%
33/143
23.08%
2/2
100%
2/56
3.57%
_
_
1/3
33.33%
10/97
10.31%
_
_
_
_
_
_
_
_
3/6
50%
9/214
4.21%
PIN
_
_
_
_
4/5
80%
34/128
26.56%
3/4
75%
10/114
8.77%
_
_
0/1
0%
0/23
0%
_
_
_
_
_
_
2/9
22.22%
6/230
2.61%
_
_
544 K. Olšavská, C.J. Löser

Homoploid and Heteroploid Hybridizations
In total, 564 pollinated capitula from experimental crosses were examined for pres-
ence of well-developed achenes. In crosses between species belonging to the same
group (either the C.triumfetti or C.montanus group), the frequency of successful
crosses (0.99 %–17.6 %) and the number of well-developed achenes was lower or
similar to crosses within species/subspecies (11.69 %–22.15 %). Both types of
artificial crosses (between and within species/subspecies) yielded lower production
of well-developed achenes than open pollination (Table 2, Fig. 2). The low percent-
age of well-developed achenes in controlled pollinations might be the consequence of
methodology (the artificial crosses did not sufficiently simulate natural pollination).
Seed sets of up to 72.4 % (Fig. 2), yielded from homoploid crossing experiments
between taxa of the C.triumfetti group, demonstrate their incomplete reproductive
isolation under experimental conditions. The success of homoploid crosses was inde-
pendent of the direction of the cross (Mann-Whitney U-test; P=0.215–0.960) but
depend significantly on the type of crosses (Kruskal-Wallis test; P<0.001). The per-
centage of well-developed achenes of most homoploid crosses was between 2.61 % and
8.87 %. The highest percentages of well-developed achenes were given by crosses C.
pinnatifidus ×C.triumfetti s. str. (18.36 %; 26.56 %; hereinafter, the two percentage
values indicate two directions of reciprocal crosses), C.dominii ×C.strictus (12.75 %;
13 %), and ‘intermediate morphotype’×C.strictus (17.6 %; 17.39 %). Crossing C.
strictus ×C.triumfetti s. str. yielded no or only few (0.99 %) well-developed achenes
(Table 2, Fig. 3). Because only a very low rate of autogamy was detected, the progeny of
homoploid crosses was regarded as being of hybrid origin.
Fig. 2 Box-and-whisker plots displaying percentages of well-developed achenes per capitulum resulting
from open pollination, isolation and hybridization experiments of the Cyanus triumfetti (TRI) and C.
montanus (MON) groups (N–number of inflorescences). Each box represents the distance between the 1st
and 3rd quartiles; horizontal line within the box is the second quartile (median); whiskers show the highest
and lowest data points or 1.5 times the box; crosses indicate outliers
Mating System and Hybridization in Cyanus 545

Table 3 Germination rate of hybrids obtained from experimental crosses of the Cyanus triumfetti and C.montanus groups. Abbreviations used: G –number of achenes used for
germination, AG –number of germinated achenes, MON –C.montanus s. str., MOL –C.mollis, TRI –C.triumfetti s. str., AXI –‘C.axillaris’, STR –C.strictus, DOM –C.
dominii subsp. dominii, SLO –C.dominii subsp. slovenicus, SOK –C.dominii subsp. sokolensis, INT –‘intermediate morphotype’,ADS–C.adscendens, PIN –C.pinnatifidus.
The achenes obtained from intraspecies crosses are indicated in italic
Maternal plant
MON
MOL
TRI
AXI
STR
DOM
SLO
SOK
INT
ADS
PIN
AG/G (%)
AG/G (%)
AG/G (%)
AG/G (%)
AG/G (%)
AG/G (%)
AG/G (%)
AG/G (%)
AG/G (%)
AG/G (%)
AG/G (%)
Pollen donor
MON
_
_
_
_
_
_
_
2/6 (33.33%)
_
_
_
MOL
_
_
0/22 (0%)
0/7 (0%)
1/1 (100%)
_
_
_
_
_
_
TRI
0/5 (0%)
_
10/47 (21.28%)
8/16 (50%)
_
3/6 (50%)
_
0/6 (0%)
_
0/2 (0%)
1/7 (14.29%)
AXI
2/3 (66.67%)
_
4/22 (18.18%)
67/131 (51.15%)
11/16 (68.75%)
1/1 (100%)
24/28 (85.71%)
21/35 (60%)
9/28 (32.15%)
0/2 (0%)
12/19 (63.16%)
STR
_
0/2 (0%)
_
16/32 (50%)
22/31 (70.97%)
_
5/13 (38.46%)
_
5/5 (100%)
_
_
DOM
_
_
9/11 (81.81%)
_
_
12/20 (60%)
3/27 (11.11%)
_
_
4/4 (100%)
_
SLO
_
_
_
10/19 (52.63%)
8/13 (61.54%)
0/4 (0%)
4/18 (22.22%)
20/30 (66.67%)
_
_
_
SOK
1/1 (100%)
_
5/17 (29.41%)
31/64 (48.44%)
_
_
9/10 (90%)
_
12/15 (80%)
_
_
INT
_
_
_
11/26 (42.31%)
_
0/1 (0%)
2/11 (18.18%)
2/11 (18.18%)
3/3 (100%)
_
_
ADS
_
_
5/31 (16.13%)
1/2 (50%)
_
3/10 (30%)
_
_
_
_
1/7 (14.29%)
PIN
_
_
1/13 (7.69%)
7/10 (70%)
_
_
_
_
_
2/6 (33.33%)
_
546 K. Olšavská, C.J. Löser

The success of homoploid crosses of Central and Western European species of the
C.triumfetti group was negatively correlated with geographic distance (Mantel
test; r=−0.75, P<0.05). However, no significant correlation between success of
homoploid crosses and geographic distances was obtained when C.adscendens
from the Dinarides and C.pinnatifidus from the Eastern Carpathians were included
(Mantel test; r=−0.14, P=0.44).
The percentage of germinated achenes from interspecies crosses of the C.trium-
fetti group varied from 7.69 % (C.pinnatifidus ×C.triumfetti s. str.) to 100 %
(‘intermediate morphotype’×C.strictus); however, these results are hard to interpret
as high variation in germination rate was also detected for seeds from intraspecific
crosses (21.28 % –70.79 %; Table 3, Fig. 3) and some hybrid combinations were
represented by few or no well-developed achenes.
After division of samples of ‘C.axillaris’into two geographical groups (Austrian-
Czech vs Hungarian-Slovak samples; Fig. 4), differences were observed among crosses
within (16.98 %, 16.45 %; hereinafter, the two percentage values indicate two directions
of reciprocal crosses) and between these groups (5.30 % , 10.53 % ), however, the
Fig. 3 Results of the crossing experiments between taxa of the Cyanus triumfetti group. For each type of
crossing, mean percentage of well-developed achenes and mean percentage of germinated achenes (in
brackets) is indicated. The results in grey are obtained from less than five capitula; thickness of arrows is
proportional to seed production. Abbreviations used: TRI –C.triumfetti s. str., AXI –‘C.axillaris’, STR –
C.strictus, DSS –C.dominii, INT –‘intermediate morphotype’
Mating System and Hybridization in Cyanus 547

difference was not statistically significant (Mann-Whitney U-test; P=0.086). Apparent
asymmetry in reciprocal crosses involving either western (Austrian-Czech samples) or
eastern ‘C.axillaris’(Hungarian-Slovak samples) and geographically adjacent taxa was
also not significant (Fig. 4, Mann-Whitney U-test; P=0.471–0.987). Only in crosses
between both geographic groups of ‘C.axillaris’, seed set was marginally significantly
higher (Mann-Whitney U-test; P=0.065) when plants from the eastern part of Central
Europe served as pollen donor. The crosses between Alpine C.triumfetti s. str. and
Austrian-Czech plants of ‘C.axillaris’yielded much more well-developed achenes
(21.26 %, 20.28 %) in comparison to crosses between Alpine C.triumfetti s. str. and
Hungarian-Slovak plants of ‘C.axillaris’(1.97 %, 2.26 %). In crosses with C.strictus,
Hungarian-Slovak plants of ‘C.axillaris’were more successful (11 %, 9.11 %) than
Austrian-Czech plants of ‘C.axillaris’(5.29 %, 5.68 %).
The success of heteroploid crosses was reduced. Only 97 (2.96 %) achenes from a total
of 3,280 achenes were well developed (Table 2). Flow cytometric screening of some
achenes (N=46; Table 4) showed that the progeny of heteroploid crosses comprised 63 %
of triploid hybrids (29 achenes) of presumable hybrid origin on tetraploid (9 achenes) as
well diploid maternal plants (20 achenes). Beside triploid, tetraploid maternal plants had
tetraploid progeny (two achenes; 4.00 %) and diploid maternal plants had diploid progeny
(five achenes; 10.87 %), for which autogamous origin is expected. In nine cases (19.57 %)
the ploidy level of embryos could not be identified by flow cytometry. Among the 47
achenes resulting from heteroploid crosses, six germinated (13 %), however, four seed-
lings died subsequently and the remaining two survivors were proven using flow
cytometry analyses to be diploid and thus originated probably from self-pollination.
Fig. 4 Results of the crossing experiments of Austrian-Czech vs Hungarian-Slovak samples of ‘Cyanus
axillaris’. For each type of crossing, mean percentage of well-developed achenes is indicated; thickness of
arrows is proportional to seed production. Abbreviations used: AXI west –samples of ‘C.axillaris’from
the Czech Republic and Austria, AXI east –samples of ‘C.axillaris’from Slovakia and Hungary, TRI –C.
triumfetti s. str., STR –C.strictus, DSS –C.dominii, INT –‘intermediate morphotype’
548 K. Olšavská, C.J. Löser

Discussion
Mating System
In this study, emasculation experiments were performed to control for apomixis in the
Cyanus triumfetti and C.montanus groups. The genus Cyanus (as Centaurea L. s.l.) was
previously reported being able to reproduce apomictically (Czapik 1996) because apospo-
rous female gametophytes were observed for Cyanus segetum Hill. by Poddubnaja-Arnoldi
(1931). However, Noyes (2007) concluded that because evidence of effective apomixis is
lacking and C.segetum is diploid (apomicts are restricted to polyploids) apomictic
reproduction is probably not present in the genus Cyanus. As expected, no evidence of
apomictic seed formation was found within both studied groups in the present study.
All investigated taxa should be considered to be allogamous with a functional self-
incompatibility system with a negligible rate of autogamous seed production. This
confirms previous results for C.montanus s. str. This species was referred to as strictly
allogamous by Briquet (1902), and Gonnet (1992,1996) obtained no autogamous
Table 4 Number, germination and ploidy level of achenes from successful heteroploid crosses of the
Cyanus triumfetti and C.montanus groups. A –number of all achenes, AW –number of well-developed
achenes, G –number of achenes used for germination, AG –number of germinated achenes
Maternal plant Pollen donor AW/A AG/G Progeny ploidy levels
2x3x4xUndetected by FCM
MON 1/4 (4x) TRI 62/6 (2x) 1/29 ––1––
MON 1/4 (4x) TRI 51/3 (2x) 2/36 ––2––
MON 1/5 (4x) TRI 51/2 (2x) 1/27 1(0)/1 ––– –
MON 3A/2 (4x) TRI 40/4 (2x) 1/32 –––1–
MON 3B/20 (4x) TRI 62/6 (2x) 8/25 0/5 –3––
MON 3B/19 (4x) TRI 62/6 (2x) 1/41 ––1––
MOL 2/1 (4x) TRI 62/6 (2x) 1/44 –––1–
MOL 2/I (4x) TRI 39/19 (2x) 2/13 0/2 ––– –
MOL 4/5 (4x) TRI 23/3 (2x) 2/32 ––2––
TRI 38/2 (2x) MON 6/1 (4x) 5/40 –32––
TRI 41/4 (2x) MON 3B/19 (4x) 9/69 0/5 –3–1
TRI 49/3 (2x) MON 1/4 (4x) 3/18 2/3 (2) –– –
TRI 51/2 (2x) MON 1/5 (4x) 6/18 2(0)/6 ––– –
TRI 62/6 (2x) MON 3B/19(4x) 1/40 ––1––
TRI 62/6 (2x) MON 1/4 (4x) 13/48 0/6 –7––
TRI 62/6 (2x) MON 1/4 (4x) 8/70 0/6 –2––
TRI 62/6 (2x) MON 3B/20 (4x) 20/59 0/10 –5––
TRI 15/2 (2x) MOL 3/1 (4x) 2/22 0/2 ––– –
TRI 39/19 (2x) MOL 2 (4x) 1/28 1(0)/1 ––– –
TRI 51/4 (2x) MOL 2/IV (4x) 5/23 –––– –
TRI 62/6 (2x) MOL 2/1 (4x) 5/49 –––– 5
Mating System and Hybridization in Cyanus 549

progeny in self-pollination experiments of C.montanus s. str. Asteraceae possess a
homomorphic sporophytic self-incompatibility system (De Nettancourt 2001), which
likely holds also for Cyanus. It is typical for this system that specific proteins on the
pollen grain coat are expressed by the anther tapetum of the paternal plant and the pollen
itself (Castric and Vekemans 2004). The sporophytic system is a multi-allelic one-locus
system with a complicated hierarchy of dominance-recessive interactions between alleles
that allows only plants carrying distinct alleles to reproduce (De Nettancourt 2001).
Occasionally, self-pollination or crosses that ought to be incompatible gives a low
percentage of seeds. Such deviations from self-incompatibility could be caused by
genetic factors or induced by presence of allied/heterospecific pollen (mentor effect),
environmental variables (e.g., high temperature) and phenology (e.g., delayed pollina-
tion; Ortiz et al. 2006; Ferrer and Good-Avila 2007). Similarly, environmental factors
and phenology could be responsible for producing autogamous achenes in the isolation
experiment of the C.triumfetti and C.montanus groups. The allied pollen could
influence production of presumable autogamous achenes in heteroploid crosses ofthese
groups. Sporadic breakdown of self-incompatibility is relatively frequent (Ortiz et al.
2006) and has been observed for other Asteraceae species (e.g., Hieracium L., Mráz
2003;Picris L., Slovák et al. 2007;Teph roseri s (Rchb.) Rchb., Janišová et al. 2012).
Low incidence of autogamy has also been revealed for close relatives of the genus
Cyanus from Centaurea sect. Jacea (Mill.) DC. and Centaurea sect. Acrolophus (Cass.)
DC. (Gardou 1972;SunandRitland1998; Hardy et al. 2001;Kouteckýetal.2011).
Homoploid Hybridization and Crossability within the C. triumfetti Group
The results from the reciprocal crosses involving individuals of the C.triumfetti
group provide an insight into the evolutionary relationship of this highly polymorphic
group. Taxa of the C.triumfetti group sharing the ploidy level 2n=2x=22 hybridize
easily and produce viable progeny. Crossing and field experiments in groups related
to the genus Cyanus, such as Centaurea sect. Jacea, have also shown that hybrid-
ization within the same ploidy level is frequent and hybrids are fertile and capable of
back-crossing (Gardou 1972; Hardy et al. 2001;Štěpánek and Koutecký 2004).
For most of the C.triumfetti group differentiation has not resulted in a complete
reproductive isolation. Differences in the percentage of well-developed achenes per
capitulum obtained from interspecific crosses among members of this group suggested
different levels of reproductive isolation. Obtained patterns of reproduction isolation
do not just reflect geographic distance. For example, high percentages of well-
developed achenes were obtained in crosses between Alpine C.triumfetti s. str. and
geographically remote Eastern Carpathian C.pinnatifidus, and Slovenian C.adscen-
dens, but also among geographically close C.dominii,C.strictus and an ‘intermediate
morphotype’from the Western Carpathians. While allopatry predicts the strong
reproductive barrier of C.triumfetti s. str. with C.strictus, the ‘intermediate morpho-
type’and Hungarian-Slovak samples of ‘C.axillaris’, the high success of crosses
between C.triumfetti s. str. Austrian-Czech samples of ‘C.axillaris’is surprising.
Reduced reproduction rates in intraspecies crosses between the two geographic and
genetic groups of ‘C.axillaris’and high success observed in Hungarian-Slovak
samples of ‘C.axillaris’with Pannonian C.strictus line up with results of the previous
AFLP study (Olšavská et al. 2011). Genetic differentiation connected with spatial
550 K. Olšavská, C.J. Löser

segregation may be considered as the most important factors preventing hybridization
within the C.triumfetti group.
Worthy of notice are two divergent chloroplast haplotypes found in the C.trium-
fetti group: the common haplotype is shared by C.triumfetti s. str., C.adscendens, the
west samples of ‘C.axillaris’and some individuals of C.pinnatifidus; while all
remaining samples, including the eastern range of ‘C.axillaris’, possess the other
haplotype (Löser, unpubl. data). Interestingly, most crosses between taxa/groups of
samples with the same chloroplast haplotype yielded high seed sets. Observed
asymmetry of some crosses between samples belonging to different cpDNA lineages
(for example C.triumfetti s. str. × C.dominii, Austrian-Czech samples of ‘C.
axillaris’× Hungarian-Slovak samples of ‘C.axillaris’) indicates that incompatibility
may be connected to cytoplasmic factors. Interaction between autosomal loci and
uniparentally inherited factors (including mitochondria, chloroplasts and maternal
transcripts) are specific to a particular direction of hybridization and therefore
contribute to asymmetric reproduction isolation (Turelli and Moyle 2007).
Heteroploid Hybridization between the C. triumfetti and C. montanus Groups
The C.triumfetti and C.montanus groups belonging to different cytotypes are repro-
ductively well isolated. Only a few triploid hybrids were found in the crossing experi-
ments while tetraploid and diploid progeny could be assigned to autogamy. Only a low
number of seeds germinated, and no triploid seedlings were found. During sample
preparation for flow cytometry analyses we observed that some seeds that proved to
be triploid contained an aborted embryo. Others produced no peaks for the embryo and it
was not possible to detect its ploidy level (Table 4). We did not detect the presence of
endosperm nuclei in triploid achenes using flow cytometry, which should indicate
poorly developed endosperm (but this could be caused by the long time between
ripening and ploidy level measurement, too). Aborted embryos of triploids and absence
of triploid seedlings from experimental crosses indicate that post-zygotic barriers exist
between diploid species of the C.triumfetti group and tetraploids of the C.montanus
group. Hence, gene flow between them is highly unlikely even in the extreme/theoretical
case when only pollen of different ploidy level is available. Heteroploid crosses achieve
fertilization but the resulting embryo does not develop into a mature and viable seed,
because deviation in maternal:paternalgenome ratio could halt endosperm development
and seed ripening (Bretagnolle and Thompson 1995). Guinochet (1957a,b)previously
reported successful experimental crossing between C.triumfetti s. l. and C.montanus s.
l., where two plants with intermediate morphotype with 2n=33 and 2n=44 originated.
However, regular bivalents or trivalents suggesting homologous genomes were ob-
served during metaphase of presumable interspecific hybrids and autogamous origin
was not excluded (Guinochet 1957b).
Low incidence of (triploid) hybrids in crossing experiments together with their absence
in natural populations is documented for other pairs of diploid –tetraploid taxa of subtribe
Centaureinae (Centaurea sect. Jacea;Gardou1972; Hardy et al. 2001; Koutecký 2007).
Koutecký et al. (2011) showed that mixed pollination (e.g., 2x+4xpollen), more pre-
sumable in nature, contributed to the reproductive isolation of Centaurea species belong-
ing to different cytotypes. Thus, using only one type of pollen in experimental crosses
might cause incongruence between experimental and field observation.
Mating System and Hybridization in Cyanus 551

Interspecific hybrids and introgressed forms of C.triumfetti s. str. and C.montanus s.
str. reported from the Alps (Gugler 1907;Gonnet1993) can be explained by gene flow
via unreduced ovules of diploids. However, this event might be very rare in the C.
triumfetti group because until now no triploid or tetraploid plant was found in nature in
spite of an extensive study (645 plants/141 populations analyzed in Europe; Olšavská
and Löser 2012).
Several premating isolation mechanisms may evolve to avoid interspecies hybrid-
ization. Such premating isolation between sympatric taxa of the C.triumfetti group and
of the C.montanus group is represented mainly by habitat differentiation. However, if
the studied taxa of these two groups occur in close vicinity, they have opportunity for
hybridization because an overlap in flowering time together with no pollination prefer-
ences have been recorded. In contrast to Gonnet (1993), we did not observe a shift of
flowering period of the plants of the C.triumfetti group and those of the C.montanus
group during our experiments.
Conclusions
In the present study, thorough attention has been given to the mode of reproduction and
degree of reproductive isolation within and between the diploid C.triumfetti group and the
tetraploid C.montanus group. Based on the presented results, investigated taxa of the C.
triumfetti and C.montanus groups are almost strictly allogamous. Low production of
autogamous seeds could be induced by deviations of the environmental factors or phenol-
ogy in the isolation experiments and by the presence of allied pollen in heteroploid crosses.
Reproductive barriers within the C.triumfetti group are weak. Genetic differenti-
ation may be considered the most important factor responsible for different levels of
reproductive isolation. Seed set and germination success in most interspecific crosses
suggests that the possibility of gene flow in natural populations is high but species
integrity is maintained also by geographic and ecological isolation.
The reproductive barrier connected with different chromosome number seems to be
sufficient to prevent hybridization in experimental conditions. Heteroploid crosses
resulted in a few mature triploid seeds with no or low viability. Hence, interploidy gene
flow is, beside different ecological preferences of the C.triumfetti and C.montanus
groups, limited by inconspicuous pre- (pollen incompability) or/and post-zygotic mech-
anisms (e.g., seed abortion, inviability of offspring).
Acknowledgments This study was supported by the Grant Agency of the Ministry of Education of the Slovak
Republic and the Slovak Academy of Sciences (VEGA 2/0075/11) and Research and Development Support
Agency of the Slovak Republic (APVV-0320-10). This study received funds also from the Millennium Seed Bank
of the Royal Botanic Gardens, Kew (United Kingdom). Michaela Horváthová and Lýdia Skokanová are deeply
acknowledged for help with cultivating of plants and performing experiments; Marián Perný, Iva Hodálová and
Patrik Mráz are thanked for valuable discussions and critical reading of the manuscript.
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Received: 28 April 2012 /Revised: 9 October 2012 / Accepted: 18 October 2012 /
Published online: 21 March 2013
554 K. Olšavská, C.J. Löser